MX2012005432A - Microbial engineering for the production of chemical and pharmaceutical products from the isoprenoid pathway. - Google Patents

Abstract

The invention relates to recombinant expression of a taxadiene synthase enzyme and a geranylgeranyl diphosphate synthase (GGPPS) enzyme in cells and the production of terpenoids.

Description

MICROBIAL ENGINEERING FOR THE PRODUCTION OF CHEMICAL AND PHARMACEUTICAL PRODUCTS FROM THE ROUTE OF ISOPRENOID RELATED REQUESTS This application claims the benefit under 35 USC § 1 19 (E) of the US Provisional Application Serial No. 61 / 280,877, entitled "Microbial Engineering for the Production of Chemical and Pharmaceutical Products from Isoprenoid Pathway," filed on November 10. of 2009 and the Provisional US Application Serial No. 61 / 388,543, entitled "Microbial Engineering for the Production of Chemical and Pharmaceutical Products from Isoprenoid Pathway," filed on September 30, 2010, the entire descriptions of which are incorporated as reference in the present in their totalities.

GOVERNMENT INTEREST This work was funded in part by the National Institutes of Health under Grant Number 1-R01-GM085323-01A1. The government has certain rights in this invention.

TECHNICAL FIELD The invention relates to the production of one or more terpenoids through microbial engineering.

BACKGROUND OF THE INVENTION Taxol and its structural analogs have been recognized as the most powerful and commercially successful anti-cancer drugs introduced in the last decade.1 Taxol was the first isolated first of the Pacific yew tree bark, 2 and the production methods in the early phase they required sacrificing two to four fully adult trees to supply a sufficient dose for a patient.3 The structural complexity of Taxol required a complex chemical synthesis path that required 35-51 steps with the highest yield of 0.4% .4, 56 However a semi-synthetic route was devised with which the baccatin III biosynthetic intermediate was first isolated from two plant sources and subsequently converted to Taxol.7 While this approach and subsequent production efforts based on plant cell cultures have decreased the need to harvest the yew, production still depends on plant-based procedures8 with the accompanying limitations of productivity and scalability, and limitations in the number of Taxol derivatives that can be synthesized in the search for more effective drugs.9,10. 9, 10 BRIEF DESCRIPTION OF THE INVENTION Recent developments in metabolic engineering and synthetic biology offer new possibilities for the overproduction of complex natural products through more technically docile microbial hosts.11, 2 Although exciting progress has been made in clarifying the biosynthetic mechanism of Taxol in Taxus, 13"16 the commercially relevant Taxol producing strains have eluded previous attempts that point to the transfer of this complex biosynthetic machinery into a microbial host.17 '18 Still, as with other natural products, microbial production through of metabolically designed strains, offers an attractive economy and great potential to synthesize a diverse series of new compounds with activity against cancer and another pharmaceutical.19, 20 The metabolic pathway for Taxol and its analogues consists of an upstream pathway of isoprenoid that is native to E. coli, and a heterologous pathway downstream of terpenoid (Fig. 6). The routes of upstream mevalonic acid (MVA) or methyleritritol phosphate (MEP) can produce the two common building blocks, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), of which Taxol and other isoprenoid compounds are trained.12 Recent studies have highlighted the engineering of previous upstream routes to support the biosynthesis of heterologous isoprenoids such as lycopene and artemisinic acid.21"23 The downstream route of taxadiene has been reconstructed in E. coli, but, to date, valuations have not exceeded 1.3 mg / L.24 24 Previous approaches to rational metabolic engineering focused on either the upstream path (MVA or MEP) or the downstream terpenoid, implicitly assuming that the modifications are additive, that is, linear behavior. While this approach may yield moderate increases in flow, it generally ignores non-specific effects, such as the toxicity of intermediate metabolites, cellular effects of the vectors used for expression, and hidden unknown routes that may compete with the main route and divert the flow away from the desired target. Combinatorial approaches can avoid such problems by offering the opportunity to adequately test the parameter space and clarify these complex nonlinear interactions 21,28, 2930 However, they require a high performance test, which is often not available for many natural products desirable.31 Yet another class of route optimization methods have explored the combinatorial space of different sources of the heterologous genes that comprise the route of interest.32 Still dependent on a high throughput assay, these methods generally ignore the need to determine a optimal level of expression for the individual gene pathway and, as such, has shown less effectiveness in structuring an optimal pathway.

In the present work, as an example of aspects of the invention, we focus on the optimal balance between the upstream route, the IPP forming route and the downstream path of the taxadiene synthesis terpenoid. This is achieved by grouping the nine-enzyme pathway into two modules - a four-gene, upstream, native (MEP) path module and a downstream, two-gene pathway for taxadiene (Fig. 1A and 1B ). Using this basic configuration, parameters such as the effect of plasmid copy number on cell physiology, gene order and promoter strength in an expression cassette, and chromosomal integration are evaluated with respect to their effect on taxadiene production. This modular and multivariable combinatorial approach allows us to efficiently test the main parameters that affect the flow of the route without the need for a high performance test. The multivariate search through multiple promoters and copy numbers for each route module reveals a highly non-linear landscape of taxadiene flow with a global maximum that exhibits a 15,000-fold increase in taxadiene production over the control, yielding 300 mg / L of taxadiene production in small scale fermentations. In addition, we designed the oxidation chemistry based on P450 in the biosynthesis of Taxol in E. coli, with our strains designed to improve the production of taxadien-5a-ol 2400 times over the previous technique. These improvements unlock the potential for large-scale production of thousands of valuable terpenoids by well-established microbial systems.

The aspects of the invention relate to methods that involve recombinantly expressing a taxadiene synthase enzyme and a geranylgeranyl diphosphate enzyme (GGPPS) enzyme in a cell that overexpresses one or more components of the non-mevalonate (MEP) pathway. In some embodiments the cell is a bacterial cell like an Escherichia coli cell. In some embodiments, the bacterial cell is a Gram-positive cell such as a Bacillus cell. In some embodiments, the cell is a yeast cell such as a Saccharomyces cell or a Yarrowia cell. In some embodiments, the cell is an alga cell or a plant cell.

In some embodiments, the enzyme taxadiene synthase is a Taxus enzyme such as an enzyme from Taxus brevifolia. In some embodiments, the GGPPS enzyme is a Taxus enzyme such as an enzyme from Taxus canadenis. In some embodiments, the gene encoding the enzyme taxadiene synthase and / or the gene encoding the GGPPS enzyme and / or the genes encoding the one or more components of the MEP pathway are expressed from one or more plasmids In some embodiments, the gene encoding the enzyme taxadiene synthase and / or the gene encoding the GGPPS enzyme and / or the genes encoding the one or more components of the MEP are integrated into the genome of the cell.

In some embodiments, one or more components of the non-mevalonate (MEP) pathway is selected from the group consisting of dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, ispA and ispB. In certain modalities, dxs, idi, ispD and ispF are overexpressed. For example, dxs, idi, ispD and ispF can be overexpressed in the dxs-idi-idpDF operon. In some embodiments, the gene encoding the enzyme taxadiene synthase and the gene encoding the GGPPS enzyme are expressed together in an operon.

In some embodiments, the cell also expresses a taxadiene 5a-hydroxylase (T5ctOH) or a catalytically active portion thereof. In certain embodiments, the T5aOH enzyme or a catalytically active portion thereof is fused to a cytochrome P450 reductase enzyme or a catalytically active portion thereof. For example, the T5aOH enzyme can be At2T5a or H-tTC P R.

The expression of the enzyme taxadiene synthase, the GGPPS enzyme and the one or more components of the MEP route can be balanced to maximize the production of taxadiene. The methods associated with the invention may further comprise culturing a cell to produce taxadiene or Taxadien-5a-ol. In some embodiments, at least 10 mg L'1 of taxadiene is produced. In certain embodiments, at least 250 mg L -1 of taxadiene is produced.In some embodiments, at least 10 mg L "1 of Taxadien-5a-ol is produced. In certain embodiments, at least 50 mg L'1 of Taxadien-5a-ol is produced. In some embodiments, the conversion ratio of taxadiene to Taxadien-5a-ol and byproduct 5 (12) -Oxa-3 (11) -cyclotaxane is at least 50%, at least 75% or at least 95% .

The methods associated with the invention may comprise also recover the taxadiene or Taxadien-5a-ol cell culture. In some embodiments, taxadiene or Taxadien-5a-ol is recovered from the gas phase while in other embodiments, an organic layer is added to the cell culture, and taxadiene or Taxadien-5a-ol is recovered from the organic layer.

Aspects of the invention relate to cells overexpressing one or more components of the non-mevalonate (MEP) pathway, and that recombinantly expresses a taxadiene synthase enzyme and a geranylgeranyl diphosphate synthase enzyme (GGPPS). In some embodiments the cell is a bacterial cell like an Escherichia coli cell. In some embodiments, the bacterial cell is a Gram-positive cell such as a Bacillus cell. In some embodiments, the cell is a yeast cell such as a Saccharomyces cell or a Yarrowia cell. In some embodiments, the cell is an alga cell or a plant cell.

In some embodiments, the enzyme taxadiene synthase is a Taxus enzyme such as an enzyme from Taxus brevifolia. In some embodiments, the GGPPS enzyme is a Taxus enzyme such as an enzyme from Taxus canadenis. In some embodiments, the gene encoding the enzyme taxadiene synthase and / or the gene encoding the GGPPS enzyme and / or the genes encoding the one or more components of the MEP pathway are expressed from one or more plasmids In some embodiments, the gene encoding the enzyme taxadiene synthase and / or the gene encoding the GGPPS enzyme and / or the genes encoding the one or more components of the MEP are integrated into the genome of the cell.

In some embodiments, the one or more components of the non-mevalonate pathway (MEP) is selected from the group consisting of dxs, spC, ispD, ispE, ispF, ispG, ispH, idi, ispA and ispB. In certain modalities, dxs, ¡di, ispD and ispF are overexpressed. For example, dxs, idi, ispD and ispF can be overexpressed in the dxs-idi-idpDF operon. In some embodiments, the gene encoding the enzyme taxadiene synthase and the gene encoding the GGPPS enzyme are expressed together in an operon. In some embodiments, the expression of the enzyme taxadiene synthase, the GGPPS enzyme and the one or more components of the MEP pathway are balanced to maximize the production of taxadiene.

In some embodiments, the cell further expresses a taxadiene 5a-hydroxylase (T5aOH) or a catalytically active portion thereof. In certain embodiments, the T5aOH enzyme or a catalytically active portion thereof is fused to a cytochrome P450 reductase enzyme or a catalytically active portion thereof. For example, the T5aOH enzyme can be At2T5aOH-tTCPR. In some embodiments, the cell produces taxadiene and / or taxadiene-5a-ol.

Aspects of the invention relate to methods for selecting a cell that exhibits the increased production of a terpenoid, including creating or obtaining a cell that overexpresses one or more components of the non-mevalonate (MEP) pathway, producing terpenoid the cell, comparing the amount of terpenoid produced from the cell to the amount of terpenoid produced in a control cell, and selecting a first improved cell that produces a higher amount of terpenoid than a control cell, wherein a first improved cell which produces a higher amount of terpenoid than the control cell is a cell that exhibits the increased production of terpenoid.

In some embodiments, the cell recombinantly expresses a terpenoid synthase enzyme and / or a geranylgeranyl diphosphate synthase enzyme (GGPPS). The methods may further comprise altering the level of expression of one or more of the components of the non-mevalonate (MEP) pathway, the terpenoid synthase enzyme and / or the geranylgeranyl diphosphate enzyme (GGPPS) enzyme in the first cell improved to produce a second improved cell, and compare the amount of terpenoid produced from the second improved cell to the amount of terpenoid produced in the first improved cell, wherein a second improved cell produces a higher amount of terpenoid than the first cell Improved is a cell that exhibits increased production of terpenoid. In some embodiments, the enzyme terpenoid synthase is a taxadiene enzyme synthase. The cell can also express recombinantly any of the polypeptides associated with the invention.

Aspects of the invention relate to isolated polypeptides comprising a taxadiene enzyme 5a-hydroxylase (T5aOH) or a catalytically active portion thereof fused to a cytochrome P450 reductase enzyme or a catalytically active portion thereof. In some embodiments, the cytochrome P450 reductase enzyme is a Taxus cytochrome P450 reductase (TCPR). In certain embodiments, the taxadiene 5a-hydroxylase and the TCPR are linked by a linker such as GSTGS (SEQ ID NO: 50). In some embodiments, the taxadiene 5a-hydroxylase and / or TCPR are truncated to remove all or part of the transmembrane region. In certain embodiments, 8, 24, or 42 amino acids of the amino terminus of taxadiene 5a-hydroxylase are truncated. In certain embodiments, 74 amino acids of TCPR are truncated. In some embodiments, an additional peptide is fused to the taxadiene 5a-hydroxylase. In certain embodiments, the additional peptide is bovine 17a-hydroxylase. In certain embodiments, the peptide is MALLLAVF (SEQ ID NO: 51). In certain embodiments, the isolated polypeptide is At24T5aOH-tTCPR. Aspects of the invention also encompass nucleic acid molecules that encode any of the polypeptides associated with the invention and cells that recombinantly express any of the polypeptides associated with the invention.

Aspects of the invention relate to methods for increasing the production of terpenoid in a cell that produces one or more terpenoids. The methods include controlling the accumulation of indole in the cell or in a culture of the cells, thereby increasing the production of terpenoid in a cell. Any of the cells described herein can be used in the methods, including bacterial cells, such as Escherichia coli cells; Gram-positive cells, such as Bacillus cells; yeast cells, such as Saccharomyces cells or Yarrowia cells; alga cells; plant cell; and any of the designed cells described herein.

In some embodiments, the step of controlling indole accumulation in the cell or in a culture of the cells includes balancing the upstream path of the non-mevalonate isoprenoid pathway with the downstream product synthesis pathways and / or modifying or regulating the indole route. In other embodiments, the step of controlling indole accumulation in the cell or in a culture of the cells also includes or includes removing the accumulated indole from fermentation by chemical methods, such as using absorbents or captors.

The one or more terpenoids produced by the cells or in the culture may be a monoterpenoid, a sesquiterpenoid, a diterpenoid, a triterpenoid or a tetraterpenoid. In certain embodiments, the terpenoid is taxadiene or any precursor of taxol.

Aspects of the invention relate to methods that include measuring the amount or concentration of indole in a cell that produces one or more terpenoids or in a culture of cells that produce one or more terpenoids. Methods may include measuring the amount or concentration of indole two or more times. In some embodiments, the measured amount or concentration of indole is used to guide a process to produce one or more terpenoids. In some embodiments, the measured amount or concentration of indole is used to guide the construction of strains.

These and other aspects of the invention, as well as various embodiments thereof, will become more apparent with respect to the drawings and detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS The attached drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in several figures is represented by a similar numeral. For purposes of clarity, not every component can be marked in each drawing. In the drawings: Figures 1A and 1B The engineering of the modular multivariate isoprenoid pathway reveals strong nonlinear response in terpenoid accumulation. To increase the flow through the upstream route of MEP, we concentrated the enzymatic steps of reported bottleneck (dxs, idi, ispD and ispF) for overexpression by an operon (dxs-idi-ispDF) .28 To channel the Excess capacity flow of the universal isoprenoid precursors, IPP and DMAPP, towards the biosynthesis of Taxol, a synthetic downstream synthase operon of GGPP (G) and taxadiene (T) 16 synthase genes was constructed. The upstream and synthetic isoprenoid routes downstream of taxadiene were placed under the control of inducible promoters to control their relative expression of genes. (Figure 1A) Diagram of the two modules, the native upstream MEP sioprenoid pathway (left) and the taxadiene synthetic pathway (right). In the biosynthetic network of E. coli, the isoprenoid route of MEP is initiated by the condensation of glyceraldehyde-3 phosphate (G3P) and pyruvate (PYR) precursors of glycolysis. The bifurcation of the Taxol route starts from the universal isoprenoid precursors IPP and DMAPP to first form the "linear" precursor of Geranylgeranyl diphosphate, and then the "cyclic" taxadiene, a committed and key intermediary for Taxol. The cyclic olefinic taxadiene undergoes multiple series of stereospecific oxidations, acylations, benzoylation with the secondary chain assembly, and finally, forms Taxol. (Figure 1B) Schematic of the multivariate modular isoprenoid route design approach to test the nonlinear response in the terpenoid accumulation of cells designed in the 5 'and 3' direction path. The expression of the upstream and downstream routes is modulated by varying the strength of the promoter (Trc, T5 and T7) or by increasing the number of copies using different plasmids. The variation of upstream and downstream path expression gives different maxima in taxadiene accumulation.

Figures 2A-2D Optimization of taxadiene production by regulating the expression of the upstream and downstream modular routes. (Figure 2A) Response in the accumulation of taxadiene to the increase in the forces of the upstream path for constant values of the downstream path. (Figure 2B) the dependence of the downstream path for constant increases in force on the upstream path. Multiple local maxima observed in the taxadiene response depend on the increase in expression strength of the upstream or downstream path. (Figure 2C) taxadiene response of designed strains (17-24) with high overexpression in the upstream path (20-100) with two different downstream expressions (~ 30 and ~ 60) to identify the taxadiene response with balanced expressions . The expression of the downstream path of the low copy plasmid (p5 and p10) under strong operon T7TG promoter was used to modulate these expressions. Note that on both upstream and downstream routes expressed from different plasmids with different promoters may impose metabolic loading of plasmid birth. (Figure 2D) Modulating the upstream path with increasing strength of the chromosome promoter with two different downstream expressions (~ 30 and ~ 60) to identify the lost search space with reduced toxic effects (strains 25-32).

Figures 3A and 3B The metabolite correlates inversely with the production of taxadiene. (Figure 3A) mass spectrum of the metabolite that was detected correlates inversely with the production of taxadiene in the strain constructs of Figs. 2A-2D. The observed peaks characteristic of the metabolite are 233, 207, 178, 117, 89 and 62. (Figure 3B) Correlation between the isoprenoid by-product of Fig. 3A and taxadiene. Strains 26-29 and 30-32, all with chromosomatically integrated pathway expression upstream, were chosen for consistent comparison.

In strains 26-29 and 30-32, upstream expression increased by shifting to the Trc promoters, at T5 and T7 respectively. The two sets of strains vary only in the expression of the downstream path with the second set (30-32) having twice the level of expression of the first. With the first set, the optimal equilibrium is achieved with strain 26, using the Trc promoter for upstream path expression and also showing the lowest accumulation of metabolite. With strains 30-32, strain 31 shows the lowest metabolite accumulation and highest taxadiene production. The data demonstrate the inverse correlation observed between the unknown production of metabolite and taxadiene.

Figures 4A-4D Transcriptional gene expression levels of upstream and downstream pathways and changes in cell physiology of designed strains. The relative expression of the first genes in the operon of the upstream path (DXS) and downstream (T) is quantified by qPCR. Similar profiles of expression were observed with the genes downstream of the operons. The corresponding strain numbers are shown in the graph. (Figure 4A) DXS gene expression of quantified relative transcript level of different upstream expressions modulated using promoters and plasmids under two different downstream expressions. (Figure 4B) TS gene expression of quantified relative transcript level of two different downstream expressions modulated using plasmids p517 and p10T7 under different upstream expressions. Our analysis of gene expression directly supports the hypothesis, with increase in plasmid copy number (5, 10 and 20) and promoter strength (Trc, T5 and T7) the expression of upstream and downstream pathways can be modulated. (Figure 4C) Cell growth of the strains designed 25-29. The growth phenotype was affected by the activation of isoprenoid metabolism (strain 26), expression of recombinant protein (strain 25) and metabolic load of plasmid birth (strain against control designed) and (Figure 4D) growth phenotypes of strains 17, 22, 25-32. The black lines are the designed strains that produce the taxadiene and the gray lines are the control strains without downstream expression that carry an empty plasmid with promoter and multiple cloning sites. The growth was correlated to the activation of terpenoid metabolism, metabolic load of plasmid birth as well as the expression of recombinant protein.

Figures 5A-5D Design of the oxidation chemistry of Taxol p450 in E. coli. (Figure 5A) Schemes of the conversion of taxadiene to taxadiene 5a-ol to Taxol. (Figure 5B) Transmembrane design and construction of a taxane component protein taxane 5a-ol hydroxylase (T5aOH) and Taxus cytochrome P450 reductase (TCPR). 1 and 2 represent the full-length proteins of T5aOH and TCPR identified with TM regions of 42 and 74 amino acids respectively, 3-chimera enzymes generated from the three different designed T5aOH constructions of TM, (At8T5aOH, At24T5aOH and At42T5aOH constructed by fusing a peptide synthetic from 8 residues (A) to truncated T5aOH of 8, 24 and 42 AA) by a truncated TCPR (tTCPR) translational fusion of 74 AA using a 5 residue GSTGS linker peptide. (Figure 5C) functional activity of the constructs 5At8T5aOH-tTCPR, At24T5aOH-tTCPR and At42T5aOH-tTCPR transformed into strain 18 which produces taxadiene. (Figure 5D) Time course profile of accumulation and growth profile of taxadien-5a-ol of strain 18-At24T5aOH-tTCPR fermented in a 1L bioreactor.

Figure 6 Biosynthetic scheme for the production of taxol in £. coli Schemes of the two modules, native isoprenoid route upstream (left) and synthetic Taxol route (right). In the biosynthetic network of E. coli, the divergence of the MEP isoprenoid pathway starts from the glyceraldehyde-3 phosphate (G3P) and pyruvate (PYR) precursors of glycolysis (l-V). The bifurcation of the Taxol route starts from the isoprenoid precursor of E. coli IPP and DMAPP to the "linear" precursor of Geranilgeranil diphosphate (VIII), "cyclic" taxadiene (IX), 5a-ol "oxidized" taxadiene (X) to multiple series of stereospecific oxidations, acylations, benzoylations and epoxidation for the early precursor Baccatin III (XII) and finally with the secondary chain assembly to Taxol (XIII). DXP-1-deoxy-D-xylulose-5-phosphate, MEP-2C-methyl-D-erythritol-4-phosphate, CDP-ME-4-diphosphocytidyl-2-methyl-D-erythritol, CDP-MEP-4-diphosphocytidyl- 2C-methyl-D-erythritol-2-phosphate, ME-cPP-2C-methyl-D-erythritol-2,4-cyclodiphosphate, IPP-isopentenyl diphosphate, DMAPP-dimethylallyl diphosphate. Genes involved biosynthetic pathways of G3P and PYR to Taxol. DXS-1-deoxy-D-xylulose-5-phosphate synthase, ispC-1-Deoxy-D-xylulose-5-phosphate reductoisomerase, lspD-4-diphosphocytidyl-2C-methyl-D-erythritol synthase, lspE-4- diphosphoc Tidyl-2-C-methyl-D-erythritol kinase, lspF-2C-Methyl-D-erythritol-2,4-cyclodiphosphate Synthase, lspG-1-hydroxy-2-methyl-2 - (/ =) - butenyl- 4- diphosphate synthase, lspH-4-hydroxy-3-methyl-2- (£) -butenyl-4-diphosphate reductase IDI-isopentenyl diphosphate isomerase, GGPPS-geranyl geranyl diphosphate synthase, Taxadiene synthase, Taxoid da-hydroxylase, Taxoid-5a-0-acetyltransferase, Taxoid 13a-hydroxylase, Taxoid-β-hydroxylase, Taxoid 2a-hydroxylase, Taxoid 2-0-benzoyltransferase, Taxoid 7 -hydroxylase, Taxoid 10-O-acetyltransferase, Taxoid-β-hydroxylase * , Taxoid 9a-hydroxylase, Taxoid 9-keto-oxidase *, Taxoid C4.C20- ß-epoxidase *, phenylalanine aminomutase, secondary chain CoA-ligase *, Taxoid 13 O-phenylpropanoyltransferase, Taxoid 2'-hydroxylase *, Taxoid 3 ' -N-benzoyltransferase.216,219 * The tagged genes are pending identification ficados or characterized.

Figure 7 Times of improvements in the taxadiene production of the modular route expression search. Taxadiene response in times of improvements of all the observed maxims of Figures 2A, 2B, and 2C compared to strain 1. 2.5 times of differences between two highest maxims (strain 17 and 26) and 23 times (strain 26 and 10) with the lowest indicates that the loss of an optimal response results in appreciably lower valuations.

Figures 8A-8C The metabolite (Figure 8A) Correlation between the accumulation of taxadiene to the metabolite. The accumulation of the metabolite of the designed strain is related in an anti-proportional manner to the production of taxadiene in an exponential manner. The correlation coefficient for this ratio was determined as 0.92 (Figure 8B) Representative GC profile of strains 26-28 to demonstrate the change in accumulation of taxadiene and metabolite. The numbers in chromatogram 1 and 2 corresponding to the metabolite peak and taxadiene respectively. (Figure 8C) GC-MS profile of metabolite (1) and taxadiene (2) respectively. The observed peaks characteristic of the metabolite are 233, 207, 178, 117, 89 and 62. Taxa-4 (20), 11, 12-diene ion characteristic m / z 272 (P +), 257 (P + -CH3), 229 ( P + -C3H7); 121, 122, 123 (clustering of ring C fragment) .60 The peak marked with a star is the internal standard caryophyllene.

Figures 9A-9D. GC-MS profiles and taxadiene / taxadien-5a-ol production of artificial chimera enzyme designed in strain 26. (Figure 9A) GC profile of hexane: ether extract (8: 2) of three constructs (A -At8T5aOH-tTCPR, t24T5aOH-tTCPR and At42T5aOH-tTCPR) transferred to strain 26 and fermented for 5 days. Labels 1, 2 and 3 in the peaks correspond to taxadiene, taxadien-5a-ol and 5 (12) -Oxa-3 (11) -cyclotaxane (OCT) respectively. (Figure 9B) The production of taxa-4 (20), 11, 12-dien-5a-ol and OCT quantified all three strains. (Figure 9C) and (Figure 9D) GC-MS profile of taxa-4 (20), 11, 12-dien-5a-ol and OCT and the peaks corresponding to the fragmentation were compared with the authentic standards and previous reports 4247 The GC-MS analysis confirmed the identity of the mass spectrum with authentic taxa-4 (20), 11, 12-dien-5a-ol with characteristic ion m / z 288 (P +), 273 (P + -H20 ), 255 (P + -H20-CH3).

Figure 10 presents a scheme that represents the route terpenoid biosynthetic and the natural products produced by this route.

Figure 11 presents a scheme representing the modulation of the upstream path to amplify the production of taxadiene.

Figure 12 presents a scheme representing the modulation of the downstream path to amplify the production of taxadiene.

Figure 13 presents a diagram indicating that the divergence of the newly identified route is not the characteristic of the downstream synthetic route.

Figures 14A-14B. The strength of the route is correlated to transcriptional gene expression levels. (Figure 14A) relative expression of genes di, ispD and ispF with increase in force in the upstream path and force in the downstream path in 31 arbitrary units, and (Figure 14B) relative expression of idi genes, ispD to ispF with increase in strength in the 5 'direction route and force in the downstream route in 61 arbitrary units. As expected, gene expression increased as the strength of the upstream path increased. The corresponding strain numbers are indicated in the bar graph. Relative expression was quantified using maintenance rrsA gene expression. The data are means + / - SD for four replicas.

Figures 15A-15C The impact of byproduct accumulation of indole metabolite on taxadiene and growth production. (Figure 15A) Reverse correlation between taxadiene and indole. Strains 26 to 28 and 30 to 32, all with chromosomatically integrated pathway expression upstream, They were chosen for the consistent comparison. The two sets of strains vary only in the expression of the downstream path with the second set (30 to 32) having twice the level of expression of the first. In strains 26 to 28 and 30 to 32, upstream expression increased by shifting to the Trc promoters, at T5 and T7, respectively. With the first set, the optimal equilibrium is achieved with strain 26, using the Trc promoter for upstream path expression and also showing the lowest indole accumulation. With strains 30 to 32, strain 31 shows the lowest indole accumulation and the highest taxadiene production. The times of improvements are relative to Iña strain 25 and 29, respectively, for the two sets. (Figure 15B) Effect of indole externally introduced in the production of taxadiene for the high production strain 26. Different indole concentrations were introduced in cultures of cells grown in minimal medium with 0.5% yeast extract. The taxadiene production was appreciably reduced as the concentration of indole increased from 50 mg / L to 100 mg / L (Figure 15C) Effect of indole externally introduced into the cell growth for designed E. coli strains. The data are means + / - SD for three replicas. Strains devoid of the downstream route and with different forces from the upstream route (1, 2, 6, 21, 40 and 100) were selected. Strain 26, the high producer of taxadiene, exhibits the strongest inhibition.

Figures 16A-16E The unknown metabolite identified as indole. (Figure 16A) and (Figure 16A1) Gas chromatogram and mass spectrum of the unknown metabolite extracted using hexane from the cell culture. (Figure 16B) and (Figure 16B1) corresponds to the gas chromatogram and mass spectrum of pure indole dissolved in hexane. To further confirm the chemical identity, the metabolite was extracted from the fermentation broth using hexane extraction and purified by silica column chromatography using hexane: ethyl acetate (8: 2) as eluent. The purity of the compound was confirmed by TLC and GC-MS. The 1HRMN and 13CRMN spectra confirmed the chemical identity of the metabolite as indole. (Figure 16C) 1H NMR spectrum of indole extracted from cell culture (CDCI3, 400 MHz) d: 6.56 (d, 1 H, Ar CH), 7.16 (m, 3H, Ar CH), 7.38 (d, 1 H, Ar CH), 7.66 (d, 1 H, Ar CH), 8.05 (b, 1 H, Indol NH). (Figure 16D) 3CNMR d: 135.7, 127.8, 124.2, 122, 120.7, 119.8, 111, 102.6. (Figure 16E) is the spectrum of 1HRMN of pure indole.

Figures 17A-17D. Batch-fed culture of strains designed in a 1 L bioreactor Time-course accumulation of taxadiene (Figure 17A), cell growth (Figure 17B), accumulation of acetic acid (Figure 17C) and total substrate addition (glycerol) (Figure 17D) ) for strains 22, 17 and 26 during 5 days of cultivation in bioreactor batch fed in 1 L bioreactor containers under pH and oxygen conditions controlled with minimum media and 0.5% yeast extract. After the glycerol was depleted at -0.5 to 1 g / L in the fermenter, 3 g / L of glycerol was introduced into the bioreactor during fermentation. The data are means of two replicas of bioreactors.

DETAILED DESCRIPTION OF THE INVENTION Taxol is a powerful anticancer drug first isolated as a natural product of the Pacific tree Taxus brevifolia. However, the safe and cost-efficient production of Taxol or Taxol analogues by traditional routes of production of limited plant extracts. Here, we report a multivariate modular approach to design the metabolic pathway to amplify by -15,000 times the production of taxadiene in a designed Escherichia coli. Taxadiene, the first committed intermediary of Taxol, is the biosynthetic product of the non-mevalonate route in E. coli comprising two modules: the upstream native pathway that forms Isopentenyl pyrophosphate (IPP) and a heterologous pathway that forms a terpenoid in address 3 '. The multivariate systematic search identified the conditions that optimally balance the two route modules to minimize the accumulation of inhibitory intermediates and the flow deviation to secondary products. We also designed the next step, after taxadiene, in the biosynthesis of Taxol, a step of oxidation based on P450, which yielded > 98% substrate conversion and presents the first example of in vivo production of any functionalized intermediary of Taxol in E. coli. The design approach of the modular route not only highlights the complexity of multi-step routes, but also allowed the accumulation of high taxadiene and taxadien-5a-ol (~ 300mg / L and 60mg / L, respectively) valuations in small-scale fermentations, thus exemplifying the potential of the microbial production of Taxol and its derivatives.

This invention is not limited in its application to the details of construction and arrangement of components set forth in the foregoing description or illustrated in the drawings. The invention may have other modalities and may be practiced or performed in various ways. Also, the phraseology and terminology used herein are for the purpose of the description and should not be considered as limiting. The use of "includes," "comprises," or "has," which "contains", "implies", and the variations thereof herein, are intended to encompass the items listed below and their equivalents as well as articles additional The microbial production of terpenoids as taxadiene is demonstrated in the present. When expressed at satisfactory levels, microbial routes dramatically reduce the cost of producing such compounds. Additionally, they use cheap, abundant and renewable raw materials (such as sugars and other carbohydrates) and can be the source for the synthesis of numerous derivatives that can exhibit properties far superior to the original compound. A key element in the competitive production of compound costs of the isoprenoid route using a microbial route is the amplification of this route to allow the overproduction of these molecules. Described herein are methods that increase or amplify the flow toward terpenoid production in Escherichia coli (E. coli). Specifically, the methods are provided to amplify the metabolic flux to the synthesis of isopentenyl pyrophosphate (IPP) (a key intermediate for the production of isoprenoid compounds), dimethylallyl pyrophosphate (DMAPP), geranyl diphosphate (GPP), diphosphate farnesyl (FPP), geranylgeranyl diphosphate (GGPP), and farnesyl geranyl diphosphate (FGPP), paclitaxel (Taxol), ginkolides, geraniol, farnesol, geranilgeraniol, linalool, isoprene, monoterpenoids as menthol, carotenoids as lycopene, polyisoprenoids as polyisoprene or natural rubber, diterpenoids such as eleuterobine, and sesquiterpenoids such as artemisinin.

Aspects of the invention relate to the production of terpenoids. As used herein, a terpenoid, also referred to as an isoprenoid, is an organic chemical derived from a five carbon isoprene unit. Several non-limiting examples of terpenoids, classified based on the number of isoprene units they contain, include: hemiterpenoids (1 unit of isoprene), monoterpenoids (2 units of isoprene), sesquiterpenoids (3 units of isoprene), diterpenoids (4 units) of isoprene), sesterterpenoids (5 units of isoprene), triterpenoids (6 units of isoprene), tetraterpenoids (8 units of isoprene), and polyterpenoids with a larger number of isoprene units. In some embodiments, the terpenoid that is produced is taxadiene. In some embodiments, the terpenoid that is produced is Citronellol, Cubebol, Nootkatone, Cineol, Limonene, Eleuterobine, Sarcodictiin, Pseudopterosins, Ginkgolides, Stevioside, Rebaudioside A, sclareol, labdenediol, levopimaradiene, sandracopimaradiene or isopemaradiene.

Described herein are methods and compositions for optimizing the production of terpenoids in cells by controlling the expression of genes or proteins that take part in an upstream path and a downstream path. The upstream route involves the production of isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which can be achieved by two different metabolic pathways: the mevalonic acid route (MVA) the MEP pathway (2-C-methyl) -D-erythritol 4 phosphate), also called the MEP / DOXP pathway (2-C-methyl-D-erythritol 4-phosphate / 1-deoxy-D-xylulose 5 phosphate), the non-mevalonate pathway or the independent route of mevalonic acid.

The downstream path is a synthetic route leading to the production of a terpenoid and involves the expression of the recombinant gene of a terpenoid synthase enzyme (also referred to as terpene cyclase), and a geranylgeranyl diphosphate synthase enzyme (GGPPS). In some embodiments, a terpenoid synthase enzyme is a diterpenoid synthase enzyme. Several non-limiting examples of diterpenoid synthase enzymes include casbene synthase, taxadiene synthase, levopimaradiene synthase, abietadiene synthase, isopymaradiene synthase, e / 7f-copalyl diphosphate synthase, syn-stemar-13-ene synthase, syn-stemod-13 ( 17) -enoate synthase, syn-pimara-7,15-diene synthase, enf-sandaracopimaradiene synthase, enf-cassa-12,15-diene synthase, en.-pimara-8 (14), 15-diene synthase, kaur-15-eno synthase, enf-kaur-16-eno synthase, afidicolan-163-ol synthase, fillocladan-16a-ol synthase, fusicocca-2,10 (1) -dione, synthase, and terpentetriene cyclase.

Surprisingly, as demonstrated in the Examples section, the optimization of terpenoid synthesis by the manipulation of the upstream and downstream routes described herein was not a simple linear or additive process. Rather, by complex combinatorial analysis, optimization was achieved by balancing the components of the upstream and downstream routes. Unexpectedly, as shown in Figures 1A-1 B and 2A-2D and Table A, the accumulation of taxadiene exhibited a strong non-linear dependence on the relative strengths of the upstream MEP and downstream taxadiene synthesis routes.

TABLE A Genetic details of the strains that produce the taxadiene. 1. Ep20TrcGT 2. EC lTrc EPp20GT 3. Ep5Trc EPp20TrcGT 4. Ep10Trc EPp20TrcGT 5. Ep20TrcTG 5 6. Ep20T5GT 7. Ep20T5GTTrcT 8. ECh 1 TrcM EPp20TrcTG 9. ECh 1 TrcM EPp20T5GT 10. ECh 1TrcMEPp20T5GTTrcT 1 1. Ep5Trc EPp20TrcTG 12. Ep5Trc EPp20T5GT 13. Ep5TrcMEPp20T5GTTrct 14. Ep10TrcMEPp20TrcTG 15. Ep10TrcMEPp20T5GT 16. Ep10TrcMEPp20T5GTTrcT 17. EDE3p10TrcMEPp5T7TG 0 18. EDE3p20TrcMEPp5T7TG 19. EDE3p20T5MEPp5T7TG 20. EDE3p20T7MEPp5T7TG 21. EDE3p5TrcMEPp10T7TG 22. EDE3p20TrcMEPp10T7TG 23. EDE3p20T5MEPp10T7TG 24. EDE3p20T7MEPp10T7TG 25. EDE3p5T7TG 26. EDE3Ch1 TrcMEPp5T7TG 27. EDE3Ch 1 T5MEPp5T7TG 28. EDE3Ch1T7MEPp5T7TG (- 29. EDE3p10T7TG ¾ 30. EDE3Ch1 TrcMEPp10T7TG 31. EDE3Ch1 T5MEPp10T7TG 32. EDE3Ch1T7MEPp10T7TG The numbers corresponding to different strains and their corresponding genotype, E-E. coli K12mG1655 ArecAAendA, EDE3-E. coli K12mG1655 ArecAAendA with T7 RNA polymerase DE3 constructed on the 0 chromosome, MEP-dxs-idi-ispDF operon, GT-GPPS-TS operon, TG-TS-GPPS operon, copy on chromosome Ch1-1, Trc-Trc promoter, T5 promoter - T5, promoter T7 - T7, copy of plasmid p5, p10, p20 - ~ 5 (SC101), -10 (p15), and -20 (pBR322) Aspects of the invention are related to controlling the expression of genes and proteins in the MEP route for the optimized production of a terpenoid such as taxadiene. The optimized production of a terpenoid refers to producing a higher amount of a terpenoid followed by an optimization strategy that would be achieved in the absence of such a strategy. It should be appreciated that any gene and / or protein within the MEP pathway are encompassed by the methods and compositions described herein. In some embodiments, a gene within the MEP path is one of the following: dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, ispA or ispB. The expression of one or more genes and / or proteins within the MEP route can be upregulated and / or downregulated. In certain embodiments, up-regulation of one or more genes and / or proteins within the MEP pathway may be combined with down-regulation of one or more genes and / or proteins within the MEP pathway.

It should be appreciated that genes and / or proteins can be regulated alone or in combination. For example, the expression of dxs can be upregulated or downregulated alone or in combination with upregulation or down regulation of the expression of one or more of ispC, ispD, ispE, ispF, ispG, ispH, idi, ispA and ispB. The expression of ispC may be upregulated or downregulated alone or in combination with upregulation or down regulation of the expression of one or more of dxs, ispD, ispE, ispF, ispG, ispH, idi, ispA and ispB. The expression of ispD can be upregulated or down-regulated alone or in combination with upregulation or down-regulation of the expression of one or more of dxs, ispC, ispE, ispF, ispG, ispH, idi, ispA and ispB. The expression of ispE can be upregulated or downregulated alone or in combination with upregulation or down regulation of the expression of one or more of dxs, ispC, ispD, ispF, ispG, ispH, idi, ispA and ispB. The expression of ispF can be upregulated or downregulated alone or in combination with upregulation or down regulation of the expression of one or more of dxs, ispC, ispD, ispE, ispG, ispH, idi, ispA and ispB. The expression of ispG may be upregulated or downregulated alone or in combination with upregulation or down regulation of the expression of one or more of dxs, ispC, ispD, ispE, ispF, ispH, idi, ispA and ispB. The expression of ispH may be upregulated or downregulated alone or in combination with upregulation or down regulation of the expression of one or more of dxs, ispC, ispD, ispE, ispF, ispG, idi, ispA and ispB. The expression of idi can be upregulated or downregulated alone or in combination with upregulation or down regulation of the expression of one or more of dxs, ispC, ispD, ispE, ispF, ispG, ispH, ispA and ispB. The expression of ispA can be upregulated or downregulated alone or in combination with upregulation or down regulation of the expression of one or more of dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi and ispB. The expression of ispB can be upregulated or downregulated alone or in combination with upregulation or down regulation of the expression of one or more of dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi and ispA. In some embodiments, the expression of the gene and / or the protein of one or more of dxs, ispC, ispD, ispE, ispF, ispG, ispH, and I di is upregulated while expression of the gene and / or ispA protein and / or ispB is downgraded.

The expression of genes within the MEP pathway can be regulated in a modular method. As used herein, regulation by a modular method refers to the regulation of multiple genes together. For example, in some embodiments, multiple genes within the MEP pathway are recombinantly expressed in a contiguous region of DNA, such as an operon. It should be appreciated that a cell expressing such a module can also express one or more other genes within the MEP path either recombinantly or endogenously.

A non-limiting example of a gene module within the MEP pathway is a module containing the dxs, idi, ispD and ispF genes, as presented in the Examples section, and referred to herein as dxs-idi-ispDF . It should be appreciated that the gene modules within the MEP pathway, consistent with aspects of the invention, can contain any of the genes within the MEP pathway, in any order.

The expression of genes and proteins within the synthetic path downstream of terpenoid synthesis can also be regulated to optimize terpenoid production. The synthetic route downstream of terpenoid synthesis involves the recombinant expression of a terpenoid synthase enzyme and a GGPPS enzyme. Any terpenoid synthase enzyme, as discussed above, can be expressed with GGPPS depending on the downstream product being produced. For example, the taxadiene synthase is used for the production of taxadiene. The Recombinant expression of the enzyme taxadiene synthase and the GGPPS enzyme can be regulated independently or together. In some embodiments the two enzymes are regulated together in a modular fashion. For example, the two enzymes can be expressed in an operon in any order (GGPPS-TS, referred to as "GT," or TS-GGPPS, referred to as "TG").

Manipulation of the expression of genes and / or proteins, including modules such as the dxs-idi-ispDF operon, and like the TS-GGPPS operon, can be accomplished by methods known to one of ordinary skill in the art. For example, the expression of genes or operons can be regulated by the selection of promoters, as inducible promoters, with different forces. Several non-limiting examples of promoters include Trc, T5 and T7. Additionally, the expression of genes or operons can be regulated by manipulation of the copy number of the gene or operon in the cell. For example, in certain embodiments, a strain containing an additional copy of the dxs-idi-ispDF operon on its chromosome under control of the Trc promoter produces an increased amount of taxadiene with respect to one that overexpresses only the downstream synthetic route. In some embodiments, the expression of genes or operons can be regulated by manipulating the order of the genes within a module. For example, in certain embodiments, changing the order of genes in a synthetic operon downstream from GT to TG results in a 2-3 fold increase in taxadiene production. In some modalities, the expression of genes or operons is regulated by the integration of one or more genes or operons in a chromosome. For example, in certain embodiments, integration of the upstream operon of dxs-idi-ispDF into the chromosome of a cell results in increased production of taxadiene.

It should be appreciated that the genes associated with the invention can be obtained from a variety of sources. In some modalities, genes within the MEP pathway are bacterial genes such as Escherichia coli genes. In some embodiments, the gene encoding GGPPS is a plant gene. For example, the gene encoding GGPPS may be from a Taxus species such as Taxus canadensis (T. canadensis). In some embodiments, the gene encoding the taxadiene synthase is a plant gene. For example, the gene coding for the taxadiene synthase may be from a Taxus species such as Taxus brevifolia (G. brevifolia). GenBank representative Access numbers for T. canadensis GGPPS and taxadiene synthase of T. brevifolia is provided by AF081514 and U48796, the sequences of which are integrated as a reference herein in their totalities.

As you know one of ordinary skill in the art, homologous genes for use in methods associated with the invention may be derived from another species and may be identified by homology searches, for example by a BLAST search of protein available in the National Center for Biotechnology Information (NCBI) Internet site (www.ncbi.nlm.nih.gov). The genes and / or operons associated with the invention can be cloned, for example by PCR amplification and / or digestion restriction, of DNA from any source of DNA containing the given gene. In some embodiments, a gene and / or operon associated with the invention is synthetic. Any means of obtaining a gene and / or operon associated with the invention is compatible with the present invention.

In some embodiments, further optimization of terpenoid production is achieved by modifying a gene before it is expressed recombinantly in a cell. In some embodiments, the enzyme GGPPS has one or more of the following mutations: A162V, G140C, L182M, F218Y, D160G, C184S, K367R, A151T, M185I, D264Y, E368D, C184R, L331 I, G262V, R365S, A114D, S239C, G295D, I276V, K343N, P183S, I172T, D267G, I149V, T234I, E153D and T259A. In some embodiments, the GGPPS enzyme has a mutation in residue S239 and / or residue G295. In certain embodiments, the GGPPS enzyme has the mutation S239C and / or G295D.

In some embodiments, modification of a gene before it is expressed recombinantly in a cell involves optimization of the codon for expression in a bacterial cell. The codon uses for a variety of organisms can be accessed in the Codon Use Database (www.kazusa.or.jp/codon/). Codon optimization, including identification of the optimal codon for a variety of organisms, and methods for achieving codon optimization, is familiar to one of ordinary skill in the art, and can be achieved using standard methods.

In some embodiments, modifying a gene before it is expressed recombinantly in a cell involves making one or more mutations in the gene before it is expressed recombinantly in a cell. For example, a mutation may involve a substitution or deletion of a single nucleotide or multiple nucleotides. In some embodiments, a mutation of one or more nucleotides in a gene will result in a mutation in the produced protein of the gene, such as a substitution or deletion of one or more amino acids.

In some embodiments, it may be advantageous to use a cell that has been optimized for the production of a terpenoid. For example, in some embodiments, a cell overexpressing one or more components of the route not mevalonate (MEP) is used, at least in part, to amplify diphosphate isopentyl (IPP) and diphosphate dimethylallyl (DMAPP) , the substrates of GGPPS. In some embodiments, overexpression of one or more components of the non-mevalonate (MEP) pathway is achieved by increasing the number of copies of one or more components of the non-mevalonate (MEP) pathway. For example, the number of component copies in the rate-limiting steps in the MEP path such as (dxs, ispD, ispF, idi) can be amplified, as per additional episomal expression.

In some modalities, "rational design" participates in the construction of specific mutations in proteins such as enzymes. As used herein, "rational design" refers to integrate knowledge of the enzyme, or related, as its three-dimensional structure, their active sites, their substrates and / or interaction between the enzyme and substrate enzymes , in the design of the specific mutation. Based on a rational design approach, mutations can be created in an enzyme that can then be investigated for the increased production of a terpenoid relative to the control levels. In some modalities, mutations can be rationally designed based on the modeling of homology. As used herein, "homology modeling" refers to the method of constructing an atomic model of resolution of a protein of its amino acid sequence and a three-dimensional structure of a homologous related protein.

In some embodiments, random mutations can be made in a gene, such as a gene that codes for an enzyme, and these mutations can be investigated for the increased production of a terpenoid relative to the control levels. For example, research for mutations in components of the MEP pathway, or components of other pathways, that leads to increased production of a terpenoid can be done by a random mutagenesis test, or by the investigation of known mutations. In some embodiments, cloning of triggering genomic fragments could be used to identify the genomic regions that lead to an increase in the production of a terpenoid, by investigating cells or organisms that have those fragments for the increased production of a terpenoid. Sometimes one or more mutations they can be combined in the same cell or organism.

In some embodiments, the production of a terpenoid in a cell can be increased by manipulation of enzymes that act in the same path as the enzymes associated with the invention. For example, in some embodiments it may be advantageous to increase the expression of an enzyme or other factor acting upstream of a target enzyme such as an enzyme associated with the invention. This could be achieved by over-expressing the upstream factor using any standard method.

The optimization of protein expression can also be achieved by selection of appropriate promoters and ribosome binding sites. In some embodiments, this may include the selection of high copy number plasmids, or low or medium number copy plasmids. The step of transcription termination can also be directed for the regulation of gene expression, by the introduction or elimination of structures such as stem loops.

Aspects of the invention relate to the expression of recombinant genes in cells. The invention encompasses any type of cell that recombinantly expresses genes associated with the invention, including prokaryotic and eukaryotic cells. In some embodiments, the cell is a bacterial cell, such as Escherichia spp., Streptomyces spp., Zymonas spp., Acetobacter spp., Citrobacter spp., Synechocystis spp., Rhizobium spp., Clostridium spp., Corynebacterium spp., Streptococcus spp. , Xanthomonas spp., Lactobacillus spp., Lactococcus spp., Bacillus spp., Alcaligenes spp., Pseudomonas spp., Aeromonas spp., Azotobacter spp., Comamonas spp., Mycobacterium spp., Rhodococcus spp., Gluconobacter spp., Ralstonia spp., Acidithiobacillus spp., Microlunatus spp., Geobacter spp., Geobacillus spp., Arthrobacter spp., Flavobacterium spp., Serratia spp., Saccharopolyspora spp., Thermus spp., Stenotrophomonas spp., Chromobacterium spp., Sinorhizobium spp. , Saccharopolyspora spp., Agrobacterium spp. and Pantoea spp. . The bacterial cell can be a Gram negative cell such as an Escherichia coli cell (E. coli), or a Gram positive cell as a Bacillus species. In other embodiments, the cell is a fungal cell such as a yeast cell, eg, Saccharomyces spp., Schizosaccharomyces spp., P / cft / 'a spp., Pa / fia spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp., Yarrow / spp., And industrial strains of polyploid yeast. Preferably the yeast strain is a strain of S. cerevisiae or a strain of Ya / row / 'a spp. Other examples of fungi include Aspergillus spp., Pennicilium spp., Fusarum spp., Rhizopus spp., Acremonium spp., Neurospora spp., Sordaria spp., Magnaporthe spp., Allomyces spp., Ustilago spp., Botrytis spp., And Trichoderma spp. In other embodiments, the cell is an alga cell, or a plant cell. It should be appreciated that some cells compatible with the invention can express an endogenous copy of one or more of the genes associated with the invention as well as a recombinant copy. In some modalities, if a cell has an endogenous copy of one or more of the genes associated with the invention then the methods will not necessarily add a recombinant copy of the genes that are endogenously expressed. In some embodiments, the cell can endogenously express one or more enzymes of the routes described herein can recombinantly express one or more other enzymes of the routes described herein for the efficient production of a terpenoid.

Additional aspects of the invention relate to research for bacterial cells or strains that exhibit optimized terpenoid production. As described above, the methods associated with the invention involve generating cells that overexpress one or more genes in the MEP pathway. The production of terpenoid from the culture of such cells can be measured and can be compared to a control cell wherein a cell that exhibits a higher amount of a terpenoid production with respect to a control cell is selected as a first improved cell . The cell can be further modified by recombinant expression of a terpenoid synthase enzyme and a GGPPS enzyme. The level of expression of one or more of the components of the non-mevalonate pathway (MEP), the terpenoid synthase enzyme and / or the GGPPS enzyme in the cell can then be manipulated and the terpenoid production can be measured. time, leading to the selection of a second improved cell that produces larger quantities of a terpenoid than the first improved cell. In some embodiments, the enzyme terpenoid synthase is a taxadiene enzyme synthase.

Additional aspects of the invention relate to the identification and characterization (through GC-MS) of a previously unknown metabolite in bacterial E. coli cells (Figures 3A-3B and 6). The level of accumulation of the newly identified metabolite, indole, can be controlled by genetically manipulating the microbial pathway by overexpression, down-regulation or mutation of the genes of the isoprenoid pathway. The indole metabolite is anti-correlated as a direct variable to the production of taxadiene in designed strains (Figures 3A-3B, 6 and 15A-15C). Also controlling the accumulation of indole to improve the flow towards terpenoid biosynthesis in bacterial systems (specifically in cells, such as E. coli cells) or other cells, can be achieved by balancing the upstream route of non-mevalonate isoprenoid with the routes downstream of product synthesis or by modifications or regulation of the indole route. In doing so, the skilled person can reduce or control the accumulation of indole and thereby reduce the inhibitory effect of indole in the production of taxadiene, and other terpenoids derived from the routes described, such as: monoterpenoids, sesquiterpenoids (including amorphadiene), diterpenoids (including levopimaradiene), triterpenes, and tetraterpenes. Other methods to reduce or control indole accumulation include removing the accumulated indole from fermentation by chemical methods such as using absorbers, captors, etc.

In other embodiments, methods are provided which include measuring the amount or concentration of indole in a cell that produces one or more terpenoids or in a culture of cells that produce one or more terpenoids. The amount or concentration of indole can be measured once, or two or more times, as convenient, using methods known in the art and those described herein. Such methods can be used to guide processes to produce one or more terpenoids, for example, in the improvement of the process. Such methods can be used to guide the construction of strains, for example, for the improvement of strains.

The identification of the means to achieve this balance yielded an improvement of 1, 5000 times in the overproduction of terpenoids as taxadiene, compared with the wild type bacterial cells, expressed with a heterologous biosynthetic taxadiene pathway. The production was further increased by the modified fermentation methods that yielded concentrations of approximately 2g / L, which is 1500 times higher compared to any previously reported taxadiene production. As demonstrated herein, by genetically engineering the non-mevalonate soprenoid pathway in E. coli, the accumulation of this metabolite can now be controlled by regulating the flow toward isoprenoid biosynthesis in E. coli bacterial cells.

It is also demonstrated herein that channeling the production of taxadiene in the next key precursor to Taxol, taxadien-5a-ol, achieved by designing the oxidation chemistry for Taxol biosynthesis. Example 5 presents the first successful extension of the taxadiene synthetic route to taxadien-5-ol. Similar to most other terpenoids, Taxol biosynthesis follows the unified mode of biosynthetic "two phase" procedure, (i) the "cyclase phase" of linear association of prenyl precursors (IPP and DMAPP) to GGPP followed by molecular cyclization and rearrangement for the committed precursor taxadiene (Figure 6, VIII-IX) .57, 58 After the committed precursor, (ii) the "oxidation phase", the olefinic cyclic structure of the taxadiene core is then functionalized by seven cytochrome P450 oxygenases together with their redox partners, decorated with two acetate groups and a benzoate group by acyl-aroyl-CoA-dependent transferases, keto group by keto-oxidase, and epoxide group by epoxidase carry the last intermediate baccatin III, that the C13 secondary chain is bound for Taxol ((Figure 6, X-XIII) .15 Although an approximate sequential order of the oxidation phase early reactions is predicted, programming / order In precise part of the hydroxylations, acylations and benzoylation reactions are uncertain. However, it is clear that the early bifurcation starts from the hydroxylation mediated by the cytochrome p450 of the taxadiene nucleus at position C5 followed by downstream hydroxylations using a homologous family of cytochrome p450 enzymes with high similarity deduced from each other (> 70% ) but with limited similarity (< 30%) to other plant p450.41 59 Furthermore, structural and functional diversity with possible evolutionary analysis implies that the taxadien-5a-ol gene may be the parental sequence from which the other Hydroxylase genes evolved in the Taxol biosynthetic pathway, reflecting the order of hydroxylations.15 Additional aspects of the invention relate to chimeric P450 enzymes. Functional expression of plant cytochrome P450 has been considered challenging due to the inherent limitations of bacterial platforms, such as the absence of electron transfer machinery, of cytochrome P450 reductases, and of the translational incompatibility of signal modules of membrane of P450 enzymes due to the lack of an endoplasmic reticulum.

In some embodiments, the taxadiene-5a-hydroxylase associated with the methods of the invention is optimized by the N-terminal transmembrane design and / or the generation of chimeric enzymes by translational fusion with a redox partner of CPR. In some embodiments, the redox partner of CPR is a Taxus cytochrome P450 reductase (TCPR, Figure 5B). In certain embodiments, the cytochrome P450 taxadien-5a-hydroxylase (? Da ??) is obtained from Taxus cuspidate (GenBank accession number AY289209, the sequence of which is incorporated herein by reference). In some embodiments, the NADPH: cytochrome P450 reductase (TCPR) is obtained from Taxus cuspidate (GenBank accession number AY571340, the sequence of which is incorporated herein by reference).

The 5a-hydroxylase taxane and the TCPR can be linked by a linker as GSTGS (SEQ ID NO: 50). In some embodiments, the taxa 5a-hydroxylase and / or TCPR are truncated to remove all or part of the transmembrane region of one or both proteins. For example, the Taxadien 5a-hydroxylase in some embodiments is truncated to remove the 8, 24, or 42 N-terminal amino acids. In some embodiments, the 74 amino acids of the amine end of TCPR are truncated. An additional peptide can also be fused to taxadiene 5a-hydroxylase. For example, one or more bovine 17a hydroxylase amino acids can be added to taxane 5a-hydroxylase. In certain embodiments, the MALLLAVF peptide (SEQ ID NO: 51) is added to taxadiene 5a-hydroxylase. A non-limiting example of a polypeptide comprising the oxadiene 5a-hydroxylase fused to TCPR is At24T5aOH-tTCPR.

In some embodiments, the chimeric enzyme can carry out the first oxidation step with conversion of taxadiene of more than 10% to and by-product 5 (12) -Oxa-3 (11) -cyclotaxane. For example, the percent conversion of taxadiene to taxadiene-5a-ol and byproduct 5 (12) -Oxa-3 (11) -cyclotaxane can be at least 20%, at least 30%, at least 40% %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, approximately 99% or approximately 100 %.

In certain embodiments, the chimeric enzyme is At245aOH-tTCPR which was found capable of carrying out the first oxidation step with more than 98% conversion of taxadiene to taxadiene-5a-ol and to byproduct 5 (12) -Oxa-3 (11) -cyclotaxane (OCT; Figure 9A). The passage design of the taxadien-5a-ol production is critical in the production of Taxol and was found to be limited in previous efforts to build this route in the yeast. The designed construct developed herein showed more than 98% conversion of taxadiene in vivo with a 2400 fold improvement over the previous heterologous expression in the yeast. Thus, in addition to synthesizing appreciably larger amounts of key Taxol intermediates, this study also provides the basis for the synthesis of subsequent metabolites in the pathway by the similar P450 chemistry.

As used herein, the terms "protein" and "polypeptide" are used interchangeably and thus the term polypeptide can be used to refer to a full-length polypeptide and can also be used to refer to a fragment of a polypeptide of longitude complete As used herein with respect to polypeptides, proteins, or fragments thereof, "isolated" means separated from its native environment and present in sufficient quantity to permit its identification or use. "Isolated", when referring to a protein or polypeptide, means, for example: (i) selectively produced by expression cloning or (ii) purified as by chromatography or electrophoresis. The isolated proteins or polypeptides can be, but should not be, substantially pure. The term "substantially pure" means that the proteins or polypeptides are in essence free of other substances with which they can be found in production, nature, or systems to some extent practical and appropriate for their intended use. Substantially pure polypeptides can be obtained naturally or can be produced using the methods described herein and can be purified by techniques well known in the art. And that an isolated protein may be mixed with other components in a preparation, the protein may comprise only a small percentage by weight of the preparation. The protein however is isolated in that it has been separated from the substances with which it may be associated in living systems, ie isolated from other proteins.

The invention also encompasses nucleic acids that encode any of the polypeptides described herein, libraries that contain any of the nucleic acids and / or polypeptides described herein, and compositions that contain any of the nucleic acids and / or polypeptides described in the present.

In some embodiments, one or more of the genes associated with the invention is expressed in a recombinant expression vector. As used herein, a "vector" can be any of several nucleic acids in which a desired sequence or sequences can be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically DNA compounds, although RNA vectors are also available. Vectors include, but are not limited to: plasmids, fosmids, phagemids, virus genomes and artificial chromosomes.

A cloning vector is one that can autonomously replicate or integrate into the genome in a host cell, and that is further characterized by one or more restriction endonuclease sites in which the vector can be cut in a determinable manner and in the that a desired DNA sequence can be ligated so that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence can occur many times as the plasmid increases in the number of copies within the host cell as a host bacterium or only once per host before the host reproduces by mitosis. In the case of phage, replication can occur actively during a lytic phase or passively during a lysogenic phase.

An expression vector is one in which a desired sequence of DNA can be inserted by restriction and ligation so that it is operably linked to regulatory sequences and can be expressed as an RNA transcript. The vectors may further contain one or more proprietary marker sequences for use in the identification of cells that have been or have not been transformed or transfected with the vector. Markers include, for example, genes that encode proteins that increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes that encode enzymes whose activities are detectable by standard assays known in the art (e.g. -galactosidase, luciferase or alkaline phosphatase), and genes that visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (eg, green fluorescent protein). Preferred vectors are those capable of autonomous replication and expression of the structural products of the gene present in the DNA segments to which they are operably linked.

As used herein, a coding sequence and regulatory sequences are said to be "operably" linked when they are covalently linked in order to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, it is said that two sequences of DNA are operably linked if the induction of a promoter in the 5 'regulatory sequences results in the transcription of the coding sequence and if the nature of the union between the two DNA sequences is not (1) results in the introduction of a frame change mutation, (2) it interferes with the ability of the promoter region to direct the transcription of the coding sequences, or (3) it interferes with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably linked to a coding sequence if the promoter region were able to perform the transcription of that DNA sequence so that the resulting transcript could be translated into the desired protein or polypeptide.

When the nucleic acid molecule encoding any of the claimed enzymes of the invention is expressed in a cell, a variety of transcription control sequences (eg, promoter / enhancer sequences) can be used to direct their expression. The promoter may be a native promoter, i.e., the promoter of the gene in its endogenous context, which provides for normal regulation of gene expression. In some embodiments, the promoter can be constitutive, i.e., the promoter is not regulated allowing continuous transcription of its associated gene. A variety of conditional promoters can also be used, as promoters controlled by the presence or absence of a molecule.

The precise nature of the regulatory sequences necessary for gene expression may vary between the species or the type of the cell, but generally includes, as necessary, 5 'untranscribed and 5' untranslated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, cap sequence, CAAT sequence, and the like. In particular, such non-transcribed 5 'regulatory sequences will include a promoter region that includes a promoter sequence for the transcriptional control of the operably linked gene. Regulatory sequences may also include enhancer sequences or upstream activating sequences as desired. The vectors of the invention may optionally include 5 'leader or signal sequences. The choice and design of an appropriate vector are within the capacity and discretion of one of ordinary skill in the art.

Expression vectors containing all the elements necessary for expression are commercially available and are known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into cells of heterologous DNA (RNA). That heterologous DNA (RNA) is placed under operable control of transcription elements to allow expression of the heterologous DNA in the host cell. The heterologous expression of genes associated with the invention, for the production of a terpenoid, such as taxadiene, is demonstrated in the Examples section using E. coli. The novel method for producing terpenoids can also be expressed in other bacterial cells, fungi (including yeast cells), plant cells, etc.

A nucleic acid molecule encoding an enzyme associated with the invention can be introduced into a cell or cells using the methods and techniques that are standard in the art. For example, nucleic acid molecules can be introduced by standard protocols such as transformation including transformation and chemical electroporation, transduction, particle bombardment, etc. Expressing the nucleic acid molecule encoding the claimed enzymes of the invention can also be achieved by integrating the nucleic acid molecule into the genome.

In some embodiments, one or more genes associated with the invention are recombinantly expressed in a bacterial cell. The bacterial cells according to the invention can be cultured in media of any type (rich or minimal) and any composition. As will be comprised by one of ordinary skill in the art, routine optimization would allow the use of a variety of media types. The selected medium can be supplemented with several additional components. Some non-limiting examples of supplemental components include glucose, antibiotics, IPTG for gene induction, ATCC Trace Mineral Supplement, and glycolate. Likewise, other aspects of the medium, and of growth conditions of the cells of the invention can be optimized by routine experimentation. For example, pH and temperature are non-limiting examples of factors that can be optimized. In some modalities, factors such as choice of media, media supplements, and temperature can influence the production levels of terpenoids, such as taxadiene. In some modalities the concentration and amount of a supplementary component can be optimized. In some embodiments, the frequency in which the media is supplemented with one or more supplementary components is optimized, and the amount of time the media is cultivated before harvesting a terpenoid, such as taxadiene.

According to aspects of the invention, high titers of a terpenoid such as taxadiene, are produced by the recombinant expression of genes associated with the invention, in a cell. As used in the present "high valuation" refers to a valuation on a scale of milligrams per liter (mg L "1) The valuation produced for a given product will be influenced by multiple factors including choice of means. the total valuation of taxadiene is at least 1 mg L ~ 1. In some embodiments, the total valuation of taxadiene is at least 10 mg L'1. In some embodiments, the total valuation of taxadiene is at least 250 mg L "1. For example, the total valuation of taxadiene may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 , 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800 , 825, 850, 875, 900 or more than 900 mg l / 1 includes intermediate value. In some modalities, the total valuation of taxadiene can be at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or more of 5.0 g L "1 including any intermediate value.

In some embodiments, the total valuation of taxa 5a-ol is at least 1 mg L "1. In some embodiments, the total valuation of taxa 5a-ol is at least 10 mg L" 1. In some embodiments, the total valuation of taxa 5a-ol is at least 50 mg L'1. For example, the total assessment of taxa 5a-ol can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or more than 70 mg L "1 including any intermediate value.

The liquid cultures that are used to grow cells associated with the invention can be housed in any of the culture vessels known and used in the art. In some embodiments, large scale production in an aerated reaction vessel such as a stirred tank reactor can be used to produce large quantities of terpenoids, such as taxadiene, which can be recovered from the cell culture. In some embodiments, the terpenoid is recovered from the gas phase of the cell culture, for example by adding an organic layer such as dodecane to the cell culture and recovering the terpenoid from the organic layer.

The terpenoids, such as taxadiene, produced by the methods described herein have broad applications including pharmaceuticals such as paclitaxel (Taxol), artemisinin, ginkolides, eleuterobine and pseudopterosins, and many other potential pharmaceutical compounds. Additional applications include compounds used in flavors and cosmetics such as geraniol, farnesol, geranligeraniol, linalool, limonene, pinene, cineole and isoprene. Additional applications include compounds for use as biofuels such as alcohols of 5, 10, and 15 carbon atoms in length. It is appreciated that the aforementioned compounds are currently produced as extracts of several plants. The methods based on plant extracts are tedious, yield very small amounts and are limited in terms of the true molecules that can be obtained, namely, do not allow the easy production of derivatives that may possess properties much higher than the original compounds.

EXAMPLES Methods Strains, plasmids, oligonucleotides and genes E.coli strain K12 MG1655 was used as the host strain of the entire taxadiene strain construct. Strains of E. coli K12MG 1655 A (recA, endA) and E. coli K12MG 1655? (recA, endA) ED3 were provided by the laboratory of Professor Kristala Prather at MIT (Cambridge, MA). The detail of all the plasmids constructed for the study is shown in Table 2. All the oligonucleotides used in this study are contained in Table 3.

The geranylgeranyl pyrophosphate synthase sequences (GGPPS), 50 taxadiene synthase (TS), 51 cytochrome P450 axadiene 5a-hydroxylase. { ? da ??) and Taxus NADPH itchrome P450 reductase (TCPR 6 were obtained from Taxus canadensis, Taxus brevifolia, Taxus cuspidate (Genbank Access Codes: AF081514, U48796, AY289209 and AY571340) .The genes were synthesized to measure using the plasmids and the protocols reported by Kodumal et al.52 (Supplementary Appendix of details 1) to incorporate the E. coli translation codon and eliminate restriction sites for cloning purposes. nucleotides corresponding to amino acids 98 and 60 N-terminal of GGPPS and TS (plastid transit peptide) were removed and the sequence of insertion of translation Met was inserted.17 Construction of the MEP route (dxs-idi-idpDF operon) The dxs-idi-ispDF operon was initially constructed by cloning each of the E. coli K12 MG1655 genome genes using the primers, dxs (s), dxs (a), idi (s), idi (a), ispDF ( s) and ispDFI (a) under plasmid pET21 C + with the promoter T7 (p20T7MEP) .53 Using the primers dxsidiispDFNcol (s) and dxsidiispDFKpnl (a) the dxs-idi-ispDF operon was sub-cloned and the plasmid pTrcHis2B (Invitrogen) was then digested with Ncol and Kpnl for the pTrcMEP plasmid (p20TrcMEP) . Plasmid p20TrcMEP was digested with Mlul and Pmel and cloned into plasmid pACYC184-melA (P2A) digested with Mlul and Pmel to construct plasmid plOTrcMEP. Plasmid pTrcMEP was digested with BstZ17I and Seal and cloned into plasmid pCL1920 digested with Pvull to construct the plasmid p5TrcMEP. For the construction of the p20T5MEP plasmid, initially the dxs-idi-ispDF operon was cloned into the plasmid pQE with the T5 promoter (pQE-MEP) using the primers dxsidiispDFNcol (s) and dxsidiispDFXhol (a). A fraction of the T5 promoter operon DNA was amplified using the T5Agel (s) and T5Nhel (a) primers of the pQEMEP plasmid. The DNA fragment was digested with Agel / Nhel and cloned into the p20T7MEP plasmid digested with SGrAI / Nhel enzymes.

Construction of the taxadiene route foperones GT v TG) The downstream routes of taxadiene (GT and TG operon) were constructed by PCR cloning of GGPS and TS fragments at Noel sites -EcoRI and EcoRI-Sal from plasmid pTrcHIS2B to create p20TrcGT and p20TrcTG using the primers GGPPSNcol (s), GGPPSEcoRI ( a), TSEcoRI (s), TSsall (a), TSNcol (s) TSEcoRI (a) GGPPSEcoRI (s) and GGPPSSall (a). To construct p20T5GT, initially the operon was amplified with primers GGPPSNcol (s) and TSXhol (a) and cloned in a plasmid of pQE under T5 promoter digested with Ncol / Xhol. The sequence was then digested with XbaI and XhoI and cloned into the structure of the amplified pTrc plasmid using the primers pTrcSal (s) and pTrcXba (a). P10T7TG was constructed by subcloning the TG operon digested with Ncol / Sall from p20TrcTG into plasmid pACYC-DUET1 digested with Ncol / Sall. P5T7TG was constructed by cloning the fragment digested with BspEI / Xbal to DNA digested with Xbal / Bsp amplified from plasmid pCL1920 using the primers pCLBspEI (s) and pCLXbal (a).

Construction of chromosomal integration MEP pathway plasmids To construct the plasmids with the FRP-Km-FRP cassette to amplify the sequence for integration, p20T7MEP and p20T5MEP were digested with Xhol / Scal. The FRP-Km-FRP cassette was amplified from the Km cassette with the FRP sequence of plasmid pkD 3 using the primers KmFRPXhol (s) and KmFRPScal (a). The amplified DNA was digested with XhoI / Scal and cloned into the plasmid p20T7MEP and p20T5MEP digested with XhoI / Scal (p20T7MEPKmFRP and p20T5MEPKmFRP). Also the plasmid p20TrcMEP was digested with Sacl / Scal and the amplified DNA using the primers KmFRPSacl (s) and KmFRPScal (a) was digested, cloned in the plasmid p20TrcMEP (p20TrcMEPKm-FRP).

Chromosomal integration of the MEP route cassette (Laclq-MEP-FRP-Km-FRP cassette) The MEP routes constructed under the T7, T5 and Trc promoters were localized to the region of the ara operon on the chromosome with the Kan marker. PCR fragments were amplified from p20T7MEPKmFRP, p20T5MEPKmFRP and p20TrcMEPKm-FRP using the primers lntT7T5 (s), IntTrc (s) and lnt (a) and then electroporated into cells of E. coli MG1655 recA-end- and E. coJi MG1655 recA-end-EDE3 for chromosomal integration by the Red recombination technique54. The site-specific localization was confirmed and the km marker was removed by the action of FLP recombinase after successful integration of the gene.

Construction of 5a-ol taxadien route The transmembrane (TM) region of the taxa 5a-ol hydroxylase (T5aOH) and Cytochrome P450 Taxus reductase (TCPR) was identified using the software (www.predictprotein.org55). For the transmembrane design, selective truncation of amino acid residues 8, 24 and 42 was carried out in the N-terminal transmembrane region of taxadiene 5a-ol hydroxylase (T5aOH) and amino acid region 74 in TCPR. The elimination of the N-terminal amino acid residues 8, 24 and 42 of the taxane 5a-ol hydroxylase (T5aOH) incorporates a MALLLAVF peptide (SEQ ID NO: 51) of residue 8 at the amine end of bovine 17a hydroxylase substituted in an amino acid to the 44 T5aOH sequences truncated at the amino terminus and the GSTGS peptide linker was made using the primer CYP17At8AANdel (s), CYP17At24AANdel (s), CYP17At42AANdel (s) and CYPLinkBamHI (a). Using these primers each modified DNA was amplified, digested with Ndel / BamHI and cloned into plasmid pACYC DUET1 digested with Ndel / BamHI to construct the plasmids p10At8T5aOH, p10At24T5aOH and p10At42T5aOH. The truncated TCPR sequence (tTCPR) at amino acid 74 was amplified using primers CPRBamHI (s) and CPRSall (a). The amplified sequence of tTCPR and the plasmids, p10At8T5aOH, p10At24T5aOH and p10At42T5aOH, was digested with BamHI / Sall and cloned to construct the plasmids p10At8T5aOH-tTCPR, p10At24T5aOH-tTCPR and p10At42T5aOH-tTCPR Cell growth for the analysis of taxadiene and taxadien analysis - 5a-ol Unique transformants of pre-designed E. coli strains that harbor the appropriate upstream plasmid (MEP), downstream pathway of taxadiene and taxadien-5a-ol were grown for 18h at 30 ° C in Luria-Bertani (LB) medium (supplemented with appropriate antibiotics, 100 mg / mL of carbenecillin, 34 mg / mL of chloramphenicol, 25 mg / mL of kanamycin or 50 mg / mL of streptomycin). For small-scale cultures to investigate the strains designed, these pre-inoculated were used to plant 2 mL of fresh rich medium (5g / L of yeast extract, 10g / L of Trypton, 15 g / L of glucose, 10 g / L of NaCl, HEPS 100mM, 3 mL / L of Antifoam B, pH 7.6, 100 ug / mL of Carbenicillin and 34 ug / mL of chloramphenicol), at a starting A6oo of 0.1. The culture was maintained with appropriate antibiotics and 100 mM of IPTG for the induction of genes at 22 ° C for 5 days.

Experiments in bioreactor for the production strain of taxadien 5a-ol.

The 3 L bioreactor from Bioflo (New Brunswick) was assembled according to the manufacturer's instructions. One liter of medium rich with 1% glycerol (VA /) was inoculated with 50 mL of culture 8h (A6oo of ~ 2.2) of strain 26-At24T5aOH-tTCPR grown in LB medium containing antibiotics (100 mg / mL carbenicillin , 34 mg / mL of chloramphenicol) in the same concentrations. 1L bioreactors with biphasic liquid-liquid fermentation using 20% dodecane v / v. Oxygen was supplied as filtered air at 0.5 v / v / m and agitation was adjusted to maintain dissolved oxygen levels above 50%. The pH of the crop was controlled in 7.0 using 10% NAOH. The temperature of the culture in the thermenator was controlled at 30 ° C until the cells grew at an optical density of about 0.8, measured at a wavelength of 600 nm (OD600). The temperature of the fermenter was reduced to 22 ° C and the cells were induced with 0.1 mM of IPTG. The dodecane was added aseptically to 20% (v / v) of the volume of the medium. During the course of the fermentation the glycerol concentration and acetate accumulation was monitored at constant intervals of time. During the fermentation as the glycerol concentration was depleted below 0.5 G / L, the glycerol (3 g / L) was introduced into the bioreactor.

The fermentation was further optimized using a batch-fed culture with a defined feed medium containing 0.5% yeast extract and 20% (v / v) dodecane (13.3 g / L KH2P04, 4 g / L (NH4) ) 2HP04, 1.7 g / L of citric acid, 0.0084 g / L of EDTA, 0.0025 g / L of CoCl2l 0.015 g / L of MnCI2l 0.0015 g / L of CuCI2, 0.003 g / L of H3B03, 0.0025 g / L of Na2Mo04 ) 0.008 g / L of Zn (CH3COO) 2, 0.06 g / L of Fe citrate (lll), 0.0045 g / L of thiamine, 1.3 g / L of MgSO4, 10 g / L glycerol, 5 g / L of extract of yeast, pH 7.0). The same medium composition was used for the fermentation of strains 17 and 26 with appropriate antibiotics (strain 17: 100 pg / mL of carbenicillin and 50 pg / mL of streptomycin, strain 26: 50 pg / mL of streptomycin).

For the strain that produces taxadie-5a-ol, one liter of complex medium with 1% glycerol (v / v) was inoculated with 50 mL of a culture of 8h (OD of -2.2) of strain 26-At24T5aOH-tTCPR grown in LB medium containing 50 pg / mL of spectinomycin and 34 pg / mL of chloramphenicol). Oxygen was supplied as filtered air at 0.5 (wm) and the stir was adjusted to maintain dissolved oxygen levels above 30%. The pH of the culture was controlled at 7.0 using 10% NaOH. The temperature of the culture in the fermentor was controlled at 30 ° C until the cells grew at an optical density of about 0.8, measured at a wavelength of 600 nm (OD600). The temperature of the fermentor was reduced to 22 ° C and the route was induced with 0.1 mM of IPTG. The dodecane was added aseptically to 20% (v / v) of the volume of the medium. During the course of the fermentation, the concentration of glycerol and acetate accumulation was monitored with constant intervals of time. During fermentation, since the glycerol concentration was depleted by 0.5-1 g / L, 3 g / L of glycerol was introduced into the bioreactor.

Analysis of GC-MS of taxadiene and taxadien-5a-ol For the analysis of small-scale culture taxadiene accumulation, 1.5 ml_ of the culture was vortexed with 1 mL of hexane for 30 min. The mixture was centrifuged to separate the organic layer. For the bioreactor 1 uL of the dodecane layer was diluted to 200 uL using hexane. 1 uL of the hexane layer was analyzed by GC-MS (Varian Saturn 3800 GC connected to a Varian 2000 MS). The sample was injected into a HP5ms column (30m X 250 uM X 0.25uM thick) (Agílent Technologies USA). Helium (ultra pure) at a flow rate of 1.0 ml / min was used as a carrier gas. The oven temperature was first maintained constant at 50 ° C for 1 min, and then increased to 220 ° C in increments of 10 ° C / min, and finally maintained at this temperature for 10 min. The temperatures of the injector and transfer lines were set at 200 ° C and 250 ° C, respectively.

Standard compues of biological or synthetic sources for taxadiene and taxa 5a-ol was not commercially available. In this way we carry out fermentations of E. coli that produces taxadiene in a 2L bioreactor to extract pure material. The taxadiene was extracted by solvent extraction using hexane, followed by multiple series of silica column chromatography to obtain the pure material to construct a standard curve for GC-MS analysis. We have compared the GC and MS profile of pure taxadiene with the literature reported to confirm the authenticity of the compound60. To verify the purity we have made 1 HRMN of taxadiene. Since the accumulation of taxadien-5a-ol was very low, we used taxadiene as a measure to quantify the production of this molecule and the mass spectrum fragmentation characteristics of previous reports42.

QPCR measurements for the transcriptional analysis of the strains designed The expression levels of the transcriptional gene of each gene were detected by qPCR in the mRNA isolated from the appropriate strains. To prevent degradation, the RNA was stabilized before lysis of the cells using bacterial reagent RNAprotect (Qiagen). Subsequently, the total RNA was isolated using the RNeasy mini kit (Qiagen) combined with nuclease-based elimination of the genomic cDNA contaminants. The cDNA was amplified using the iScript cDNA synthesis kit (Biorad). The qPCR was carried out in an iCycler of Bio-Rad using ¡Q SYBR Green Supermix (Biorad). The expression level of the rrsA gene, which is not subject to variable expression, was used for the normalization of qPCR values.56 Table 3 has the primers used for the qPCR. For each primer pair, a standard curve was constructed with E. coli mRNA as the template.

EXAMPLE 1 The accumulation of taxadiene exhibits strong non-linear dependence on the relative strengths of the upstream MEP routes and synthetic taxadiene in the 3 'direction Fig. 1B depicts the various ways in which promoters and copy numbers of the gene were combined to modulate relative flow (or strength) by the upstream and downstream pathways of taxadiene synthesis. A total of 16 strains was constructed for the de-bottling of the MEP route, as well as to optimally balance it with the downstream taxadiene route. Figs. 2A-2B summarizes the results of accumulation of taxadiene in each of these strains, with Fig. 2A emphasizing the dependency of taxadiene accumulation in the upstream path for constant values of the downstream path, and Fig. 2B the dependence in the downstream path for the constant force in the upstream path (see also Table 1 for the calculation of upstream and downstream path expression of the reported promoter forces and plasmid copy numbers33"36). Clearly, there are maxima exhibited with respect to both upstream and downstream path expressions.For constant downstream path expression (Fig. 2A), as the upstream path expression increases from very low levels, the production of taxadiene increases initially due to an increased supply of precursors to the general route. intermediate value, additional increments of upstream route can not be accommodated by the capacity of the downstream route. This imbalance of the route leads to the accumulation of an intermediary (see the following) which may be either inhibitory of the cells or simply indicating flow deviation to a competing route, ultimately resulting in the reduction of taxadiene accumulation.

For the upstream constant path expression (Fig. 2B), a maximum is also observed with respect to the downstream path expression level. This is attributed to an initial limitation of taxadiene production by low expression levels of the downstream path, which is thus speed limiting with respect to taxadiene production. At high levels of downstream path expression we are probably seeing the negative effect of high copy number on cell physiology, hence, a maximum exists with respect to downstream path expression. These results demonstrate that dramatic changes in taxadiene accumulation can be obtained from changes within a narrow window of expression levels for the upstream and downstream routes. For example, a strain containing an additional copy of the upstream path in its chromosome under control of Trc promoter (Strain 8, Fig. 2A) yielded 2000 times more taxadiene than one overexpressing only the downstream synthetic pathway (Strain 1, Fig. 2A). In addition, changing the order of genes in the synthetic operon downstream from GT (GPPS-T) to TG (TS-GPPS) resulted in a 2-3 fold increase (strains 1-4). compared to 5, 8, 11 and 14). The observed results show that the key to overproduction of taxadiene is the broad downstream pathway capacity and careful balancing between the upstream path of the precursor with the synthetic path downstream of taxadiene. Together, the designed strains established that the MEP route flow can be substantial, if a wide variety of expression levels for the upstream and downstream synthetic endogenous pathway are searched simultaneously.

EXAMPLE 2 Chromosomal integration and fine tuning of the upstream and downstream routes further increases the production of taxadiene To provide broad downstream path strength by minimizing plasmid birth load of plasmids, two new sets of 4 strains were each designed (strains 25-28 and 29-32) in which the downstream path was placed under the control of a strong promoter (T7) maintaining a relatively low number of 5 and 10 copies, respectively. It can be seen (Fig. 2C) that while the maximum taxadiene is maintained at high strength downstream (strain 21-24), a monotonic response is obtained at the lowest point of downstream path strength (strains 17-20, Fig. 2C). This observation prompted the construction of two additional sets of 4 strains each which maintained the same level of force downstream of the route as before but very low levels expressed from the upstream route (strains 25-28 and 29-32, Fig. 2D ). Additionally, the operon of the route upstream of the last strain set was chromosomally integrated. It can be seen that not only is the maximum taxadiene recovered, albeit at very low levels of upstream route, but a much higher maximum of taxadiene was reached (300 mg / L). We believe that this significant increase can be attributed to a decrease in the metabolic load of the cell. This was accomplished 1) by eliminating the dependence on the plasmid by integration of the path in the chromosome and 2) reaching a fine balance between the expression in the upstream and downstream path.

The 32 recombinant constructs allowed us to adequately test the modular path expression space and amplify ~ 1 5000 times the improvement in taxadiene production. This is by far the highest terpenoid production of the MEP isoprenoid pathway in E. coli reported (Fig. 3A). Additionally, the improvements in numbers of times observed in terpenoid production are appreciably higher than those of the reported combinatorial metabolic engineering approaches that sought an extensive genetic space comprising up to a trillion combinatorial vanants of the isoprenoid pathway. suggests that route optimization depends much more on the fine balance of route module expression than on combinatorial gene optimization from multiple sources. The multiple maxima exhibited in the phenotypic landscape of Figs. 1A-1B subqualifies the importance of testing the expression space at a sufficient resolution to identify the region of optimal overall performance of the route. Fig. 7 represents the number of times of the improvements in taxadiene production of the modular search of route expression.

EXAMPLE 3 The metabolite correlates inversely with the production of taxadiene and identification of the metabolite The Metabolomics analysis of two previously designed strains identified a yet unknown, metabolite by-product that strongly correlates with levels of pathway expression and taxadiene production (Figs 3A-3B and Figs 8A-8C). Although the chemical identity of the metabolite was unknown, we hypothesized that it is a secondary product of isoprenoid, resulting from the route deviation and has been anti-correlated as a direct variable to the production of taxadiene (Figs 3A-3B and Figs. 8A-8C) of the strains designed. A critical attribute of our optimal strains is the fine balance that relieves the accumulation of this metabolite, resulting in the highest production of taxadiene. This equilibrium can be modulated at different levels of the chromosome, or from different copy number plasmids, using different promoters, with appreciably different accumulation of taxadiene.

Subsequently the corresponding peak in the gas chromatography-mass spectrometry chromatogram (GC-MS) was identified as indole by GC-MS studies, and nuclear magnetic resonance (NMR) spectroscopy of 1H and 3C (Figs. 16E). We found that the taxadiene synthesis by strain 26 is severely inhibited by the exogenous indole at indole levels higher than -100 mg / L (Fig. 15B). An additional increase in indole concentration also inhibited cell growth, with the level of inhibition being highly dependent on the strain (Fig. 15C). Although the biochemical mechanism of indole interaction with the isoprenoid pathway is not currently clear, the result in Figs. 15A-15C suggests a possible synergistic effect between the indole and terpenoid compounds of the isoprenoid pathway to inhibit cell growth. Without knowing the specific mechanism, it seems that strain 26 has mitigated the effect of indole, which we conducted for further study.

EXAMPLE 4 Cultivation of strains designed To explore the potential for taxadiene production under controlled conditions for the strains designed, batch-fed cultures of the three strains that accumulate most taxadiene (~ 60 mg / L of strain 22; -125 mg / L of strain 17; 300 mg / L of strain 26) were carried out in 1 L bioreactors (Figs 17A-17D). The batch-fed culture studies were carried out as a two-phase liquid-liquid fermentation using a 20% (v / v) dodecane cover. The organic solvent was introduced to prevent air purification of the taxadiene secreted from the fermentation medium, as indicated by preliminary conclusions. In defined media with glycerol controlled feeding, the productivity of taxadiene increased to 174 ± 5 mg / L (SD), 210 ± 7 mg / L (SD), and 1020 ± 80 mg / L (SD), respectively for the strains 22, 17 and 26 (Fig. 17A). Additionally, taxadiene production significantly affected the growth phenotype, acetate accumulation and glycerol consumption (Fig. 17B-17D).

Fig. 17C shows that acetate accumulates in all strains initially, but then after -60 hrs the acetate decreases in strains 17 and 26 while it continues to increase in strain 22. This phenomenon highlights the differences in the central metabolism of carbon between the high-flow MEP strains (26 and 17) and the low-flow MEP strain (22). Additionally, this observation is another illustration of the good physiology that characterizes a well-balanced functional strain. Acetic acid, as a product of excessive capacity metabolism, is initially produced by all strains due to the high initial concentrations of glycerol used in these fermentations and the corresponding high glycerol route flow. This flow is sufficient to also supply the MEP pathway, as well as the other metabolic pathways in the cell.

In ~ 48 hours, the initial glycerol is depleted, and the culture changes to a batch mode of feeding, during which low but constant levels of glycerol are maintained. This results in a lower overall glycerol flow, which, for strains with high MEP flow (strains 26 and 17), is mostly diverted to the MEP route while minimizing excessive metabolic capacity. As a result, the production of acetic acid is reduced or even completely eliminated. With respect to the decrease in the concentration of acetic acid, it is possible that the assimilation of acetic acid may have happened to a certain extent, although this was not further investigated from a flow analysis point of view. Some evaporation and dilution due to the glycerol feed also contributes to the observed decrease in acetic acid concentration. In contrast, for strains with low MEP flow (strain 22), the flow deviation to the MEP route is not very significant, so the glycerol flow still supplies all the necessary carbon and energy requirements. The metabolism of excessive capacity continues to occur leading to an acetate secretion.

Clearly the high productivity and more robust growth of strain 26 allowed a very high accumulation of taxadiene. Additional improvements should be possible by optimizing the conditions in the bioreactor, balancing nutrients in the medium of growth, and optimizing carbon delivery.

EXAMPLE 5 Levels of expression in the upstream and downstream path and cell growth reveal underlying complexity For a more detailed understanding of the equilibrium designed in the expression of route, we quantified the expression levels of transcriptional gene of dxs (upstream route) and TS (downstream path) for the strains of higher production of taxadiene and neighboring strains of Figs. 2C and 2D (strains 17, 22 and 25-32) (Fig. 4A-4B). As in our hypothesis, the expression of the upstream path increased monotonically with promoter strength and copy number for the MEP vector from: native promoter, Trc, T5, T7, and plasmids of 10 copies and 20 copies, as seen in the expression of DXS (Fig. 4A). Thus we find that the expression level of dxs correlates well with force in the upstream path. Similar correlations were found for the other genes of the upstream path, iD, ispD and ispF (Fig. 14A-14B). In downstream gene expression, a ~ 2-fold mensor was quantified after transferring the plasmid route from 5 to 10 copies (series 25-28 and series 29-32) (Fig. 4B).

While the effects of promoter and number of copies influenced the expressions of the gene, side effects in the expression of the other route were also prominent. Fig. 4A shows that for the same dx expression cassettes, increasing the copy number of the TS plasmid from 5 to 10, the expression of dxs was increased. Interestingly, the 5-copy TS plasmid (strain 25-28) contained substantially higher taxadiene yields (Fig. 2D) and less growth (Figs. 4C-4D) than 10-copy TS plasmid. Control plasmids that did not contain the heterologous taxadiene pathway grew twice the highest densities, implying that growth inhibition in strains 25-28 is directly related to the metabolic pathway of taxadiene and the accumulation of taxadiene and its direct intermediaries (Fig. 4C). However, strain 29-32 showed only modest increases in growth performance when comparing empty control plasmids to strains expressing taxadiene (Fig. 4D). This interaction between growth, taxadiene production, and level of expression can also be seen with expression vectors based on upstream plasmid (strains 17 and 22). The inhibition of growth was much greater in copy 10, strain of high production of taxadiene (strain 17) compared to copy 20, strain of lower production of taxadiene (strain 22) (Fig. 4D). Therefore the toxicity of the product and carbon deviation to the heterologous route probably prevent growth, rather than the maintenance of the plasmid.

Also unexpected was the deep effect of the upstream expression vector on downstream expression. Fig. 4B would have two straight lines, if there was no crossing between the routes. However, ~ 3-fold changes in TS expression are observed for different MEP expression vectors. This is probably due to the significant competition for resources (raw material and energy) that are extracted from host metabolism by overexpression of both the four upstream genes and the two downstream genes.38 Compared to control strain 25c, a 4-fold growth inhibition It was sometimes observed with strain 25 indicating that the high overexpression of the synthetic taxadiene pathway induced toxicity by altering the growth phenotype compared to overexpression of the native route (Fig. 4C). However, as the upstream expression increased, the downstream expression was reduced, inadvertently in our case, to desirable levels to balance the upstream and downstream routes, minimizing growth inhibition (strain 26).

At the extreme of protein overexpression, the MEP path driven by the T7 promoter resulted in severe inhibition of growth, due to the synthesis of four high level proteins (strains 28 and 32). The expression of the TS genes by T7 does not seem to have such a drastic effect on its own. The high protein synthesis rates of T7-induced expression (Fig. 4A-4B) could lead to underregulation of the protein synthesis machinery including the components of the maintenance genes of the early growth phase damaging cell growth and decreases the increase in biomass.39, 0 We hypothesized that our observed complex phenotypes of growth are cumulative effects of (1) the toxicity induced by the activation of isoprenoid / taxadiene metabolism, and (2) and the effects of the high recombinant expression of proteins. Overall, our modular multivariate pathway design approach generated an unexpected diversity in terpenoid metabolism and its correlation to cell expression and physiology. The rational design of microbes for the secondary production of metabolite will require an understanding of path expression that goes beyond a linear / independent understanding of promoter forces and copy numbers. However, simple, multivariate approaches, as used here, can introduce the necessary diversity to both of (1) finding high producers, and (2) providing a landscape for systematic investigation of higher order effects that are dominant, more despised, in the design of the metabolic pathway.

EXAMPLE 6 Design of the oxidation chemistry based on Taxol P450 in E. coli A central feature in the Taxol biosynthesis is the oxygenation in multiple positions of the taxane core structure, the reactions that are considered to be mediated by cytochrome-dependent monooxygenases P450.41 After the completion of the compromised path cyclization step , the parental olefin, taxa-4 (5), 11 (12) -diene, is then hydroxylated at the C5 position by a cytochrome P450 enzyme, representing the first of eight oxygenation steps (from the taxane nucleus) on the route Taxol (Fig. 6) .42 Thus, a key step towards the design of Microbes that produce Taxol is the development of the oxidation chemistry based on P450 in vivo. The first step of oxygenation is catalyzed by a cytochrome P450, taxadiene 5a-hydroxylase, an exceptional monooxygenase that catalyzes the hydroxylation reaction together with the migration of the double bond in the diterpene precursor taxadiene (Fig. 5A). We report the first successful extension of the taxadiene synthetic route to taxane 5a-ol and present the first examples of the in vivo production of any functionalized intermediary of Taxol in E. coli.

In general, functional plant decitochrome P450 expression is challenging43 due to the inherent limitations of bacterial platforms, such as the absence of electron transfer machinery, cytochrome P450 reductases, and the translational incompatibility of membrane signal modules. the P450 enzymes due to the lack of an endoplasmic reticulum. Recently, due to the design of the transmembrane (TM) and the generation of P450 chimera enzymes and CPR reductases, some plant P450 have been expressed in E. coli for the biosynthesis of functional molecules22,44 Yet, each cytochrome p450 of plant is unique in its transmembrane signal sequence and the electron transfer characteristics of its reductase counterpart.45 Our initial studies were focused on optimizing the expression of synthetic taxadienase-hydroxylase by N-terminal transmembrane engineering and generating enzymes chimera by translational fusion with the redox partner CPR of the Taxus species, Taxus cytochrome P450 reductase (TCPR) (Fig. 5B) .42,44,46 One of the generated chimera enzymes, At24T5aOH-tTCPR was highly efficient in carrying out the first oxidation step with conversion of taxadiene of more than 98% to taxadiene-5a-ol and to by-product 5 (12) -Oxa-3 (11) -cyclotaxane (OCT) (Fig. 9A).

Compared to the other chimeric P450s, At24T5aOH-tTCPR yielded twice the production of taxadiene-5a-ol twice (21 mg / L). Also, the weaker activity of At8T5aOH-tTCPR and At24T5aOH-tTCPR resulted in the accumulation of a recently characterized byproduct, a new complex structural arrangement of taxadiene in the ether 5 (12) -Oxa-3 (11) -cyclic cyclist ( OCT) (Figs 9A-9D) .47 The byproduct accumulated in amounts approximately equal to the desired product taxadien-5a-ol. The formation of OCT was mediated by an unprecedented reaction sequence of Taxus cytochrome P450 involving oxidation and subsequent cyclizations.47 Thus, it seems likely that by the protein design of the taxa-5a-hydroxylases, the termination of the reaction before the Cyclization will prevent the accumulation of such unwanted byproduct and channeling the flow to taxa 5a-ol could be achieved.

The productivity of strain 26-At24T5aOH-tTCPR was significantly reduced relative to that of taxadiene production by parental strain 26 (~ 300 mg / L) with a concomitant increase in accumulation of the above-described uncharacterized metabolite. No accumulation of taxadiene was observed. Apparently, the introduction of an additional medium copy plasmid (copy 10, p10T7) carrying the At24T5aOH-tTCPR construction perturbed the carefully designed equilibrium in the upstream and downstream path of strain 26. Small-scale fermentations were carried out in bioreactors to quantify alcohol production by strain 26-At24T5aOH-tTCPR. The time course profile of the accumulation of taxadie-5a-ol (Fig. 5D) indicates the production of alcohold e up to 58 ± 3 mg / L with an equal amount of the produced OCT byproduct. The observed alcohol production was ~ 2400 times higher than the previous production in S. cerevisiae Additional increases in the production of taxadien-5a-ol are probably possible due to route optimization and protein design.

The multivariate modular route optimization approach has yielded very high production strains of a critical Taxol precursor. In addition, recombinant constructs have been equally effective in redirecting the flow towards the synthesis of other complex pharmaceutical compounds, such as mono-, sesqui- and di-terpene (geraniol, linalool, amorphadiene and levopimaradiene) products designed from the same route (results not published). Thus, our route design opens new avenues to bio-synthesize natural products, especially in the context of microbially derived terpenoids for use as chemical and renewable resource fuels. Focusing on the universal terpenoid precursors IPP and DMAPP, it was possible, first, to define the critical modules of the route and then modulate the expression to optimally balance the route modules for the continuous precursor conversion and minimum intermediary accumulation. This approach seems to be more effective than combinatorial searches of large genetic spaces and also does not depend on a high performance test.

The MEP route is energetically balanced and thus more efficient overall in converting either glucose or glycerol to isoprenoids. Yet, over the past 10 years, many attempts to design the MEP route in E. coli to increase the supply of the key precursors IPP and D APP for the overproduction of carotenoid28, 7, sesquiterpenoid23 and diterpenoid61 had limited success. This inefficacy was attributed to unknown regulatory effects associated specifically with the expression of the MEP pathway in E. coli23. Here we provide evidence that such limitations are correlated with the accumulation of the indole metabolite, due to the non-optimal expression of the route, which inhibits the activity of the isoprenoid pathway. The overproduction of taxadiene (under conditions of suppression of indole formation), establishes the route of MEP as a very efficient route for the biosynthesis of pharmaceutical products and chemicals of the isoprenoid family. One simply needs to carefully balance the modular routes as suggested by our multivariate modular route design approach.

For the successful microbial production of Taxol, the demonstration of the chemical decoration of the taxadiene nucleus by the oxidation chemistry based on P450 is essential.41 Cytochrome P450 monooxygenases constitute about half of the 19 different enzymatic steps in the path biosynthetic of Taxol. Typically, these genes show an unusually high sequence similarity to one another (> 70%) but low similarity (<30%) with other plant P450s.14 Because of the apparent similarity between Taxol monooxygenases, expressing the Appropriate activity to carry out the specific oxidation chemistry of P450 was a particular challenge. By engineering TM and constructing an artificial chimera enzyme with redox partner (TCPR), the Taxol cytochrome P450, the taxa 5a-hydroxylase was functionally expressed in E. coli and showed efficient conversion of taxadiene to the corresponding alcohol product in vivo Previous in vitro studies have described the mechanism of converting taxadiene to taxadien-5a-ol by the native enzyme of taxa 5a-hydroxylase, but has not discussed the same conversion in vivo.42 This oxygenation reaction and the new arrangement involves abstraction of hydrogen from the C20 position of the taxadiene to form an intermediate allylic radical, followed by insertion of specific regio-and stereo oxygen at the C5 position to yield the alcohol derivative (Fig. 5A). The modest abundance observed for the enzyme in Taxus cells, and the low values of Kcafsugirieron that the 5a-hydroxylation step of Taxol biosynthesis is slow with respect to oxygenations and acylations downstream in the Taxol route.41 , the design of this step is key to the synthesis of Taxol, especially in the context of the functional design of the Taxol P450 in the prokaryotic host such as E. coli. In addition, this step limited in previous efforts the construction of the route in the yeast.17 The construction designed in this study showed > 98% conversion of taxadiene in vivo with product accumulation to ~ 60 mg / L, a 2400 fold improvement over previous heterologous expression in the yeast. This study has therefore been successful not only in synthesizing appreciably larger amounts of key Taxol intermediates but also providing the basis for the synthesis of subsequent metabolites en route by the similar chemistry of P450.

Previous studies on the structure-activity relationship in Taxol have shown that the modifications made either by elimination or addition of some of its functional groups did not substantially change the activity of Taxol.1,8 However, such studies were limited due to the Restricted capacity to introduce changes by chemical synthesis. The availability of a microbial pathway for the synthesis of Taxol will drastically expand the space of chemical modifications that can be examined, thus increasing the likelihood of identifying more powerful candidate drugs. This offers exciting new opportunities for drug development, especially when taking into account that such drug candidates will also be associated with an efficient production route.

In the past decades, Taxol has spread more interest within the scientific and public communities than any other natural product drug candidate.10 A major supply crisis is predicted from the projected increase in the use of Taxol or Taxol analogues for cancer chemotherapy, requiring new production routes, such as the design of Taxol biosynthetic machinery in microbes.8 While a few endophytic fungi of the Taxus species have been isolated capable of producing Taxol naturally, these microbial systems still have to demonstrate convenience for the sustainable production of the drug.49 The results reported here represent a disruptive step towards a Taxol or precursor of Taxol derived microbially, removing the bottlenecks in the committed precursor pathway. In addition, the assembly of a synthetic route offers new possibilities to tailor Taxol analogues by selectively designing the route, thereby altering the structure of the taxane. These developments raise optimism for a microbial route for the profitable production of Taxol or convenient precursors of Taxol.

Table 1 Classification of the expression of the upstream and downstream path in arbitrary units (a.u.). The levels of expression of the MEP route and the taxadiene / synthase synthase pathway of GGPP were estimated using published values of promoter forces and copy number. The forces of the promoter were calculated as trc = 1, T5 = 1.96, T7 = 4.97, based on Brosius et. to the. and Brunner et. al.33 3 The copy number of the gene was assigned by published copy numbers for the origin of the replica for the different plasmids used, and a copy was used for the integrations.35"37 The total expression was calculated as the product of the strength of the promoter and copy number of the gene The native expression of the MEP path was arbitrarily assigned a value of one, and changing the order of the GGPP synthase operon and taxadiene was assumed to affect the expression of taxadiene synthase by 20% .35 These estimates of total expression guided design efforts. E - E coli K12 MG1655 with two deletions ArecAAendA; EDE3 - K12 MG1655 ArecAAndAnd with an 11 RNA polymerase (DE3) integrated; MEP operon - dxs-idi-ispDF; operon GT-GPPS-TS; TG operon - TS-GPPS; Ch1 - 1 chromosome copy; Trc-trc promoter; Promoter T5 - T5; promoter T7 - T7; copy p5-5 of the plasmid (pSC101); copy p10 - -10 of the plasmid (p15A); and copy p20--20 of the plasmid (pBR322).

TABLE 1 * A value of 1 was given to justify the native copies of the MEP route.

* The construction of MEP is located on the chromosome.

# P20T5GT-TrcT - An additional copy of the T gene under separate control of the promoter (Trc) next to the GT operon (under promo T5) in the same plasmid. For the calculation of force, we have added the value as equivalent of two separate operons (TrcT + T5GT = (20 * 1.96 + 20x1 = 59)) since our studies show that the expression of T was limited in comparison to G.

TABLE 2 Detail of all the plasmids built for the study TABLE 3 Details of the primer used for the cloning of plasmids. chromosomal supply of the MEP route and qPCR measurements TABLE 4 Optimized protein and codon nucleotide sequences Synthesis of GGPP MFDFNEYMKSKAVAVDAALDKAIPLEYPEKIHESMRYSLLAGGKRVRPALCIAACELVGGSQDLA MPTACAMEMIHT SLIHDDLPCMDNDDFRRGKPTNHKVFGEDTAVLAGDALLSFAFEHIAVATSK TVPSDRTLRVISELGKTIGSQGLVGGQVVDITSEGDANVDLKTLEWIHIHKTAVLLECSVVSGGILG GATEDEIARIRRYARCVGLLFQWDDILDVTKSSEELGKTAGKDLLTDKATYPKLMGLEKAKEFAA ELATRAKEELSSFDQIKAAPLLGLADYIAFRQN (SEQ ID NO: 42) ATGTTTGATTTCAATGAATATATGAAAAGTAAGGCTGTTGCGGTAGACGCGGCTCTGGATAAA GCGATTCCGCTGGAATATCCCGAGAAGATTCACGAATCGATGCGCTACTCCCTGTTAGCAGG AGGGAAACGCGTTCGTCCGGCATTATGCATCGCGGCCTGTGAACTCGTCGGCGGTTCACAG GACTTAGCAATGCCAACTGCTTGCGCAATGGAAATGATTCACACAATGAGCCTGATTCATGAT GATTTGCCTTGCATGGACAACGATGACTTTCGGCGCGGTAAACCTACTAATCATAAGGTTTTT GGCGAAGATACTGCAGTGCTGGCGGGCGATGCGCTGCTGTCGTTTGCCTTCGAACATATCG CCGTCGCGACCTCGAAAACCGTCCCGTCGGACCGTACGCTTCGCGTGATTTCCGAGCTGGG AAAGACCATCGGCTCTCAAGGACTCGTGGGTGGTCAGGTAGTTGATATCACGTCTGAGGGTG ACGCGAACGTGGACCTGAAAACCCTGGAGTGGATCCATATTCACAAAACGGCCGTGCTGCTG GAATGTAGCGTGGTGTCAGGGGGGATCTTGGGGGGCGCCACGGAGGATGAAATCGCGCGT ATTCGTCGTTATGCCCGCTGTGTTGGACTGTTATTTCAGGTGGTGGATGACATCCTGGATGTC ACAAAATCCAGCGAAGAGCTTGGCAAGACCGCGGGCAAAGACCTTCTGACGGATAAGGCTA CATACCCGAAATTGATGGGCTTGGAGAAAGCCAAGGAGTTCGCAGCTGAACTTGCCACGCG GGCGAAGGAAGAACTCTCTTCTTTCGATCAAATCAAAGCCGCGCCACTGCTGGGCCTCGCCG ATTACATTGCGTTTCGTCAGAAC (SEQ ID NO: 43) Taxadiene Synthase MSSSTGTSKVVSETSSTIVDDIPRLSANYHGDLWHHNVIQTLETPFRESSTYQERADELVVKIKD FNALGDGDISPSAYDTAWVARLATISSDGSEKPRFPQALNWVFNNQLQDGSWGIESHFSLCDRL LNTTNSVIALSVWKTGHSQVQQGAEFIAENLRLLNEEDELSPDFQIIFPALLQKAKALGINLPYDLPF IKYLSTTREARLTDVSAAADNIPAN LNALEGLEEVIDWNKIMRFQSKDGSFLSSPASTACVLMNT GDEKCFTFLNNLLDKFGGCVPC YSIDLLERLSLVDNIEHLGIGRHFKQEIKGALDYVYRHWSERG IGWGRDSLVPDLNTTALGLRTLRMHGYNVSSDVLNNFKDENGRFFSSAGQTHVELRSVVNLFRA SDLAFPDERA DDARKFAEPYLREALATKISTNTKLFKEIEYWEYPWHMSIPRLEARSYIDSYDD NYVWQRKTLYRMPSLSNSKCLELAKLDFNIVQSLHQEELKLLTRWWKESGMADINFTRHRVAEV YFSSATFEPEYSATRIAFTKIGCLQVLFDDMADIFATLDELKSFTEGVKRWDTSLLHEIPECMQTCF KVWFKLMEEVNNDVVKVQGRDMLAHIRKPWELYFNCYVQEREWLEAGYIPTFEEYLKTYAISVGL GPCTLQPILLMGELVKDDVVEKVHYPSNMFELVSLSWRLTNDTKTYQAEKARGQQASGIACY K DNPGATEEDAIKHICRWDRALKEASFEYFKPSNDIPMGCKSFIFNLRLCVQIFYKFIDGYGIANEEI KDYIRKVYIDPIQV (SEQ ID NO: 44) ATGTCTAGCTCTACGGGTACGTCTAAAGTCGTGAGTGAAACCTCATCGACGATCGTGGA CGATATTCCACGCTTGTCGGCGAACTATCATGGAGATCTGTGGCATCATAACGTCATTC AGACATTGGAAACCCCGTTTCGCGAAAGTAGCACCTACCAGGAACGGGCAGATGAATTA GTCGTGAAAATCAAAGATATGTTTAATGCATTAGGAGATGGAGACATCTCGCCCAGCGC ATATGATACGGCGTGGGTGGCTCGGTTGGCCACGATTAGCTCCGATGGCAGTGAAAAG CCGCGTTTCCCGCAGGCGCTGAACTGGGTGTTTAATAATCAATTGCAGGATGGCAGCT GGGGCATTGAATCTCACTTTAGCCTCTGTGACCGGTTACTCAACACGACAAACTCCGTA ATTGCGTTGTCAGTTTGGAAAACGGGCCATAGCCAGGTTCAACAGGGCGCGGAATTTAT CGCTGAAAATCTGCGCCTGCTGAACGAGGAGGACGAACTGTCACCCGATTTTCAGATTA TTTTTCCGGCTTTACTCCAGAAAGCCAAAGCCTTAGGCATCAACCTGCCATATGATCTGC CGTTCATCAAGTATCTGTCTACTACCCGCGAAGCCCGTCTCACTGACGTCTCTGCGGCG GCGGACAATATTCCAGCGAACATGCTGAACGCACTGGAAGGGCTGGAAGAGGTTATCG ACTGGAATAAAATCATGCGCTTCCAAAGCAAGGACGGTAGCTTCTTAAGCAGCCCAGCA TCTACTGCTTGTGTTCTGATGAATACCGGAGACGAAAAGTGCTTTACGTTTCTGAACAAT CTGCTGGACAAATTTGGGGGTTGTGTTCCTTGTATGTATTCCATTGATCTGTTGGAACGT CTGTCGCTGGTCGATAACATTGAACACTTAGGTATCGGCCGCCACTTCAAACAAGAAAT CAAGGGGGCGTTGGATTATGTATACCGTCATTGGAGCGAGCGTGGTATTGGTTGGGGG CGCGATAGCTTGGTACCTGATCTGAACACCACTGCTTTGGGACTGCGCACTCTTCGTAT GCACGGATACAACGTTAGTTCCGATGTCCTCAATAATTTCAAGGACGAGAACGGCCGTT TTTTCAGCTCGGCCGGTCAGACGCATGTTGAACTGCGGTCCGTAGTCAATCTCTTTCGC GCTAGTGATCTGGCCTTCCCCGACGAGCGCGCTATGGACGATGCACGGAAGTTTGCCG AGCCGTATCTCCGCGAAGCCCTGGCCACCAAAATTTCAACCAACACCAAGCTTTTCAAA GAAATTGAGTATGTAGTAGAGTATCCGTGGCATATGTCTATTCCGCGCCTGGAAGCCCG CTC GTAT ATCG ATTCTT AC G ATG AC AATTATGTGTGG C AAC GC AAAAC ACTGT AC C GT AT GCCCAGCCTGTCAAATAGTAAGTGTCTGGAGCTGGCGAAACTGGATTTCAACATTGTGC AATCCCTGCACCAAGAAGAGCTGAAATTACTGACTCGCTGGTGGAAGGAATCCGGCAT GGCAGACATCAATTTTACGCGTCACCGTGTTGCAGAGGTGTACTTCTCCTCGGCGACCT TTGAGCCGGAGTATTCGGCCACACGTATTGCATTTACCAAGATTGGCTGCCTTCAGGTG CTTTTTGACGATATGGCGGATATTTTTGCGACACTTGATGAGCTTAAATCATTTACCGAA GGCGTGAAGCGTTGGGATACCTCTCTGTTGCATGAAATCCCCGAATGTATGCAGACCTG CTTCAAAGTTTGGTTCAAACTGATGGAAGAAGTGAACAACGACGTCGTGAAAGTTCAGG GTCGTGATATGTTAGCACACATCCGCAAGCCGTGGGAACTCTATTTCAATTGCTATGTG CAGGAGCGTGAATGGTTAGAAGCGGGCTACATTCCTACCTTCGAAGAGTACTTAAAAAC CTATGCCATTTCCGTCGGTTTAGGCCCGTGCACTCTGCAGCCTATCTTGCTGATGGGTG AGCTGGTAAAGGATGATGTGGTGGAAAAAGTTCACTACCCGTCGAATATGTTTGAACTG GTAAGTCTGAGTTGGCGTCTGACAAACGACACCAAAACGTACCAGGCAGAAAAGGCAC GTGGGCAACAGGCAAGCGGTATCGCGTGTTATATGAAGGATAATCCGGGCGCTACTGA GGAAGATGCCATTAAGCATATCTGCCGTGTTGTGGATCGCGCTCTTAAAGAAGCGTCAT TCGAATATTTTAAACCTAGTAATGATATTCCGATGGGTTGTAAGTCATTCATTTTCAATCT TCGCCTGTGCGTGCAMTTTTTTACAAATTTATTGACGGCTACGGAATCGCCAACGAAGA AATCAAAGACTATATTCGTAAAGTTTACATCGATCCAATCCAGGTC (SEQ ID NO: 45) Cytochrome P450 Taxadiene 5a-hydroxylase (? Da ??) MDALYKSTVAKFNEVTQLDCSTESFSIALSAIAGILLLLLLFRSKRHSSLKLPPGKLGIPFI GESFIFLRALRSNSLEQFFDERVKKFGLVFKTSLIGHPTWLCGPAGNRLILSNEEKLVQ MSWPAQFMKLMGENSVATRRGEDHIVMRSALAGFFGPGALQSYIGKMNTEIQSHI NE KWKGKDEVNVLPLVRELVFNISAILFFNIYDKQEQDRLHKLLETILVGSFALPIDLPGFGF HRALQGRAKLNKIMLSLIKKRKEDLQSGSATATQDLLSVLLTFRDDKGTPLTNDEILDNF SSLLHASYDTTTSPMALIFKLLSSNPECYQKWQEQLEILSNKEEGEEITWKDLKA KYT WQVAQETLRMFPPVFGTFR AITDIQYDGYTIPKGW LLWTTYSTHPKDLYFNEPEKF MPSRFDQEGKHVAPYTFLPFGGGQRSCVGWEFSKMEILLFVHHFVKTFSSYTPVDPD EKISGDPLPPLPSKGFSIKLFPRP (SEQ ID NO: 46) ATGGATGCCCTCTATAAGTCTACCGTGGCGAAATTTAACGAAGTAACCCAGCTGGA TTGCAGCACTGAGTCATTTAGCATCGCTTTGAGTGCAATTGCCGGGATCTTGCTGT TGCTCCTGCTGTTTCGCTCGAAACGTCATAGTAGCCTGAAATTACCTCCGGGCAAA CTGGGCATTCCGTTTATCGGTGAGTCCTTTATTTTTTTGCGCGCGCTGCGCAGCAA TTCTCTGGAACAGTTCTTTGATGAACGTGTGAAGAAGTTCGGCCTGGTATTTAAAAC GTCCCTTATCGGTCACCCGACGGTTGTCCTGTGCGGGCCCGCAGGTAATCGCCTC ATCCTGAGCAACGAAGAAAAGCTGGTACAGATGTCCTGGCCGGCGCAGTTTATGA AGCTGATGGGAGAGAACTCAGTTGCGACCCGCCGTGGTGAAGATCACATTGTTAT GCGCTCCGCGTTGGCAGGCTTTTTCGGCCCGGGAGCTCTGCAATCCTATATCGGC AAGATGAACACGGAAATCCAAAGCCATATTAATGAAAAGTGGAAAGGGAAGGACGA GGTTAATGTCTTACCCCTGGTGCGGGAACTGG I I I I I AACATCAGCGCTATTCTGT TCTTTAACATTTACGATAAGCAGGAACAAGACCGTCTGCACAAGTTGTTAGAAACCA TTCTGGTAGGCTCGTTTGCCTTACCAATTGATTTACCGGGTTTCGGGTTTCACCGC GCTTTACAAGGTCGTGCAAAACTCAATAAAATCATGTTGTCGCTTATTAAAAAACGT AAAGAGGACTTACAGTCGGGATCGGCCACCGCGACGCAGGACCTGTTGTCTGTGC TTCTGACTTTCCGTGATGATAAGGGCACCCCGTTAACCAATGACGAAATCCTGGAC AACTTTAGCTCACTGCTTCACGCCTCTTACGACACCACGACTAGTCCAATGGCTCT GATTTTCAAATTACTGTCAAGTAACCCTGAATGCTATCAGAAAGTCGTGCAAGAGCA ACTCGAGATTCTGAGCAATAAGGAAGAAGGTGAAGAAATTACCTGGAAAGATCTTA AGGCCATGAAATACACGTGGCAGGTTGCGCAGGAGACACTTCGCATGTTTCCACC GGTGTTCGGGACCTTCCGCAAAGCGATCACGGATATTCAGTATGACGGATACACA ATCCCGAAAGGTTGGAAACTGTTGTGGACTACCTATAGCACTCATCCTAAGGACCT TTACTTCAACGAACCGGAGAAATTTATGCCTAGTCGTTTCGATCAGGAAGGCAAAC ATGTTGCGCCCTATACCTTCCTGCCCTTTGGAGGCGGTCAGCGGAGTTGTGTGGG TTGGGAGTTCTCTAAGATGGAGATTCTCCTCTTCGTGCATCATTTCGTGAAAACATT TTCGAGCTATACCCCGGTCGATCCCGATGAAAAAATTTCCGGCGATCCACTGCCG CCGTTACCGAGCAAAGGGTTTTCAATCAAACTGTTCCCTCGTCCG (SEQ ID NO: 47) Taxus NADPH: cytochrome P450 reductase (TCPR) MQANSNTVEGASQGKSLLDIS LDHIFALLLNGKGGDLGAMTGSALILTENSQNLMILTTA LAVLVACVFFFVWRRGGSDTQKPAVRPTPLVKEEDEEEEDDSA KKVTIFFGTQTGTAEG FAKALAEEAKARYEKAVFKWDLDNYAADDEQYEEKLKKEKLAFFMLATYGDGEPTDNAA RFY WFLEGKEREPWLSDLTYGVFGLGNRQYEHFNKVAKAVDEVLIEQGAKRLVPVGLG DDDQCIEDDFTAWREQVWPELDQLLRDEDDEPTSATPYTAAIPEYRVEIYDSWSVYEET HALKQNGQAVYDIHHPCRSNVAVRRELHTPLSDRSCIHLEFDISDTGLIYETGDHVGVHTE NSIETVEEAAKLLGYQLDTIFSVHGDKEDGTPLGGSSLPPPFPGPCTLRTALARYADLLNP PRKAAFLALAAHASDPAEAERLKFLSSPAGKDEYSQWVTASQRSLLEIMAEFPSAKPPLG VFFAAIAPRLQPRYYSISSSPRFAPSRIHVTCALVYGPSPTGRIHKGVCSNWMKNSLPSEE THDCSWAPVFVRQSNFKLPADSTTPIVMVGPGTGFAPFRGFLQERAKLQEAGEKLGPAV LFFGCRMRQMDYIYEDELKGYVEKGILTNLIVAFSREGATKEYVQHKMLEKASDTWSLIAQ GGYLYVCGDAKGMARDVHRTLHTIVQEQESVDSSKAEFLVKKLQMDGRYLRDIW (SEQ ID NO: 48) ATGCAGGCGAATTCTAATACGGTTGAAGGCGCGAGCCAAGGCAAGTCTCTTCTGGAC ATTAGTCGCCTCGACCATATCTTCGCCCTGCTGTTGAACGGGAAAGGCGGAGACCTT GGTGCGATGACCGGGTCGGCCTTAATTCTGACGGAAAATAGCCAGAACTTGATGATT CTGACCACTGCGCTGGCCGTTCTGGTCGCTTGCG I I I I I I I I TTCGTTTGGCGCCGTG GTGGAAGTGATACACAGAAGCCCGCCGTACGTCCCACACCTCTTGTTAAAGAAGAGG ACGAAGAAGAAGAAGATGATAGCGCCMGAAAAAGGTCACAATA7TTTTTGGCACCCA GACCGGCACCGCCGAAGGTTTCGCAAAGGCCTTAGCTGAGGAAGCAAAGGCACGTT ATGAAAAGGCGGTATTTAAAGTCGTGGATTTGGATAACTATGCAGCGGATGACGAACA GTACGAAGAGAAGTTGAAAAAGGAAAAGCTAGCGTTCTTCATGCTCGCCACCTACGG TGACGGCGAACCGACTGATAATGCCGCTCGCTTTTATAAATGGTTTCTCGAGGGTAAA GAGCGCGAGCCATGGTTGTCAGATCTGACTTATGGCGTGTTTGGCTTAGGTAACCGT CAGTATGAACACTTTAACAAGGTCGCGAAAGCGGTGGACGAAGTGCTCATTGAACAA GGCGCCAAACGTCTGGTACCGGTAGGGCTTGGTGATGATGATCAGTGCATTGAGGAC GACTTCACTGCCTGGAGAGAACAAGTGTGGCCTGAGCTGGATCAGCTCTTACGTGAT GAAGATGACGAGCCGACGTCTGCGACCCCGTACACGGCGGCTATTCCAGAATACCG GGTGGAAATCTACGACTCAGTAGTGTCGGTCTATGAGGAAACCCATGCGCTGAAACA AAATGGACAAGCCGTATACGATATCCACCACCCGTGTCGCAGCAACGTGGCAGTACG TCGTGAGCTGCATACCCCGCTGTCGGATCGTAGTTGTATTCATCTGGAATTCGATATT AGTGATACTGGGTTAATCTATGAGACGGGCGACCACGTTGGAGTTCATACCGAGAAT TCAATTGAAACCGTGGAAGAAGCAGCTAAACTGTTAGGTTACCAACTGGATACAATCT TCAGCGTGCATGGGGACAAGGAAGATGGAACACCATTGGGCGGGAGTAGCCTGCCA CCGCCGTTTCCGGGGCCCTGCACGCTGCGGACGGCGCTGGCACGTTACGCGGACC TGCTGAACCCTCCGCGCAAAGCCGCCTTCCTGGCACTGGCCGCACACGCGTCAGAT CCGGCTGMGCTGAACGCCTTAAATTTCTCAGTTCTCCAGCCGGAAAAGACGAATACT CACAGTGGGTCACTGCGTCCCAACGCAGCCTCCTCGAGATTATGGCCGAATTCCCCA GCGCGAAACCGCCGCTGGGAGTGTTTTTCGCCGCAATAGCGCCGCGCTTGCAACCT AGGTATTATAGCATCTCCTCCTCCCCGCGTTTCGCGCCGTCTCGTATCCATGTAACGT GCGCGCTGGTCTATGGTCCTAGCCCTACGGGGCGTATTCATAAAGGTGTGTGCAGCA ACTGGATGAAGAATTCTTTGCCCTCCGAAGAAACCCACGATTGCAGCTGGGCACCGG TCTTTTGTGCGCCAGTCAAACTTTAAACTGCCCGCCGATTCGACGACGCCAATCGTGAT GGTTGGACCTGGAACCGGCTTCGCTCCATTTCGCGGCTTCCTTCAGGAACGCGCAAA ACCGCCAGATGGATTACATCTATGAAGATGAGCTTAAGGGTTACGTTGAAAAAGGTAT TCTGACGAATCTGATCGTTGCATTTTCACGAGAAGGCGCCACCAAAGAGTATGTTCAG CACAAGATGTTAGAGAAAGCCTCCGACACGTGGTCTTTAATCGCCCAGGGTGGTTAT CTGTATGTTTGCGGTGATGCGAAGGGTATGGCCAGAGACGTACATCGCACCCTGCAT 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Nucleic acids research 32, W321 (2004). 56. Shalel-Levanon, S., San, K. Y. & Bennett, G. N. Effect of ArcA and FNR on the expression of genes related to the oxygen regulation and the glycolysis pathway in Escherichia coli under microaerobic growth conditions. Biotechnology and bioengineering 92 (2005). 57. Heinig, U. & Jennewein, S. Taxol: A natural diterpenoid complex product with an evolutionarily obscure origin. African Journal of Biotechnology 8, 1370-1385 (2009). 58. Walji, A. M. & MacMillan, D. W. C. Strategies to Bypass the Taxol Problem. Enantioselective Cascade Catalysis, a New Approach for the Efficient Construction of Molecular Complexity. Synlett 18, 1477-1489 (2007). 59. Chau, M., Jennewein, S., Walker, K. & Croteau, R. Taxol biosynthesis: Molecular cloning and characterization of a cytochrome P450 taxoid 7 beta-hydroxylase. Chem Biol 11, 663-72 (2004). 60. Williams, D.C. et al. Heterologous expression and characterization of a "Pseudomature" form of taxa and synthase involved in paclitaxel (Taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction. Arch Biochem Biophys 379, 137-46 (2000). 61. Morrone D. et al. Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering. Appl Microbiol Biotechnol. 85 (6): 1893-906 (2010) Thus having described various aspects of at least one embodiment of this invention, it will be appreciated that various alterations, modifications, and improvements will readily occur to those skilled in the art. It is intended that such alterations, modifications and improvements be part of this disclosure and are intended to be within the spirit and scope of the invention. Accordingly, the above description and drawings are by way of example only. Those skilled in the art will recognize, or may be successful using, no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Said equivalents are projected to be comprised by the following claims.

All references described herein are incorporated by reference in their entirety for the specific purpose mentioned herein.

Claims (99)

NOVELTY OF THE INVENTION CLAIMS

1. - A method comprising: recombinantly expressing a taxadiene synthase enzyme and a geranylgeranyl diphosphate enzyme (GGPPS) enzyme in a cell that overexpresses one or more components of the non-mevalonate (MEP) pathway.

2 - . 2 - The method according to claim 1, further characterized in that the cell is a bacterial cell.

3. - The method according to claim 2, further characterized in that the cell is an Escherichia coli cell.

4. - The method according to claim 2, further characterized in that the cell is a Gram positive cell.

5. - The method according to claim 4, further characterized in that the cell is a Bacillus cell.

6. - The method according to claim 1, further characterized in that the cell is a yeast cell.

7. - The method according to claim 6, further characterized in that the yeast cell is a cell of Saccharomyces.

8. - The method according to claim 6, further characterized in that the yeast cell is a Yarrowia cell.

9. - The method according to claim 1, further characterized in that the cell is an algae cell.

10. - The method according to claim 1, further characterized in that the cell is a plant cell.

11- The method according to claim 1, further characterized in that the enzyme taxadiene synthase is a Taxus enzyme.

12. - The method according to claim 1, further characterized in that the enzyme taxadiene synthase is an enzyme of Taxus brevifolia.

13. - The method according to any of claims 1-12, further characterized in that the GGPPS enzyme is a Taxus enzyme.

14. - The method according to claim 13, further characterized in that the GGPPS enzyme is an enzyme of Taxus canadenis.

15. - The method according to any of claims 1-14, further characterized in that the gene coding for the enzyme taxadiene synthase and / or the gene encoding the GGPPS enzyme and / or the genes coding for the one or more components of the MEP pathway are expressed from one or more plasmids.

16. - The method according to any of claims 1-15, further characterized in that the gene coding for the enzyme taxadiene synthase and / or the gene encoding the GGPPS enzyme and / or the genes coding for the one or more MEP components are integrated into the cell genome.

17. - The method according to any of claims 1-16, further characterized in that the one or more components of the non-mevalonate route (MEP) is selected from the group consisting of dxs, spC, ispD, ispE, ispF, ispG , ispH, ¡di, ispA and ispB.

18. - The method according to claim 17, further characterized in that dxs, dI, ispD and ispF are overexpressed.

19. - The method according to claim 18, further characterized in that dxs, dI, ispD and ispF are overexpressed in the dxs-idi-idpDF operon.

20. - The method according to any of claims 1-9, further characterized in that the gene encoding the enzyme taxadiene synthase and the gene encoding the GGPPS enzyme are expressed together in an operon.

21. The method according to any of claims 1-20, further characterized in that the cell further expresses a taxadiene 5a-hydroxylase (T5aOH) or a catalytically active portion thereof.

22. The method according to claim 21, further characterized in that the T5aOH enzyme or a catalytically active portion thereof is fused to a cytochrome P450 reductase enzyme or a catalytically active portion thereof.

23. - The method according to claim 22, further characterized in that the T5aOH enzyme is At24T5aOH-tTCPR.

24. - The method according to any of claims 1-23, further characterized in that the expression of the enzyme taxadiene synthase, the GGPPS enzyme and the one or more components of the MEP route are balanced to maximize the production of the taxadiene.

25. - The method according to any of claims 1-24, further characterized in that it further comprises culturing the cell.

26. - The method according to any of claims 1-25, further characterized in that the cell produces taxadiene or taxadiene-5a-ol.

27. - The method according to claim 26, further characterized in that at least 10 mg L "1 of taxadiene is produced.

28. - The method according to claim 27, further characterized in that at least 250 mg L 1 of taxadiene is produced.

29. - The method according to claim 26, further characterized in that at least 10 mg L "1 of taxadiene-5a-ol is produced.

30. - The method according to claim 29, further characterized in that at least 50 mg L "1 of taxadiene-5a-ol is produced.

31. - The method according to claim 26, 29 or 30, further characterized in that the conversion ratio of taxadiene to taxadiene-5a-ol and byproduct 5 (12) -Oxa-3 (11) -cyclotaxane is at least 50 %.

32. - The method according to claim 31, further characterized in that the conversion ratio of taxadiene to taxadiene-5o ol and byproduct 5 (12) -Oxa-3 (11) -cyclotaxane is at least 75%.

33. The method according to claim 32, further characterized in that the conversion ratio of taxadiene to taxadiene-5a-ol and byproduct 5 (12) -Oxa-3 (11) -cyclotaxane is at least 95%.

34. - The method according to any of claims 25-33, further characterized in that it additionally comprises recovering the taxadiene or taxadiene-5a-ol from the cell culture.

35. - The method according to claim 34, further characterized in that the taxadiene or taxadiene-5a-ol is recovered from the gas phase.

36. - The method according to claim 34, further characterized in that an organic layer is added to the cell culture, and the taxadiene or taxadiene-5a-ol is recovered from the organic layer.

37. - A cell that overexpresses one or more components of the non-mevalonate (MEP) pathway, and that recombinantly expresses a taxadiene synthase enzyme and a geranylgeranyl diphosphate enzyme (GGPPS).

38. - The cell according to claim 37, further characterized in that the cell is a bacterial cell.

39. - The cell according to claim 38, further characterized in that the cell is an Escherichia coli cell.

40. - The cell according to claim 38, further characterized in that the cell is a Gram positive cell.

41. - The cell according to claim 40, further characterized in that the cell is a Bacillus cell.

42. - The cell according to claim 37, further characterized in that the cell is a yeast cell.

43. - The cell according to claim 42, further characterized in that the yeast cell is a Saccharomyces cell.

44. - The cell according to claim 42, further characterized in that the yeast cell is a Yarrowia cell.

45. - The cell according to claim 37, further characterized in that the cell is an alga cell.

46. - The cell according to claim 37, characterized further because the cell is a plant cell.

47 -. 47. The cell according to claim 37, further characterized in that the enzyme taxadiene synthase is a Taxus enzyme.

48. - The cell according to claim 47, further characterized in that the enzyme taxadiene synthase is an enzyme of Taxus brevifolia.

49. - The cell according to any of claims 37-48, further characterized in that the GGPPS enzyme is a Taxus enzyme.

50. - The cell according to claim 49, further characterized in that the GGPPS enzyme is an enzyme of Taxus canadenis.

51. - The cell according to any of claims 37-50, further characterized in that the gene coding for the enzyme taxadiene synthase and / or the gene encoding the GGPPS enzyme and / or the genes coding for the one or more components of the MEP pathway are expressed from one or more plasmids.

52. - The cell according to any of claims 37-51, further characterized in that the gene coding for the enzyme taxadiene synthase and / or the gene encoding the GGPPS enzyme and / or the genes coding for the one or more MEP components are integrated into the cell genome.

53. - The cell according to any of claims 37-52, further characterized in that the one or more components of the non-mevalonate route (MEP) is selected from the group consisting of dxs, ispC, ispD, ispE, ¡spF, ispG , ispH, idi, ispA and ispB.

54. - The cell according to claim 53, further characterized in that the dxs, i.d, ispD and ispF are overexpressed.

55. - The cell according to claim 54, further characterized in that the dxs, idi, ispD and ispF are overexpressed in the dxs-idi-idpDF operon.

56. - The cell according to any of claims 37-55, further characterized in that the gene coding for the enzyme taxadiene synthase and the gene encoding the GGPPS enzyme are expressed together in an operon.

57. - The cell according to any of claims 37-56, further characterized in that the cell further expresses a taxadiene 5a-hydroxylase (T5aOH) or a catalytically active portion thereof.

58. - The cell according to claim 57, further characterized in that the T5aOH enzyme or a catalytically active portion thereof is fused to a cytochrome P450 reductase enzyme or a catalytically active portion thereof.

59. - The cell according to claim 58, further characterized in that the T5aOH enzyme is expressed as At24T5aOH-tTCPR.

60. - The cell according to any of claims 37-59, further characterized in that the expression of the enzyme taxadiene synthase, the GGPPS enzyme and the one or more components of the MEP route are balanced to maximize the production of the taxadiene.

61. - The cell according to any of claims 37-60, further characterized in that the cell produces taxadiene or taxadiene-5a-ol.

62. - A method for selecting a cell that exhibits the increased production of a terpenoid, the method comprises: creating or obtaining a cell that overexpresses one or more components of the non-mevalonate (MEP) pathway, produces terpenoid from the cell, compares the amount of terpenoid produced from the cell to the amount of terpenoid produced in a control cell, and selecting a first improved cell that produces a higher amount of terpenoid than a control cell, wherein a first improved cell produces a higher amount of terpenoid that the control cell is a cell that exhibits increased production of terpenoid

63. - The method according to claim 62, further characterized in that the cell recombinantly expresses a terpenoid synthase enzyme.

64. - The method according to claim 62 or 63, further characterized in that the cell recombinantly expresses a geranylgeranyl diphosphate enzyme (GGPPS) enzyme.

65. The method according to any of claims 62-64, further characterized in that it additionally comprises altering the level of expression of one or more of the components of the non-mevalonate (MEP) pathway, the terpenoid synthase enzyme and / or the enzyme geranylgeranyl diphosphate synthase (GGPPS) in the first improved cell to produce a second improved cell, and compare the amount of terpenoid produced from the second improved cell to the amount of terpenoid produced in the first improved cell, wherein a second improved cell that produces a higher amount of terpenoid than the first improved cell is a cell that exhibits increased production of terpenoid.

66. The method according to any of claims 62-65, further characterized in that the enzyme terpenoid synthase is a taxadiene synthase enzyme.

67. An isolated polypeptide comprising a taxadiene 5a-hydroxylase enzyme (T5aOH) or a catalytically active portion thereof fused to a cytochrome P450 reductase enzyme or a catalytically active portion thereof.

68. The isolated polypeptide according to claim 67, further characterized in that the cytochrome P450 reductase enzyme is a reductase enzyme of the Taxus cytochrome P450 (TCPR).

69. The isolated polypeptide according to claim 68, further characterized in that the taxadiene 5a-hydroxylase and the TCPR are linked by a linker.

70. The isolated polypeptide according to claim 69, further characterized in that the linker is GSTGS (SEQ ID NO: 50).

71. The polypeptide isolated according to any of claims 67-70, further characterized in that the taxadiene 5a-hydroxylase and / or the TCPR are truncated to remove all or part of the transmembrane region.

72. - The isolated polypeptide according to the claim 71, further characterized in that the N-terminal amino acids 8, 24, or 42 of the taxadiene-hydroxylase are truncated.

73. The isolated polypeptide according to claim 71 or 72, further characterized in that 74 amino acids of the TCPR are truncated.

74. The isolated polypeptide according to any of claims 67-73, further characterized in that an additional peptide is fused to the taxadienase-hydroxylase.

75. The polypeptide isolated according to claim 74, further characterized in that the additional peptide is bovine 17a hydroxylase.

76. The isolated polypeptide according to claim 75, further characterized in that the peptide is MALLLAVF (SEQ ID NO: 5).

77. The isolated polypeptide according to claim 67, further characterized in that the isolated polypeptide is At24T5aOH-tTCPR.

78. - A nucleic acid molecule encoding the polypeptide of any of claims 67-77.

79. - A cell that recombinantly expresses a polypeptide of any of claims 67-77.

80. A method for increasing the production of terpenoid in a cell that produces one or more terpenoids, which comprises controlling the accumulation of indole in the cell or in a culture of the cells, thereby increasing the production of terpenoid in a cell.

81. - The method according to claim 80, further characterized in that the cell is a bacterial cell.

82. - The method according to claim 81, further characterized in that the cell is an Escherichia coli cell.

83. - The method according to claim 81, further characterized in that the cell is a Gram positive cell.

84. - The method according to claim 83, further characterized in that the cell is a Bacillus cell.

85. - The method according to claim 80, further characterized in that the cell is a yeast cell.

86. - The method according to claim 85, further characterized in that the yeast cell is a cell of Saccharomyces.

87. - The method according to claim 85, further characterized in that the yeast cell is a Yarrowia cell.

88. The method according to claim 80, further characterized in that the cell is an algae cell.

89. - The method according to claim 80, further characterized in that the cell is a plant cell.

90. - The method according to claim 80, further characterized in that the cell is a cell as recited in any of claims 37-61.

91 -. 91 - The method according to any of claims 80-90, further characterized in that the step of controlling the accumulation of indole in the cell or in a culture of the cells comprises balancing the upstream path of non-mevalonate isoprenoid with the routes downstream of product synthesis and / or modifying or regulating the indole route.

92. - The method according to any of claims 80-91, further characterized in that the step of controlling the accumulation of indole in the cell or in a culture of the cells comprises or additionally comprises removing the accumulated indole from the fermentation by chemical methods, using optionally absorbers or captors.

93. - The method according to any of claims 80-92, further characterized in that the one or more Terpenoids are a monoterpenoid, a sesquiterpenoid, a diterpenoid, a triterpenoid or a tetraterpenoid.

94. - The method according to claim 93, further characterized in that the one or more terpenoids is taxadiene or any precursor of taxol.

95. - A method comprising measuring the amount or concentration of indole in a cell that produces one or more terpenoids or in a culture of the cells that produces one or more terpenoids.

96. - The method according to claim 95, further characterized in that the method comprises measuring the amount or concentration of indole two or more times.

97. - The method according to claim 95 or claim 96, further characterized in that the measured amount or concentration of indole is used to guide a process of producing one or more terpenoids.

98. - The method according to claim 95 or claim 96, further characterized in that the measured amount or concentration of indole is used to guide the construction of the strain.

99. - The method according to any of claims 62-66, further characterized in that the cell also recombinantly expresses a polypeptide of any of claims 67-77.