WO2018129795A1 - 一种生产β-胸苷的工程菌株及其应用 - Google Patents

一种生产β-胸苷的工程菌株及其应用 Download PDF

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WO2018129795A1
WO2018129795A1 PCT/CN2017/075586 CN2017075586W WO2018129795A1 WO 2018129795 A1 WO2018129795 A1 WO 2018129795A1 CN 2017075586 W CN2017075586 W CN 2017075586W WO 2018129795 A1 WO2018129795 A1 WO 2018129795A1
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gene
strain
thymidine
engineered
engineered strain
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施明安
王涛
基拉奇克⋅K
布拉奇克⋅M
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上海创诺医药集团有限公司
赤峰制药股份有限公司
赤峰蒙广生物科技有限公司
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    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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Definitions

  • the present invention relates to the field of biotechnology, and more particularly to an engineered strain for producing ⁇ -thymidine and its use.
  • Beta-thymidine (hereinafter referred to as thymidine) is a key intermediate for anti-AIDS zidovudine.
  • Zidovudine is the world's first anti-AIDS drug approved by the US FDA. Because of its exact efficacy, it is the most basic combination of "cocktail" therapy.
  • thymidine Due to the dramatic increase in the number of AIDS patients, inexpensive methods for large-scale production of thymidine have received widespread attention.
  • the production of thymidine by biological fermentation mainly uses the biosynthetic route of microbial strains to produce thymidine.
  • the use of biological method to produce thymidine has the advantages of simple process and low cost of subsequent separation, especially the current genetic engineering breeding technology and high-yield optimization control technology.
  • the adoption of the fermentation method greatly reduces the production cost of the fermentation method, so the construction of a high-yield thymidine engineering strain has a good application prospect.
  • Escherichia coli is the most detailed prokaryotic bacteria ever studied. It is a safe Gram-negative model strain. Its chromosomal DNA has been sequenced, and the biological functions of most of the genes have been identified. Because E. coli breeds rapidly and culture metabolism is easy to control, the molecular genetic background of E. coli is quite clear, and it has perfect genetic manipulation technology. Moreover, when the Escherichia coli is cultured in aerobic fermentation, the substrate consumption rate is fast, so that the product productivity can be obtained with a wide range of substrates, and the inexpensive raw materials can be used to produce the target product, thereby reducing the production cost. Therefore, rational transformation of strains by metabolic engineering methods has broad prospects and practical significance.
  • the intracellular synthesis of thymidine is tightly regulated, and in the wild, there is almost no accumulation of thymidine in the cells.
  • the BLdtug RPA24 strain constructed by the Lee group has accumulated 5.2 g/L thymidine in a pH-stat fed-batch fermentation with glycerol as a carbon source.
  • the fermentation production process using glycerol as a carbon source has problems such as high cost and long fermentation cycle.
  • an engineered strain is provided, the strain being Escherichia coli BL21 (Escherichia coli), and the deoA gene (thymidine phosphorylase gene), the tdk gene (thymidine kinase gene), and the udp gene (uridine phosphorylase gene) in the strain were inactivated or knocked out.
  • Escherichia coli BL21 Escherichia coli
  • the deoA gene thymidine phosphorylase gene
  • the tdk gene thymidine kinase gene
  • udp gene uridine phosphorylase gene
  • the inactivation comprises gene knockout, gene disruption or gene insertion.
  • the inactivation comprises the gene not expressing, or expressing an inactive protein.
  • the pgi gene in the strain is also inactivated or knocked out.
  • the promoter of the pyrA gene, the pyrBI gene, and/or the pyrC gene in the genome of the strain is replaced with a T7 phage strong promoter.
  • the pyrA negative regulatory gene, the pyrL negative regulatory gene, and/or the purR negative regulatory gene in the strain are inactivated or knocked out.
  • the promoter of the pyrA gene in the genome of the strain is replaced with a strong promoter of the T7 phage; the pyrA negative regulatory gene in the strain is inactivated or knocked out.
  • the promoter of the pyrBI gene in the genome of the strain is replaced with a strong promoter of the T7 phage; the pyrL negative regulatory gene in the strain is inactivated or knocked out.
  • the promoter of the pyrC gene in the genome of the strain is replaced with a strong promoter of the T7 phage; the purR negative regulatory gene in the strain is inactivated or knocked out.
  • the promoter of the pyrA gene and the pyrBI gene in the genome of the strain is replaced with a T7 phage strong promoter; the pyrA negative regulatory gene and the pyrL negative regulatory gene in the strain are inactivated or knocked out.
  • the promoters of the pyrA gene, the pyrBI gene, and the pyrC gene in the genome of the strain are replaced with a T7 phage strong promoter; the pyrA negative regulatory gene, the pyrL negative regulatory gene, and the purR in the strain. Negative regulatory genes are inactivated or knocked out.
  • the gene encoding the DE3 T7 RNA polymerase and the lactose operon lac I gene are integrated into the genome of the strain.
  • the gene encoding DE3 T7 RNA polymerase is integrated at a position after knocking out the udp gene.
  • the genome of the strain is integrated with a gene selected from the group consisting of a td gene (thymidylate synthase gene), a nrdC gene (thioredoxin reductase gene), and a nrdAB gene (nucleoside) A diphosphate reductase gene), a dTMPase gene (deoxythymidylate phosphohydrolase gene), or a combination thereof.
  • a gene selected from the group consisting of a td gene (thymidylate synthase gene), a nrdC gene (thioredoxin reductase gene), and a nrdAB gene (nucleoside) A diphosphate reductase gene), a dTMPase gene (deoxythymidylate phosphohydrolase gene), or a combination thereof.
  • the td gene and the nrdC gene are derived from a T4 phage.
  • the nrdAB gene is derived from E. coli.
  • the dTMPase gene is derived from phage PBS1.
  • a foreign gene expression cassette is integrated into the genome of the strain, and the exogenous gene expression cassette tandemly expresses a protein encoded by the td gene, the nrdC gene, the nrdAB gene, and/or the dTMPase gene.
  • the exogenous gene expression cassette has a strong promoter element.
  • the strong promoter element is a T7 phage strong promoter.
  • the exogenous gene expression cassette is integrated at a position after knocking out the deoA gene.
  • the strain down-regulates a negative regulatory gene of the carAB gene, a negative regulatory gene of the pyrBI gene, and/or a negative regulatory gene of the pyrC gene.
  • the negative regulatory genes of the carAB gene include an IHF gene (integration host factor), a PepA gene (aminopeptidase A), a PurR gene (purine-mediated regulation), and/or an ArgR gene (hexameric arginine repressor).
  • the negative regulatory gene of the pyrBI gene includes the pyrL gene.
  • the negative regulatory gene of the pyrC gene includes the purR gene.
  • beta-thymidine comprising the steps of:
  • the engineered strain expresses ⁇ -thymidine under the induction of lactose or IPTG.
  • the ⁇ -thymidine production of the strain is increased by at least 10%; preferably at least 10-50%; more preferably, at least 50%-500% compared to the starting strain.
  • the engineered strain of claim 1 is cultured in a fermentation medium in which the carbon source is glucose.
  • a third aspect of the invention there is provided the use of the engineered strain of the first aspect, which is used as an engineered strain for the production of ⁇ -thymidine by fermentation.
  • the present invention obtains a stable thymidine producing strain by modifying the genome of the strain itself.
  • the use of glucose as a carbon source fermentation process greatly reduces production costs, subsequent separation costs are low, and environmental pollution is reduced.
  • Figure 1 shows the determination of thymidine content of each mutant in Example 2.
  • Figure 2 shows the genetic modification of the pyrA, pyrBI, and pyrC operons in Example 4.
  • A shows the pyrA gene and its negative regulatory region to be knocked out
  • B shows the pyrBI gene and its negative regulatory region to be knocked out
  • C shows the pyrC gene and its negative regulatory region to be knocked out.
  • Figure 3 shows the integrated expression of the DE3 T7 RNA polymerase gene in Example 5.
  • A shows the left and right arm regions of the homologous recombination remaining on both sides after the udp gene knockout
  • B shows the recombinant integration of the DE3 gene at the udp::FRT position
  • C shows the elimination of the kanamycin screening marker. .
  • Figure 4 shows the nrdC+ECnrdAB+td+dTMPase tandem gene expression cassette and the base mutation modification inside the EC-nrdA gene in Example 6.
  • Figure 5 shows the integrated expression of the OPEC expression cassette in Example 6.
  • A shows the OPEC tandem gene fragment and the upstream and downstream homology arms after the deoA gene knockout
  • B shows the recombinant integration of the OPEC expression cassette
  • C shows the integrated expression of the OPEC expression cassette on the chromosome
  • D shows the Kana. Elimination of the selection marker for the mycin.
  • Figure 6 is a graph showing the results of a glucose fermentation process of strain BL21UDT2PpyrAIIBCgDE3gOPEC, wherein blue-green: thymidine (g/L); black: stirring speed (rpm); red: pH (pH); orange: glucose (g/ L); green: OD600; gray: dissolved oxygen DO (%); pink: glycerol (g/L); blue: IPTG addition amount ( ⁇ M).
  • the inventors have extensively and intensively studied, and for the first time, unexpectedly discovered a recombinant bacterium for producing ⁇ -thymidine.
  • the related genes affecting the metabolism of ⁇ -thymidine were modified to form ⁇ -thymidine from the breeding, fermentation and separation and purification of the complete fermentation process. Craft route.
  • the method of the invention has good repeatability and can realize large-scale industrial production.
  • the fermentation process using glucose as a carbon source is used, the production cost is greatly reduced, the subsequent separation cost is low, and environmental pollution is reduced. On the basis of this, the present invention has been completed.
  • the present invention provides a novel microorganism strain which produces ⁇ -thymidine highly. New strains pass the following The program is obtained:
  • a new thymidine strain was obtained by genetically modifying three genes of thymidine phosphorylase (deoA), thymidine kinase (tdk) and uridine phosphorylase (udp) by knockout of E. coli BL21 pyrimidine supplementation pathway.
  • deoA thymidine phosphorylase
  • tdk thymidine kinase
  • udp uridine phosphorylase
  • the genetically modified thymidine producing strain can also knock out the EMP pathway pgi gene to provide more precursors for the synthesis of thymidine.
  • the genetically modified thymidine producing strain wherein the promoters of the carAB (pyrA), pyrBI, and pyrC operons are endogenously replaced, and replaced with a T7 phage strong promoter for efficient expression, and simultaneously The negative regulatory region of the operon was knocked out, and the regulation was suppressed.
  • the promoters of the carAB (pyrA), pyrBI, and pyrC operons are endogenously replaced, and replaced with a T7 phage strong promoter for efficient expression, and simultaneously The negative regulatory region of the operon was knocked out, and the regulation was suppressed.
  • the genetically modified thymidine producing strain comprises integrating a gene encoding DE3 T7 RNA polymerase and a lactose operon lac I gene into the chromosome of the strain, in order to obtain a BL21 strain containing stable DE3 for use in dependence Expression of the T7 promoter transcribed by T7 RNA polymerase.
  • the genetically modified thymidine producing strain which comprises co-expression of four key enzymes of the thymidine pathway, specifically refers to tandem expression under the strong promoter of T7 phage, and the entire expression cassette has been integrated into the chromosome of the strain. on.
  • the four key enzyme genes include T1 phage thymidylate synthase td, T4 phage-derived thioredoxin reductase nrdC, Escherichia coli-derived nucleoside diphosphate reductase nrdAB, phage PBS1 derived deoxythymidine Acid phosphate hydrolase dTMPase gene.
  • the host strain is Escherichia coli BL21 (F-ompThsdSB (rB-, mB-) gal dcm).
  • the genetic modification refers to gene knockout or gene integration on the chromosome of the strain.
  • the present invention obtains the accumulation of thymidine by cutting off the decomposition pathway of ⁇ -thymidine by performing gene knocking or gene integration on the chromosome of Escherichia coli E. coli BL21.
  • the new strains all showed stable thymidine production capacity.
  • the thymidine production strain provided by the invention adopts a fermentation process using glucose as a carbon source, replaces glycerin as a carbon source with cheap glucose, greatly reduces production cost, and has simple process and good repeatability, and can realize large-scale industrial production.
  • the deoA gene is a gene encoding E. coli in the E. coli BL21 pyrimidine replenishment pathway.
  • the tdk gene is a gene encoding E. coli in addition to the E. coli BL21 pyrimidine replenishment pathway.
  • the udp gene is a coding gene in the E. coli pyrimidine complement pathway of E. coli.
  • the pgi gene is a gene encoding the E. coli EMP pathway in E. coli. Knocking out the pgi gene prevents glucose-6-phosphate from being isomeric as fructose-6-phosphate, allowing glucose-6-phosphate to flow to the pentose phosphate pathway, increasing the flux of the pentose phosphate pathway, providing more for the synthesis of thymidine Precursor.
  • the td gene As used herein, the td gene, the thymidylate synthase gene, derived from T4 phage, is a key enzyme in the thymidine synthesis pathway.
  • the nrdC gene As used herein, the nrdC gene, the thioredoxin reductase gene, derived from T4 phage, is a key enzyme in the thymidine synthesis pathway.
  • the nrdAB gene the nucleoside diphosphate reductase gene, derived from E. coli, is a key enzyme in the thymidine synthesis pathway.
  • the dTMPase gene is derived from phage PBS1 and is a key enzyme in the thymidine synthesis pathway.
  • the carA gene and the carB gene respectively encode two subunits contained in a carbamyl phosphate synthase.
  • the first reaction of the pyrimidine nucleotide synthesis pathway is to use CO 2 and glutamine as raw materials, and to supply carbamoyl phosphate under the catalysis of carbamyl phosphate synthase.
  • Carbamoyl phosphate is a common precursor for the synthesis of pyrimidine nucleotides and arginine, and thus the expression of carbamyl phosphate synthase is regulated by the pyrimidine nucleotide and arginine synthesis pathways.
  • the carAB gene is a gene encoding a carbamyl phosphate synthase and is the first key enzyme encoding gene in the de novo synthesis pathway of E. coli pyrimidine nucleotides.
  • the carAB operon has two promoters, P1 and P2, and the transcription of the carAB operon is controlled by these two promoters.
  • the upstream promoter P1 is regulated by intracellular purines and pyrimidines and is regulated by at least five transcription factors, namely IHF (integration host factor), two PepA (aminopeptidase A), and PurR (purine-mediated regulation).
  • the downstream promoter P2 is negatively regulated by two ArgR (hexameric arginine repressors) (shown in Figure 2A).
  • the pyrA gene is a gene encoding a carbamyl phosphate synthase and is the first key enzyme encoding gene in the de novo synthesis pathway of E. coli pyrimidine nucleotides.
  • the pyrBI gene the aspartic acid carbamoyltransferase gene, is the second key enzyme encoding gene in the de novo synthesis pathway of E. coli pyrimidine nucleotides.
  • the pyrC gene is the coding gene for the third key enzyme in the de novo synthesis pathway of E. coli pyrimidine nucleotides.
  • the carAB gene and the pyrA, pyrBI gene, and pyrC genes are the three key enzymes responsible for the reaction of the de novo synthesis pathway of pyrimidine nucleotides, and are important reaction enzymes constituting the pyrimidine ring.
  • T7 promoter and "T7 phage strong promoter” are used interchangeably and are a strong promoter derived from phage.
  • the powerful T7 promoter is completely exclusively controlled by T7 RNA polymerase, while the highly active T7 RNA polymerase synthesizes mRNA five times faster than E. coli RNA polymerase - when both are present, the host's own gene Transcription competition is not the T7 expression system, almost all cell resources are used to express the protein of interest; only a few hours after induction of expression, the target protein can usually account for more than 50% of the total protein. Since E. coli itself does not contain T7 RNA polymerase, it is necessary to introduce exogenous T7 RNA polymerase into the host strain.
  • the regulation mode of T7 RNA polymerase determines the regulation mode of T7 system - under non-inducing conditions, it can make the purpose
  • the gene is completely silent and not transcribed, thereby avoiding the influence of the genotoxicity of the target on the stability of the host cell and the plasmid; by controlling the induction condition to control the amount of T7 RNA polymerase, the amount of product expression can be controlled.
  • Escherichia coli BL21 is a phage DE3 lysogenic strain and is a preferred starting strain of the engineered strain of the present invention.
  • the constructed expression vector can be directly transferred into the expression strain, and the induction regulation mode is the same as lac. IPTG induction.
  • the starting strain of the engineered strain of the present invention may also be Escherichia coli without T7 RNA polymerase, and the T7 promoter is induced by genetic manipulation integration of T7 RNA polymerase.
  • the invention adopts a series of genetic modification with Escherichia coli BL21 which does not accumulate thymidine as a starting strain, and obtains an engineering strain with high yield of ⁇ -thymidine.
  • the engineering strain of the invention has simple fermentation process and can only use glucose as carbon. Source, complete thymidine fermentation, greatly reducing production costs.
  • the "engineering strain”, “thymidine producing strain”, and “recombinant bacteria” are used interchangeably and refer to the engineered strain for producing ⁇ -thymidine of the present invention, that is, the first aspect of the present invention. Said strain.
  • the method of the present invention has good repeatability and can realize large-scale industrial production.
  • the method of the present invention uses a fermentation process using glucose as a carbon source, which greatly reduces production costs, reduces subsequent separation costs, and reduces environmental pollution.
  • the present invention obtains a stable thymidine-producing strain by modifying the genome of the strain itself.
  • knocking out the BL21 pyrimidine replenishing pathway encodes three genes of thymidine phosphorylase (deoA), thymidine kinase (tdk) and uridine phosphorylase (udp), and truncated thymidine
  • decomposition pathway allows thymidine to accumulate.
  • E. coli BL21 was used as the original strain, and the mutant strain BL21-U2 obtained by knocking out the udp single gene was knocked out, and the mutant strain BL21-UD2 obtained by knocking out the udp and deoA genes was superimposed, and the udp, deoA and tdk genes were superimposed and knocked out.
  • the mutant strain BL21-UDT2 (see Table 1) was cultured as follows for thymidine production.
  • the E. coli BL21 strain and each mutant were inoculated in 5 ml of M9-enriched medium (addition of 0.1 M K 2 HPO 4 , 0.01 M Fe 2 (SO 4 ) 3 and 2 g/L of amino acid) overnight at 37 °C. 40 ul of overnight cultured cells were inoculated into 4 ml of M9 enriched medium for 5 h (220 rpm) at 37 ° C while using blank M9 enriched medium as a control.
  • the cultured cells were centrifuged (3500 rpm, 10 min, 4 ° C) to collect the supernatant, and the contents of thymidine, thymine, 2-deoxyuridine, uridine and uracil in the supernatant were detected. Table 2.
  • thymidine content determination The results of thymidine content determination are shown in Figure 1.
  • M9 enriched medium was used as control.
  • deoA and tdk genes were knocked out in BL21 original strain, 6.5 mg/L thymidine was accumulated and knocked out.
  • the accumulation of thymidine was not detected in the mutant strain BL21-U2, and the results showed that the strain could detect the accumulation of thymidine outside the cell after superimposing the three genes in the thymidine replenishment pathway.
  • the pgi gene of BL21-UDT2 strain in Example 2 was knocked out by genetic recombination method to obtain BL21-UDT2P, and thymidine content was measured after incubation for 72 hours in a 50 ml centrifuge tube, and the strain chest was compared before and after pgi gene knockout.
  • the effect of the glycoside content is shown in Table 3.
  • Table 3 The ability of strains to produce thymidine before and after pgi gene knockout
  • Escherichia coli BL21-UDT2PpyrA1 is an endogenous replacement of the carAB operon itself promoter as a T7 phage strong promoter, while removing the sequence including the above negative regulatory region, releasing the feedback repression of the carAB operon, while The carAB operon is constitutively expressed (shown in Figure 2A).
  • the endogenous promoter of the pyrA gene of BL21UDT2P strain in Example 4 was replaced by gene recombination method, and the expression of pyrA gene was enhanced by T7 phage strong promoter, and the negative regulatory gene of pyrA was knocked out, and the obtained strain BL21-UDT2PpyrA1 was obtained.
  • the thymidine content was greatly improved in the fermentation culture test, and the results are shown in Table 4.
  • the first key enzyme (aspartate ammonia) was encoded.
  • Feedback repression of the pyrBI operon of formyltransferase) removed the pyrL sequence of the regulatory gene upstream of pyrB, and released the feedback repression of pyrBI by UTP, while endogenously replacing the promoter of the pyrBI operon itself with the T7 phage strong promoter.
  • the pyrBI operon is constitutively expressed (shown in Figure 2B).
  • the endogenous promoter of the pyrB gene of the above BL21-UDT2PpyrA1 strain was replaced with a strong promoter of T7 phage by gene recombination, and the negative regulatory gene pyrL of the pyrBI operon was knocked out, and the obtained strain BL21-UDT2PpyrAB was detected by fermentation culture.
  • the thymidine content was greatly improved on the basis of the BL21-UDT2PpyrA1 strain, and the thymidine content was 3.2 times that of the original strain BL21-UDT2P. The results are shown in Table 4.
  • pyrC is a gene encoding dihydroorotase in the pyrimidine nucleotide synthesis pathway.
  • the E.coli BL21-UDT2PpyrABC is an endogenous replacement of the promoter of the pyrC operon itself as a strong promoter of the T7 phage, and the sequence including the above negative regulatory region of purR is removed, and the pyrC operon is constitutively expressed (Fig. 2C)).
  • the negative regulatory gene purR of pyrC was further knocked out, and the endogenous promoter of pyrC gene was replaced.
  • the transcriptional expression of pyrC gene was enhanced by T7 phage strong promoter to obtain strain BL21-UDT2PpyrABC.
  • Table 4 The results of thymidine content determination are shown in Table 4.
  • Table 4 shows the ability of the pyrA, pyrBI, and pyrC operon gene-modified strains to synthesize thymidine
  • test is performed after 72 hours of incubation in a 50 ml centrifuge tube.
  • the genetic modification of the E. coli BL21-UDT2PpyrABC strain of Example 4 also includes the expression of the gene encoding DE3T7 RNA polymerase and the expression of the lactose operon lac I gene (shown in Figure 3), which is integrated on the chromosome of BL21.
  • a BL21 strain containing stable DE3 which is suitable for expression of a T7 promoter containing T7 RNA polymerase-dependent transcription, or expression of a protein of interest can be induced using lactose or IPTG.
  • the integration region of the DE3 T7 RNA polymerase gene of the present invention is located on the chromosome of BL21, specifically referring to an FRT position left in the elimination of the resistance gene after udp gene knockout in Example 2.
  • FRT is a 39 bp sequence that is a recognition sequence for FLP recombinase.
  • FLP recombinase intramolecular recombination can occur to remove the DNA sequence between two FRTs, leaving only one FRT.
  • the resistance gene screening marker of DE3 T7 RNA polymerase was continuously eliminated, and finally the strain BL21-UDT2PpyrABCgDE3 (see Fig. 3) was obtained, which can better enhance the gene expression of the T7 promoter.
  • thymidylate synthase td derived from T4 phage
  • thioredoxin reductase nrdC derived from T4 phage
  • nucleoside II derived from Escherichia coli Phosphoreductase nrdAB
  • TMP deoxythymidyl phosphate hydrolase dTMPase
  • the genes are constructed in tandem on a vector to form a co-expressed gene fragment and co-expressed under a strong T7 promoter.
  • the constructed plasmid is named pET29C+T4-td/EC-nrdAB/T4-nrdC/PBS1- TMP (as shown in Figure 4).
  • the nucleoside diphosphate reductase nrdAB gene is derived from Escherichia coli, but not the T4 phage source. Further, a base mutation modification was performed on the dTTP interaction binding site inside the EC-nrdA gene (shown in Fig. 4).
  • the thymidine-producing strains obtained by genetic modification include T4 phage-derived thymidylate synthase td under T7 strong promoter, T4 phage-derived thioredoxin reductase nrdC, Escherichia coli-derived nucleoside diphosphate Reductase nrdAB, a co-expression of the phage PBS1-derived deoxythymidylate phosphate hydrolase dTMPase gene, which has been integrated into the chromosome of BL21 in order to obtain a BL21 strain stably expressing four tandem genes.
  • the tandem gene fragment T4-td/EC-nrdAB/T4-nrdC/PBS1-TMP (referred to as OPEC) was integrated into Example 2 by genetic recombination. After the deoA gene was knocked out, one FRT position left in the elimination of the resistance gene, the FLP recombinase was used to continue to eliminate the OPEC resistance gene screening marker, and finally the thymidine high-yield strain BL21UDT2PpyrAIIBCgDE3gOPEC was obtained (see Fig. 5). The results in Fig.
  • test is performed after 72 hours of incubation in a 50 ml centrifuge tube.
  • Example 6 demonstrates the possibility of using glucose as a carbon source to produce large amounts of thymidine.
  • the BL21UDT2PpyrAIIBCgDE3gOPEC strain of Example 6 was cultured in a 5 L fermentor in the medium of Table 6 according to the following method (see Fig. 6 for the fermentation process).

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Abstract

提供了一种生产β-胸苷的工程菌株，所述菌株为大肠杆菌BL21，而且该菌株中的deoA基因、tdk基因和unp基因被失活或敲除。还提供了利用所述工程菌株生产β-胸苷的方法，以及所述菌株用于发酵生产β-胸苷的用途。

Description

一种生产β-胸苷的工程菌株及其应用 技术领域
本发明涉及生物技术领域，更具体地涉及一种生产β-胸苷的工程菌株及其应用。
背景技术
β-胸苷(以下简称胸苷)是抗艾滋病齐多夫定的关键中间体。齐多夫定是全球首例获得美国FDA批准生产的抗艾滋病药品，因其疗效确切，成为“鸡尾酒”疗法最基本的组合成分。
由于艾滋病病人数量的急剧增加，廉价的、能大规模生产胸苷的工艺方法得到了广泛的关注。生物发酵法生产胸苷，主要是利用微生物菌株的生物合成途径来生产胸苷，使用生物法生产胸苷具有工艺简单、后续分离成本低廉等优势，特别是目前基因工程育种技术及高产优化控制技术的采用，使发酵法生产成本大大降低，因此构建高产胸苷的工程菌株具有良好的应用前景。
大肠杆菌Escherichia coli是迄今为止研究的最为详尽的原核细菌，是一种安全的革兰氏阴性模式菌株，其染色体DNA已测序完毕，其中大部分基因的生物功能已被鉴定。由于大肠杆菌繁殖迅速，培养代谢易于控制，大肠杆菌的分子遗传学背景已相当明了，具备完善的基因操作技术。而且，大肠杆菌在有氧发酵培养时，底物消耗速率快，从而可以获得较为理想的产物生产率，底物范围广，可以利用廉价的原料来生产目的产物，降低生产成本。因此，通过代谢工程方法对菌株进行合理定向的改造具有广泛的前景与现实意义。
胸苷在胞内的合成受到严密的调控，在野生状态下，胞内几乎没有胸苷积累。目前通过代谢工程方法构建胸苷生产菌株的研究较少，其中报道的Lee课题组构建的BLdtugRPA24菌株在以甘油为碳源的pH-stat流加发酵中积累5.2g/L胸苷。但以甘油为碳源的发酵生产工艺存在成本高，发酵周期长等问题。
因此，本领域迫切需要开发高产、经济的β-胸苷生产菌株及其应用。
发明内容
本发明的目的在于提供一种生产β-胸苷的工程菌株及其应用。
在本发明的第一方面，提供一种工程菌株，所述菌株为大肠杆菌 BL21(Escherichia coli)，并且所述菌株中的deoA基因(胸苷磷酸化酶基因)、tdk基因(胸苷激酶基因)和udp基因(尿苷磷酸化酶基因)被失活或敲除。
在另一优选例中，所述的失活包括基因剔除、基因中断或基因插入。
在另一优选例中，所述的失活包括所述基因不表达，或表达没有活性的蛋白。
在另一优选例中，所述菌株中的pgi基因也被失活或敲除。
在另一优选例中，所述菌株基因组中的pyrA基因、pyrBI基因、和/或pyrC基因的启动子替换为T7噬菌体强启动子。
在另一优选例中，所述菌株中的pyrA负调控基因、pyrL负调控基因、和/或purR负调控基因被失活或敲除。
在另一优选例中，所述菌株基因组中的pyrA基因的启动子替换为T7噬菌体强启动子；菌株中的pyrA负调控基因被失活或敲除。
在另一优选例中，所述菌株基因组中的pyrBI基因的启动子替换为T7噬菌体强启动子；菌株中的pyrL负调控基因被失活或敲除。
在另一优选例中，所述菌株基因组中的pyrC基因的启动子替换为T7噬菌体强启动子；菌株中的purR负调控基因被失活或敲除。
在另一优选例中，所述菌株基因组中的pyrA基因和pyrBI基因的启动子替换为T7噬菌体强启动子；所述菌株中的pyrA负调控基因和pyrL负调控基因被失活或敲除。
在另一优选例中，所述菌株基因组中的pyrA基因、pyrBI基因、和pyrC基因的启动子替换为T7噬菌体强启动子；所述菌株中的pyrA负调控基因、pyrL负调控基因、和purR负调控基因被失活或敲除。
在另一优选例中，所述菌株的基因组中整合有编码DE3 T7 RNA聚合酶的基因和乳糖操纵子lac I基因。
在另一优选例中，所述的编码DE3 T7 RNA聚合酶的基因整合于敲除所述udp基因后的位置。
在另一优选例中，所述菌株的基因组中整合有选自下组的基因：td基因(胸苷酸合成酶基因)、nrdC基因(硫氧还蛋白还原酶基因)、nrdAB基因(核苷二磷酸还原酶基因)、dTMPase基因(脱氧胸苷酸磷酸水解酶基因)、或其组合。
在另一优选例中，所述的td基因和nrdC基因来源于T4噬菌体。
在另一优选例中，所述的nrdAB基因来源于大肠杆菌。
在另一优选例中，所述的dTMPase基因来源于噬菌体PBS1。
在另一优选例中，所述菌株的基因组中整合有外源基因表达盒，所述的外源基因表达盒串联表达td基因、nrdC基因、nrdAB基因和/或dTMPase基因所编码的蛋白。
在另一优选例中，所述的外源基因表达盒具有强启动子元件。
在另一优选例中，所述的强启动子元件为T7噬菌体强启动子。
在另一优选例中，所述的外源基因表达盒整合于敲除所述deoA基因后的位置。
在另一优选例中，所述菌株下调了carAB基因的负调控基因、pyrBI基因的负调控基因、和/或pyrC基因的负调控基因。
在另一优选例中，所述的carAB基因的负调控基因包括IHF基因(integration host factor)、PepA基因(aminopeptidase A)、PurR基因(purine-mediated regulation)、和/或ArgR基因(hexameric arginine repressor)。
在另一优选例中，所述的pyrBI基因的负调控基因包括pyrL基因。
在另一优选例中，所述的pyrC基因的负调控基因包括purR基因。
本发明的第二方面，提供一种生产β-胸苷的方法，包括步骤：
(i)培养第一方面所述的工程菌株，从而获得含产β-胸苷的发酵产物；和
(ii)从所述发酵产物中分离出产β-胸苷。
在另一优选例中，所述的工程菌株在乳糖或IPTG诱导下，表达β-胸苷。
在另一优选例中，与出发菌株相比，所述菌株的β-胸苷产量提高了至少10％；较佳地至少10-50％；更佳地，至少50％-500％。
在另一优选例中，在发酵培养基中培养权利要求1所述的工程菌株，所述发酵培养基中碳源为葡萄糖。
本发明的第三方面，提供第一方面所述的工程菌株的用途，所述菌株被用作发酵生产β-胸苷的工程菌。
本发明通过对菌株自身基因组的修饰，获得了稳定的胸苷生产菌株。使用了以葡萄糖为碳源的发酵工艺，大大降低了生产成本，后续分离成本低廉，且减少了环境污染。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了实施例2中各突变株胸苷含量的测定。
图2显示了实施例4中pyrA、pyrBI、pyrC操纵子的基因修饰。其中，A显示了pyrA基因以及其要敲除的负调控区域，B显示了pyrBI基因以及其要敲除的负调控区域，C显示了pyrC基因以及其要敲除的负调控区域。
图3显示了实施例5中DE3 T7 RNA聚合酶基因的整合表达。其中，A显示了udp基因敲除后两侧剩余的同源重组左臂和右臂区域，B显示了DE3基因在udp::FRT位置的重组整合，C显示了卡那霉素筛选标记的消除。
图4显示了实施例6中nrdC+ECnrdAB+td+dTMPase串联基因表达盒以及对EC-nrdA基因内部的碱基突变修饰。
图5显示了实施例6中OPEC表达盒的整合表达。其中，A显示了OPEC串联基因片段以及deoA基因敲除后的上下游同源臂，B显示了OPEC表达盒的重组整合，C显示了OPEC表达盒在染色体上的整合表达，D显示了卡那霉素筛选标记的消除。
图6示出菌株BL21UDT2PpyrAIIBCgDE3gOPEC的葡萄糖发酵工艺结果图，其中，蓝绿色：胸苷(g/L)；黑色：搅拌转速(rpm)；红色：酸碱值(pH)；橘色：葡萄糖(g/L)；绿色：OD600；灰色：溶解氧DO(％)；粉红色：甘油(g/L)；蓝色：IPTG添加量(μM)。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现一种用于生产β-胸苷的重组细菌。通过对大肠杆菌代谢途径的分析，结合基因操作手段，对影响β-胸苷代谢的相关基因进行了修饰，从而形成了从菌种选育、发酵到分离纯化完整的发酵法生产β-胸苷的工艺路线。本发明的方法重复性好，可实现规模化工业生产。而且，使用了以葡萄糖为碳源的发酵工艺，大大降低了生产成本，后续分离成本低廉，且减少了环境污染。在此基础上，完成了本发明。
具体地，本发明提供了一种高产β-胸苷的微生物新菌株。新菌株通过下列 方案获得：
通过组合敲除E.coli BL21嘧啶回补途径胸苷磷酸化酶(deoA)、胸苷激酶(tdk)和尿苷磷酸化酶(udp)的三个基因，经过基因修饰获得胸苷新菌株。
所述经过基因修饰获得的胸苷生产菌株，还可以再对EMP途径pgi基因进行敲除，为胸苷的合成提供了更多的前体物。
所述经过基因修饰获得的胸苷生产菌株，其中是对carAB(pyrA)、pyrBI、pyrC操纵子自身的启动子进行了内源替换，替换为T7噬菌体强启动子进行高效表达，且同时对上述操纵子的负调控区域进行了敲除，解除调控抑制。
所述经过基因修饰获得的胸苷生产菌株，包括将编码DE3 T7 RNA聚合酶的基因以及乳糖操纵子lac I基因整合于菌株的染色体上，为的是获得含有稳定DE3的BL21菌株，用于依赖T7 RNA聚合酶转录的T7启动子的表达。
所述经过基因修饰获得的胸苷生产菌株，其中包括对胸苷途径4个关键酶的共表达，具体是指在T7噬菌体强启动子下进行串联表达，且整个表达框已经整合到菌株的染色体上。4个关键酶基因包括T4噬菌体的胸苷酸合成酶td，T4噬菌体来源的硫氧还蛋白还原酶nrdC，大肠埃希氏菌来源的核苷二磷酸还原酶nrdAB，噬菌体PBS1来源的脱氧胸苷酸磷酸水解酶dTMPase基因。
所述的宿主菌株为大肠埃希氏菌BL21(Escherichia coli BL21(F–ompThsdSB(rB–,mB–)gal dcm)。
所述的基因修饰是指在菌株染色体上进行基因敲除或基因整合。
本发明通过对大肠埃希氏菌E.coli BL21进行菌株染色体上的基因敲除或基因整合的基因修饰，从而截断β-胸苷的分解途径，获得胸苷的积累。新菌株均表现为稳定的胸苷高生产能力。
本发明提供的胸苷生产菌株采用了以葡萄糖为碳源的发酵工艺，用廉价的葡萄糖替代甘油作为碳源，大大降低了生产成本，且工艺简单，重复性好，可以实现规模化的工业生产
定义
如本文所用，所述的deoA基因，即胸苷磷酸化酶基因，是大肠杆菌除E.coli BL21嘧啶回补途径中的编码基因。
如本文所用，所述的tdk基因，即胸苷激酶基因，是大肠杆菌除E.coli BL21嘧啶回补途径中的编码基因。
如本文所用，所述的udp基因，即尿苷磷酸化酶基因，是大肠杆菌E.coli嘧啶回补途径中的编码基因。
如本文所用，所述的pgi基因，即磷酸葡萄糖异构酶基因，是大肠杆菌E.coliEMP途径中的编码基因。敲除pgi基因能够阻止葡萄糖-6-磷酸异构为果糖-6-磷酸，使葡糖-6-磷酸流向磷酸戊糖途径，增加磷酸戊糖途径的通量，为胸苷的合成提供更多的前体物。
如本文所用，所述的td基因，即胸苷酸合成酶基因，来源于T4噬菌体，是胸苷合成途径中的关键酶。
如本文所用，所述的nrdC基因，即硫氧还蛋白还原酶基因，来源于T4噬菌体，是胸苷合成途径中的关键酶。
如本文所用，所述的nrdAB基因，即核苷二磷酸还原酶基因，来源于大肠杆菌E.coli，是胸苷合成途径中的关键酶。
如本文所用，所述的dTMPase基因，即脱氧胸苷酸磷酸水解酶基因，来源于噬菌体PBS1，是胸苷合成途径中的关键酶。
如本文所用，所述的carA基因和carB基因分别编码氨甲酰磷酸合成酶包含的2个亚基。嘧啶核苷酸合成途径的第一个反应是由CO2和谷氨酰胺为原料，由ATP供能，在氨甲酰磷酸合成酶催化下，合成氨甲酰磷酸。氨甲酰磷酸是合成嘧啶核苷酸和精氨酸的共同前体，因此氨甲酰磷酸合成酶的表达受到嘧啶核苷酸和精氨酸合成途径的共同调控。
如本文所用，所述的carAB基因，即pyrA基因,是编码氨甲酰磷酸合成酶的基因，是大肠杆菌E.coli嘧啶核苷酸从头合成途径中第一个关键酶的编码基因。carAB操纵子有2个启动子P1和P2，carAB操纵子的转录受到这2个启动子的控制。上游启动子P1受到胞内嘌呤和嘧啶的共同调控，受到至少5个转录因子的调控，分别是IHF(integration host factor)，2个PepA(aminopeptidase A)，PurR(purine-mediated regulation)。下游启动子P2受到2个ArgR(hexameric arginine repressor)的负调控(附图2A所示)。
如本文所用，所述的pyrA基因，即carAB基因,是编码氨甲酰磷酸合成酶的基因,是大肠杆菌E.coli嘧啶核苷酸从头合成途径中第一个关键酶的编码基因。
如本文所用，所述的pyrBI基因，即天冬氨酸氨甲酰基转移酶基因，是大肠杆菌E.coli嘧啶核苷酸从头合成途径中第二个关键酶的编码基因。
如本文所用，所述的pyrC基因，即二氢乳清酸酶基因，是大肠杆菌E.coli嘧啶核苷酸从头合成途径中第三个关键酶的编码基因。
carAB基因与pyrA、pyrBI基因、pyrC基因是负责编码嘧啶核苷酸从头合成途径反应的三个关键酶，是构成嘧啶环的重要反应酶。
如本文所用，所述的“T7启动子”、“T7噬菌体强启动子”可互换使用，是一种来源于噬菌体的强启动子。强大的T7启动子完全专一受控于T7 RNA聚合酶，而高活性的T7 RNA聚合酶合成mRNA的速度比大肠杆菌RNA聚合酶快5倍——当二者同时存在时，宿主本身基因的转录竞争不过T7表达系统，几乎所有的细胞资源都用于表达目的蛋白；诱导表达后仅几个小时目的蛋白通常可以占到细胞总蛋白的50％以上。由于大肠杆菌本身不含T7 RNA聚合酶，需要将外源的T7 RNA聚合酶引入宿主菌，因而T7 RNA聚合酶的调控模式就决定了T7系统的调控模式——非诱导条件下，可以使目的基因完全处于沉默状态而不转录，从而避免目的基因毒性对宿主细胞以及质粒稳定性的影响；通过控制诱导条件控制T7 RNA聚合酶的量，就可以控制产物表达量，
如本文所用，大肠杆菌BL21是噬菌体DE3溶源化的菌株，是本发明工程菌株的一种优选的出发菌株，构建好的表达载体可以直接转入表达菌株中，诱导调控方式和lac一样都是IPTG诱导。本发明工程菌株的出发菌株也可以是不含T7 RNA聚合酶的大肠杆菌，通过基因操作整合T7 RNA聚合酶实现T7启动子的诱导。
本发明以不积累胸苷的大肠杆菌BL21为出发菌株，进行了一系列的基因改造，得到一株高产β-胸苷的工程菌株，本发明的工程菌株发酵工艺简单，可以仅以葡萄糖为碳源，完成胸苷发酵，大大降低了生产成本。
如本文所用，所述的“工程菌株”、“胸苷生产菌株”、“重组细菌”可互换使用，均指本发明的用于生产β-胸苷的工程菌株，即本发明第一方面所述的菌株。
本发明的主要优点包括：
(a)本发明的方法重复性好，可实现规模化工业生产。
(b)本发明的方法使用了以葡萄糖为碳源的发酵工艺，大大降低了生产成本，后续分离成本低廉，且减少了环境污染。
(c)本发明通过对菌株自身基因组的修饰，获得了稳定的胸苷生产菌株。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方 法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1
采用E.coli BL21做为原始菌株，敲除BL21嘧啶回补途径编码胸苷磷酸化酶(deoA)、胸苷激酶(tdk)和尿苷磷酸化酶(udp)的三个基因，截断胸苷的分解途径，使胸苷能够积累。
实施例2
采用E.coliBL21做为原始菌株，对分别敲除了udp单个基因获得的突变株BL21-U2，叠加敲除了udp和deoA基因获得的突变株BL21-UD2，叠加敲除了udp、deoA和tdk基因获得的突变株BL21-UDT2(见表1)，如下进行胸苷生产的培养。
表1E.coli BL21各突变株基因型
Figure PCTCN2017075586-appb-000001
将E.coliBL21菌株和各突变株接种在5ml的M9富集培养基中(补加0.1M K2HPO4,0.01M Fe2(SO4)3以及2g/L的氨基酸)37℃培养过夜。接种40ul过夜培养细胞到4ml的M9富集培养基中37℃培养5h(220rpm)，同时以空白M9富集培养基为对照。在完成培养后，培养细胞经离心(3500rpm，10min，4℃)收集上清液， 检测上清液中胸苷、胸腺嘧啶、2-脱氧尿苷、尿苷和尿嘧啶的含量，检测结果见表2。
表2BL21各突变株尿嘧啶、尿苷、2-脱氧尿苷、胸腺嘧啶、胸苷含量测定
Figure PCTCN2017075586-appb-000002
胸苷含量的测定结果如图1所示，以M9富集培养基为对照，BL21原始菌株中叠加敲除udp、deoA和tdk基因后，能够积累6.5mg/L的胸苷，而敲除单个基因的突变株BL21-U2中检测不到胸苷的积累，结果显示叠加敲除胸苷回补途径中的3个基因后，菌株可以在细胞外检测到胸苷的积累。
实施例3
通过基因重组的方法对实施例2中BL21-UDT2菌株的pgi基因进行敲除，获得BL21-UDT2P，在50ml离心管中培养72小时后进行胸苷含量测定，比较pgi基因敲除前后对菌株胸苷含量的影响，结果如表3所示。
表3pgi基因敲除前后菌株产生胸苷的能力
菌株 胸苷含量(mg/L)
BL21-UDT2 115
BL21-UDT2P 400
实施例4
大肠埃希氏菌E.coli BL21-UDT2PpyrA1是内源替换了carAB操纵子自身的启动子为T7噬菌体强启动子，同时除去包括上述负调控区域的序列，解除对carAB操纵子的反馈阻遏，同时使carAB操纵子组成型表达(附图2A所示)。
通过基因重组的方法对实施例4中BL21UDT2P菌株pyrA基因的内源启动子进行替换,利用T7噬菌体强启动子增强pyrA基因的表达，同时敲除pyrA的负调控基因，获得的菌株BL21-UDT2PpyrA1经发酵培养检测，与出发菌株BL21-UDT2P相比，其胸苷含量有较大的提高，结果如表4所示。
随后，为了解除嘧啶核苷酸从头合成途径中编码第一个关键酶(天冬氨酸氨 甲酰基转移酶)的pyrBI操纵子的反馈阻遏，去除了pyrB上游的调控基因pyrL序列，解除UTP对pyrBI的反馈阻遏，同时内源替换了pyrBI操纵子自身的启动子为T7噬菌体强启动子，使pyrBI操纵子组成型表达(附图2B所示)。通过基因重组的方法将上述BL21-UDT2PpyrA1菌株的pyrB基因内源启动子替换为T7噬菌体强启动子，同时敲除pyrBI操纵子的负调控基因pyrL，获得的菌株BL21-UDT2PpyrAB经发酵培养检测，其胸苷含量在BL21-UDT2PpyrA1菌株的基础上有较大的提高，胸苷含量是出发菌株BL21-UDT2P的3.2倍，结果如表4所示。
pyrC是嘧啶核苷酸合成途径中编码二氢乳清酸酶的基因。所述的E.coli BL21-UDT2PpyrABC是内源替换了pyrC操纵子自身的启动子为T7噬菌体强启动子，同时去除了包括purR上述负调控区域的序列，使pyrC操纵子组成型表达(附图2C所示)。在菌株BL21-UDT2PpyrAB的基础上，继续敲除pyrC的负调控基因purR，另外对pyrC基因的内源启动子进行了替换，利用T7噬菌体强启动子增强pyrC基因的转录表达,获得菌株BL21-UDT2PpyrABC，其胸苷含量测定结果见表4。
表4对pyrA、pyrBI、pyrC操纵子基因修饰后菌株合成胸苷的能力
Figure PCTCN2017075586-appb-000003
注：试验是在50ml离心管中培养72小时后进行的含量测定。
实施例5
对实施例4中E.coli BL21-UDT2PpyrABC菌株的基因修饰还包括编码DE3T7 RNA聚合酶的基因以及乳糖操纵子lac I基因的表达(附图3所示)，该区域整合于BL21的染色体上，为的是获得含有稳定DE3的BL21菌株，适用于含有依赖T7RNA聚合酶转录的T7启动子的表达，或可以使用乳糖或IPTG诱导进行目的蛋白的表达。
本发明所述的DE3 T7 RNA聚合酶基因的整合区域位于BL21的染色体上，具体指的是实施例2中udp基因敲除后在消除抗性基因时留下的一个FRT位置。 FRT是一个39bp的序列，是FLP重组酶的识别序列，在FLP重组酶存在的情况下，能够发生分子内重组而将两个FRT间的DNA序列移除，最后只剩下一个FRT。根据上述的重组原理继续消除DE3 T7 RNA聚合酶的抗性基因筛选标记，最终获得菌株BL21-UDT2PpyrABCgDE3(见图3)，可以更好的增强带有T7启动子的基因表达。
实施例6
体外构建表达胸苷合成途径中的4个关键酶基因，包括来源于T4噬菌体的胸苷酸合成酶td，T4噬菌体来源的硫氧还蛋白还原酶nrdC，大肠埃希氏菌来源的核苷二磷酸还原酶nrdAB，噬菌体PBS1来源的脱氧胸苷酸磷酸水解酶dTMPase(简称TMP)基因。所述基因被串联构建在一个载体上，形成共表达基因片段，且在一个T7强启动子下进行共表达，构建的质粒名称为pET29C+T4-td/EC-nrdAB/T4-nrdC/PBS1-TMP(如图4所示)。
所述的核苷二磷酸还原酶nrdAB基因来源于大肠埃希氏菌，而非T4噬菌体来源。而且对EC-nrdA基因内部的dTTP作用结合位点进行了碱基突变修饰(附图4所示)。
经过基因修饰获得的胸苷生产菌株包括在T7强启动子下T4噬菌体来源的胸苷酸合成酶td，T4噬菌体来源的硫氧还蛋白还原酶nrdC，大肠埃希氏菌来源的核苷二磷酸还原酶nrdAB，噬菌体PBS1来源的脱氧胸苷酸磷酸水解酶dTMPase基因的共表达，该串联基因表达盒已经整合于BL21的染色体上，为的是获得稳定表达4个串联基因的BL21菌株。
具体地，对实施例5中E.coli BL21-UDT2PpyrABCgDE3菌株，通过基因重组的方法将串联基因片段T4-td/EC-nrdAB/T4-nrdC/PBS1-TMP(简称OPEC)整合到实施例2中deoA基因敲除后在消除抗性基因时留下的一个FRT位置，利用FLP重组酶继续消除OPEC的抗性基因筛选标记，最终获得胸苷高产菌株BL21UDT2PpyrAIIBCgDE3gOPEC(见图5)。图5的结果显示：串联过表达上述4个基因后菌株产苷能力增强，在IPTG诱导条件下产苷效果增强明显，在0.5mM的IPTG诱导下，BL21UDT2PpyrAIIBCgDE3gOPEC菌株的胸苷产量在50ml离心管内可以达到2g/L以上。
表5OPEC表达盒整合菌株合成胸苷的能力
Figure PCTCN2017075586-appb-000004
Figure PCTCN2017075586-appb-000005
注：试验是在50ml离心管中培养72小时后进行的含量测定。
实施例7
该实施例证实了利用葡萄糖作为碳源生产大量胸苷的可能。根据以下方法，将实施例6中的BL21UDT2PpyrAIIBCgDE3gOPEC菌株在表6培养基中进行5L发酵罐的培养(发酵工艺见图6)。
表6发酵培养基配方
培养基成分 g/L
六偏磷酸钠 1～10
磷酸二氢钾 1～10
磷酸氢二氨 1～10
氯化钙 0～1
柠檬酸 0～1
甘氨酸 1～5
酵母粉 1～50
大豆 1～50
胰蛋白胨 1～10
硼酸 0～1
氯化钙 0～1
氯化钴 0～1
硫酸铜(5水) 0～1
硫酸锰(5水) 0～1
钼酸钠(2水) 0～1
硫酸锌 0～1
氯化铁(FeCl3) 0～1
硫酸亚铁 0～1
七水硫酸镁 1～10
甘油 1～50
葡萄糖 1-200
通过在初始培养基中加入50g/L的葡萄糖和少量的甘油以促进细胞的生产，在后续发酵过程中继续补加碳源葡萄糖，同时加入10x 250μM IPTG诱导宿主细胞中胸苷的积累，最终在5L发酵罐中胸苷的最高产量达到6.5g/L(见表7)。
表7菌株的5L发酵罐验证
菌株 IPTG诱导 胸苷最高产量
BL21UDT2PpyrAIIBCgDE3gOPEC 10x 250μM 6.5g/L
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种工程菌株，其特征在于，所述菌株为大肠杆菌BL21，并且所述菌株中的deoA基因、tdk基因和udp基因被失活或敲除。
  2. 如权利要求1所述的工程菌株，其特征在于，所述菌株中的pgi基因也被失活或敲除。
  3. 如权利要求1所述的工程菌株，其特征在于，所述菌株基因组中的pyrA基因、pyrBI基因、和/或pyrC基因的启动子替换为T7噬菌体强启动子。
  4. 如权利要求1所述的工程菌株，其特征在于，所述菌株中的pyrA负调控基因、pyrL负调控基因、和/或purR负调控基因被失活或敲除。
  5. 如权利要求1所述的工程菌株，其特征在于，所述菌株的基因组中整合有编码DE3 T7RNA聚合酶的基因和乳糖操纵子lac I基因。
  6. 如权利要求1所述的工程菌株，其特征在于，所述菌株的基因组中整合有选自下组的基因：td基因、nrdC基因、nrdAB基因、dTMPase基因、或其组合。
  7. 一种生产β-胸苷的方法，其特征在于，包括步骤：
    (i)培养权利要求1所述的工程菌株，从而获得含产β-胸苷的发酵产物；和
    (ii)从所述发酵产物中分离出产β-胸苷。
  8. 如权利要求7所述的方法，其特征在于，所述的工程菌株在乳糖或IPTG诱导下，表达β-胸苷。
  9. 如权利要求7所述的方法，其特征在于，在发酵培养基中培养权利要求1所述的工程菌株，所述发酵培养基中碳源为葡萄糖。
  10. 一种权利要求1所述的工程菌株的用途，其特征在于，所述菌株被用作发酵生产β-胸苷的工程菌。
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CN114410561B (zh) * 2022-01-28 2023-09-01 天津科技大学 一种生产胸苷的基因工程菌株及其构建方法与应用

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