WO2023178849A1 - 合成l-缬氨酸的大肠杆菌及其构建方法与应用 - Google Patents

合成l-缬氨酸的大肠杆菌及其构建方法与应用 Download PDF

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WO2023178849A1
WO2023178849A1 PCT/CN2022/099472 CN2022099472W WO2023178849A1 WO 2023178849 A1 WO2023178849 A1 WO 2023178849A1 CN 2022099472 W CN2022099472 W CN 2022099472W WO 2023178849 A1 WO2023178849 A1 WO 2023178849A1
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valine
escherichia coli
coli
transcription
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饶志明
郝亚男
徐美娟
潘学玮
尤甲甲
杨套伟
张显
邵明龙
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江南大学
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Definitions

  • the invention relates to the field of biotechnology, and in particular to an E. coli that synthesizes L-valine and its construction method and application.
  • L-Valine is an essential amino acid for the human body and a precursor of important cellular components and chemical substances. It has broad application potential in many fields such as medicine, food, and feed. L-valine is also one of the four limiting amino acids in crude protein feed for piglets and broilers, and its long-term deficiency will have adverse effects on animal growth performance. In recent years, the demand for L-valine as an animal feed additive has increased significantly, which has stimulated interest in more efficient and economical production of L-valine. At present, L-valine is mainly produced through microbial fermentation. Mutagenesis or metabolic engineering strategies have been used to construct L-valine-producing strains. However, most of the L-valine-producing strains reported so far still have high yields. and lower productivity.
  • Corynebacterium glutamicum is a Gram-positive bacterium and is the most commonly used industrial microorganism for the production of amino acids, including L-glutamic acid, L-lysine, and BCAAs.
  • L-valine-producing strains using Corynebacterium glutamicum as the "microbial chassis” have been obtained through mutagenesis and systemic metabolic engineering.
  • Strategies for constructing L-valine engineering strains of Corynebacterium glutamicum mainly include strengthening key enzymes in the synthesis pathway, overexpressing rate-limiting enzymes, balancing intracellular redox levels, inhibiting competition pathways, and strengthening the L-valine extracellular transport pathway. .
  • E. coli has a well-defined genetic background and is an attractive industrial production chassis for amino acid production. However, compared to Corynebacterium glutamicum, there are fewer reports on L-valine-producing E. coli strains, which may be due to the more complex regulatory mechanism of L-valine biosynthesis in E. coli.
  • Acetyl hydroxy acid synthase (AHAS) is the rate-limiting enzyme for L-valine biosynthesis.
  • Escherichia coli has three AHAS isoenzymes, encoded by ilvBN, ilvGM and ilvIH, with different properties and regulatory mechanisms. Park et al.
  • L-valine production strain was modified through systemic metabolic engineering of Escherichia coli W3110 and Escherichia coli W.
  • the final L-valine production reached 60.7g/L, and the sugar-acid conversion rate was 0.22g/g.
  • cofactor balance is also considered to be a key bottleneck in improving L-valine production. Because intracellular cofactors affect metabolic networks, signal transduction, and material transport, they further affect the physiological functions of microbial cells. In the process of chemical production by microbial fermentation, the potency and yield of chemicals are often limited by cofactor imbalance, which is mainly caused by the unbalanced expression of cofactor-dependent enzymes in the synthetic pathway. Savrasova and Stoynova et al.
  • Pyruvate is the direct precursor for L-valine synthesis and the central precursor of many metabolites, including acetyl-CoA, L-alanine, etc. Inactivating the activity of the pyruvate dehydrogenase complex or directly blocking the TCA cycle increases the carbon flux of L-valine biosynthesis. However, attempts at this method can have a large impact on cell growth. These results suggest that there is a trade-off in pyruvate distribution between L-valine biosynthesis and cell growth. Inadequate supply of pyruvate may explain the low fermentation yield of L-valine. Oxygen-limited fermentation is a more direct and effective method to weaken the TCA cycle. Under conditions of insufficient oxygen supply, E.
  • transcription factor as a tool for modifying microbial metabolic pathways, has the unique advantage of "multi-point regulation" and can make up for the shortcomings of single gene modification in metabolic engineering.
  • global transcriptional regulators gTMEs
  • gTME global transcriptional regulators
  • gTME is a method of modifying transcription factors that can trigger the reprogramming of gene networks and cellular metabolic networks, changing the transcription efficiency of cells, thereby leading to overall changes in gene expression at the transcription level. Under hypoxic conditions, many transcription factors in E.
  • coli are involved in regulating cell metabolism. Although many studies have isolated and evaluated TFs involved in the regulation of amino acids (e.g., L-glutamic acid and L-lysine) and other secondary metabolites, TFs responsive to L-valine in E. coli The possible role of has not yet been characterized.
  • amino acids e.g., L-glutamic acid and L-lysine
  • TFs responsive to L-valine in E. coli The possible role of has not yet been characterized.
  • the present invention provides an E. coli that synthesizes L-valine and its construction method and application.
  • the present invention significantly reduces the production cost of L-valine related products at the raw material level, is beneficial to enterprises in obtaining a larger market share in the fierce market competition, and provides new methods for constructing efficient and economical L-valine engineering strains. ideas and lay a theoretical foundation for the efficient synthesis of L-valine, which is of great value in both theory and practical applications.
  • the first object of the present invention is to provide an E. coli that synthesizes L-valine.
  • the E. coli is named E. coli W3110 and was deposited at the China Typical Culture Collection Center on March 18, 2022.
  • the deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei province, the collection number is CCTCC M 2022293.
  • the second object of the present invention is to provide a recombinant Escherichia coli that synthesizes L-valine.
  • the recombinant Escherichia coli uses the Escherichia coli as the starting strain and overexpresses the transcriptional regulatory factor to obtain the recombinant Escherichia coli.
  • the transcription regulator is a positive transcription regulator and/or a negative transcription regulator;
  • the positive transcription regulator is a DNA-binding transcription dual regulator pdhR, a DNA-binding transcription dual regulator crp and DNA-binding transcription dual regulator lrp;
  • the negative transcription regulator is RNA polymerase sigma factor rpoS.
  • the KEGG number of the DNA-binding and transcription dual regulator pdhR is JW0109; the KEGG number of the DNA-binding and transcription dual regulator crp is JW5702; the KEGG number of the DNA-binding and transcription dual regulator lrp is JW0872; The KEGG number of RNA polymerase sigma factor rpoS is JW1223.
  • the recombinant E. coli overexpresses dicarboxylic acid reductoisomerase gene ilvC, dihydroxy acid dehydratase gene ilvD and branched-chain amino acid aminotransferase gene ilvE.
  • the dicarboxylic acid reductoisomerase gene ilvC, the dihydroxy acid dehydratase gene ilvD and the branched-chain amino acid aminotransferase gene ilvE are expressed through the Ptrc promoter.
  • the KEGG number of the dicarboxylic acid reductoisomerase gene ilvC is JW3747; the KEGG number of the dihydroxy acid dehydratase gene ilvD is JW5605; and the KEGG number of the branched-chain amino acid aminotransferase gene ilvE for JW5606.
  • the recombinant E. coli knocks out the branched chain amino acid transporter gene brnQ, and integrates the branched chain amino acid exporter gene brnFE into the site where the branched chain amino acid transporter gene brnQ has been knocked out, And make double copies of the brnFE gene.
  • the KEGG number of the branched-chain amino acid transporter gene brnQ is JW0391; the KEGG number of the brnF gene in the gene brnFE is cg0314, and the KEGG number of the brnE gene is cg0315.
  • the recombinant Escherichia coli integrates the phosphogluconate dehydratase gene edd and the KHG/KDPG aldolase gene eda, and the phosphogluconate dehydratase gene edd and the KHG/KDPG aldolase gene eda Expression is regulated via the Ptrc promoter.
  • the KEGG number of the phosphogluconate dehydratase gene edd is ZMO0368; the KEGG number of the KHG/KDPG aldolase gene eda is ZMO0997.
  • the operon ilvIH containing a mutation point is integrated into E. coli.
  • the operon ilvIH includes ilvI (KEGG numbering JW007) and ilvH (KEGG numbering JW0077), wherein the mutation point is ilvH G41A or/and C50T in the gene; the specific mutation is to mutate G to A at position 41 and C to T at position 50 in the ilvH gene.
  • the transcription regulatory factor is overexpressed using ptrc99A or ptrc28A as a vector.
  • the third object of the present invention is to provide a method for constructing the recombinant Escherichia coli for synthesizing L-valine, which includes the following steps:
  • the transcriptional regulatory factors are overexpressed, and the transcriptional regulatory factors are positive transcriptional regulatory factors and/or negative transcriptional regulatory factors;
  • dicarboxylic acid reductoisomerase gene ilvC dihydroxy acid dehydratase gene ilvD and branched-chain amino acid aminotransferase gene ilvE;
  • knock out the branched chain amino acid transporter gene brnQ integrate the branched chain amino acid exporter gene brnFE into the site where the branched chain amino acid transporter gene brnQ has been knocked out, and make double copies of the brnFE gene;
  • the phosphogluconate dehydratase gene edd and the KHG/KDPG aldolase gene eda are integrated to obtain the recombinant Escherichia coli.
  • the positive transcription regulatory factor is one or more of the DNA-binding transcription dual regulator pdhR, the DNA-binding transcription dual regulator crp, and the DNA-binding transcription dual regulator lrp.
  • the negative transcription regulatory factor is RNA polymerase sigma factor rpoS.
  • the fourth object of the present invention is to provide the application of the above-mentioned preserved E. coli or the recombinant E. coli after genetic engineering in the fermentation and synthesis of L-valine.
  • the fermentation conditions are: using pyruvate as a precursor, dissolved oxygen content of 10-20%, fermentation time of 24-72h, temperature of 35-40°C, and rotation speed of 210 -230rpm.
  • seed culture medium g/L: Glucose 20, Yeast Extract 10, Tryptone 6, KH 2 PO 4 1.2, MgSO 4 ⁇ 7H 2 O 0.5, FeSO 4 ⁇ 7H 2 O 0.01, MnSO 4 ⁇ H 2 O 0.01, V B1 0.0013, V H 0.0003, phenol red solution volume concentration 2%, pH controlled at around 6.5, sterilized for 15 minutes at a temperature of 115°C and a pressure of 0.75MPa.
  • fermentation medium g/L: Glucose 20, Yeast Extract 2, Tryptone 4, KH 2 PO 4 2, Sodium Citrate 1, MgSO 4 ⁇ 7H 2 O 0.7, FeSO 4 ⁇ 7H 2 O 0.1, MnSO 4 ⁇ H 2 O 0.1, VB1 0.008, VH 0.0002, phenol red solution volume concentration 2%, pH controlled at around 7.0, sterilized for 15 minutes at a temperature of 121°C and a pressure of 0.75MPa.
  • the recombinant Escherichia coli that synthesizes L-valine constructs a biosensor based on the lrp-type transcriptional regulator, and obtains L-valine-producing bacteria through ARTP mutagenesis high-throughput screening to strengthen L-valine. Acid synthesis pathway and transport system to obtain chassis cells that efficiently produce L-valine, with L-valine production reaching 16.2g/L.
  • the present invention explores transcriptional regulatory factors related to L-valine metabolism through comparative transcriptome analysis of L-valine-producing strains under different dissolved oxygen conditions.
  • the recombinant E. coli that synthesizes L-valine according to the present invention ferments the test strain under slightly dissolved oxygen conditions in a 5L fermentation tank.
  • the L-valine production reaches 112g/L, and the bacterial OD is 104.
  • Escherichia coli that synthesizes L-valine.
  • the Escherichia coli is named Escherichia coli W3110. It was deposited at the China Type Culture Collection Center on March 18, 2022. The deposit address is Bayi Road, Wuchang District, Wuhan City, Hubei City. Luojia Mountain, the collection number is CCTCC M 2022293.
  • Figure 1 is a diagram showing the L-valine production of the recombinant strain CP17 identified at the 5L fermentation tank level of the present invention.
  • Seed culture medium (g/L): Glucose 20, Yeast Extract 10, Tryptone 6, KH 2 PO 4 1.2, MgSO 4 ⁇ 7H 2 O 0.5, FeSO 4 ⁇ 7H 2 O 0.01, MnSO 4 ⁇ H 2 O 0.01, VB1 0.0013, VH 0.0003, phenol red solution volume concentration 2%, pH controlled at around 6.5, sterilized for 15 minutes at a temperature of 115°C and a pressure of 0.75MPa.
  • Fermentation medium g/L: Glucose 20, Yeast Extract 2, Tryptone 4, KH 2 PO 4 2, Sodium citrate 1, MgSO 4 ⁇ 7H 2 O 0.7, FeSO 4 ⁇ 7H 2 O 0.1, MnSO 4 ⁇ H 2 O 0.1, VB1 0.008, VH 0.0002, phenol red solution volume concentration 2%, pH controlled at around 7.0, sterilized for 15 minutes at a temperature of 121°C and a pressure of 0.75MPa.
  • pREDCas9 includes the Red recombination system and Cas9 protein expression System, pGRB elimination system, spectinomycin resistance gene, temperature-sensitive system, the suitable temperature is 32°C; the pGRB plasmid contains gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance gene, the suitable temperature is 37°C.
  • the gRNA transcribed by pGRB carries the Cas9 protein to recognize the PAM (protospacer adjacent motifs) gene target site through base pairing, achieve the target DNA double-strand break, and recombine the fragment containing the PAM gene target site with the linearized vector fragment. Construction of pGRB plasmid.
  • Transform pREDCas9 plasmid Electroporate the pREDCas9 plasmid into the competent strain of the starting strain used in the present invention, resuscitate it for 2 hours and spread it on an LB plate containing spectinomycin, culture it in a 32°C incubator for 10-12 hours, and select single colonies for PCR verification. Screen positive transformants.
  • Transformation of pGRB and recombinant DNA fragments Electroporate the donor DNA and pGRB plasmid into electroporation competent cells containing pREDCas9, use 0.1mM IPTG to induce the expression of ⁇ -Red recombinase, recover for 2 hours and then spread on ampicillin-containing On LB plates of penicillin and spectinomycin, incubate at 32°C for 10-12 hours, select single colonies for PCR verification, and select positive recombinants after colony PCR verification.
  • a kind of Escherichia coli that synthesizes L-valine and its construction method specifically including the following steps:
  • the present invention first constructs a biosensor based on the Lrp-type transcriptional regulator, and then uses the ARTP mutagenesis system to construct a mutation library of E. coli W3110.
  • the time for treating wild E. coli with ARTP is set to 40 seconds, and its lethality rate is between 99.4% and 96 Approximately 5 ⁇ 100 single colonies were screened out on the well microtiter plate.
  • the L-valine production strain was obtained through multiple rounds of mutagenesis and screening.
  • the highest production strain CP1 was obtained.
  • the production strain CP1 was cultured in a seed medium at 37°C and 220 rpm for 12 hours to prepare a seed liquid.
  • the prepared seeds were The liquid was inoculated into the fermentation medium at an inoculation amount of 2% (v/v), and cultured for 24 hours at 37°C and 220 rpm to prepare a fermentation liquid.
  • the L-valine production reached 2.9g/L.
  • L-valine has achieved zero breakthrough in the production of L-valine, it is still necessary to strengthen the branch synthesis pathway from the key precursor pyruvate to L-valine.
  • the synthesis of L-valine from pyruvate involves three enzymes: dicarboxylic acid reductoisomerase (encoded by the ilvC gene), dihydroxyacid dehydratase (encoded by the ilvD gene) and branched-chain amino acid aminotransferase (encoded by the ilvE gene).
  • the segmented integration method is used to first integrate the ilvED gene, which carries exogenous cutting plasmid sgRNA at its end, and then integrate the ilvC gene at this gene site.
  • CP1 was used as the starting strain
  • P trc -ilvCDE was integrated into CP1
  • strain CP2 was constructed. After fermentation in a shake flask for 24 hours, the L-valine yield was 8.9 g/L, the bacterial OD is 36.1.
  • Efficient efflux and blocking the intracellular transport of L-valine are effective strategies to reduce intracellular L-valine content and weaken its feedback inhibition of key enzymes.
  • LivFGHMJ and LivFGHMK are two ATP-dependent high-affinity BCAA transport systems.
  • BrnQ is a low-affinity BCAA transporter. Knocking out the L-valine uptake gene brnQ to increase L-valine production.
  • BrnFE encoded by brnFE
  • Corynebacterium glutamicum has been shown to be an efficient transporter of L-valine.
  • the gene brnFE was integrated into CP2, and the upstream homology arm (UP-brnQ-S and UP-brnQ-A3) and downstream homology arm (DN-brnQ) of the gene site-brnQ were designed through primer 5.
  • UP-brnQ-S and UP-brnQ-A3 and downstream homology arm (DN-brnQ) of the gene site-brnQ were designed through primer 5.
  • -S3 and DN-brnQ-A and the target gene brnFE primers (brnFE-S1 and brnFE-A1).
  • coli W3110 genome as a template to amplify through PCR to obtain the upstream and downstream homology arms and intermediate target fragments, and then use the recovered fragments as templates to perform overlapping PCR amplification to obtain the donor DNA fragment; construct pGRB with the target site Plasmid, primers (gRNA-brnQ-S and gRNA-brnQ-A) are annealed to obtain a fragment containing the PAM gene target site, which is connected to linearized pGRB and then transferred to the E.coli DH5 ⁇ transfection competent state and picked.
  • primers gRNA-brnQ-S and gRNA-brnQ-A
  • the pGRB-brnQ plasmid was obtained from the positive colonies; the gene brnFE integrated fragment and the plasmid pGRB-brnQ were electroporated to the electroporation competent state containing the pREDCas9 plasmid, and the positive colonies containing the target gene brnFE were screened and the primers (brnFE-JD-S1 and brnFE-JD- A1) Plasmid pGRB-brnQ and pREDCas9 were double eliminated to obtain strain CP3. To further increase L-valine production. After fermentation in shake flask for 24 hours, the L-valine production was 16.2g/L, and the bacterial OD was 39.4.
  • L-valine strain CP3 was cultured horizontally in a 5L fermentor, and samples were taken to determine the transcriptome under different dissolved oxygen conditions. A 5L fermentor test was conducted, and a fed-batch fermentation was performed on a 5L bioreactor to simulate the impact of dissolved oxygen on L-valine production.
  • the ventilation is reduced, and the dissolved oxygen level in the fermentation broth is maintained at 30% by adjusting the ventilation and rotation speed.
  • the high dissolved oxygen condition remains at 30%, and the high dissolved oxygen level remains at 30%.
  • the oxygen condition is used as the control group, named G01; the low dissolved oxygen condition maintains the dissolved oxygen level in the fermentation broth at 10% by reducing ventilation and rotation speed.
  • the low dissolved oxygen condition is used as the experimental group, named G02, and the fermentation time is 48h for sampling. , and measure the transcriptome.
  • Comparative transcriptome analysis of G01 and G02 samples showed that genes with obvious differences related to E. coli metabolism were selected and overexpressed.
  • 10 transcriptional regulatory factors related to E. coli carbon metabolism were selected, and based on the genome of CP3, they were connected to the ptrc99A plasmid for overexpression to construct strains CP4-CP14 (CP4 was the control group, that is, the 99A empty plasmid was electroporated ), 24h shake flask fermentation test, the analysis results show that the positive transcription regulatory factors are the DNA-binding transcription dual regulator pdhR, the DNA-binding transcription dual regulator crp and the DNA-binding transcription dual regulator lrp, and the negative transcription regulator is the RNA polymerase ⁇ factor rpoS.
  • the strain CP9 constructed by the over-expressed gene pdhR has the most obvious increase in L-valine production, reaching 15g/L, which is 37.6% higher than the control group; the strains CP10 and CP11 constructed by the over-expressed genes crp and lrp respectively, L-valine production
  • the acid production reaches 13g/L and 12.1g/L. Compared with the control group, they increased by 19.3% and 11.1% respectively.
  • the plasmid test gene pdhR increased the L-valine production most significantly. Then the gene pdhR was integrated into the genome of CP3 to construct strain CP15, and the L-valine production reached 15.9g. /L.
  • the antisense strand of the gene rpoS is overexpressed.
  • the antisense RNA is complementary to the mRNA of the gene rpoS that is normally expressed in the cell, thereby preventing the normal translation of the gene rpoS to achieve an inhibitory effect.
  • an antisense RNA interference strategy was used to overexpress the antisense chain of gene rpoS to construct strain CP16.
  • Antisense RNA is used to inhibit the translation of the mRNA encoded by the gene rpoS into rpoS.
  • the shake flask fermentation results showed that the L-valine production reached 17.4g/L, an increase of 9.4%.
  • a synergistic strategy is proposed by constructing a balanced redox metabolic network to produce L-valine. This design should reduce cell growth stress while producing high yields of L-valine.
  • the genes P trc -edd and P trc -eda were integrated into the Escherichia coli genome CP16 to construct strain CP17.
  • the fragments P trc -edd and P trc -eda were synthesized by synthesizing the phosphogluconate dehydratase genes edd and KHG/KDPG of the Movementmonas fermentum genome.
  • the promoter was connected to the primer when designing the primer, and the L-valine production reached 19.3g/L, an increase of 10.6% compared to the control group.
  • the gene brnFE was integrated, and the upstream homology arms (UP-yjiT-S and UP-yjiT-A) and downstream homology arms (DN-yjiT-S and DN) of the pseudogene site yjiT were designed through primer 5.
  • UP-yjiT-S and UP-yjiT-A upstream homology arms
  • DN-yjiT-S and DN downstream homology arms
  • coli W3110 genome as a template to amplify through PCR to obtain the upstream and downstream homology arms and intermediate target fragments, and then use the recovered fragments as templates to perform overlapping PCR amplification to obtain the donor DNA fragment; construct pGRB with the target site Plasmid, primers (gRNA-yjiT-S and gRNA-yjiT-A) are annealed to obtain a fragment containing the PAM gene target site, which is connected to linearized pGRB and then transferred to the E.coli DH5 ⁇ transfection competent state and picked.
  • primers gRNA-yjiT-S and gRNA-yjiT-A
  • the pGRB-yjiT plasmid was obtained from the positive colonies; the integrated fragment of the gene brnFE and the plasmid pGRB-yjiT were electrotransferred to the electroporation competent state containing the pREDCas9 plasmid, and the positive colonies containing the target gene brnFE were screened and the primers (- brnFE-JD-S2 and brnFE-JD -A2) Plasmid pGRB-yjiT and pREDCas9 were double eliminated to obtain strain CP18.
  • the gene ilvIH mut was integrated, and the upstream homology arms (UP-yghX-S and UP-yghX-A) and downstream homology arms (DN-yghX-S and DN-yghX-S of the pseudogene site yghX were designed through primer 5 DN-yghX-A) and the target gene ilvIH mut primers (ilvIH mut -S1 and ilvIH mut -A1). Use the E.
  • coli W3110 genome as a template to amplify through PCR to obtain the upstream and downstream homology arms and intermediate target fragments, and then use the recovered fragments as templates to perform overlapping PCR amplification to obtain the donor DNA fragment; construct pGRB with the target site Plasmid, primers (gRNA-yghX-S and gRNA-yghX-A) are annealed to obtain a fragment containing the PAM gene target site, which is connected to linearized pGRB and then transferred to the E.coli DH5 ⁇ transfection competent state and picked.
  • primers gRNA-yghX-S and gRNA-yghX-A
  • the pGRB-yghX plasmid was obtained from the positive colonies; the gene ilvIH mut integrated fragment and the plasmid pGRB-yghX were electrotransferred to the electroporation competent state containing the pREDCas9 plasmid, and the positive colonies containing the target gene ilvIH mut were screened and the primers (UP-yghX-S and DN- yghX-A) Plasmid pGRB-yghX and pREDCas9 were double eliminated to obtain strain CP19.
  • Dissolved oxygen conditions are crucial to the fermentation of L-valine.
  • the L-valine synthesis pathway and the TCA cycle form a competitive relationship.
  • cellular aerobic respiration is inhibited, thereby saving energy.
  • the further precursor pyruvate is used to synthesize L-valine.
  • the dissolved oxygen conditions were controlled at 10-20%, and the strain was fermented with slightly dissolved oxygen in a 5L bioreactor. The fermentation cycle was 48 hours. The results are shown in Figure 1.
  • the L-valine production reached 112g/L, and the bacterial OD was the highest. Can reach 104.

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Abstract

提供了一种合成L-缬氨酸的大肠杆菌及其构建方法与应用，涉及生物技术领域。提供一种大肠杆菌命名为大肠杆菌W3110，于2022年03月18日保藏于中国典型培养物保藏中心，保藏编号为CCTCC M 2022293。以大肠杆菌W3110为出发菌株，过表达转录调控因子，得到合成L-缬氨酸的重组大肠杆菌。所述合成L-缬氨酸的重组大肠杆菌在5L发酵罐微溶氧条件下发酵测试菌株，L-缬氨酸产量达到112g/L，菌体OD为104。

Description

合成L-缬氨酸的大肠杆菌及其构建方法与应用 技术领域
本发明涉及生物技术领域，尤其涉及一种合成L-缬氨酸的大肠杆菌及其构建方法与应用。
背景技术
L-缬氨酸是一种人体必需氨基酸，也是重要的细胞成分和化学物质的前体，其在医药、食品、饲料等诸多领域具有广泛的应用潜力。L-缬氨酸还是幼仔猪和肉鸡粗蛋白饲料中四种限制性氨基酸之一，其长期缺乏对动物生长性能会产生不利影响。近年来，动物饲料添加剂对L-缬氨酸的需求显著增加，这激发了人们对于更高效、更经济生产L-缬氨酸的兴趣。目前，L-缬氨酸主要通过微生物发酵法生产，诱变或代谢工程改造策略已被用于构建L-缬氨酸生产菌株，但目前报道的大多数L-缬氨酸生产菌株仍存在产量和生产力较低等问题。
谷氨酸棒杆菌是一种革兰氏阳性细菌，是生产氨基酸(包括L-谷氨酸、L-赖氨酸和BCAAs)最常用的工业微生物。近年来，通过诱变和系统代谢工程获得了以谷氨酸棒杆菌为“微生物底盘”的L-缬氨酸生产菌株。构建谷氨酸棒杆菌L-缬氨酸工程菌的策略主要包括强化合成途径关键酶、过表达限速酶、平衡胞内氧化还原水平、抑制竞争途径和强化L-缬氨酸胞外转运途径。除了常规的代谢工程，适应性进化技术也被应用于L-缬氨酸产生菌的构建。Mahr等人建立了一种生物传感器驱动的自适应进化方法，以筛选L-缬氨酸产率提高和副产物降低的优势进化菌株。尽管在谷氨酸梭菌菌株的工程化方面取得了一些进展，但目前L-缬氨酸发酵的低产量和生产率低降低了经济竞争力。
大肠杆菌具有明确的遗传背景，是氨基酸生产中具有吸引力的工业化生产底盘。然而，相比于谷氨酸棒杆菌，生产L-缬氨酸的大肠杆菌菌株的相关报道较少，这可能是由于大肠杆菌对L-缬氨酸生物合成的调节机制更为复杂。乙酰 羟基酸合酶(AHAS)是L-缬氨酸生物合成的限速酶，大肠杆菌有三种AHAS同工酶，由ilvBN、ilvGM和ilvIH编码，具有不同的性质和调节机制。Park等人报道了通过Escherichia coli W3110和Escherichia coli W的系统代谢工程改造L-缬氨酸生产菌株，其L-缬氨酸终产量达到60.7g/L，糖酸转化率为0.22g/g。除了诱变育种和常规代谢工程修饰外，辅因子平衡也被认为是提高L-缬氨酸产量的关键瓶颈。由于胞内辅因子影响代谢网络、信号转导和物质运输，进而影响微生物细胞的生理功能。在微生物发酵生产化学品的过程中，化学品的效价和产量往往受到辅因子不平衡的限制，这主要是由合成途径中辅因子依赖酶的不平衡表达引起的。Savrasova和Stoynova等人通过用异源NADH依赖性亮氨酸脱氢酶替换天然NADPH依赖性转氨酶，构建了一株以大肠杆菌MG1655为出发菌株的L-缬氨酸工程菌。在微需氧条件下，该菌株的糖酸转化率(0.23g/g)仅为最大理论产量0.65g/g的35.4％。开发生物传感器高通量筛选方法，引入外源辅酶再生途径以平衡胞内辅因子，构建高效的工业底盘生产菌株，是本发明提出的第一个拟解决关键科学问题。
丙酮酸是L-缬氨酸合成的直接前体，也是许多代谢物的中心前体，包括乙酰辅酶A、L-丙氨酸等。灭活丙酮酸脱氢酶复合物的活性或直接阻断TCA循环会增加L-缬氨酸生物合成的碳通量。然而，该方法的尝试会对细胞生长造成较大的影响。这些结果表明，丙酮酸的分布在L-缬氨酸生物合成和细胞生长之间存在权衡。丙酮酸供应不足可以解释L-缬氨酸发酵产量低的原因。限氧条件发酵是减弱TCA循环的更直接、更有效的方法。在氧供应不足条件下，大肠杆菌在自身不生长的同时将葡萄糖代谢为有机酸，生长停滞但代谢活跃的细胞可以获得较高的产量和产率。随着后基因组研究的进展，转录因子(transcription factor，TF)作为一种修饰微生物代谢途径的工具，具有“多点调节”的独特优势，可以弥补单基因修饰在代谢工程改造中的不足。近年来，全局转录调控因子(gTME)因其在改变基因转录以获得有益的细胞表型方面的有效应用而备受关注。gTME是一种修饰转录因子的方法，可触发基因网络和细胞代谢网络的重新编程，改 变细胞的转录效率，从而在转录水平上导致基因的表达发生整体变化。在缺氧条件下，大肠杆菌中有许多转录因子参与调节细胞代谢。尽管许多研究已经分离和评估了参与调节氨基酸(如L-谷氨酸和L-赖氨酸)和其他次级代谢产物的TFs，但是，对L-缬氨酸有反应的TFs在大肠杆菌中的可能作用尚未得到表征。
发明内容
为解决上述技术问题，本发明提供了一种合成L-缬氨酸的大肠杆菌及其构建方法与应用。本发明在原材料层面使L-缬氨酸相关产品的生产成本显著降低，有利于企业在激烈的市场竞争中获得更大的市场份额，为构建高效、经济的L-缬氨酸工程菌株提供新思路，并为L-缬氨酸的高效合成奠定理论基础，在理论和实际应用方面均具有重要价值。
本发明的第一个目的是提供一种合成L-缬氨酸的大肠杆菌，所述大肠杆菌命名为大肠杆菌W3110，于2022年03月18日保藏于中国典型培养物保藏中心，保藏地址为湖北省武汉市武昌区八一路珞珈山，保藏编号为CCTCC M 2022293。
本发明的第二个目的是提供一种合成L-缬氨酸的重组大肠杆菌，所述重组大肠杆菌以所述的大肠杆菌为出发菌株，过表达转录调控因子，得到重组大肠杆菌。
在本发明的一个实施例中，所述转录调控因子为正转录调控因子和/或负转录调控因子；所述正转录调控因子为DNA结合转录双调控子pdhR、DNA结合转录双调控子crp和DNA结合转录双调控子lrp；所述负转录调控因子为RNA聚合酶σ因子rpoS。
在本发明的一个实施例中，所述DNA结合转录双调控子pdhR的KEGG编号为JW0109；DNA结合转录双调控子crp的KEGG编号为JW5702；DNA结合转录双调控子lrp的KEGG编号为JW0872；RNA聚合酶σ因子rpoS的KEGG编号为JW1223。
在本发明的一个实施例中，所述重组大肠杆菌过表达二羧酸还原异构酶基因ilvC、二羟酸脱水酶基因ilvD和支链氨基酸氨基转移酶基因ilvE。所述二羧 酸还原异构酶基因ilvC、二羟酸脱水酶基因ilvD和支链氨基酸氨基转移酶基因ilvE通过Ptrc启动子调控表达。
在本发明的一个实施例中，所述二羧酸还原异构酶基因ilvC的KEGG编号为JW3747；二羟酸脱水酶基因ilvD的KEGG编号为JW5605；支链氨基酸氨基转移酶基因ilvE的KEGG编号为JW5606。
在本发明的一个实施例中，所述重组大肠杆菌敲除支链氨基酸转运蛋白基因brnQ，并将支链氨基酸输出蛋白基因brnFE整合到已敲除支链氨基酸转运蛋白基因brnQ的位点上，并对brnFE基因进行双拷贝。
在本发明的一个实施例中，所述支链氨基酸转运蛋白基因brnQ的KEGG编号为JW0391；基因brnFE中基因brnF的KEGG编号为cg0314，基因brnE的KEGG编号为cg0315。
在本发明的一个实施例中，所述重组大肠杆菌整合磷酸葡萄糖酸脱水酶基因edd和KHG/KDPG醛缩酶基因eda，所述磷酸葡萄糖酸脱水酶基因edd和KHG/KDPG醛缩酶基因eda通过Ptrc启动子调控表达。
在本发明的一个实施例中，所述磷酸葡萄糖酸脱水酶基因edd的KEGG编号为ZMO0368；所述KHG/KDPG醛缩酶基因eda的KEGG编号为ZMO0997。
在本发明的一个实施例中，将包含突变点的操纵子ilvIH整合至大肠杆菌，操纵子ilvIH包括ilvI(KEGG编号为JW007)和ilvH(KEGG编号为JW0077)，其中所述突变位点为ilvH基因中G41A或/和C50T；具体突变为将ilvH基因中第41位G突变为A，第50位C突变为T。
在本发明的一个实施例中，所述转录调控因子以ptrc99A或ptrc28A为载体进行过表达。
本发明的第三个目的是提供一种所述的合成L-缬氨酸的重组大肠杆菌的构建方法，包括以下步骤：
以保藏的大肠杆菌为出发菌株，过表达转录调控因子，所述转录调控因子为正转录调控因子和/或负转录调控因子；
和/或，过表达二羧酸还原异构酶基因ilvC、二羟酸脱水酶基因ilvD和支链氨基酸氨基转移酶基因ilvE；
和/或，敲除支链氨基酸转运蛋白基因brnQ，并将支链氨基酸输出蛋白基因brnFE整合到已敲除支链氨基酸转运蛋白基因brnQ的位点上，并对brnFE基因进行双拷贝；
和/或，并将磷酸葡萄糖酸脱水酶基因edd和KHG/KDPG醛缩酶基因eda整合，得到所述重组大肠杆菌。
在本发明的一个实施例中，所述正转录调控因子为DNA结合转录双调控子pdhR、DNA结合转录双调控子crp和DNA结合转录双调控子lrp中的一种或多种。
在本发明的一个实施例中，所述负转录调控因子为RNA聚合酶σ因子rpoS。
本发明的第四个目的是提供上述保藏大肠杆菌或经基因工程改造后的重组大肠杆菌在发酵合成L-缬氨酸中的应用。
在本发明的一个实施例中，所述发酵的条件为：以丙酮酸为前体物，溶氧量为10-20％，发酵时间为24-72h，温度为35-40℃，转速为210-230rpm。
在本发明的一个实施例中，种子培养基(g/L)：葡萄糖20，Yeast Extract 10，Tryptone 6，KH 2PO 4 1.2，MgSO 4·7H 2O 0.5，FeSO 4·7H 2O 0.01，MnSO 4·H 2O 0.01，V B1 0.0013，V H 0.0003，苯酚红溶液体积浓度2％，pH控制在6.5左右，在温度为115℃，压力为0.75MPa的条件下灭菌15min。
在本发明的一个实施例中，发酵培养基(g/L)：葡萄糖20，Yeast Extract 2，Tryptone 4，KH 2PO 4 2，柠檬酸钠1，MgSO 4·7H 2O 0.7，FeSO 4·7H 2O 0.1，MnSO 4·H 2O 0.1，VB1 0.008，VH 0.0002，苯酚红溶液体积浓度2％，pH控制在7.0左右，在温度为121℃，压力为0.75MPa的条件下灭菌15min。
本发明的技术方案相比现有技术具有以下优点：
(1)本发明所述的合成L-缬氨酸的重组大肠杆菌基于lrp型转录调节因子构建生物传感器，通过ARTP诱变高通量筛选获得L-缬氨酸生产菌，强化L-缬 氨酸合成途径及转运系统，以获得高效生产L-缬氨酸的底盘细胞，L-缬氨酸产量达到16.2g/L。
(2)本发明所述的合成L-缬氨酸的重组大肠杆菌强化正向转录调控因子pdhR的表达；抑制负向转录调控因子rpoS的表达，L-缬氨酸产量达到17.4g/L。
(3)本发明所述的合成L-缬氨酸的重组大肠杆菌引入运动发酵单胞菌Entner-Doudoroff途径，过表达基因P trc-edd和P trc-eda，L-缬氨酸产量达到19.3g/L。
(4)本发明通过不同溶氧条件L-缬氨酸生产菌株的比较转录组分析，挖掘L-缬氨酸代谢相关的转录调控因子。
(5)本发明所述的合成L-缬氨酸的重组大肠杆菌在5L发酵罐微溶氧条件下发酵测试菌株，L-缬氨酸产量达到112g/L，菌体OD为104。
生物材料保藏
一种合成L-缬氨酸的大肠杆菌，所述大肠杆菌命名为大肠杆菌W3110，于2022年03月18日保藏于中国典型培养物保藏中心，保藏地址为湖北省武汉市武昌区八一路珞珈山，保藏编号为CCTCC M 2022293。
附图说明
为了使本发明的内容更容易被清楚地理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明，其中：
图1为本发明5L发酵罐水平上鉴定重组菌株CP17的L-缬氨酸产量图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
下述实施例中所涉及的材料与方法如下：
(1)培养基：
种子培养基(g/L)：葡萄糖20，Yeast Extract 10，Tryptone 6，KH 2PO 4 1.2，MgSO 4·7H 2O 0.5，FeSO 4·7H 2O 0.01，MnSO 4·H 2O 0.01，VB1 0.0013，VH 0.0003，苯酚红溶液体积浓度2％，pH控制在6.5左右，在温度为115℃，压力为0.75MPa 的条件下灭菌15min。
发酵培养基(g/L)：葡萄糖20，Yeast Extract 2，Tryptone 4，KH 2PO 4 2，柠檬酸钠1，MgSO 4·7H 2O 0.7，FeSO 4·7H 2O 0.1，MnSO 4·H 2O 0.1，VB1 0.008，VH 0.0002，苯酚红溶液体积浓度2％，pH控制在7.0左右，在温度为121℃，压力为0.75MPa的条件下灭菌15min。
(2)基因敲除或整合：参考CRISPR/Cas9基因编辑技术，该系统在进行基因敲除或整合时需要供体DNA片段、pREDCas9质粒、pGRB切割质粒；其中pREDCas9包含Red重组系统、Cas9蛋白表达系统、pGRB消除系统、奇霉素抗性基因、温敏系统，适宜温度为32℃；pGRB质粒包含gRNA-Cas9结合区域序列和终止子序列、氨苄青霉素抗性基因，适宜温度为37℃。pGRB转录得到的gRNA携带Cas9蛋白通过碱基配对识别PAM(protospacer adjacent motifs)基因靶位点，实现目的DNA双链断裂，将包含PAM基因靶位点的片段与线性化的载体片段重组连接的方法构建pGRB质粒。
(3)转化pREDCas9质粒：将pREDCas9质粒电转至本发明所用的出发菌株感受态中，2h复苏涂布于含奇霉素LB平板，32℃培养箱培养10-12h，挑选单菌落进行PCR验证，筛选阳性转化子。
(4)pGRB和重组DNA片段的转化：将供体DNA和pGRB质粒电转化到包含pREDCas9的电转感受态中，使用0.1mM IPTG诱导表达的λ-Red重组酶，复苏2h后涂布于含氨苄青霉素和奇霉素的LB平板上，32℃培养10-12h，挑选单菌落进行PCR验证，经菌落PCR验证挑选阳性重组子。
(5)本发明涉及的相关引物：
UP-yjiT-S：AATAGTTGTTGCCGCCTGAGT
UP-yjiT-A：
Figure PCTCN2022099472-appb-000001
brnFE-S2：
Figure PCTCN2022099472-appb-000002
brnFE-A2：
Figure PCTCN2022099472-appb-000003
DN-yjiT-S：
Figure PCTCN2022099472-appb-000004
DN-yjiT-A：CAGGGCTTCCACAGTCACAAT
brnFE-JD-S2：TTCGCTATTGTGCAGTTTCTC
brnFE-JD-A2：ATTGCAAAACAGGCAGCAAAGTCC
UP-yghX-S GCGCAACGTAGAACAGGAATT
UP-yghX-A：
Figure PCTCN2022099472-appb-000005
ilvIH mut-S1：
Figure PCTCN2022099472-appb-000006
ilvIH mut-A1：
Figure PCTCN2022099472-appb-000007
DN-yghX-S：
Figure PCTCN2022099472-appb-000008
DN-yghX-A：GAGCAGGTATTTACGTGAACCG
gRNA-yghX-S：
Figure PCTCN2022099472-appb-000009
gRNA-yghX-A:
Figure PCTCN2022099472-appb-000010
实施例1
一种合成L-缬氨酸的大肠杆菌及其构建方法，具体包括以下步骤：
1、ARTP诱变结合高通量筛选获得L-缬氨酸生产菌
本发明首先基于Lrp型转录调节因子构建生物传感器，然后使用ARTP诱变系统构建大肠杆菌W3110的突变文库，使用ARTP处理野生大肠杆菌时间设定为40s，其致死率在99.4％之间，在96孔微量滴定板上筛选出约5×100个单菌落。通过多轮诱变和筛选获得L-缬氨酸生产菌株，最高生产菌株CP1，取生产菌株CP1在37℃，220rpm条件下于种子培养基中培养12h，制备得到种子液，将制备得到的种子液按2％(v/v)的接种量接种于装有发酵培养基中，在37℃，220rpm条件下培养24h，制备得到发酵液，L-缬氨酸产量达到2.9g/L。
尽管通过ARTP诱变使得L-缬氨酸的产量实现零的突破，但仍需要通过强化从关键前提物丙酮酸到L-缬氨酸的支路合成途径。由丙酮酸合成L-缬氨酸涉及三种酶：二羧酸还原异构酶(ilvC基因编码)、二羟酸脱水酶(ilvD基因编码)和支链氨基酸氨基转移酶(ilvE基因编码)。采用分段整合方式先整合基因ilvED基因，该基因末端携带外源切割质粒sgRNA，再在该基因位点整合ilvC基因。为了强化丙酮酸池到L-缬氨酸的代谢碳流，以CP1为出发菌株，将P trc-ilvCDE整合至CP1中，构建得到菌株CP2，摇瓶发酵24h，L-缬氨酸产量为8.9g/L，菌体OD为36.1。
高效外排并阻断L-缬氨酸向胞内转运是降低细胞内L-缬氨酸含量和弱化其对关键酶反馈抑制的有效策略。大肠杆菌中BCAAs有两种不同的转运系统。LivFGHMJ和LivFGHMK是两个ATP依赖的高亲和力BCAA转运系统。BrnQ 是一种低亲和力的BCAA转运体。敲除L-缬氨酸摄取的基因brnQ以增加L-缬氨酸的产量。来自谷氨酸棒杆菌BrnFE(由brnFE编码)已被证实为L-缬氨酸的有效转运蛋白。
以CP2为出发菌株，将基因brnFE整合至CP2中，通过primer 5设计基因位点-brnQ的上游同源臂(UP-brnQ-S和UP-brnQ-A3)、下游同源臂(DN-brnQ-S3和DN-brnQ-A)及目的基因brnFE引物(brnFE-S1和brnFE-A1)。以大肠杆菌W3110基因组为模板通过PCR进行扩增，得到上、下游同源臂和中间目的片段，再以回收后的片段为模板进行重叠PCR扩增得到供体DNA片段；构建具有靶位点pGRB质粒，引物(gRNA-brnQ-S和gRNA-brnQ-A)退火制得包含PAM基因靶位点的片段，将其与线性化pGRB连接后化转至E.coli DH5α化转感受态，挑取阳性菌落得到pGRB-brnQ质粒；将基因brnFE整合片段和质粒pGRB-brnQ电转至含pREDCas9质粒的电转感受态，筛选含目的基因brnFE的阳性菌落，鉴定引物(brnFE-JD-S1和brnFE-JD-A1)质粒pGRB-brnQ和pREDCas9双消除，得到菌株CP3。以进一步提高L-缬氨酸产量。摇瓶发酵24h，L-缬氨酸产量为16.2g/L，菌体OD为39.4。
2、不同溶氧条件下L-缬氨酸生产菌株的比较转录组分析，调节转录调控因子
代谢途径中单基因转录水平的调控往往不能显著提高目标产物的效价，甚至导致碳氮代谢网络和辅因子网络的失衡。全局调控因子(gTME)可以激活或抑制特定代谢途径中多个基因的协同表达，因此可以根据不同的代谢调节要求表达具有特定功能的转录因子，从而有效增加目标代谢产物的合成。比较转录组学已被应用于分析不同转录水平，是鉴定在目标产物的合成和代谢中起重要作用的蛋白质的有效方法。
微溶氧条件可以抑制L-缬氨酸合成的竞争途径TCA循环，以强化L-缬氨酸的合成支路途径。转录组分析不同溶氧对基因表达的影响，大肠杆菌生产菌株CP在不同溶氧下表现出来明显的L-缬氨酸合成差异。为了鉴定不同溶氧条件下 基因表达水平，在5L发酵罐水平培养L-缬氨酸菌株CP3，并在不同溶氧下取样测定转录组。进行5L发酵罐测试，在5L生物反应器上进行补料分批发酵，模拟溶解氧对L-缬氨酸生产的影响。首先，在发酵罐水平培养至16h，菌体OD达到最大时，通风降低，通过调节通风和转速使发酵液中的溶氧水平维持在30％，之后高溶氧条件仍为30％，高溶氧条件作为对照组，命名为G01；低溶氧条件通过降低通风及转速使发酵液中的溶氧水平维持维持在10％，低溶氧条件作为实验组，命名为G02，发酵时间为48h采样，并测转录组。通过观察L-缬氨酸产量得知，低溶氧下L-缬氨酸产量为76g/L，高溶氧下L-缬氨酸产量为65g/L，接种30h后，低溶氧条件下L-缬氨酸合成率明显上升，菌体OD与葡萄糖消耗速率基本不变。
G01和G02样品的比较转录组分析显示，挑选个与大肠杆菌代谢相关的差异较明显基因，将其进行过表达。其中挑选与大肠杆菌碳代谢相关的10个转录调控因子，在CP3的基因组基础上，将其连接在ptrc99A质粒上进行过表达分别构建菌株CP4-CP14(其中CP4为对照组即电入99A空质粒)，24h摇瓶发酵测试，分析结果表明正转录调控因子为DNA结合转录双调控子pdhR、DNA结合转录双调控子crp和DNA结合转录双调控子lrp，负转录调控因子为RNA聚合酶σ因子rpoS。
其中过表达基因pdhR构建菌株CP9，L-缬氨酸产量提升最明显，达到15g/L，相比对照组提升了37.6％；过表达基因crp和lrp分别构建菌株CP10、CP11，L-缬氨酸产量达到13g/L和12.1g/L。分别相比对照组提升了19.3％和11.1％。为了消除质粒对于发酵过程中的影响，通过质粒测试基因pdhR提高的L-缬氨酸产量最明显，再将基因pdhR整合在在CP3的基因组上构建菌株CP15，L-缬氨酸产量达到15.9g/L。
以CP15为出发菌株，将基因rpoS的反义链过表达，该反义RNA与细胞内正常表达的基因rpoS的mRNA互补配对，从而阻止基因rpoS的正常翻译，以达到抑制效果。为了消除基因rpoS对L-缬氨酸合成的限制，利用反义RNA干 扰策略，过表达了基因rpoS的反义链构建菌株CP16。反义RNA被用来抑制基因rpoS编码的mRNA翻译为rpoS。摇瓶发酵结果显示，L-缬氨酸产量达到17.4g/L，提升了9.4％。
3、合理设计Entner-Doudoroff通路，以改善和控制NADPH再生
引入运动发酵单胞菌的Entner-Doudoroff途径，通过辅因子工程维持细胞内氧化还原平衡可以实现辅因子的自我平衡，促进目标产物的高效合成。辅因子供应不足常常影响生物催化的效率。在限氧发酵条件下，NADH的供应通过糖酵解代谢保持旺盛，甚至可能过度生产。每合成1mol L-缬氨酸需要消耗2mol NADPH，导致胞内氧化还原水平失衡，本发明系统地分析了大肠杆菌中L-缬氨酸的生产网络，设计了一条葡萄糖发酵L-缬氨酸的氧化还原平衡路线。
Glucose+2 NADPH＝L-Valine+2 NADH(Equation 1)
通过构建平衡的氧化还原代谢网络来生产L-缬氨酸，提出了一种协同策略。这种设计应在产生高产量L-缬氨酸的同时减轻细胞生长压力。
在大肠杆菌基因组CP16上整合基因P trc-edd和P trc-eda构建菌株CP17，片段P trc-edd和P trc-eda通过合成发酵运动单胞菌基因组磷酸葡萄糖酸脱水酶基因edd和KHG/KDPG醛缩酶基因eda，设计引物时将启动子连接在引物上，L-缬氨酸产量达到19.3g/L，相比于对照组提升了10.6％。
以CP17为出发菌株，整合基因brnFE，通过primer 5设计假基因位点yjiT的上游同源臂(UP-yjiT-S和UP-yjiT-A)、下游同源臂(DN-yjiT-S和DN-yjiT-A)及目的基因brnFE引物(brnFE-S2和brnFE-A2)。以大肠杆菌W3110基因组为模板通过PCR进行扩增，得到上、下游同源臂和中间目的片段，再以回收后的片段为模板进行重叠PCR扩增得到供体DNA片段；构建具有靶位点pGRB质粒，引物(gRNA-yjiT-S和gRNA-yjiT-A)退火制得包含PAM基因靶位点的片段，将其与线性化pGRB连接后化转至E.coli DH5α化转感受态，挑取阳性菌落得到pGRB-yjiT质粒；将基因brnFE整合片段和质粒pGRB-yjiT电转至含pREDCas9质粒的电转感受态，筛选含目的基因brnFE的阳性菌落，鉴定引物(- brnFE-JD-S2和brnFE-JD-A2)质粒pGRB-yjiT和pREDCas9双消除，得到菌株CP18。
以CP18为出发菌株，整合基因ilvIH mut，通过primer 5设计假基因位点yghX的上游同源臂(UP-yghX-S和UP-yghX-A)、下游同源臂(DN-yghX-S和DN-yghX-A)及目的基因ilvIH mut引物(ilvIH mut-S1和ilvIH mut-A1)。以大肠杆菌W3110基因组为模板通过PCR进行扩增，得到上、下游同源臂和中间目的片段，再以回收后的片段为模板进行重叠PCR扩增得到供体DNA片段；构建具有靶位点pGRB质粒，引物(gRNA-yghX-S和gRNA-yghX-A)退火制得包含PAM基因靶位点的片段，将其与线性化pGRB连接后化转至E.coli DH5α化转感受态，挑取阳性菌落得到pGRB-yghX质粒；将基因ilvIH mut整合片段和质粒pGRB-yghX电转至含pREDCas9质粒的电转感受态，筛选含目的基因ilvIH mut的阳性菌落，鉴定引物(UP-yghX-S和DN-yghX-A)质粒pGRB-yghX和pREDCas9双消除，得到菌株CP19。
实施例2
菌株CP19的基础上，微溶氧条件下5L发酵罐中的补料分批发酵
溶氧条件对L-缬氨酸的发酵至关重要，在大肠杆菌代谢网络中，L-缬氨酸合成途径和TCA循环形成竞争关系，通过控制溶氧条件以抑制细胞有氧呼吸，进而节省更多的前体物丙酮酸用于合成L-缬氨酸。将溶氧条件控制在10-20％，5L生物反应器中对菌株进行微溶氧发酵，发酵周期48h，结果如图1所示，L-缬氨酸产量达到112g/L，菌体OD最高可达到104。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (8)

  1. 一种合成L-缬氨酸的大肠杆菌，其特征在于，所述大肠杆菌命名为大肠杆菌W3110，于2022年03月18日保藏于中国典型培养物保藏中心，保藏地址为湖北省武汉市武昌区八一路珞珈山，保藏编号为CCTCC M 2022293。
  2. 一种合成L-缬氨酸的重组大肠杆菌，其特征在于，所述重组大肠杆菌以权利要求1所述的大肠杆菌为出发菌株，所述重组大肠杆菌过表达二羧酸还原异构酶基因ilvC、二羟酸脱水酶基因ilvD和支链氨基酸氨基转移酶基因ilvE；并敲除支链氨基酸转运蛋白基因brnQ，并将支链氨基酸输出蛋白基因brnFE整合到已敲除支链氨基酸转运蛋白基因brnQ的位点上；整合磷酸葡萄糖酸脱水酶基因edd和KHG/KDPG醛缩酶基因eda；过表达转录调控因子，得到所述重组大肠杆菌。
  3. 根据权利要求2所述的合成L-缬氨酸的重组大肠杆菌，其特征在于，所述转录调控因子为正转录调控因子和/或负转录调控因子；所述正转录调控因子为DNA结合转录双调控子pdhR、DNA结合转录双调控子crp和DNA结合转录双调控子lrp；所述负转录调控因子为RNA聚合酶σ因子rpoS。
  4. 根据权利要求2所述的合成L-缬氨酸的重组大肠杆菌，其特征在于，将包含突变点的操纵子ilvIH整合至大肠杆菌，其中所述突变位点为ilvH基因G41A或/和C50T。
  5. 根据权利要求2所述的合成L-缬氨酸的重组大肠杆菌，其特征在于，所述转录调控因子以ptrc99A或ptrc28A为载体。
  6. 一种权利要求2-5任一项所述的重组大肠杆菌的构建方法，其特征在于，包括以下步骤：
    以权利要求1所述的大肠杆菌为出发菌株，过表达转录调控因子，所述转录调控因子为正转录调控因子和/或负转录调控因子；所述正转录调控因子为DNA结合转录双调控子pdhR、DNA结合转录双调控子crp和DNA结合转录双 调控子lrp；所述负转录调控因子为RNA聚合酶σ因子rpoS；
    和/或，过表达二羧酸还原异构酶基因ilvC、二羟酸脱水酶基因ilvD和支链氨基酸氨基转移酶基因ilvE；
    和/或，敲除支链氨基酸转运蛋白基因brnQ，并将支链氨基酸输出蛋白基因brnFE整合到已敲除支链氨基酸转运蛋白基因brnQ的位点上，并对brnFE基因进行双拷贝；
    和/或，并将磷酸葡萄糖酸脱水酶基因edd和KHG/KDPG醛缩酶基因eda整合，得到所述重组大肠杆菌。
    和/或，将包含突变点的操纵子ilvIH整合至大肠杆菌，其中所述突变位点为ilvH基因G41A或/和C50T。
  7. 一种权利要求1所述的大肠杆菌或权利要求2-5任一项所述的重组大肠杆菌在发酵合成L-缬氨酸中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述发酵的条件为：以丙酮酸为前体物，溶氧量为10-20％，发酵时间为24-72h，温度为35-40℃，转速为210-230rpm。
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