[go: up one dir, main page]

WO2015198219A1 - Procédé de biotransformation d'alcanes linéaires - Google Patents

Procédé de biotransformation d'alcanes linéaires Download PDF

Info

Publication number
WO2015198219A1
WO2015198219A1 PCT/IB2015/054697 IB2015054697W WO2015198219A1 WO 2015198219 A1 WO2015198219 A1 WO 2015198219A1 IB 2015054697 W IB2015054697 W IB 2015054697W WO 2015198219 A1 WO2015198219 A1 WO 2015198219A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
cells
biotransformation
medium
polysaccharide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2015/054697
Other languages
English (en)
Inventor
Oluwafemi Ayokunle OLAOFE
Caryn J FENNER
Susan T L HARRISON
Murray Peter MEISSNER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Cape Town
Original Assignee
University of Cape Town
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Cape Town filed Critical University of Cape Town
Publication of WO2015198219A1 publication Critical patent/WO2015198219A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
    • C12Y114/15003—Alkane 1-monooxygenase (1.14.15.3)

Definitions

  • the invention relates to a method of whole-cell catalysed biotransformation of linear alkanes to oxygenated products in actively dividing cells capable of expressing a biocatalyst that catalyses conversion of a linear alkane into an oxygenated product, comprising incubating the actively dividing cells in a biotransformation medium comprising a linear alkane, thereby catalysing the conversion of the linear alkane into the oxygenated product.
  • the invention relates to a method of whole-cell catalysed biotransformation of linear alkanes to oxygenated products in actively dividing cells capable of expressing a biocatalyst that catalyses conversion of a linear alkane into an oxygenated product, comprising (i) incubating actively dividing cells in a growth medium including (a) a polysaccharide and (b) a polysaccharide-hydrolysing enzyme that hydrolyses the polysaccharide into a growth substrate for the actively dividing cells at a controlled rate and (ii) incubating the actively dividing cells in a biotransformation medium comprising a linear alkane, thereby catalysing the conversion of the linear alkane into the oxygenated product.
  • the linear alkanes from expanding activities in the petrochemical industry potentially provide an inexpensive hydrocarbon feedstock for the production of high value oxygenated products such as alcohols, ketones, aldehydes, hydroxyacids, dicarboxylic acids, and the like by means of biotransformation.
  • the application of whole-cells as biocatalysts in biotransformation is the preferred approach to guarantee the continuous regeneration of reducing equivalents from cofactors such as NAD(P)H. It also ensures the structural organisation and stable environment required for the biotransformation reaction in organic solvents (Duetz et al., 2001). In living cells, however, various cellular reactions compete for such cofactors, including oxidative phosphorylation.
  • Biotransformation reactions are usually performed in a batch process.
  • the batch operation is a discontinuous or two-phase process, where there is first a growth phase in which the cells are grown to the desired biomass concentration, followed by a separate bioconversion or biotransformation phase.
  • resting cells that have been harvested from the growth phase are provided with a bioreaction mixture consisting of the resting cell suspension (biocatalyst), carbon and an energy source to enable co-factor regeneration, but not active cell division (aqueous phase), as well as an alkane substrate (organic phase) and incubated for a period of time for the biotransformation process.
  • bioreaction mixture consisting of the resting cell suspension (biocatalyst), carbon and an energy source to enable co-factor regeneration, but not active cell division (aqueous phase), as well as an alkane substrate (organic phase) and incubated for a period of time for the biotransformation process.
  • These reactants are initially introduced into the reaction vessel and operate as a partially closed system, except for exchange of
  • the batch reaction is agitated to promote oxygen and substrate mass transfer in the two-phase biocatalytic system.
  • the batch system suffers from disadvantages such as high initial concentration of the carbon source and toxicity that may arise from the high concentration of the organic substrate at the start of biotransformation. This encourages uncontrolled process resulting in overflow metabolism, carbon wastage and biocatalyst inhibition.
  • the fed-batch mode of operation provides a means of controlling cell metabolism of the resting cells.
  • the growth-limiting substrate usually the carbon source
  • the growth-limiting substrate is supplied into the reactor at a predetermined rate which allows better control of cell metabolism.
  • Glucose was constantly supplied at a rate of 0.15 g min -1 to provide carbon and energy for the cell metabolism (but not active cell division), under biotransformation conditions.
  • a similar feeding regime has been demonstrated by Vallon et al.
  • biotransformation method could be developed; in particular a system that could be used both during the process development phase and during the scale-up phase of biotransformation. It would furthermore be useful if such a process was able to be performed in a single bioreactor, thereby reducing the complexity and cost of the unit operations required.
  • a method of whole-cell catalysed biotransformation of linear alkanes to oxygenated products in actively dividing cells capable of expressing a biocatalyst that catalyses conversion of a linear alkane into an oxygenated product comprising incubating the actively dividing cells in a biotransformation medium comprising a linear alkane, thereby catalysing the conversion of the linear alkane into the oxygenated product.
  • the method of whole-cell catalysed biotransformation of linear alkanes to oxygenated products in actively dividing cells capable of expressing a biocatalyst that catalyses conversion of a linear alkane into an oxygenated product comprises: (i) incubating actively dividing cells in a growth medium including:
  • the method comprises the steps of:
  • introducing the cells of (i) into a growth medium that supports active cell division including (a) a polysaccharide and (b) a polysaccharide-hydrolysing enzyme wherein the enzyme hydrolyses the polysaccharide into a substrate for growth of the actively dividing cells at a controlled rate and maintaining active cell division at a controlled rate in the growth medium;
  • steps (ii) to (vi) are performed in a single bioreactor.
  • the method may comprise a further step (iA), wherein prior to step (ii), the cells of step (i) are cultured in an initial growth medium that supports active cell division.
  • the initial growth medium may be a conventional growth medium known to those skilled in the art), or a growth medium including (a) a polysaccharide and (b) a polysaccharide- hydrolysing enzyme wherein the enzyme hydrolyses the polysaccharide into a substrate for growth of the actively dividing cells at a controlled rate.
  • the initial growth medium and the growth medium of step (ii) may have a similar composition, apart from the growth medium of step (ii) comprising any one or more of: a different concentration of the polysaccharide-hydrolysing enzyme, a growth-limiting nutrient, or an inducing agent for enzyme expression, compared with the initial growth medium.
  • the step (ii) may comprise a step of inoculating the cells cultured in step (iA) into the growth medium of step (ii).
  • step (ii) may comprise a step of supplementing the cells cultured in step (iA) with growth medium components to produce the growth medium of step (ii).
  • a person skilled in the art may, rather than by use of a polysaccharide and polysaccharide-hydrolysing enzyme to control the rate of growth of the actively dividing cells, use a fed-batch system where a substrate for growth of the actively dividing cells is fed into the system at a controlled rate, thereby to control the rate of growth of the actively dividing cells during the method.
  • the oxygenated product may be an alcohol, a ketone, an aldehyde, a hydroxyacid, a dicarboxylic acid or the like.
  • the polysaccharide may include starch, amylose, amylopectin, dextrin, cellulose, or hemicellulose and derivatives thereof known to those skilled in the art.
  • the growth substrate may be sucrose, fructose, glucose or another readily assimilable compound released from the polysaccharide including maltose, or maltotriose.
  • the growth substrate is glucose.
  • the polysaccharide-hydrolysing enzyme may be a single enzyme or a cocktail of enzymes. Typically, the enzyme would be selected depending on the polysaccharide used and the metabolically active growth substrate desired.
  • the enzyme may be an amylase, a glucoamylase, an isoamylase, a beta-glucosidase, a cellulolytic enzyme, or others known to those skilled in the art.
  • the enzyme is a glucoamylase.
  • the linear alkane may be an n-alkane selected from any one or more of Ci to C36 alkanes.
  • n-alkane may be octane and the octane may be converted into 1-octanol.
  • the biocatalyst may be an oxygenase.
  • the biocatalyst is a hydroxylase.
  • the biocatalyst is AlkB or cytochrome P450.
  • the cytochrome P450 is CYP153, CYP102, or CYP 52.
  • the cytochrome P450 is CYP153A6.
  • the cell may be a bacterial or a fungal cell, including a yeast cell.
  • the bacterial cell may be Escherichia. coli, Pseudomonas sp. , including Pseudomonas putida, Rhodococcus sp. , Act netobacter sp., Bacillus sp. , including Bacillus megate um, Mycobacterium sp, Arthrobacter sp., Streptomyces sp., Marinobacter aquaelolei, or Geobacillus thermodenitrificans.
  • the fungal cell may be a yeast cell, including Candida sp., Yarrowia or Arxula.
  • the cell may further express one or more electron transfer protein(s) for providing reducing equivalents for biotransformation.
  • the electron transfer protein(s) may be Ferrodoxin or Ferrodoxin Reductase, or both, or Rubredoxin, or Rubredoxin Reductase or both, or Cytochrome P450 Reductase, or FAD/FMN Reductase.
  • the cell may further express a transport protein for transporting hydrophobic substrates or products or both across the cell membrane.
  • the transport protein may be AlkL.
  • the cell may further express or over-express a dehydrogenase enzyme, including a glucose dehydrogenase or glycerol dehydrogenase enzyme.
  • a dehydrogenase enzyme including a glucose dehydrogenase or glycerol dehydrogenase enzyme.
  • the cell may be genetically engineered to express any one or more of the biocatalyst, the electron transfer protein(s), the transport protein, or the dehydrogenase enzyme.
  • the genetically engineered cell may be Escherichia coli.
  • the method at step (iii) may further comprise inducing the cells to express the biocatalyst.
  • the cells may be induced by addition of Isopropyl ⁇ -D-l-thiogalactopyranoside (IPTG), n-alkane or lactose.
  • IPTG Isopropyl ⁇ -D-l-thiogalactopyranoside
  • n-alkane or lactose n-alkane
  • lactose lactose
  • the growth medium of step (ii) may comprise a booster solution comprising complex additives, yeast extract and tryptone.
  • the growth medium of step (iv) may be formulated to exclude complex additives.
  • step (v) may be performed at from about 20 °C to about 37 °C, preferably from about 20 °C to about 25°C and most preferably at about 20 °C.
  • the steps (iii) and (iv) may be performed at a temperature of about 37 °C and the incubation step (v) at a temperature of from about 20 °C to about 25°C, preferably about 20 °C.
  • the biotransformation medium may further comprise an organic solvent to serve as a product sink, wherein during step (vi) the oxygenated product is drawn out of the aqueous phase into the product sink for recovery.
  • the solvent may be 1-hexadecanol or bis(2-ethylhexyl) phthalate (BEHP).
  • the oxygenated product is octanol
  • the solvent is BEHP.
  • a method of whole-cell catalysed biotransformation of linear alkanes to oxygenated products wherein at least from step (ii) onward of the method is performed in a bioreactor such as a stirred tank reactor or an orbital shaking reactor, or others known to those skilled in the art.
  • the orbital shaking reactor may comprise a helical track.
  • the entire method may be performed in a bioreactor.
  • FIG. 1 shows Octane bioconversion using metabolically active resting cells and growing whole cell biocatalysts cultured on chemically defined glucose medium.
  • the biomass was resuspended (to 3.5 gDcw LBRM "1 ) in 200 mM sodium phosphate buffer (pH 7.2) consisting of glucose.
  • the same biomass was resuspended in glucose-based defined medium (pH 7.2) to a starting concentration of 2.5 gDcw LBRM "1 .
  • the vials were opened intermittently for glucose addition and also to allow inlet of air to avoid oxygen limitation.
  • the octanol (A), P450 concentration (B), acetate formation (C) and reaction pH (D) were determined.
  • Figure 2 shows the influence of glucoamylase activity on glucose release from starch polymer in terms of (a) resultant glucose concentration and (b) glucose release rate using the EnBase® medium.
  • the data was collected in the absence of biomass addition.
  • the glucoamylase concentration was in the range 0.6 to 12 U L 1 .
  • the data represents an average of values from studies with and without octane.
  • Figure 3 shows the initial rate of glucose release from the polysaccharide substrate through varying the concentration of gluco-amylase concentration. The reaction was carried out in the presence of octane over the duration of 10 h.
  • Figure 4 shows the continuous supply of carbon and energy source in whole-cell hydroxylation of n-octane through the activity of glucoamylase in a starch hydrolysed medium. The (a) octanol formation (b) and pH of the reaction mixture were determined. The biomass was cultured on EnBase® medium, harvested by centrifugation and resuspended in starch solution to a concentration of 6.95 gDcw L "1 .
  • the bio-reaction mixture was supplemented with glucoamylase in the range of 0.6 to 12 U L 1 and 1 ml of the broth was introduced into 60 ml reaction vials, followed by the addition of 200 ⁇ octane and 100 ⁇ BEHP.
  • the reaction was mixed on an orbital shaker at 200 rpm and 20°C and the vials were opened intermittently to allow air inlet to prevent oxygen limitation.
  • Figure 5 shows whole cell biotransformation with growing biocatalyst cultured on
  • EnBase® medium consisting of the complex booster component.
  • the glucoamylase concentration was 0.6 U L 1 while process temperature was varied from 20 to 30°C, and compared with growing cell on chemically defined medium.
  • Figure 6 shows the effect of glucoamylase concentration across the range 0.6 - 12 U
  • Figure 7 shows octane oxidation as a function of glucose release rate, varied by varying the glucoamylase concentration. Following 22 h of cell growth and 1 h of protein induction, 1 ml of cell broth was introduced into 60 ml vials and glucoamylase was added. Thereafter, octane and BEHP were added for biotransformation. The reaction duration was 9 h.
  • Figure 8 shows (a) whole cell oxidation of octane using growing biocatalyst on
  • EnBase® medium (b) the production of acetic acid during biotransformation.
  • E. coli was grown on EnBase® medium for 12 h at 30°C using starting enzyme concentration of 0.6 U L 1 prior to induction. Following 3 h of induction, 1 ml aliquots were distributed into 60 ml reaction vessel with glucoamylase added in the range 0.6 to 12 U L "1 . Thereafter, 200 ⁇ of octane and 100 ⁇ of BEHP (product sink) were added to commence biotransformation at 20°C and agitation at 200 rpm.
  • Figure 9 shows (a) the oxidation of n-octane using growing whole cell E. coli (expressing CYP153A6) in EnBase® Flo Zero.
  • the biomass was grown on EnBase® for 22 h, induced and incubated at 20°C for 3 h, harvested and re- suspended (1-1.5 gDCW L "1 ) in EnBase® Flo Zero supplemented with IPTG, ⁇ -ALA and thiamine.
  • a 1 ml aliquot of this mixture was introduced into the 60 ml amber vials, glucoamylase was added at varied concentration and 30% v/v organic phase consisting of 200 ⁇ octane and 100 ⁇ BEHP was included to start biotransformation; and (b) the biomass formation.
  • Overflow metabolism was monitored via (c) acetic acid determination followed by (d) detection of medium pH (e) and the excess (un-used) glucose was determined in the reaction mixture.
  • the current invention provides a method of whole-cell catalysed biotransformation of linear alkanes to oxygenated products in actively dividing cells capable of expressing a biocatalyst that catalyses conversion of a linear alkane into an oxygenated product, comprising incubating the actively dividing cells in a biotransformation medium comprising a linear alkane, thereby catalysing the conversion of the linear alkane into the oxygenated product.
  • the invention relates to a method of whole-cell catalysed biotransformation of linear alkanes to oxygenated products in actively dividing cells capable of expressing a biocatalyst that catalyses conversion of a linear alkane into an oxygenated product, comprising (i) incubating actively dividing cells in a growth medium including (a) a polysaccharide and (b) a polysaccharide-hydrolysing enzyme that hydrolyses the polysaccharide into a growth substrate for the actively dividing cells at a controlled rate and (ii) incubating the actively dividing cells in a biotransformation medium comprising a linear alkane, thereby catalysing the conversion of the linear alkane into the oxygenated product.
  • a person skilled in the art may, rather than by use of a polysaccharide and polysaccharide- hydrolysing enzyme to control the rate of growth of the actively dividing cells, use a fed- batch system where a substrate for growth of the actively dividing cells is fed into the system at a controlled rate, thereby to control the rate of growth of the actively dividing cells during the method.
  • the term “growing cell” refers to a cell where growth is supported by a medium that supports active cell division
  • the term “resting cell” refers to a cell in a medium that does not support active cell division, although the cell may still be metabolically active.
  • bioreactor refers to a system in which a biological conversion is effected. Accordingly, a bioreactor may range from a small-scale system such as a vial or micro-titre plate to a mid-scale bioreactor such as a flask, to a large fermentation chamber capable of holding many cubic meters of fluid.
  • the linear alkane is converted into a primary alcohol
  • the method of the invention may be used for conversion of linear alkanes into a variety of commercially useful oxygenated products, including alcohols, ketones, aldehydes, hydroxyacids, dicarboxylic acids, and the like known to those skilled in the art.
  • the applicants have investigated whether this technology might also be applicable to the field of biotransformation for use during the biotransformation reaction in coversion of linear alkanes to high value oxygenated products, in particular, with the use of growing (actively dividing) cells, rather than resting cells as traditionally used.
  • the applicants have determined whether such technology could be used during the biotransformation phase of the biocatalytic reaction involving an organic phase.
  • the applicants have investigated whether the technology may be useful to facilitate the use of a single bioreactor, or equivalent small scale system such as a vial, microtitre plate or flask system for the complete duration of the two-phase biotransformation process.
  • the method comprises the steps of:
  • introducing the cells of (i) into a growth medium that supports active cell division including (a) a polysaccharide and (b) a polysaccharide-hydrolysing enzyme wherein the enzyme hydrolyses the polysaccharide into a substrate for growth of the actively dividing cells at a controlled rate and maintaining active cell division at a controlled rate in the growth medium;
  • steps (ii) to (vi) are performed in a single bioreactor.
  • the step (ii) may comprise a step of inoculating cells grown in an initial growth medium which may either be a conventional growth medium or a medium including (a) a polysaccharide and (b) a polysaccharide-hydrolysing enzyme wherein the enzyme hydrolyses the polysaccharide into a substrate for growth of the actively dividing cells at a controlled rate into the growth medium of (ii).
  • the step (ii) may comprise a step of supplementing the cells grown in the initial growth medium with the growth medium components of (ii).
  • the initial growth medium will differ from the growth medium of (ii) in that although they may both comprise a polysaccharide-hydrolysing enzyme wherein the enzyme hydrolyses a polysaccharide into a substrate for growth of the actively dividing cells at a controlled rate, the concentration of the polysaccharide-hydrolysing enzyme in the growth medium of (ii) is different to that of the first medium.
  • the growth medium of (ii) may further comprise a growth-limiting nutrient and/or at least one inducing agent for enzyme expression.
  • the inducing agent may be IPTG or lactose, although others may be used that are known to those skilled in the art and may be selected depending on the host organism used for biotransformation.
  • the polysaccharide is a starch that can be digested by an enzyme into a readily assimilable substrate by the cells.
  • the substrate could be amylose, amylopectin, dextrin, cellulose, hemicellulose and derivatives thereof known to those skilled in the art.
  • the growth substrate selected would typically depend on the cell to be cultured and the growth substrate for that cell.
  • the growth substrate could be sucrose, fructose, glucose or another readily assimilable compound hydrolysed from the polysaccharide including maltose, or maltotriose as desired for growth.
  • the polysaccharide-hydrolysing enzyme may be a single enzyme or a cocktail of enzymes. Typically, the enzyme would be selected depending on the polysaccharide used and the growth substrate desired to be hydrolysed from the polysaccaride.
  • the enzyme may be an amylase, a glucoamylase, an isoamylase, a beta-glucosidase, a cellulolytic enzyme, or others known to those skilled in the art.
  • the enzyme is glucoamylase.
  • the glucoamylase concentration during the biotransformation stage of step (iv) is in the range of 0.6 to 6 units per liter (U L 1 ). Optimally, the glucoamylase concentration is 0.6 U L "1 .
  • the medium comprising the polysaccharide and enzyme would typically be a mineral salt medium (MSM).
  • the medium may comprise 2 g/L Na 2 S0 4 , 2.68 g/L (NH 4 ) 2 S0 4 , 0.5 g/L NH 4 CI, 14.6 g/L K 2 HP0 4 , 3.6 g/L NaH 2 PO 4 .H 2 0, 1.0 g/L (NH 4 ) 2 -H- citrate and 1.5 M MgS0 4 .
  • low amounts of tryptone (0.24 g/L) and yeast extract (0.48 g/L) may be added (complex additives).
  • the medium of step (ii) is Enbase ® Flo medium.
  • tryptone, peptone and yeast extract may be added as a booster to the medium at step (ii).
  • the base MSM is supplemented with 3mM MgSCU, 2 ml/L of trace element solution and 0.1 g/l thiamine hydrochloride.
  • the MSM is supplemented with MOPS-buffer at pH 7.
  • the cells are harvested, washed and resuspended in an MSM comprising a metabolically inactive substrate and enzyme but without complex additives, such as peptone.
  • the medium used is the Enbase ® Flo Zero medium (obtained from BioSilta Oy, Oulu, Finland).
  • the medium prior to step (iv) is simply supplemented to obtain the MSM comprising the metabolically inactive substrate and enzyme, without complex additives. In this method, use of a single bioreactor or similar small-scale system is facilitated.
  • the polysaccharide In a preferred embodiment of the invention where the cell to be used in the biotransformation method is Escherichia coli, the polysaccharide and is digested by the polysaccharide hydrolysing enzyme, glucoamylase (or EnZ 'lm), thereby releasing the growth substrate, glucose.
  • the method may be used for any linear alkane and the biocatalyst would be selected depending on the alkane to be converted and the desired catalytic reaction.
  • any n-alkane from a Ci - C36 alkane may be used.
  • biocatalysts may be used, although typically the biocatalyst is an oxygenase.
  • the biocatalyst is a hydroxylase.
  • the biocatalyst may be AlkB or a cytochrome P450 like CYP153, CYP102 or CYP52.
  • the cytochrome P450 is CYP153A6.
  • the n-alkane is octane and the octane is catalysed by the cytochrome P450 CYP153A6 into 1 -octanol.
  • the method may be used with various cells capable of growth in cell culture.
  • the method would optimally be used with a bacterial or a fungal cell, particularly a yeast cell.
  • the cell selected for use may depend on the alkane feedstock to be converted and the biocatalytic enzyme(s) intrinsically expressed by the cell.
  • the bacterial cell may be Pseudomonas sp. , including Pseudomonas putida, Rhodococcus sp. , Acinetobacter sp., Bacillus sp., including Bacillus megaterium, Mycobacterium sp, Arthrobacter sp., Streptomyces sp., Marinobacter aquaelolei, or Geobacillus thermodenitrificans.
  • Pseudomonas sp. including Pseudomonas putida, Rhodococcus sp. , Acinetobacter sp., Bacillus sp., including Bacillus megaterium, Mycobacterium sp, Arthrobacter sp., Streptomyces sp., Marinobacter aquaelolei, or Geobacillus thermodenitrificans.
  • the fungal cell may be a yeast cell including Candida sp., Yarrowia or Arxula.
  • the cell does not intrinsically express a biocatalytic enzyme, but has been genetically engineered to express a desired biocatalyst.
  • the genetically engineered cell is Escherichia coli (E. coli).
  • E. coli Escherichia coli
  • Various methods of generating recombinant E. coli cells to express heterologous proteins including biocatalyst enzymes are well known to those skilled in the art.
  • the cell may further express or be genetically engineered to express one or more electron transfer protein(s) for providing reducing equivalents for biotransformation.
  • the electron transfer protein selected would depend on the orgin of the chosen P450 biocatalyst.
  • the electron transfer protein may be any one or more of Ferrodoxin, Ferrodoxin Reductase, Rubredoxin, or Rubredoxin Reductase, Cytochrome P450 Reductase, or FAD/FMN Reductase.
  • the cell may further express or be genetically engineered to express a transport protein for transporting hydrophobic substrates or products or both across the cell membrane for recovery.
  • a transport protein for transporting hydrophobic substrates or products or both across the cell membrane for recovery.
  • a transport protein that may be used is AlkL, although others known to those skilled in the art may be selected.
  • the cell may further express or be genetically engineered to express or over- express a dehydrogenase enzyme, including a glucose dehydrogenase or glycerol dehydrogenase enzyme or others known to those skilled in the art to produce an overexpression of reducing power.
  • a dehydrogenase enzyme including a glucose dehydrogenase or glycerol dehydrogenase enzyme or others known to those skilled in the art to produce an overexpression of reducing power.
  • the cells may constitutively express the biocatalyst, it is more typical that the method at step (iii) would comprise a method of inducing the cells to express the biocatalyst.
  • the cells would be induced during their mid- exponential growth phase (A578 of about 1.2 - 1.5).
  • the cell may be an E. coli cell expressing ⁇ - galactosidase, and the induction may be by addition of IPTG or lactose.
  • the cell medium is supplemented at induction with 0.25 mM ⁇ -aminolevulinic acid ( ⁇ -ALA) and 50 ⁇ FeCI 3 .6H 2 0.
  • the biotransformation medium may further comprise an organic solvent, thereby to produce a product sink, wherein during step (vi) the oxygenated product is drawn into the product sink for recovery.
  • the product sink may be selected by those skilled in the art depending on the oxygenated product to be recovered, the toxicity level of the solvent sink and the effect of the solvent sink on the particular organism used.
  • the linear alkane itself may serve as the organic solvent during two-phase biotransformation.
  • the solvent may be 1-hexadecanol or bis(2-ethylhexyl) phthalate (BEHP).
  • BEHP bis(2-ethylhexyl) phthalate
  • Step (v) may be performed at from about 20 to about 37 °C. Optimally however, step (v) is performed at from about 20 °C to about 25 °C.
  • the method is versatile in that it may be performed on a small scale such as with the use of vials or microtitre plates and the like, or the method may be scaled up for use in a bioreactor.
  • steps (ii) onward of the method can be performed in a bioreactor such as a stirred tank reactor or an orbital shaking reactor, or others known to those skilled in the art.
  • a bioreactor such as a stirred tank reactor or an orbital shaking reactor, or others known to those skilled in the art.
  • the entire method may be performed in a bioreactor.
  • the orbital shaking reactor would comprise a helical track with dimensions selected according to standard methods known to those skilled in the art.
  • conditions including the type of bioreactor, agitation rate, aeration rate and the like may be optimised depending on the type of bioreactor selected, and the desired size and capacity of the reactor according to standard methods known to those skilled in the art.
  • Table 1 Overview of experimental approach using growing cells as biocatalyst in the terminal hydroxylation of octane to 1 -octanol.
  • the EnBase® medium is a minimal salt medium containing a starch polysaccharide as substrate for glucose release, vitamins, trace elements and complex nutrient components, referred to as booster (Krause et al., 2010).
  • the addition of a glucoamylase, the polysaccharide-degrading enzyme (EnZ 'Im) responsible for the release of glucose is specified.
  • a variety of the EnBase® preparation, EnBase® Flo Zero is devoid of complex nutrients (e.g. peptone).
  • the complete EnBase® media packs (Flo and Flo Zero) were purchased from BioSilta Oy (Oulu, Finland). For cultivations in baffled shake flasks, 0.1 ml L "1 antifoam was added to prevent foaming and all media contained 30 ⁇ g ml -1 kanamycin for maintenance of plasmid stability through the extended growing phase.
  • the seed cultures were prepared by inoculating LB broth supplemented with kanamycin (30 ⁇ g ml -1 ) with E. coli cells from glycerol stocks previously maintained at -60°C. This was incubated on an orbital shaker at 37°C for 6 to 8 h at an agitation speed of 200 rpm.
  • a 4 ml inoculum was used to inoculate the glucose-based defined medium (200 ml in 2 L flask, 2% v/v). Selective pressure was maintained by adding 30 ⁇ g ml "1 kanamycin and the culture was incubated at 30°C with constant shaking at 160 rpm. At mid-exponential phase of cell growth (A578 of 1.2 - 1.5), the medium was supplemented with 0.25 mM ⁇ - aminolevulinic acid ( ⁇ -ALA), 50 ⁇ FeCl3.6H 2 0 and 0.25 mM isopropyl ⁇ -D- thiogalactopyranoside (IPTG).
  • ⁇ -ALA ⁇ - aminolevulinic acid
  • IPTG isopropyl ⁇ -D- thiogalactopyranoside
  • Process B Octane biotransformation using resting cells cultivated on
  • a 1 ml aliquot of the preculture was used to inoculate 50 ml of the EnBase ® Flo medium (2%, v/v) containing the complex polysaccharide which had been introduced into a sterile 500 ml baffled flask.
  • the starch-degrading enzyme (EnZ I'm) was added to the medium at a concentration of 0.6 U L "1 .
  • An AirOtop membrane (Thomson Instrument Company, USA) was used to cover the baffled flask for improved air inflow and enhanced oxygen transfer.
  • the culture was incubated on an orbital shaker at 30°C and 200 rpm. At specified time intervals (8, 17 or 22 h) representing different phases of cell growth (As78nm of 2.6.
  • the broth was supplemented with 0.5 mM IPTG (for induction of protein expression) and 0.5 mM ⁇ -ALA (for heme synthesis).
  • the booster component of the EnBase ® medium was also added for optimal pH conditions (Yun et ai, 1996) and an additional 0.6 U L 1 of glucoamylase was included. This cultivation was allowed to continue for a total duration of 48 h and the P450 concentration determined.
  • the cells were harvested through centrifugation (13 000 g for 10 mins at 4°C), washed and resuspended in 200 mM sodium phosphate buffer (pH 7.2) to a concentration range of 3.5 - 4 gpcw LBRM "1 for octane biotransformation as resting whole-cell biocatalysts.
  • the bioreaction mixture (1 ml) in 60 ml reaction vials was supplemented with glucose (5 g L 1 ) as the carbon and energy source.
  • the biomass was resuspended in a 30 g L 1 starch solution and supplemented with glucoamylase (0.6 U L 1 ).
  • reaction vials were agitated on an orbital shaker at 200 rpm under varied temperature conditions (20, 25 and 30°C). Vials were removed at specified time intervals across the range 3 to 120 hours for product extraction.
  • the biotransformation reactions were stopped by adding 100 ⁇ 5 M HCI to each vial and the octanol concentration was quantified using GC analysis.
  • EnBase ® medium consisting of the complex polysaccharide was introduced into sterile 60 ml reaction vial.
  • the glucoamylase (EnZ I'm) concentration was varied from 0.6 to 12 U L "1 and incubated at 20°C.
  • 200 ⁇ of octane was added to the vial to investigate the possible inhibiting effect of the substrate on EnZ I'm.
  • Samples were taken at specified intervals and the reaction terminated by the addition of 50 ⁇ 5M HCI.
  • Glucose formation was analysed by determining the generation of H2O2 spectrophotometrically, following reaction catalysed by glucose oxidase using a glucose oxidase kit (Roche, Germany).
  • EnBase® medium was investigated in 60 ml vials under varied glucoamylase activities (0.6 to 12 U L 1 ).
  • the EnBase® medium 50 ml was inoculated with the seed culture (2% v/v). A 1 ml aliquot of the well mixed broth was immediately transferred to each vial and the specified concentration of glucoamylase added.
  • the capped vial was incubated at 20°C and agitated at 200 rpm.
  • the reaction vial was periodically (6-8 hourly) opened to air under sterile conditions to avoid oxygen limitation. Samples were taken at specified time intervals by sacrificing a vial set. Cell growth was monitored through absorbance reading at As ⁇ and the pH of the medium was followed offline with a pH electrode (Cyber Scan 2500, Eutech Instruments).
  • the culture was induced with 0.5 mM IPTG (for protein expression) and 0.5 mM ⁇ -ALA (for heme synthesis) after 12 - 14 h (As78nm of 6.2).
  • the culture was further incubated for 3 h (at 20°C) in preparation for the biotransformations (Process D).
  • a 1 ml portion of the cell broth (6 g Dew L "1 ) was pipetted into each 60 ml vial under varying concentrations of EnZ I'm (0.6 to 12 U L 1 ).
  • the biotransformation was initiated with the addition of octane (200 ⁇ ) and BEHP (100 ⁇ ).
  • the culture was induced with the same concentration of IPTG and ⁇ -ALA at the 24 th hour (A 57 8nm of 16.5) and further incubated for 3 h at 20°C.
  • the biomass was harvested through centrifugation (7000 rpm for 10 mins at 4°C), washed with sterile distilled water and resuspended in EnBase ® Flo Zero to a concentration of 1 to 1.5 g L "1 (Process E).
  • the broth was supplemented with 0.5 mM IPTG and ⁇ -ALA including 100 ⁇ of thiamine solution included in the BioSilta pack.
  • reaction vials were agitated on an orbital shaker at 200 rpm and 20°C. Vials were removed over the duration of 120 hours (where the full contents of one vial represented a single sample) and the reaction terminated by adding 100 ⁇ 5 M HCI prior to product extraction and quantification.
  • the pH of the reaction mixture was measured offline with a pH electrode (Cyber Scan 2500, Eutech Instruments).
  • Biomass concentration was increased from 2.5 to 4.8 g L 1 at the end of biotransformation.
  • a similar final volumetric concentration (4.5 g LBRM "1 ) was observed at 140 h with resting whole cells, although with reduced maximum volumetric productivity and lower biomass concentration. This resulted in higher octanol yield on biocatalyst,
  • the P450 concentration profile in Figure 1 b shows that growing biocatalysts were able to synthesise the heterologous CYP153A6 under biotransformation conditions. While the active properly folded P450 concentration was depleting under resting whole cell biocatalysis, it was increasing in the growing system up to 25 h. The reduced efficiency of the growing whole cell biocatalyst after 25 h may likely be due to acetic acid accumulation (Figure 1c) and the resulting acidification of the reaction mixture ( Figure 1d). The maximum volumetric production rate of acetic acid was 0.08 and 0.02 g LBRM "1 IT 1 in growing and resting whole cell biotransformation respectively.
  • the EnBase ® delivery system is based on the controlled glucose release into the biotransformation mixture through enzymatic degradation of the starch polymer.
  • concentration of the polysaccharide degrading enzyme glucoamylase was varied to vary the rate of glucose release. This, in turn, informs the optimum concentration that favours cell growth and octane oxidation.
  • Figure 2 shows that glucose accumulation and the associated glucose release rate are a function of the glucoamylase concentration over the range, 0.6 to 12 U L "1 .
  • the results also show that presence of octane at 30%, v/v did not significantly affect glucoamylase activity.
  • the initial glucose release rate (from Figure 2b) over the first 10 h shows a linear relationship with the corresponding increase in gluco-amylase concentration (Figure 3). It is expected that this correlation will be maintained provided glucose accumulation (that prompts feedback inhibition) or overflow metabolism is avoided during cell cultivation or biotransformation. This result indicates that the gluco-amylase can function in the two phase biocatalytic system involving aqueous and organic phase.
  • EnBase ® medium a technology that allows the supply of glucose in a controlled release fashion as is typical of the controlled feed in a fed- batch process.
  • Initial studies were performed using resting cells grown on EnBase ® medium and compared with the whole cell activities reported for cells grown on LB and glucose based chemically defined medium (data not shown).
  • Optimum conditions for maximum gene expression were established through induction studies. It was observed that the addition of IPTG and amino-levulinic acid (heme precursor) at 8, 17 and 22 h did not negatively impact the final biomass concentration from cell cultivation (Table 3).
  • the culture reached a final biomass concentration in the range of 10.5 to 12 g Dew L "1 .
  • the bioconversion was carried out in phosphate buffer with glucose addition.
  • Results presented in Table 3 show that protein induction at the 22 h gave the best result in terms of P450 expression, whole-cell biocatalyst activity, biocatalyst efficiency and volumetric octanol concentration.
  • the maximum biocatalyst activity achieved at 20 and 37°C show a 25% increase over the maximum value previously reported with the use of LB medium under the same conditions (data not shown).
  • a final octanol yield of 1.93 g gpcw "1 was observed after 94 h of biotransformation, representing a 1.5 to 2 fold increase over the concentration achieved with resting biocatalyst expressing CYP153A6 cultured on LB medium (data not shown).
  • Biomass grown on EnBase ® was washed and resuspended in sodium phosphate buffer supplemented with glucose. Biomass concentration was in the range 3.7 - 4.3 g LBRM "1 . Biotransformation were carried out in 1 ml volumes in 60 ml vials.
  • the biomass from the optimised induction condition i.e. protein induced at 22 h
  • the continuous supply of glucose was guaranteed through glucoamylase activity.
  • the biocatalyst activity across all glucoamylase concentrations proceeded at an initial and maximum rate of 0.20 g octanol formed L 1 h "1 (3.95 ⁇ gDcw "1 min -1 ).
  • hydroxylation of n-octane was carried out by growing E. coli cells cultured on EnBase ® medium following induction under the optimal conditions determined previously (i.e. induction of protein expression at 22 h of cell growth).
  • the biotransformation study was carried out under temperatures of 20, 25 and 30°C. The results are presented in Figure 5 and compared with standard biotransformation condition using growing biocatalyst in defined medium at 20°C, described above. As with previous studies, the reaction at lower temperature (20°C) gave the highest biocatalyst efficiency compared to higher temperature conditions at 25 and 30°C.
  • biomass accumulation was highest with the maximum glucose release rate. This increased by 2 fold from starting concentration of 6 g L 1 while only 50% increase was observed at glucoamylase level of 0.6 U L 1 . This indicates that a substantial part of the released glucose at high glucoamylase level was used for biomass formation.
  • Octanol formation was slowed down and finally inhibited by acetic acid formation and the resulting medium acidification below pH of 6 (Table 5).
  • This and the usage of "excess" glucose for other cell functions including biomass formation led to reduced biocatalyst efficiency at higher glucoamylase concentration (0.40 g octanol gocw "1 ). Specific octanol formation was higher at lower glucoamylase concentration, suggesting that the limited glucose release contributed to the efficient biotransformation of octane to octanol under this condition.
  • the acetic acid profile suggests that increasing glucoamylase concentration (and thereby initial glucose release rate and potentially residual glucose concentration) encourages overflow metabolism.
  • the reactions with a higher level of glucoamylase produced over 4 g L 1 of acetic acid which was apparently inhibitory to the whole cell biocatalyst. This corresponded to the time at which octanol production began to level off.
  • the acetic acid accumulated in the presence of 0.6 U L 1 glucoamylase remained in the range 1.5 to 1.8 g L 1 throughout the reaction phase, permitting continued production of octanol.
  • the accumulation of acetic acid reduced the pH of the biotransformation mixture (Figure 9d).
  • the glucose concentration was maintained in a limited fashion at glucoamylase level below 1.2 U L 1 and much increased at higher amylase concentrations, 3 to 12 U L "1 . This was apparently responsible for overflow metabolism and contributed substantially to the significant level of acetic acid detected at glucoamylase concentration in the range 3 to 12 U L "1 .
  • Biocatalyst efficiency was 2.1 g octanol gDcw " , representing over 2 fold increase over that achieved with growing cells in glucose-based chemically defined medium operated as a batch process.
  • acetic acid and possibly other acidic metabolites
  • acetic acid also impacts synthesis of native and recombinant proteins (CYP153A6) which are essential in maintaining cell integrity for biotransformation.
  • This unfavourable condition is possibly caused by overflow metabolism (of the carbon source) as oxygen depletion was discouraged by intermittent (6 to 8 hourly) opening of the vials to allow air inflow.
  • overflow metabolism constitutes energy drain thereby reducing NAD(P)H yield, causing metabolic shifts, uncoupling of proton gradient and ATP synthesis.
  • Table 7 Summary of results achieved under varied biotransformation conditions in processes A to E. This is limited to conditions under similar glucoamylase concentration (0.6 U L "1 ) and process temperature (20°C).
  • the novel EnBase ® technology is an enzyme based system that releases glucose from starch polymer through glucoamylase activity (Panula-Perala et al., 2008). It was specifically designed for small scale agitated systems (such as shake flask and microtiter plate) but has been shown to be applicable in large scale bioreactors (Glazyrina et al., 2012, Siurkus et al., 2010).
  • This system has been useful in improving biomass accumulation on small scale while also enhancing protein expression per unit biomass (Glazyrina et al., 2010, Hortsch and Weuster- Botz, 2011 , Siurkus and Neubauer, 201 1). However, it has not been tested under biotransformation conditions, particularly a two-phase biocatalytic reaction involving an organic phase. Glucoamylase concentration can be optimised to determine the preferred glucose release rate which in turn impacts the cell growth rate and biomass accumulation.
  • the envisaged major advantage is the continuous and stoichiometric supply of glucose for energy and cofactor regeneration during biotransformation.
  • EnBase® grown cells resuspended in starch solution and used as resting biocatalyst under glucoamylase influence showed relatively lower activity and efficiency when compared to values achieved with glucose- supplemented biotransformation using cells grown on EnBase® (Table 7). This may indicate the preference of the cells for refined glucose relative to the one released from the starch polysaccharide.
  • process D the cells were induced at 14 h, incubated for another 2 - 3 h and used for biotransformation as the cells were at the exponential phase of growth.
  • the octanol accumulation over the initial phase of 9 h did not correspond to the initial glucose release rate at high amylase concentration. This indicates that some of the glucose were utilised for other cell functions including biomass formation and production of side products such as acetic acid.
  • this maximum value was lower than other values in process A to C. This may have been due to the induction of P450 expression at sub-optimal conditions (12 - 14 h), even though it was considered that exponentially growing cells may enhance octanol production.
  • EnBase® Flo Zero was considered as it allows the cells to be resuspended in fresh medium lacking complex additives (process E).
  • the cells were cultivated in EnBase® medium, induced at 22 h and incubated for another 3 h.
  • the harvested biomass was resuspended in EnBase® Flo Zero to a concentration of 1 to 1.2 g L 1 and supplemented with glucoamylase 0.6 to 12 U L 1 to ensure the continuous supply of carbon for energy and cofactor regeneration.
  • the biocatalyst efficiency and activity of the growing cells were enhanced under the influence of slowly released glucose in the range 0.04 to 0.08 g L 1 hr 1 , giving of 0.02 to 0.03 hr 1 .
  • the residual glucose concentration was maintained at ⁇ 0.5 g L 1 through the bioconversion phase which discouraged overflow metabolism and relatively stabilised the reaction pH.
  • this reduces biomass accumulation which is a requirement for increased volumetric concentration of octanol. This may be resolved in a controlled bioreactor environment where adequate nutrient and oxygen can be provided to improve biomass accumulation while maintaining a slow and linear growth rate under biotransformation conditions.
  • EnBase ® without complex additives has shown the importance of slowly growing cells as a means of increasing the specific octanol yield and reducing carbon wasting in form of overflow metabolism and by-product formation.
  • CYP153A6 was expressed by the insertion of a plasmid, pET28b(+), encoding the complete operon from Mycobacterium sp. HXN-1500 as well as the ferrodoxin reductase (FdR) and ferrodoxin (Fdx) redox partner proteins.
  • Pre-culture inoculum 50 mL in a 500 mL flask was prepared by supplementing LB medium (10 g L "1 tryptone, 5 g L “1 , NaCI and 5 g L “1 yeast extract) with 30 ⁇ yg mL "1 kanamycin and E. coli BL21 (DE3) pET28b-PFR1500 stock.
  • the pre-culture was incubated at 30 °C and 160 rpm for at least 12 h before being used as an inoculum.
  • EnBase® Flo BioSilta liquid growth medium (50 mL in a 500 mL baffled flask, 1.54 L in the stirred reactor (STR) or 0.924 L in the shaking reactor according to the recommended EnBase® protocol for growth and induction.
  • the medium was inoculated with 2% (v/v) cells from the pre-culture inoculum (at an OD578 nm 5, 3.2 gDCW L 1) and the EnBase® components (0.6 ⁇ "1 EnBase® enzyme (EnZ I'm), 0.1 g L "1 thiamine and 6.14 mM MgS04) as well as 30 ⁇ g mL "1 kanamycin were included at startup. Growth was carried out at 20 °C and 200 rpm for the vial apparatus, 860 rpm and 1 vvm for the STR or 175 rpm and 1.2 L total volume for the shaking reactor.
  • the growth medium was supplemented with 0.5 mM isopropyl ⁇ -D-l- thiogalactopyranoside (IPTG), 0.5 mM d-aminolevulinic acid ( ⁇ -ALA) and 50 ⁇ FeC ⁇ 6H2O as well as an additional 0.084 units EnZ I'm and 5 mL, 154 mL or 92.3 mL depending on the growth apparatus (10% of the working volume) EnBase® booster solution as recommended in the EnBase® Flo protocol. During cultivation samples were periodically taken for the determination of the growth rate and cell concentration.
  • IPTG isopropyl ⁇ -D-l- thiogalactopyranoside
  • ⁇ -ALA d-aminolevulinic acid
  • FeC ⁇ 6H2O 50 ⁇ FeC ⁇ 6H2O
  • Biomass was quantified by optical density (OD) measurement at 578 nm.
  • the dry cell mass was determined from a 2 ml sample of cell broth by drying the cell pellet after centrifugation at 80 °C for > 24 h, followed by desiccation in a desiccator to cool to room temperature before being weighed to four decimal places.
  • CYP153A6 protein concentration analysis The amount of active CYP153A6 protein inside the cells was quantified using the CO-difference spectra method described by Guengerich et al. (2009).
  • the volumetric concentration of P450, CP45O [nmolp45o mL -1 ] can be determined using an extinction coefficient of 91 mM "1 cm -1 as follows:
  • the orbital shaking reactor was characterized with and without the helical track modification at shaking frequencies of 150, 175 and 200 rpm and working volumes of 40%, 50% and 60% (corresponding to 0.8, 1.0 and 1.2 L respectively). In all instances when working with the shaking reactors, the aeration rate into the headspace was kept minimal at 0.5 L min -1 to mimic passive diffusion of air into the sealed vessel.
  • DOT Dissolved oxygen tension
  • the gassing out method for K/_a determination was also employed in both reactor setups in both the presence (23% v/v) and absence of an organic phase.
  • the same operating conditions described in the hydrogen peroxide method agitation and aeration were used for the gassing out method.
  • the Oxygen Utilisation Rate (OUR) of the growing culture was established using the dynamic gassing out method.
  • the protocol is the same as that of the gassing out method, but instead of sparging the medium with nitrogen, aeration was interrupted and the culture slowly used up the residual oxygen. The rate at which this oxygen was depleted is the OUR.
  • the DOT was maintained above 30% air saturation to prevent the culture from becoming oxygen limited.
  • the hydrogen peroxide method for Kia determination was employed in the stirred reactor setup.
  • the gassing out method was also employed and the results of the two methods were compared.
  • the hydrogen peroxide method underestimated the Kia when compared to those results generated from the gassing out method under the same conditions.
  • the reactor oxygen transfer characterizations were therefore determined with the gassing out method. For the sake of consistency and accuracy, results for Kua determination using the gassing out method are reported including the probe response lag constant.
  • the OTR characteristics of the STR are shown in Figure 1 1 as a function of agitation rate, aeration rate and organic loading.
  • the organic loading was kept at 23% (v/v).
  • the OTR trends in the STR at this organic loading are shown in Figure 11 b.
  • Variance in the data was elevated at higher values of Kia as a result of the error of regression introduced when including the probe response time.
  • the effect of increasing the aeration rate above 1 vvm had little effect on the oxygen transfer of the system. This was most likely a result of flooding of the impeller causing no further mass transfer advantages.
  • the effectiveness of shaking reactors is more dependent on the physical reactor characteristics such as reactor geometry and filling volume.
  • the OTR was much lower in the shaking reactor than in the STR, however still in a reasonable range for growth ( /_a > 5 h "1 ).
  • the oxygen sensor placement in the shaking reactor would pose the worst-case scenario for oxygen transfer as it was in the least homogenous region of the reactor, at the center of the vortex. For this reason, the effective Kia values of the shaking reactor may be underestimated when compared to the STR.
  • Shaking reactors are heavily influenced by scale. As is evident from Table 8, lower specific power inputs are required at larger filling volumes. The shaking reactor also had an overall lower specific power requirement than the STR.
  • Table 8 S ecific power inputs of the shaking reactor and STR.
  • pH control of the reactors was more refined in the second run. pH control via base addition was applied to the STR as only a decrease in pH was expected with the formation of any acetic acid. It was observed that after the addition of booster solution at the point of induction, the pH increased to values above 8 (in the first experimental run). The high nitrogen content of the booster solution may have caused ammonium-based product formation compensations from the cells, causing the pH of the medium to increase. The high pH was remedied by the manual addition of 30% H 3 P0 4 (Panke et al., 2002). Manual pH control was performed in the shaking reactor at each sample time, using the STR control patterns as a guide. Any differences in results between first and second experimental runs may be attributed to this inadequate pH control.
  • results of this study are compared to that of previous studies in Table 10.
  • Results from the STR and shaking reactor present the highest P450 enzyme concentrations (with that of the shaking reactor being over 2-fold higher than previously reported values) as well as the highest biocatalyst effiencies to date.
  • Glazyrina J., Materna, E.-M., Dreher, T., Storm, D., Junne, S., Adams, T., Greller, G. and Neubauer, P. (2010). High cell density cultivation and recombinant protein production with Escherichia coli in a rocking-motion-type bioreactor. Microbial Cell Factories, 9, 42.
  • Glazyrina J., Krause, M., Junne, S., Glauche, F., Strom, D. and Neubauer, P. (2012). Glucose-limited high cell density cultivations from small to pilot plant scale using an enzyme-controlled glucose delivery system. New Biotechnology, 29, 235- 242.
  • Shaken helical track bioreactors Providing oxygen to high density cultures of mammalian cells at volumes up to 1000 L by surface aeration with air. New Biotechnology, 25(1):68-75.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de biotransformation catalysée par cellule entière d'alcanes linéaires en produits oxygénés dans des cellules se divisant activement aptes à exprimer un biocatalyseur qui catalyse la conversion d'un alcane linéaire en un produit oxygéné, comprenant l'incubation des cellules se divisant activement dans un milieu de biotransformation contenant un alcane linéaire, ce qui permet de catalyser la conversion de l'alcane linéaire en produit oxygéné. En particulier, l'invention concerne un procédé de biotransformation catalysée par cellule entière d'alcanes linéaires en produits oxygénés dans des cellules se divisant activement, comprenant (i) l'incubation de cellules se divisant activement dans un milieu de croissance contenant (a) un polysaccharide et (b) une enzyme hydrolysant le polysaccharide qui hydrolyse le polysaccharide en un substrat de croissance pour les cellules se divisant activement à une vitesse contrôlée et (ii) l'incubation des cellules se divisant activement dans un milieu de biotransformation comprenant un alcane linéaire, ce qui permet de catalyser la conversion de l'alcane linéaire en produit oxygéné.
PCT/IB2015/054697 2014-06-24 2015-06-23 Procédé de biotransformation d'alcanes linéaires Ceased WO2015198219A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB1411177.7A GB201411177D0 (en) 2014-06-24 2014-06-24 Method of biotransforma of linear alkanes
GB1411177.7 2014-06-24

Publications (1)

Publication Number Publication Date
WO2015198219A1 true WO2015198219A1 (fr) 2015-12-30

Family

ID=51410017

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2015/054697 Ceased WO2015198219A1 (fr) 2014-06-24 2015-06-23 Procédé de biotransformation d'alcanes linéaires

Country Status (2)

Country Link
GB (1) GB201411177D0 (fr)
WO (1) WO2015198219A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107287256A (zh) * 2016-03-31 2017-10-24 南京诺云生物科技有限公司 全细胞催化合成l-2-哌啶甲酸的方法
CN111808784A (zh) * 2020-08-04 2020-10-23 中国海洋大学 一株产耐高温α-淀粉酶的热反硝化地芽孢杆菌Y62及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013110557A1 (fr) * 2012-01-25 2013-08-01 Evonik Industries Ag Procédé d'oxydation d'alcènes utilisant la mono-oxygénase alkb de pseudomonas putida gpol
WO2015014644A1 (fr) * 2013-07-31 2015-02-05 Basf Se Procédé pour la bioconversion d'alcanes c3-c13 en alcools primaires c3-c13

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013110557A1 (fr) * 2012-01-25 2013-08-01 Evonik Industries Ag Procédé d'oxydation d'alcènes utilisant la mono-oxygénase alkb de pseudomonas putida gpol
WO2015014644A1 (fr) * 2013-07-31 2015-02-05 Basf Se Procédé pour la bioconversion d'alcanes c3-c13 en alcools primaires c3-c13

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
GUDIMINCHI ET AL: "Cofactor regeneration during whole-cell biotransformation of n-octane using E. coli cells expressing the CYP153A6 operon", CONFERENCE PAPER, 2011, pages 61 - 67, XP002743908, Retrieved from the Internet <URL:http://nature.cc.hirosaki-u.ac.jp/lab/2/celltech2/gazou2/Kudo-2011.pdf> [retrieved on 20150902] *
GUDIMINCHI ET AL: "Whole-cell hydroxylation of n-octane by Escherichia coli strains expressing the CYP153A6 operon", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 96, 2012, pages 1507 - 1516, XP035139562 *
HORTSCH ET AL: "Growth and recombinant protein expression with Escherichia coli in different batch cultivation media", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 90, 2011, pages 69 - 76, XP002743973 *
LUNDEMO ET AL: "Guidelines for development and implementation of biocatalytic P450 processes", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 99, 5 February 2015 (2015-02-05), pages 2465 - 2483, XP035459157 *
OLAOFE ET AL: "The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6", MICROBIAL CELL FACTORIES, vol. 12, 2013, pages 1 - 12, XP021141767 *
SUMIRE HONDA MALCA: "Substrate characterization and protein engineering of bacterial cytochrome P450 monooxygenases for the bio-based synthesis of omega-hydroxy aliphatic compounds", THESIS, 2013, Stuttgart, Germany, pages 1 - 147, XP002743907, Retrieved from the Internet <URL:http://elib.uni-stuttgart.de/opus/volltexte/2013/8378/pdf/Dissertation_with_Cover_Sumire_Honda_A4_Format.pdf> [retrieved on 20150902] *
VALLON ET AL: "Production of 1-octanol from n-octane by Pseudomonas putida KT2440", CHEMIE INGENIEUR TECHNIK, vol. 6, 2013, pages 841 - 848, XP002743909 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107287256A (zh) * 2016-03-31 2017-10-24 南京诺云生物科技有限公司 全细胞催化合成l-2-哌啶甲酸的方法
CN111808784A (zh) * 2020-08-04 2020-10-23 中国海洋大学 一株产耐高温α-淀粉酶的热反硝化地芽孢杆菌Y62及其应用
CN111808784B (zh) * 2020-08-04 2021-06-08 中国海洋大学 一株产耐高温α-淀粉酶的热反硝化地芽孢杆菌Y62及其应用

Also Published As

Publication number Publication date
GB201411177D0 (en) 2014-08-06

Similar Documents

Publication Publication Date Title
Goldberg et al. Biocatalytic ketone reduction—a powerful tool for the production of chiral alcohols—part I: processes with isolated enzymes
Kataoka et al. Novel bioreduction system for the production of chiral alcohols
Angenent et al. Chain elongation with reactor microbiomes: open-culture biotechnology to produce biochemicals
Wichmann et al. Cofactor regeneration at the lab scale
Baldwin et al. On oxygen limitation in a whole cell biocatalytic Baeyer–Villiger oxidation process
Eckstein et al. Recent developments in NAD (P) H regeneration for enzymatic reductions in one-and two-phase systems
Schroer et al. Continuous asymmetric ketone reduction processes with recombinant Escherichia coli
Gong et al. Bio-resolution of a chiral epoxide using whole cells of Bacillus megaterium ECU1001 in a biphasic system
Kuhn et al. Production host selection for asymmetric styrene epoxidation: Escherichia coli vs. solvent-tolerant Pseudomonas
Tüllinghoff et al. Maximizing photosynthesis-driven Baeyer–Villiger oxidation efficiency in recombinant Synechocystis sp. PCC6803
Van Hecke et al. Bubble‐free oxygenation of a bi‐enzymatic system: Effect on biocatalyst stability
Buergler et al. Process intensification for cytochrome P450 BM3‐catalyzed oxy‐functionalization of dodecanoic acid
Hoschek et al. Stabilization and scale‐up of photosynthesis‐driven ω‐hydroxylation of nonanoic acid methyl ester by two‐liquid phase whole‐cell biocatalysis
Solé et al. Enzymatic synthesis of trimethyl-ε-caprolactone: process intensification and demonstration on a 100 L scale
Dowdells et al. Gluconic acid production by Aspergillus terreus
Meyer et al. Process and catalyst design objectives for specific redox biocatalysis
Luo et al. Accelerating itaconic acid production by increasing membrane permeability of whole-cell biocatalyst based on a psychrophilic bacterium Shewanella livingstonensis Ac10
Hoschek et al. In situ O2 generation for biocatalytic oxyfunctionalization reactions
Li et al. Continuous-flow microreactor-enhanced clean NAD+ regeneration for biosynthesis of 7-oxo-lithocholic acid
Schäfer et al. Rational engineering of a multi‐step biocatalytic cascade for the conversion of cyclohexane to polycaprolactone monomers in Pseudomonas taiwanensis
Hollmann et al. Coupled chemoenzymatic transfer hydrogenation catalysis for enantioselective reduction and oxidation reactions
Hagström et al. Chemo‐enzymatic epoxidation–process options for improving biocatalytic productivity
Vlaeminck et al. Pressure fermentation to boost CO2-based poly (3-hydroxybutyrate) production using Cupriavidus necator
US6991926B2 (en) Method comprising the indirect electrochemical regeneration of NAD(P)H
Xu et al. Development of a biocatalytic cascade for synthesis of 2-oxo-4-(hydroxymethylphosphinyl) butyric acid in one pot

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15741339

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15741339

Country of ref document: EP

Kind code of ref document: A1