CN1587383A - New method for modifying clavulanic acid producing bacteria and high yield clavulanic acid producing bacteria - Google Patents

Abstract

The invention provides a new method for improving clavulanic acid production capacity of clavulanic acid producing bacteria and obtained high-yielding clavulanic acid producing bacteria. The present invention utilizes the method of pathway engineering, by interrupting or knocking out the lat gene in the cephamycin C metabolic pathway, or excising the related cvm gene cluster of the branch pathway of the clavulanic acid synthesis pathway, and simultaneously increasing the positive regulatory gene CcaR of the clavulanic acid synthesis , ClaR and the key enzyme gene Cas2 copy number in the clavulanic acid synthesis pathway, so that the strain does not contain a complete metabolic pathway of cephamycin C, or does not synthesize or synthesizes very few types of clavulanic acid metabolites, so that the regulatory protein The important enzymes in clavulanic acid synthesis and metabolism can be produced in large quantities, thereby increasing the yield of clavulanic acid strains and obtaining high-yielding clavulanic acid producing strains.

Description

A new method for improving clavulanic acid producing bacteria and high-yielding clavulanic acid producing bacteria

technical field

The invention provides a new method for improving clavulanic acid production capacity of clavulanic acid producing bacteria and obtained high-yielding clavulanic acid producing bacteria. The present invention utilizes the method of pathway engineering, by interrupting or knocking out the lat gene in the metabolic pathway of cephamycin C, or excising the related cvm gene cluster of the branch pathway of clavulanic acid synthesis pathway, or increasing the positive regulatory gene CcaR of clavulanic acid synthesis , ClaR and the key enzyme gene Cas2 copy number in the synthesis pathway of clavulanic acid, so that the strain does not contain a complete metabolic pathway of cephamycin C, or does not synthesize or synthesize few types of clavulanic acid metabolites, or regulatory proteins The important enzymes in clavulanic acid synthesis and metabolism can be produced in large quantities, thereby increasing the yield of clavulanic acid strains and obtaining high-yielding clavulanic acid producing strains.

Background technique

At present, bacterial resistance to widely used antibiotics has become a major threat to clinical treatment. One of the drug resistance mechanisms is that the β-lactamase produced by bacteria destroys the β-lactam ring of β-lactam antibiotics and makes them inactive. Clavulanic acid was first researched and developed by GlaxoSmithKline. It is an extended-spectrum β-lactamase inhibitor, which can irreversibly bind to β-lactamase to protect antibiotic activity. It is mainly used in combination with antibiotics such as amoxicillin. So far, it is still an important clinical anti-infective drug. The clavulanic acid raw materials used in my country are all imported from abroad, and the main reason for its inability to produce is the low yield of strains.

Human beings have been working hard to make organisms have desired genetic characteristics. Strain selection mainly utilizes traditional ultraviolet mutagenesis and chemical mutagenesis methods, which are time-consuming, highly random, and heavy work. Moreover, many biological mutant populations with genetic and metabolic advantages are still difficult to identify and isolate due to the randomness of mutations.

With the development and mutual penetration of molecular biology, genetics, genetic engineering technology and metabolic engineering and other disciplines, the third generation of genetic engineering technology "pathway engineering" came into being. Pathway engineering is a new applied discipline or technology that uses the principles of molecular biology to systematically analyze cell metabolic networks, rationally designs cell metabolic pathways and genetic modifications through DNA recombination technology, and then completes the transformation of cell characteristics. It aims at the expression and regulatory properties of each reaction of the cell metabolic pathway at the gene level, using DNA recombination technology to amplify, delete, implant, transfer and regulate the genes related to the coding pathway, and then screen out engineering bacteria with excellent genetic characteristics. This method of breeding high-yield strains is highly targeted, time-saving and labor-saving, but it has certain requirements for the background research of strains.

The clavulanic acid producing bacteria used in this study is a strain with clear genetic background. The metabolic pathways, competitive metabolic pathways, related structural genes and regulatory genes and their regulatory modes of clavulanic acid are very clear. Therefore, it is very feasible and promising to use pathway engineering to carry out directional transformation and screen high-yield producers. The use of pathway engineering to breed high-yielding bacteria has great application prospects. There are already successful examples in foreign countries, but it is still in its infancy in our country. The breakthrough of this technology will greatly promote the development of my country's traditional pharmaceutical industry and bring huge economic profits.

Contents of the invention

The invention provides a new method for improving clavulanic acid production capacity of clavulanic acid producing bacteria and obtained high-yielding clavulanic acid producing bacteria. The present invention utilizes the method of pathway engineering, by interrupting or knocking out the lat gene in the cephamycin C metabolic pathway, or excising the related cvm gene cluster of the branch pathway of the clavulanic acid synthesis pathway, and simultaneously increasing the positive regulatory gene CcaR of the clavulanic acid synthesis , ClaR and the key enzyme gene Cas2 copy number in the synthesis pathway of clavulanic acid, so that the strain does not contain a complete metabolic pathway of cephamycin C, or does not synthesize or synthesize few types of clavulanic acid metabolites, or regulatory proteins The important enzymes in clavulanic acid synthesis and metabolism can be produced in large quantities, thereby increasing the yield of clavulanic acid strains and obtaining high-yielding clavulanic acid producing strains.

The method for transforming clavulanic acid-producing bacteria by utilizing pathway engineering technology in the present invention is not only a new technology in strain transformation, but also has very important commercial utilization value. The transformation and utilization of clavulanic acid-producing bacteria has always been the focus of my country's development support, and it was once listed as a national key project in the "Seventh Five-Year Plan" and "Eighth Five-Year Plan". However, the transformation technology of clavulanic acid producing bacteria has been limited to traditional techniques such as ultraviolet mutagenesis and chemical mutagenesis. This traditional mutagenesis method has the non-directionality of the mutation direction, which results in long work time, high randomness and heavy tasks. Moreover, many biological mutant populations with genetic and metabolic advantages are still difficult to identify and isolate due to the randomness of mutations. The method of transforming clavulanic acid-producing bacteria by using pathway engineering technology has the directionality of mutation, thereby reducing the workload, saving time and labor to achieve the purpose of transforming strains, and obtaining the target high-yield clavulanic acid-producing bacteria.

On the basis of the research background of existing clavulanic acid strains, the present invention utilizes pathway engineering technology to transform clavulanic acid producing bacteria. Pathway engineering is a new applied discipline or technology that uses the principles of molecular biology to systematically analyze cell metabolic networks, rationally designs cell metabolic pathways and genetic modifications through DNA recombination technology, and then completes the transformation of cell characteristics. It aims at the expression and regulatory properties of each reaction of the cell metabolic pathway at the gene level, using DNA recombination technology to amplify, delete, implant, transfer and regulate the genes related to the coding pathway, and then screen out engineering bacteria with excellent genetic characteristics.

In the present invention, the lat gene in the cephamycin C metabolic pathway is inserted or knocked out, so that the lysine aminoacyltransferase cannot be expressed, so that the cephamycin C precursor substance lysine cannot be catalyzed to convert downward. , blocked the metabolic synthesis of cephamycin C, and relieved the competition of cephamycin C metabolic pathway for clavulanic acid production. (The metabolic diagram of cephamycin C is shown in Figure 1)

The present invention interrupts one or more of the cvm gene clusters in the clavane metabolic pathway by means of gene insertion or knockout, so that 2-carboxyclavane, 2-hydroxymethyl clavane, 2-formyl One or more of methoxy clavane, hydroxyethyl clavane, alanyl clavane, etc., so as to cut or reduce clavulanic acid metabolism to clavanes, so that a large amount of substances and energy are produced to clavulanic acid accumulation. (The metabolic diagram of clavulanic acid and clavanes is shown in accompanying drawing 2).

The invention also relates to the high expression of regulatory genes CcaR and ClaR in clavulanic acid producing bacteria. Fish CcaR and ClaR genes from the genome of clavulanic acid producing bacteria, and insert them into stable high-copy plasmids, or insert CcaR and ClaR genes into vectors with λ phage junction attB sites, and enter by protoplast transformation The clavulanic acid-producing bacteria can highly express regulatory genes, thereby promoting the mass production of clavulanic acid. (The location and regulation mechanism of the gene cluster where the CcaR and ClaR regulated genes are located are shown in Figure 3).

The present invention also includes the high expression of the rate-limiting step enzyme Cas2 in the clavulanic acid production metabolic pathway. Fish the Cas2 gene from the clavulanate-producing bacteria genome and insert it into a stable high-copy plasmid, or insert the Cas2 gene into the vector with the λ phage junction attB site, and enter the clavulanic acid-producing bacteria through protoplast transformation , so that the regulatory genes are highly expressed, thereby promoting the mass production of clavulanic acid.

The invention first applies the pathway engineering technology to the transformation of clavulanic acid production strains, and has very important scientific and application value. This technology enables the development of multiple disciplines such as molecular biology, genetics, genetic engineering technology and metabolic engineering to interact and infiltrate. By using the principles of molecular biology to systematically analyze the cell metabolic network, and using genetic engineering technology to design reasonable cell metabolic pathways, targeting at cell metabolism The expression and regulatory properties of each reaction in the pathway at the gene level, using DNA recombination technology to amplify, delete, implant, transfer and regulate the genes related to the coding pathway, so as to complete the transformation of cell characteristics and improve the ability of the strain to produce clavulanic acid. Pathway engineering is the third generation of genetic engineering, which has more important scientific and application value than the first and second generation genetic engineering. The development and utilization of the initial genetic engineering technology was limited to the expression of specific proteins or polypeptides in organisms such as Escherichia coli, yeast and mammalian cells, that is, relatively simple expression of exogenous gene products, such as human growth hormone and human proinsulin. , human tissue plasminogen activator, some toxin polypeptides and other substances with application value. The biggest feature of exogenous gene expression in microorganisms, animal and plant cells is that the expression of exogenous genes is independent and does not act on or participate in or regulate the biosynthesis pathway or related pathways of the target product. The third-generation genetic engineering is a multidisciplinary technology, which can use recombinant DNA technology to modify the biosynthesis pathway of the target product or related pathways, thereby increasing the yield of the target product, and finally obtaining high-yield industrial production strains.

The reason why the present invention has very important industrial application value is not only because it can apply the latest pathway engineering technology to the transformation of clavulanic acid producing bacteria to improve the yield of clavulanic acid, but also because clavulanic acid is a solution to the clinical problem of bacterial drug resistance. of the best medicines. In my country, all clavulanic acid is imported and cannot be produced by itself, mainly because the ability of producing strains to produce clavulanic acid is too low to compete with foreign countries. Therefore, the present invention relates to a novel method for modifying clavulanic acid producing bacteria. The method provided by the present invention can interrupt one or more genes of the competitive metabolic pathway related to the clavulanic acid synthesis pathway, increase the copy number of the regulation gene for clavulanic acid synthesis, and increase the rate-limiting gene in the clavulanic acid synthesis pathway. The gene copy number of the step enzymes, so that the biosynthetic pathway of clavulanic acid can be more energy-efficient operation.

The present invention also provides the screening marker used for interrupting the clavulanic acid gene, the recombinant DNA cloning vector, and the high-yield clavulanic acid producing bacteria obtained by the pathway engineering technology.

Description of drawings

Accompanying drawing 1 is the metabolic pathway of cephamycin C.

Accompanying drawing 2 is the synthetic metabolic pathway of clavulanic acid and clavulanes.

Accompanying drawing 3 is the location and regulation mechanism of the gene cluster where CcaR and ClaR regulate genes.

Accompanying drawing 4 is the construction of recombinant vector and its interruption to the lat gene of S.clavuligerus bacterial strain.

Accompanying drawing 5 is to carry out PCR verification to the S.c XAL 863 strain obtained by screening, with the original strain as the control strain. Using lat-up and lat-down as upstream and downstream primers for PCR amplification, the size of the gene fragment amplified by the original strain is about 1.8kb, and the size of the gene fragment amplified by S.cXAL 863 is about 3.7kb. This is due to the addition of a 1.9kb apr resistance gene at the KpnI restriction site in the lat gene fragment. From right to left: 1. Marker, 2. Original strain, 3. Double crossover strain.

Accompanying drawing 6 is Southern hybridization analysis lat:apr interruption strain. 1 and 2 are respectively the genomic DNA of S.clavuligerus and S.c XAL863 digested with BamHI, and the size of the hybridization band was determined by λDNA Marker (not shown).

Accompanying drawing 7 is the comparison of the ability of S.clavuligerus wild bacteria and S.c XAL 863 modified bacteria to synthesize clavulanic acid at different culture times. 1, 2 are S.clavuligerus wild strains; 3, 4 are S.c XAL 863 modified strains.

Accompanying drawing 8 is the flowchart of Cvm1 interruption.

Accompanying drawing 9 is CcaR, ClaR regulation gene and Cas2 enzyme gene plasmid construction diagram.

Accompanying drawing 10 is the content of clavulanic acid in the fermented liquid of the transformed strain S.c XAL 863-2 detected by HPLC.

Accompanying drawing 11 is the content of clavulanic acid in the original strain fermentation sample detected by HPLC.

Detailed ways

The following examples will help those of ordinary skill in the art to further understand the present invention, but do not limit the present invention in any form.

Example 1

Cloning of lat gene-related fragments by PCR and construction of interruption vector

Using the total DNA of S. clavuligerus as a template, lat-up and lat-down as primers, amplify the lat gene-related fragments, then double-digest with EcorI and BamHI, and insert them into the PIJ2925 plasmid cut with the same two enzymes, Then, the plasmid PIJ2926 was extracted from the screened positive clones, and EcorI and BamHI double enzyme digestion was performed, and the size of the double enzyme digestion product was detected by electrophoresis as expected about 1.8kb. The plasmid PHP45Ωaacc4 was digested with KpnI, the apr resistance gene fragment was recovered, and then inserted into the KpnI site of the lat fragment in the PIJ2926 plasmid to obtain the plasmid PIJ2927. From the positive clones obtained by screening, the plasmid PIJ2927 was extracted, and the detected plasmid size was in line with the expected The size is consistent with that, and identified by BglII enzyme digestion, a band of about 3.7kb and about 2.7kb is obtained, which is consistent with the expected result. Plasmid PIJ2927 was digested with BglII, and a gene fragment of about 3.7 kb was recovered and inserted into the BamHI site of pSET151 plasmid to obtain a constructed interruption vector PXAL1, which was about 10 kb (Fig. 4). Or choose pHJL401, and insert the lat fragment with apr at the EcoRI and Xbal sites.

Example 2

lat gene double exchange strain screening

The plasmid PXAL1 was transformed into Escherichia coli ET12567 (pUZ8002), and then transferred to the original starting strain S.clavuligerus through intergeneric parental conjugation, and the zygotes were picked and transferred to the nalidixic acid medium containing 20ul/ml for Streptomyces Purification of zygotes. The purified zygote is then cultured on the antibiotic-free MM medium to promote the exchange of the homologous regions of the genome of the interrupted vector and the original strain, and to promote the loss of the non-exchanged foreign plasmid. Spores were collected and double-crossover strains were screened with apr and str antibiotics, respectively. Select the appropriate concentration of spores and spread them on the YD medium containing 20ul/ml apr antibiotics, and then replicate them on the medium containing str antibiotics, and screen out the apr ^R tsr ^S strain, which is the strain with double crossover of the gene. This is because the lat gene with the apr resistance gene fragment in the interruption vector PXAL1 and the chromosomal homologous region of the original starting bacterium undergo homologous double exchange (accompanying drawing 4). Finally, a double-crossover strain Sc XAL 863 was screened.

Example 3

lat double exchange strain verification

1) Perform PCR verification on the screened S.c XAL 863 strain, and use the original strain as the control strain. Using lat-up and lat-down primers for PCR amplification, the size of the gene fragment amplified by the original strain is about 1.8kb, and the size of the gene fragment amplified by S.cXAL 863 is about 3.7kb ( Accompanying drawing 5), mainly because a 1.9kb apr resistance gene is added at the KpnI restriction site in the lat gene fragment.

2) According to the method provided in the literature (K.Madduri et al, 1989, 299-302, Journal of Bacteriology), the activity of lysine aminoacyltransferase was detected, and it was found that the original wild strain had lysine aminoacyltransferase activity during fermentation , while the S.cXAL 863 strain did not detect this enzyme activity, indicating that the lat gene was interrupted.

3) Southern hybridization verification. The Southern hybridization technique was used to verify whether gene replacement occurred in the screened strain S.c XAL 863. Two probes are used for hybridization, and the difference between the two probes is that one does not contain the apr fragment and the other contains the apr fragment. The results are shown in Figure 6. After the genomes of S.clavuligerus and S.c XAL 863 were digested with BamHI and hybridized with double probes, the former had a band of about 9kb, and the latter had two bands of about 4.7kb and 1.8kb, which were in line with expectations The results are consistent, indicating that S.c XAL 863 is a lat gene disrupted strain.

From the above three methods, it can be confirmed that the strain is a lat interrupted strain.

Example 4

Fermentation test of original strain and S.c XAL 863 strain

Fermentation research was carried out on the screened double-crossover strain S.c XAL 863 to explore the ability of the strain to synthesize clavulanic acid. Soybean medium was selected as the fermentation medium, and the original strain Sclavuligerus was used as the control strain, cultured at 28°C and 200rpm, and samples were taken at different times to detect the content of clavulanic acid. The inoculum volume is 5%, and the liquid volume in the shake flask is 1:10. Each experiment has two parallel samples.

As can be seen from Figure 7 of the accompanying drawing, compared with the original bacterial strain S.clavuligerus, the transformed strain S.c XAL 863 has a significantly increased yield. At 54h, the content of clavulanic acid has increased by 3.6 times, 66h by 2.1 times, and 72h by 2.1 times. increased by 1.8 times. It shows that compared with the original strain, the modified strain has changed its metabolic pathway or the content of substances in the metabolic pathway stream.

Example 5

PCR cloning of cvm1, cvm2, cvm3, cvm4, cvm5 gene-related fragments and construction of interrupt vectors

Using the total genome of S. clavuligerus as a template, the cvm1, cvm2, cvm3, cvm4, and cvm5 genes were fished out by PCR technology, and connected to the EcoRI and BamHI sites of pBLKS, respectively. Then digest at the EoRV and BamHI sites respectively, and connect to pOJ466 at the same restriction site to obtain plasmids cvm1X, cvm2X, cvm3X, cvm4X, cvm5X, respectively, and then respectively in different plasmids cvm1X, cvm2X, cvm3X, cvm4X, cvm5X The resistance genes neo, hyg, str, tsr and kana were inserted into the different restriction sites SalI, NocI and BsaBI. The plasmids cvm11X, cvm22X, cvm33X, cvm44X, cvm55X were obtained respectively.

Using the total genome of S. clavuligerus as a template, cvm123 (including cvm1, cvm2 and cvm3 full-length genes) and cvm45 (including cvm4 and cvm5 full-length genes) were fished, respectively, and connected to the pBLKS plasmid to obtain plasmids pcvm123 and pcvm45 respectively , Digest the pcvm123 plasmid with NocI and BsaBI, get a larger fragment by electrophoresis, and insert a neo-resistant fragment at the NocI and BsaBI site to obtain the plasmid pcvm123X. Plasmid pcvm45 was digested with NocI, a larger fragment was recovered by electrophoresis, and the hyg resistance gene was inserted to obtain plasmid pcvm45X. (Attachment 8 only lists the cvm1 interrupt map)

Example 6

Screening and validation of cvm cluster gene double-crossover strains

According to the method similar to Example 2-4, double-crossover strains were screened according to different resistance genes inserted. And by detecting the output of different clavanes, we will investigate the disruption of cvm cluster genes. One or more interrupted strains in the cvm cluster, the strains were not detectable by HPLC to produce 2-carboxyclavane, 2-hydroxymethylclavane, 2-formylmethoxyclavane, hydroxyethylclavane, One or more of alanyl clavanes, etc., while the wild strain was still able to detect clavanes.

Example 7

Regulatory genes CcaR, ClaR, and rate-limiting step enzyme gene cas2 vector construction

Using the total genome of S. clavuligerus as a template, PCR technology was used to capture the regulatory genes CcaR, ClaR and the rate-limiting step enzyme gene Cas2, and then all three genes were simultaneously connected to the pIg699 plasmid (Fig. 9). Alternatively, the three genes were ligated into the pSET152 plasmid.

Example 8

Protoplast selection of transformed strains

Add YEME+TSBY (1:1) and 0.2% glycine to an Erlenmeyer flask equipped with a stainless steel spring, inoculate 100 ul of spore suspension, and incubate at 30° C., 200 rpm for 36h-40h. Then the mycelium was collected, and the collected mycelium was washed with 10.3% sucrose solution for three times, and the mycelium was collected by centrifugation at 3000 rpm for 10 min. Take the mycelium, add lysozyme P buffer (Hooper, Practical Streptomyces Genetics), and bathe in water at 30°C for 30-60min until the supernatant becomes milky. Filter with a test tube equipped with absorbent cotton, transfer the filtrate into a sterile universal, and centrifuge at 3000rpm for 7min to collect the protoplast precipitate, which is yellow. Gently break up the protoplasts, wash with P buffer to remove lysozyme, centrifuge under the same conditions, collect the protoplasts, remove the supernatant, break up the protoplasts with a gun, aliquot, and store at -70°C.

Take 5ul of DNA, add it to a 50ul protoplast tube, pipette 200ul of 25% PEG4000, wash the DNA into the protoplast, pump carefully several times, and mix well. Then the mixture was spread on R5 plate (without antibiotics), and after culturing at 30° C. for 14-20 h, 20 ul/ml antibiotic (str) was added, and the culturing was continued, and smaller transformants could be seen. This transformant is the strain that has been transformed into the plasmid.

Example 9

According to the method of Example 1-8, the strains with lat interruption, cvm1 and cvm4, cvm5 interruption, and high copy CcaR, ClaR and Cas2 genes were obtained. Fermentation studies showed that the yield of clavulanic acid of the transformed strain (S.c XAL 863-2) (accompanying drawing 10) was 4 times higher than that of the original strain (accompanying drawing 11).

Claims (9)

1. high clavulanic acid producing (clavulanic acid) is produced strain cell, the pathways metabolism that does not contain complete cephamycin C, or the not synthetic or synthetic seldom excellent alkanoic acid metabolite of kind, the important enzyme in modulin and the clavulanic acid anabolism can produce in a large number simultaneously; Be that high clavulanic acid producing is produced strain cell, be by interrupting or knocking out lat gene in the cephamycin C pathways metabolism, perhaps excise the relevant cvm gene cluster of branch's approach of clavulanic acid route of synthesis, perhaps increase the key gene Cas2 copy number in clavulanic acid synthetic positive regulating gene CcaR, ClaR and the clavulanic acid route of synthesis, thereby the energy metabolism that changes original clavulanic acid bacterium flows to, and clavulanic acid is accumulated in a large number.

2. produce mycetocyte according to the high clavulanic acid producing of claim 1, can produce the clavulanic acid of high yield.

3. produce the preparation method of bacterium according to the high clavulanic acid producing of one of right 1 to 2, comprising:

A) interrupt one or more among gene lat, cvm1, cvm2, cvm3, cvm4, the cvm5, be connected to and engage on the carrier.

B) copy number of one or more genes among increase Cas2, CcaR, the ClaR is connected on streptomycete-shuttle vehicle.

C) by the carrier that a) is obtained, engaged cultivation with transforming bacterial strain, choose zygote

D) by b) carrier that obtained, enter by protoplast transformation and to be transformed bacterial strain, choose transformant.

4. according to the method for claim 4, the resistance segment of wherein interrupting the lat gene is a kind of among apr, hyg, neo, str, the tsr.

5. according to the method for claim 4, the resistance segment of interrupting cvml, cvm2, cvm3, cvm4, cvm5 gene is one or more among apr, hyg, neo, str, the tsr.

6. according to the method for claim 4, selected carrier is pIg2925, pSET151, pOJ446, pHJL401.

7. according to claim 2, the clavulanic acid output that high clavulanic acid producing is produced bacterium is not less than 2500ug/ml.

8. according to claim 2, the clavulanic acid output that high clavulanic acid producing is produced bacterium is not less than 3500ug/ml.

9. according to claim 2, the clavulanic acid output that high clavulanic acid producing is produced bacterium is not less than 5000ug/ml.

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