WO2023240794A1 - 一种生产l-高丝氨酸的重组大肠杆菌及其应用 - Google Patents

一种生产l-高丝氨酸的重组大肠杆菌及其应用 Download PDF

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WO2023240794A1
WO2023240794A1 PCT/CN2022/114482 CN2022114482W WO2023240794A1 WO 2023240794 A1 WO2023240794 A1 WO 2023240794A1 CN 2022114482 W CN2022114482 W CN 2022114482W WO 2023240794 A1 WO2023240794 A1 WO 2023240794A1
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gene
homoserine
encoding gene
aspartate
seq
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French (fr)
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饶志明
蔡萌萌
徐美娟
杨套伟
张显
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江南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention relates to the field of biotechnology, and in particular to a recombinant Escherichia coli that produces L-homoserine and its application.
  • L-homoserine is a valuable non-protein amino acid with important physiological functions and application value. It is the precursor substance for the synthesis of essential amino acids L-threonine and L-methionine. In addition, it has the basic skeleton of L-type- ⁇ -amino acid, and its ⁇ -hydroxyl group has various chemical activities and can be used as Intermediates in the synthesis of many important chemicals.
  • L-homoserine is mainly synthesized through chemical methods. The process requires the use of iodide and a large amount of organic solvents. The reaction process is complex, costly, and will pollute the environment. The microbial fermentation method has the advantages of low cost, simple process, mild conditions, and relatively small impact on the environment, and is more suitable for large-scale industrial production of L-homoserine.
  • Li et al. Li N, Xu S, Du G, et al. Efficient production of L-homoserine in Corynebacterium glutamicum ATCC 13032 by redistribution of metabolic flux. Biochemical Engineering Journal, 2020, 161:107665.
  • Taking Corynebacterium glutamicum as the The starting strain was used to construct L-homoserine-producing bacteria, with a final yield of 8.8g/L.
  • Mu et al. Mu Q, Zhang S, Mao X, et al.
  • Zhang et al. (Zhang Y, Wei M, Zhao G, et al. High-level production of L-homoserine using a non-induced, non-auxotrophic Escherichia coli chassis through metabolic engineering. Bioresource Technology, 2021, 327(4):124814 .)
  • a non-inducible, non-auxotrophic, plasmid-free L-homoserine production strain was constructed in Escherichia coli, with a final yield of 60.1g/L. Although this strain has the advantages of being non-inducible and non-auxotrophic, its production capacity needs to be further improved if it is to meet the requirements of industrial production.
  • the present invention uses metabolic engineering methods to modify L-homoserine synthesis-related genes in E. coli at the genome level, weakening the flux of the L-threonine synthesis pathway in the degradation pathway and strengthening the L-homoserine synthesis pathway.
  • the metabolic flow of the serine synthesis pathway enhancing the supply of the precursors oxaloacetate and L-aspartic acid, promoting the extracellular transport of L-homoserine, and synergistically utilizing cofactors, resulted in a strain with a clear genetic background and no plasmid carrying , a genetically engineered bacterium that can produce L-homoserine stably and efficiently without induction, and has application potential for large-scale production.
  • the first object of the present invention is to provide a recombinant Escherichia coli that produces L-homoserine.
  • the recombinant Escherichia coli uses Escherichia coli as the starting strain, knocks out the lactose operon repressor protein encoding gene lacI, and weakens the homoserine kinase encoding gene thrB Expression, overexpression of aspartate kinase I/homoserine dehydrogenase I encoding gene thrA, phosphoenolpyruvate carboxylase encoding gene ppc, aspartate aminotransferase encoding gene aspC, aspartate ammonia cleavage
  • the synthase encoding gene aspA, the threonine and homoserine efflux system encoding gene rhtA and the pyridine nucleotide transhydrogenase encoding gene pntAB were introduced
  • the expression of the homoserine kinase-encoding gene thrB is weakened by replacing the original promoter of thrB with the promoter P fliC ;
  • the gene aspA, the pyridine nucleotide transhydrogenase encoding gene pntAB, the aspartate kinase encoding gene lysC, the aspartate semialdehyde dehydrogenase encoding gene asd and the aspartate dehydrogenase encoding gene aspdh pass through the promoter P trc regulates expression;
  • the gene rhtA encoding the threonine and homoserine efflux system regulates expression through the promoter P lpp .
  • thrA controlled by promoter P trc was integrated into the ycgH, ydeU, yjhE and tfaD gene loci respectively.
  • ppc controlled by promoter P trc was integrated into the yeeL gene site.
  • aspC controlled by promoter P trc was integrated into the ylbE gene site.
  • aspA controlled by promoter P trc was integrated into the ycdN gene site.
  • lysC controlled by promoter P trc was integrated into the ycjV gene site.
  • pntAB controlled by promoter P trc was integrated into the ilvG and ygaY gene loci respectively.
  • rhtA controlled by promoter P lpp was integrated into the yjiP gene site.
  • promoter P trc Asd controlled by promoter P trc was integrated into the yeeP gene site.
  • nucleotide sequence of promoter P fliC is shown in SEQ ID NO.1
  • nucleotide sequence of promoter P trc is shown in SEQ ID NO.2
  • nucleotide sequence of promoter P lpp is shown in SEQ Shown as ID NO.3.
  • nucleotide sequence of the homoserine kinase encoding gene thrB is shown in SEQ ID NO.4
  • nucleotide sequence of the aspartokinase I/homoserine dehydrogenase I encoding gene thrA is shown in SEQ ID NO.5
  • nucleotide sequence of the phosphoenolpyruvate carboxylase encoding gene ppc is shown in SEQ ID NO.6
  • nucleotide sequence of the aspartate aminotransferase encoding gene aspC is shown in SEQ ID NO.7
  • nucleotide sequence of aspartate ammonia lyase encoding gene aspA is shown in SEQ ID NO.
  • nucleotide sequence of aspartate kinase encoding gene lysC is shown in SEQ ID NO.9, threonine
  • nucleotide sequence of the amino acid semialdehyde dehydrogenase encoding gene asd is shown in SEQ ID NO.12
  • nucleotide sequence of the aspartate dehydrogenase encoding gene aspdh is shown in SEQ ID NO.13.
  • the lysC is derived from Corynebacterium glutamicum; asd is derived from Tistrella mobilis; aspdh is derived from Pseudomonas aeruginosa.
  • the starting strain was Escherichia coli W3110.
  • the second object of the present invention is to provide a method for constructing the above-mentioned recombinant E. coli that produces L-homoserine, including the following steps in any order:
  • ppc, aspC and aspA are respectively controlled by promoter P trc and overexpressed on the genome;
  • the present invention uses Escherichia coli, which has a clear metabolic pathway and simple genetic manipulation, as a starting strain, starts from the genetic engineering transformation of the L-homoserine biosynthetic pathway and related metabolic pathways, analyzes and reconstructs the overall metabolic pathway, and obtains a strain with a clear genetic background. , a genetically engineered strain that does not carry plasmids and can produce L-homoserine stably and efficiently.
  • the genetically engineered bacteria obtained by the present invention weaken the degradation of L-homoserine, increase the synthesis flux and precursor supply of L-homoserine, promote the transport of L-homoserine, and enhance the regeneration of NADPH and the introduction of exogenous sources.
  • NADH dehydrogenase is used to regulate intracellular cofactor levels, thereby effectively increasing the production of L-homoserine.
  • the third object of the present invention is to provide the application of the above-mentioned recombinant Escherichia coli in producing L-homoserine.
  • L-homoserine was produced by fermentation with the above recombinant Escherichia coli.
  • the activated strain is cultured at 35-37°C and 180-250r/min to obtain a seed liquid, and the seed liquid is inoculated into the fermentation medium at an inoculation amount of 10-20%.
  • the fermentation temperature is 35-37°C
  • the rotation speed is 180-250r/min
  • the pH is controlled at 7.0-7.2.
  • the fermentation is maintained by adding 60% (m/v) glucose solution.
  • the fermentation time is preferably 24-48h, more preferably 36h.
  • composition of the seed culture medium is: 20-30g/L glucose, 5-10g/L yeast powder, 1-5g/L (NH 4 ) 2 SO 4 , 1-5g/L KH 2 PO 4 , 1-5g /L MgSO 4 ⁇ 7H 2 O, 1-5g/L sodium citrate, 5-15mg/L FeSO 4 ⁇ 7H 2 O, 0.5-2mg/L V H and 0.5-2mg/L V B1 .
  • composition of the fermentation medium is: 10-20g/L glucose, 1-5g/L yeast powder, 1-5g/L (NH 4 ) 2 SO 4 , 1-5g/L KH 2 PO 4 , 1-5g /L MgSO 4 ⁇ 7H 2 O, 1-5g/L sodium citrate, 20-30mg/L FeSO 4 ⁇ 7H 2 O, 0.5-2mg/L V H and 0.5-2mg/L V B1 .
  • the recombinant Escherichia coli is activated and cultured in a seed medium to obtain a seed liquid, and then the seed liquid is inoculated into the fermentation medium at an inoculation amount of 10-20% for fermentation culture.
  • the culture temperature is 35-37°C
  • the pH is 7.0-7.2
  • the dissolved oxygen is controlled to 25-30% by adjusting the stirring speed and ventilation volume
  • the fermentation temperature is 35-37 °C
  • pH is 7.0-7.2
  • the dissolved oxygen is controlled to be 25-30%
  • the glucose residual sugar concentration in the fermentation broth is controlled to be 0.05-5g/L by feeding in batches.
  • the fermentation time is preferably 36-60h, more preferably 48h.
  • composition of the seed culture medium is: 25-35g/L glucose, 5-10g/L yeast powder, 1-5g/L peptone, 1-5g/L KH 2 PO 4 , 0.5-2g/L MgSO 4 ⁇ 7H 2 O, 1-5g/L citric acid or sodium citrate, 5-10mg/L FeSO 4 ⁇ 7H 2 O, 5-10mg/L MnSO 4 ⁇ H 2 O, 0.2-2mg/L V H and 0.5-2mg/ L V B1 .
  • composition of the fermentation medium is: 10-20g/L glucose, 10-15g/L corn steep liquor, 1-5g/L yeast powder, 1-5g/L peptone, 1-5g/L KH 2 PO 4 , 0.5-3g/L MgSO 4 ⁇ 7H 2 O, 1-5g/L citric acid or citrate, 10-30mg/L FeSO 4 ⁇ 7H 2 O, 10-20mg/L MnSO 4 ⁇ H 2 O, 0.2- 2 mg/L V H and 0.3-1 mg/L V B1 , with or without betaine, preferably, 1-3 g/L betaine is added.
  • a culture medium is fed or not fed.
  • the composition of the culture medium is: 1-10g/L yeast powder, 1-10g/L peptone, 1-10g/L KH 2 PO 4 , 1 -5g/L MgSO 4 ⁇ 7H 2 O, 1-5g/L citric acid, 5-15mg/L FeSO 4 ⁇ 7H 2 O, 5-15mg/L MnSO 4 ⁇ H 2 O, 0.1-1mg/L V H and 0.1-1mg/L VB1 .
  • the culture medium was fed during fermentation for 14-20 h.
  • the present invention at least has the following advantages:
  • the invention provides a genetic engineering strain for producing L-homoserine.
  • the genetic engineering strain uses Escherichia coli as the starting strain, knocks out the lacI gene, weakens the homoserine kinase encoding gene thrB, and strengthens the aspartate kinase I/high
  • the gene encoding serine dehydrogenase I, thrA, the gene encoding phosphoenolpyruvate carboxylase, ppc, the gene encoding aspartate aminotransferase, aspC, and the gene encoding aspartate ammonia lyase, aspA were introduced from C.
  • the aspartate kinase encoding gene lysC cgl was overexpressed, the threonine and homoserine efflux system encoding gene rhtA and the pyridine nucleotide transhydrogenase encoding gene pntAB were introduced, and the aspartate half-molecule derived from T. mobilis was introduced.
  • the aldehyde dehydrogenase encoding gene asd tmo and the aspartate dehydrogenase encoding gene aspdh pae derived from P. aeruginosa performed well in the fermentation production of L-homoserine.
  • the product concentration was as high as 42g/L after 36 hours of shake flask fermentation.
  • the product concentration of tank fermentation for 48 hours is as high as 120g/L, and the sugar-acid conversion rate reaches 60%, which has good industrial application prospects.
  • Figure 1 shows the shake flask fermentation results of the L-homoserine genetic engineering strain
  • Figure 2 shows the fed-batch fermentation process curve of strain E.coli HOM10 in a 5L fermenter.
  • upstream homology arm primers lacI-1, lacI-2
  • downstream homology arm primers lacI-3, lacI-4
  • lacI-1, lacI-2 downstream homology arm primers
  • lacI-3, lacI-4 downstream homology arm primers
  • the knockout fragments of plasmid pGRB-lacI and lacI genes were simultaneously electrotransformed into electrotransfection competent cells of E.coli W3110 containing pREDCas9 to obtain positive transformants. After eliminating the plasmid, the E.coli W3110 ⁇ lacI strain was obtained.
  • the upstream homology arm primers (P thrB -1, P thrB -2) and the downstream homology arm primers (P thrB -3, P thrB -4), the P fliC sequence (SEQ ID NO. 1) was designed on the primers P thrB -2 and P thrB -3, and its upstream and downstream homology arm fragments were amplified by PCR, and then used as a template to carry out Fusion PCR was used to obtain the replacement fragment of the P thrB gene.
  • the DNA fragment obtained after annealing the primers gRNA-P thrB -1 and gRNA-P thrB -2 was connected to plasmid pGRB to construct plasmid pGRB-P thrB .
  • the replacement fragments of plasmid pGRB-P thrB and P thrB genes were simultaneously electrotransformed into E. coli W3110 ⁇ lacI electrotransfection competent cells containing pREDCas9, and positive transformants were obtained.
  • the strain HOM1 was obtained after eliminating the plasmid.
  • the DNA fragment obtained after annealing the primers gRNA-ycgH-1 and gRNA-ycgH-2 was connected to plasmid pGRB to construct plasmid pGRB-ycgH.
  • the integrated fragments of plasmid pGRB-ycgH and P trc -thrA genes were simultaneously electrotransformed into electrotransfection competent cells of HOM1 containing pREDCas9, and positive transformants were obtained.
  • the strain HOM2-1 was obtained after eliminating the plasmid.
  • ydeU-3 and ydeU-4 in which the P trc promoter sequence is designed on primers ydeU-2 and ydeU-3 (primer ydeU-3 is consistent with the above-mentioned ycgH-3 sequence), and each fragment is obtained by PCR amplification, and then Using it as a template, fusion PCR was performed to obtain the integrated fragment of P trc -thrA gene.
  • the DNA fragment obtained after annealing the primers gRNA-ydeU-1 and gRNA-ydeU-2 was connected to plasmid pGRB to construct plasmid pGRB-ydeU.
  • the integrated fragments of plasmid pGRB-ydeU and P trc -thrA genes were simultaneously electrotransformed into the electroporated competent cells of HOM2-1 containing pREDCas9, and positive transformants were obtained.
  • the strain HOM2-2 was obtained after eliminating the plasmid.
  • yjhE-3 and yjhE-4 in which the P trc promoter sequence is designed on primers yjhE-2 and yjhE-3 (primer yjhE-3 is consistent with the above-mentioned ycgH-3 sequence), and each fragment is obtained by PCR amplification, and then Using it as a template, fusion PCR was performed to obtain the integrated fragment of P trc -thrA gene.
  • the DNA fragment obtained after annealing the primers gRNA-yjhE-1 and gRNA-yjhE-2 was connected to plasmid pGRB to construct plasmid pGRB-yjhE.
  • tfaD-3 and tfaD-4 in which the P trc promoter sequence is designed on primers tfaD-2 and tfaD-3 (primer tfaD-3 is consistent with the above-mentioned ycgH-3 sequence), and each fragment is obtained by PCR amplification, and then Using it as a template, fusion PCR was performed to obtain the integrated fragment of P trc -thrA gene.
  • the DNA fragment obtained after annealing the primers gRNA-tfaD-1 and gRNA-tfaD-2 was connected to plasmid pGRB to construct plasmid pGRB-tfaD.
  • the DNA fragment obtained after annealing the primers gRNA-P ppc -1 and gRNA-P ppc -2 was connected to plasmid pGRB to construct plasmid pGRB-P ppc .
  • the DNA fragment obtained after annealing the primers gRNA-yeeL-1 and gRNA-yeeL-2 was connected to plasmid pGRB to construct plasmid pGRB-yeeL.
  • the integrated fragments of plasmid pGRB-yeeL and P trc -ppc genes were simultaneously electrotransformed into HOM3-1 electrocompetent cells containing pREDCas9 to obtain positive transformants. After eliminating the plasmid, strain HOM3-2 was obtained.
  • the DNA fragment obtained after annealing the primers gRNA-ylbE-1 and gRNA-ylbE-2 was connected to plasmid pGRB to construct plasmid pGRB-ylbE.
  • the integrated fragments of plasmid pGRB-ylbE and P trc -aspC genes were simultaneously electrotransformed into HOM3-2 electrocompetent cells containing pREDCas9 to obtain positive transformants. After eliminating the plasmid, strain HOM4 was obtained.
  • the DNA fragment obtained after annealing the primers gRNA-ycdN-1 and gRNA-ycdN-2 was connected to plasmid pGRB to construct plasmid pGRB-ycdN.
  • the integrated fragments of plasmids pGRB-ycdN and P trc -aspA genes were simultaneously electrotransformed into electrocompetent cells of HOM4 containing pREDCas9, and positive transformants were obtained. After eliminating the plasmid, strain HOM5 was obtained.
  • the upstream homology arm primers (ycjV-1, ycjV-2) and downstream homology arm primers (ycjV-5, ycjV-6) were designed based on the ycjV gene sequence.
  • C. glutamicum13032 The genome was used as a template, and primers ycjV-3 and ycjV-4 were designed based on the lysC cgl gene sequence.
  • the P trc promoter sequence was designed on primers ycjV-2 and ycjV-3, and each fragment was obtained through PCR amplification, and then used As a template, fusion PCR was performed to obtain the integrated fragment of P trc -lysC cgl gene.
  • the DNA fragment obtained after annealing the primers gRNA-ycjV-1 and gRNA-ycjV-2 was connected to plasmid pGRB to construct plasmid pGRB-ycjV.
  • the upstream homology arm primers (yjiP-1, yjiP-2) and downstream homology arm primers (yjiP-7, yjiP-8) were designed based on the yjiP gene sequence.
  • the P lpp gene sequence SEQ ID NO.3
  • Design primers yjiP-3 and yjiP-4, design primers yjiP-5 and yjiP-6 according to the rhtA gene sequence obtain each fragment through PCR amplification, and then use it as a template to perform fusion PCR to obtain P Integrated fragment of lpp -rhtA gene.
  • the DNA fragment obtained after annealing the primers gRNA-yjiP-1 and gRNA-yjiP-2 was connected to plasmid pGRB to construct plasmid pGRB-yjiP.
  • the integrated fragments of plasmids pGRB-yjiP and P lpp -rhtA genes were simultaneously electrotransformed into electroporated competent cells of HOM6 containing pREDCas9 to obtain positive transformants. After eliminating the plasmid, strain HOM7 was obtained.
  • the DNA fragment obtained after annealing the primers gRNA-ilvG-1 and gRNA-ilvG-2 was connected to plasmid pGRB to construct plasmid pGRB-ilvG.
  • the integrated fragments of plasmid pGRB-ilvG and P trc -pntAB genes were simultaneously electrotransformed into electroporated competent cells of HOM7 containing pREDCas9 to obtain positive transformants. After eliminating the plasmid, strain HOM8-1 was obtained.
  • the upstream homology arm primers (ygaY-1, ygaY-2) and downstream homology arm primers (ygaY-5, ygaY-6) were designed based on the ygaY gene sequence, in which P trc was activated
  • the subsequences were designed on primers ygaY-2 and ilvG-3, and each fragment was obtained through PCR amplification, and then used as a template to perform fusion PCR to obtain the integrated fragment of the P trc -pntAB gene.
  • the DNA fragment obtained after annealing the primers gRNA-ygaY-1 and gRNA-ygaY-2 was connected to plasmid pGRB to construct plasmid pGRB-ygaY.
  • the integrated fragments of plasmid pGRB-ygaY and P trc -pntAB genes were simultaneously electrotransformed into electroporated competent cells of HOM8-1 containing pREDCas9, and positive transformants were obtained. After eliminating the plasmid, strain HOM8-2 was obtained.
  • the upstream homology arm primers (yeeP-1, yeeP-2) and downstream homology arm primers (yeeP-5, yeeP-6) were designed based on the yeeP gene sequence, which were derived from T. mobilis
  • the aspartate semialdehyde dehydrogenase encoding gene asd tmo was synthesized by the company after codon optimization.
  • Primers yeeP-3 and yeeP-4 were designed according to its sequence. Among them, the P trc promoter sequence was designed between primers yeeP-2 and yeeP-4.
  • each fragment was obtained through PCR amplification, and then used as a template to perform fusion PCR to obtain the integrated fragment of the P trc -asd tmo gene.
  • the DNA fragment obtained after annealing the primers gRNA-yeeP-1 and gRNA-yeeP-2 was connected to plasmid pGRB to construct plasmid pGRB-yeeP.
  • the integrated fragments of plasmids pGRB-yeeP and P trc -asd tmo genes were simultaneously electrotransformed into HOM8-2 electroporated competent cells containing pREDCas9 to obtain positive transformants. After eliminating the plasmid, strain HOM9 was obtained.
  • the aspdh pae gene derived from P. aeruginos is integrated into the yghX locus
  • upstream homology arm primers yghX-1, yghX-2
  • downstream homology arm primers yghX-5, yghX-6
  • yghX-1, yghX-2, yghX-6 were designed based on the yghX gene sequence, which were derived from P. aeruginos
  • the aspartate dehydrogenase encoding gene aspdh pae was synthesized by the company after codon optimization.
  • Primers yghX-3 and yghX-4 were designed based on its sequence.
  • the P trc promoter sequence was designed between primers yghX-2 and yghX- 3, each fragment was obtained through PCR amplification, and then used as a template to perform fusion PCR to obtain the integrated fragment of the P trc -aspdh pae gene.
  • the DNA fragment obtained after annealing the primers gRNA-yghX-1 and gRNA-yghX-2 was connected to plasmid pGRB to construct plasmid pGRB-yghX.
  • Example 2 Using genetically engineered bacteria HOM10 to produce L-homoserine through shake flask fermentation
  • Seed culture Take the -80°C preserved strain and inoculate it into the slant medium by streaking it, culture it at 37°C for 12 hours, and passage it once. Then, use an inoculation loop to scrape a ring of slant seeds and inoculate it into 30 mL of seed medium. Put it in a 500mL round-bottomed Erlenmeyer flask, seal it with nine layers of gauze, and incubate it at 37°C and 220rmp for 8-10h.
  • the composition of the seed medium is: 30g/L glucose, 10g/L yeast powder, 4g/L (NH 4 ) 2 SO 4 , 3g/L KH 2 PO 4 , 2g/L MgSO 4 ⁇ 7H 2 O, 2g/L lemon Sodium acid, 5mg/L FeSO 4 ⁇ 7H 2 O, 0.5mg/L V H and 0.5mg/L V B1 , the rest is water, pH 7.0-7.2.
  • Fermentation culture Inoculate the seed liquid into a 500mL baffled triangular flask containing 30mL of fermentation medium at an inoculation amount of 15%, seal it with nine layers of gauze, and culture at 37°C and 240r/min. During the fermentation process, phenol red is used. As an indicator, control the pH at 7.0-7.2 by adding 25% ammonia water. When the glucose in the culture medium is exhausted, maintain the fermentation by adding 60% (m/v) glucose solution; the fermentation cycle is 36 hours.
  • the composition of the fermentation medium is: 10g/L glucose, 5g/L yeast powder, 5g/L (NH 4 ) 2 SO 4 , 3g/L KH 2 PO 4 , 2g/L MgSO 4 ⁇ 7H 2 O, 2g/L lemon Sodium acid, 30mg/L FeSO 4 ⁇ 7H 2 O, 0.5mg/L V H , 0.5mg/L V B1 and 8mg/L phenol red, the rest is water, pH 7.0-7.2.
  • Example 3 Using genetically engineered bacteria HOM10 to ferment and produce L-homoserine in a 5L fermentation tank
  • Seed culture pour an appropriate amount of sterile water into the slope, use an inoculation loop to suspend the bacterial cells, and then inoculate the bacterial suspension into the seed medium for culture.
  • the culture temperature is 37°C
  • the initial ventilation volume is 2L/min
  • the initial stirring speed is 200r/min.
  • the pH of the culture medium is controlled to 7.0-7.2 by automatically adding 25% ammonia water, and the dissolved oxygen is controlled at 25-30 through stirring and ventilation. %, when the OD 600 reaches 15-20, prepare to insert the fermentation medium.
  • the composition of the seed culture medium is: 30g/L glucose, 5g/L yeast powder, 3g/L peptone, 1.5g/L KH 2 PO 4 , 0.5g/L MgSO 4 ⁇ 7H 2 O, 1g/L sodium citrate, 10mg /L FeSO 4 ⁇ 7H 2 O, 10mg/L MnSO 4 ⁇ H 2 O, 1mg/L V H and 0.5mg/L V B1 , the rest is water, pH 7.0-7.2.
  • Fermentation culture Inoculate the seed liquid into the fermentation medium at an inoculation amount of 15%.
  • the culture temperature is 37°C.
  • the pH of the medium is controlled to 7.0-7.2 by automatically adding 25% ammonia water. It is controlled by stirring and ventilation.
  • the dissolved oxygen is 25-30%.
  • 80% glucose solution is automatically added to control the residual glucose concentration in the fermentation broth to 0.05-5g/L.
  • the composition of the fermentation medium is: 10g/L glucose, 10g/L corn steep liquor, 4g/L yeast powder, 3g/L peptone, 4g/L KH 2 PO 4 , 1g/L MgSO 4 ⁇ 7H 2 O, 2g/L lemon Sodium phosphate, 10mg/L FeSO 4 ⁇ 7H 2 O, 10mg/L MnSO 4 ⁇ H 2 O, 0.2mg/L V H and 0.3mg/L V B1 , the rest is water, pH 7.0-7.2.
  • Seed culture pour an appropriate amount of sterile water into the slope, use an inoculation loop to suspend the bacterial cells, and then inoculate the bacterial suspension into the seed culture medium for culture.
  • the culture temperature is 37°C
  • the initial ventilation volume is 2L/min
  • the initial stirring speed is 200r/min
  • the pH of the culture medium is controlled to 7.0-7.2 by automatically adding 25% ammonia water
  • the dissolved oxygen is controlled at 25-30 through stirring and ventilation. %, when the OD 600 reaches 15-20, prepare to insert the fermentation medium.
  • the composition of the seed medium is: 35g/L glucose, 5g/L yeast powder, 3g/L peptone, 1.5g/L KH 2 PO 4 , 0.5g/L MgSO 4 ⁇ 7H 2 O, 1g/L citric acid, 10mg/L L FeSO 4 ⁇ 7H 2 O, 10 mg/L MnSO 4 ⁇ H 2 O, 1 mg/L V H and 0.5 mg/L V B1 , the rest is water, pH 7.0-7.2.
  • Fermentation culture Inoculate the seed liquid into the fermentation medium at an inoculation amount of 20%.
  • the culture temperature is 37°C.
  • the pH of the medium is controlled to 7.0-7.2 by automatically adding 25% ammonia water. It is controlled by stirring and ventilation.
  • the dissolved oxygen is 25-30%.
  • 80% glucose solution is automatically added to control the residual glucose concentration in the fermentation broth to 0.05-5g/L.
  • the composition of the fermentation medium is: 10g/L glucose, 12g/L corn steep liquor, 4g/L yeast powder, 3g/L peptone, 4g/L KH 2 PO 4 , 1g/L MgSO 4 ⁇ 7H 2 O, 2g/L lemon Acid, 10mg/L FeSO 4 ⁇ 7H 2 O, 10mg/L MnSO 4 ⁇ H 2 O, 0.2mg/L V H and 0.3mg/L V B1 , the rest is water, pH 7.0-7.2, and add 1g/L with the sugar flow L betaine.
  • Seed culture pour an appropriate amount of sterile water into the slope, use an inoculation loop to suspend the bacterial cells, and then inoculate the bacterial suspension into the seed medium for culture.
  • the culture temperature is 37°C
  • the initial ventilation volume is 2L/min
  • the initial stirring speed is 200r/min.
  • the pH of the culture medium is controlled to 7.0-7.2 by automatically adding 25% ammonia water, and the dissolved oxygen is controlled at 25-30 through stirring and ventilation. %, when the OD 600 reaches 15-20, prepare to insert the fermentation medium.
  • the composition of the seed medium is: 35g/L glucose, 5g/L yeast powder, 3g/L peptone, 1.5g/L KH 2 PO 4 , 0.5g/L MgSO 4 ⁇ 7H 2 O, 1g/L citric acid, 10mg/L L FeSO 4 ⁇ 7H 2 O, 10 mg/L MnSO 4 ⁇ H 2 O, 1 mg/L V H and 0.5 mg/L V B1 , the rest is water, pH 7.0-7.2.
  • Fermentation culture Inoculate the seed liquid into the fermentation medium at an inoculation amount of 20%.
  • the culture temperature is 37°C.
  • the pH of the medium is controlled to 7.0-7.2 by automatically adding 25% ammonia water. It is controlled by stirring and ventilation. Dissolved oxygen is 25-30%.
  • 80% glucose solution is automatically added to control the residual glucose concentration in the fermentation broth to 0.05-5g/L. Automatically added during 14-20 hours of fermentation. culture medium.
  • the composition of the fermentation medium is: 10g/L glucose, 12g/L corn steep liquor, 4g/L yeast powder, 3g/L peptone, 4g/L KH 2 PO 4 , 1g/L MgSO 4 ⁇ 7H 2 O, 2g/L lemon Acid, 10mg/L FeSO 4 ⁇ 7H 2 O, 10mg/L MnSO 4 ⁇ H 2 O, 0.2mg/L V H and 0.3mg/L V B1 , the rest is water, pH 7.0-7.2, and add 1g/L with the sugar flow L betaine.
  • the composition of fed-batch medium is: 4g/L yeast powder, 3g/L peptone, 4g/L KH 2 PO 4 , 1g/L MgSO 4 ⁇ 7H 2 O, 2g/L citric acid, 10mg/L FeSO 4 ⁇ 7H 2 O, 10 mg/L MnSO 4 ⁇ H 2 O, 0.5 mg/L V H and 0.5 mg/L V B1 .

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Abstract

本发明涉及一种生产L-高丝氨酸的重组大肠杆菌及其应用，通过代谢工程的方法，在基因组层面对大肠杆菌中L-高丝氨酸合成相关基因进行改造，通过弱化降解途径L-苏氨酸合成途径的通量、强化L-高丝氨酸合成途径的代谢流、增强前体物草酰乙酸和L-天冬氨酸的供应、促进L-高丝氨酸的胞外转运，以及促进辅因子协同利用，得到一株遗传背景清晰、不携带质粒、无需诱导且能稳定高效生产L-高丝氨酸的基因工程菌，具有大规模生产的应用潜力。

Description

一种生产L-高丝氨酸的重组大肠杆菌及其应用 技术领域
本发明涉及生物技术领域，尤其涉及一种生产L-高丝氨酸的重组大肠杆菌及其应用。
背景技术
L-高丝氨酸是一种有价值的非蛋白质氨基酸，具有重要的生理学功能和应用价值。它是必需氨基酸L-苏氨酸和L-甲硫氨酸合成的前体物质，另外，它具有L-型-α-氨基酸的基本骨架，并且其γ-羟基具有多样的化学活性，可作为许多重要化学品合成的中间体。目前，L-高丝氨酸主要通过化学法合成，其过程需要使用碘化物和大量有机溶剂，反应过程复杂、成本高，且会对环境造成污染。而微生物发酵法具有成本低、工艺简单、条件温和、对环境影响相对较小等优势，更适合用于L-高丝氨酸的大规模工业生产。
目前，对于微生物发酵生产L-高丝氨酸的研究主要集中在谷氨酸棒状杆菌和大肠杆菌中。Li等(Li N，Xu S，Du G，et al.Efficient production of L-homoserine in Corynebacterium glutamicum ATCC 13032 by redistribution of metabolic flux.Biochemical Engineering Journal，2020，161:107665.)以谷氨酸棒状杆菌为出发菌株构建L-高丝氨酸生产菌，最终产量为8.8g/L。Mu等(Mu Q，Zhang S，Mao X，et al.Highly efficient production of L-homoserine in Escherichia coli by engineering a redox balance route.Metab Eng.2021，67:321-329.)通过在大肠杆菌中设计一个氧化还原平衡路线来实现L-高丝氨酸的高效生产，最终产量达到84.1g/L。上述菌株均是利用质粒为载体来表达关键基因，发酵过程中质粒多拷贝对菌体生长会造成一定的负担，且在生产过程中质粒表达载体容易丢失，导致发酵不稳定，或者需要添加一定的选择压力来维持质粒，造成生产成本过高，最终导致这些L-高 丝氨酸菌株难以投入到工业化生产中。Zhang等(Zhang Y，Wei M，Zhao G，et al.High-level production of L-homoserine using a non-induced，non-auxotrophic Escherichia coli chassis through metabolic engineering.Bioresource Technology，2021，327(4):124814.)在大肠杆菌中构建了一株非诱导、非营养缺陷型、无质粒的L-高丝氨酸生产菌株，最终产量达到60.1g/L。虽然该菌株具有非诱导和非营养缺陷型的优点，但若要满足工业化生产的要求，其生产能力还有待进一步提高。
发明内容
为解决上述技术问题，本发明通过代谢工程的方法，在基因组层面对大肠杆菌中L-高丝氨酸合成相关基因进行改造，通过弱化降解途径L-苏氨酸合成途径的通量、强化L-高丝氨酸合成途径的代谢流、增强前体物草酰乙酸和L-天冬氨酸的供应、促进L-高丝氨酸的胞外转运，以及协同利用辅因子，得到一株遗传背景清晰、不携带质粒、无需诱导且能稳定高效生产L-高丝氨酸的基因工程菌，具有大规模生产的应用潜力。
本发明的第一个目的是提供一种生产L-高丝氨酸的重组大肠杆菌，该重组大肠杆菌以大肠杆菌为出发菌株，敲除乳糖操纵子阻遏蛋白编码基因lacI，弱化高丝氨酸激酶编码基因thrB的表达，过表达天冬氨酸激酶I/高丝氨酸脱氢酶I编码基因thrA、磷酸烯醇式丙酮酸羧化酶编码基因ppc、天冬氨酸转氨酶编码基因aspC、天冬氨酸氨裂合酶编码基因aspA、苏氨酸和高丝氨酸外排系统编码基因rhtA和吡啶核苷酸转氢酶编码基因pntAB，引入异源天冬氨酸激酶编码基因lysC、天冬氨酸半醛脱氢酶编码基因asd和天冬氨酸脱氢酶编码基因aspdh；
其中，
通过将thrB的原启动子替换为启动子P fliC弱化高丝氨酸激酶编码基因thrB的表达；
所述天冬氨酸激酶I/高丝氨酸脱氢酶I编码基因thrA、磷酸烯醇式丙酮酸羧化酶编码基因ppc、天冬氨酸转氨酶编码基因aspC、天冬氨酸氨裂合酶 编码基因aspA、吡啶核苷酸转氢酶编码基因pntAB、天冬氨酸激酶编码基因lysC、天冬氨酸半醛脱氢酶编码基因asd和天冬氨酸脱氢酶编码基因aspdh通过启动子P trc调控表达；
所述苏氨酸和高丝氨酸外排系统编码基因rhtA通过启动子P lpp调控表达。
进一步地，将启动子P trc控制的thrA分别整合至ycgH、ydeU、yjhE和tfaD基因位点。
进一步地，将启动子P trc控制的ppc整合至yeeL基因位点。
进一步地，将启动子P trc控制的aspC整合至ylbE基因位点。
进一步地，将启动子P trc控制的aspA整合至ycdN基因位点。
进一步地，将启动子P trc控制的lysC整合至ycjV基因位点。
进一步地，将启动子P trc控制的pntAB分别整合至ilvG和ygaY基因位点。
进一步地，将启动子P lpp控制的rhtA整合至yjiP基因位点。
进一步地，将启动子P trc控制的asd整合至yeeP基因位点。
进一步地，将启动子P trc控制的aspdh整合至yghX基因位点。进一步地，启动子P fliC的核苷酸序列如SEQ ID NO.1所示，启动子P trc的核苷酸序列如SEQ ID NO.2所示，启动子P lpp的核苷酸序列如SEQ ID NO.3所示。
进一步地，高丝氨酸激酶编码基因thrB的核苷酸序列如SEQ ID NO.4所示，天冬氨酸激酶I/高丝氨酸脱氢酶I编码基因thrA的核苷酸序列如SEQ ID NO.5所示，磷酸烯醇式丙酮酸羧化酶编码基因ppc的核苷酸序列如SEQ ID NO.6所示，天冬氨酸转氨酶编码基因aspC的核苷酸序列如SEQ ID NO.7所示，天冬氨酸氨裂合酶编码基因aspA的核苷酸序列如SEQ ID NO.8所示，天冬氨酸激酶编码基因lysC的核苷酸序列如SEQ ID NO.9所示，苏氨酸和高丝氨酸外排系统编码基因rhtA的核苷酸序列如SEQ ID NO.10所示，吡啶核苷酸转氢酶编码基因pntAB的核苷酸序列如SEQ ID NO.11所示，天冬氨酸半醛脱氢酶编码基因asd的核苷酸序列如SEQ ID NO.12所示，天冬氨酸 脱氢酶编码基因aspdh的核苷酸序列如SEQ ID NO.13所示。
其中，所述lysC来源于Corynebacterium glutamicum；asd来源于Tistrella mobilis；aspdh来源于Pseudomonas aeruginosa。
进一步地，出发菌株为Escherichia coli W3110。
本发明的第二个目的是提供上述生产L-高丝氨酸的重组大肠杆菌的构建方法，包括任意顺序的以下步骤：
(1)敲除大肠杆菌的lacI基因，并将thrB的原启动子替换为弱启动子P fliC；
(2)将P trc-thrA在基因组上进行四拷贝，分别整合至ycgH、ydeU、yjhE和tfaD基因位点；
(3)将ppc、aspC和aspA分别由启动子P trc控制，在基因组上进行过表达；
(4)将P trc-lysC和P lpp-rhtA分别整合到基因组上；
(5)将P trc-pntAB在基因组上进行双拷贝，分别整合至ilvG和ygaY基因位点；
(6)在基因组上整合P trc-asd和P trc-aspdh，构建得到上述重组大肠杆菌。
本发明以代谢途径清晰、遗传操作简单的大肠杆菌为出发菌株，从L-高丝氨酸生物合成途径以及相关代谢途径的基因工程改造出发，对整体代谢途径进行分析重构，得到一株遗传背景清晰、不携带质粒且能稳定高效生产L-高丝氨酸的基因工程菌株。
本发明获得的基因工程菌弱化了L-高丝氨酸的降解、提高了L-高丝氨酸的合成通量和前体物供应、促进了L-高丝氨酸的转运，并通过增强NADPH再生以及引入外源利用NADH的脱氢酶来调节胞内的辅因子水平，从而有效地提高了L-高丝氨酸的生产。
本发明的第三个目的是提供上述重组大肠杆菌在生产L-高丝氨酸中的应用。
进一步地，以葡萄糖为底物，通过上述重组大肠杆菌发酵生产L-高丝氨酸。
进一步地，摇瓶发酵时，将活化后的菌株在35-37℃、180-250r/min培养得到种子液，按10-20％的接种量将种子液接种至发酵培养基中，发酵温度为35-37℃，转速为180-250r/min，控制pH在7.0-7.2，当培养基中葡萄糖耗尽时，通过补加60％(m/v)的葡萄糖溶液维持发酵进行。
进一步地，发酵时间优选为24-48h，更优选为36h。
进一步地，种子培养基组成为：20-30g/L葡萄糖，5-10g/L酵母粉，1-5g/L(NH 4) 2SO 4，1-5g/L KH 2PO 4，1-5g/L MgSO 4·7H 2O，1-5g/L柠檬酸钠，5-15mg/L FeSO 4·7H 2O，0.5-2mg/L V H和0.5-2mg/L V B1。
进一步地，发酵培养基组成为：10-20g/L葡萄糖，1-5g/L酵母粉，1-5g/L(NH 4) 2SO 4，1-5g/L KH 2PO 4，1-5g/L MgSO 4·7H 2O，1-5g/L柠檬酸钠，20-30mg/L FeSO 4·7H 2O，0.5-2mg/L V H和0.5-2mg/L V B1。
进一步地，发酵罐发酵时，将重组大肠杆菌活化后在种子培养基中培养得到种子液，再将种子液按10-20％的接种量接种至发酵培养基中进行发酵培养。其中，种子培养基中培养时，培养温度为35-37℃，pH为7.0-7.2，通过调节搅拌转速和通风量控制溶氧为25-30％；发酵培养过程中，发酵温度为35-37℃，pH为7.0-7.2，控制溶氧为25-30％，分批补料控制发酵液中葡萄糖残糖浓度为0.05-5g/L。
进一步地，发酵时间优选为36-60h，更优选为48h。
进一步地，种子培养基组成为：25-35g/L葡萄糖，5-10g/L酵母粉，1-5g/L蛋白胨，1-5g/L KH 2PO 4，0.5-2g/L MgSO 4·7H 2O，1-5g/L柠檬酸或柠檬酸钠，5-10mg/L FeSO 4·7H 2O，5-10mg/L MnSO 4·H 2O，0.2-2mg/L V H和0.5-2mg/L V B1。
进一步地，发酵培养基的组成为：10-20g/L葡萄糖，10-15g/L玉米浆，1-5g/L酵母粉，1-5g/L蛋白胨，1-5g/L KH 2PO 4，0.5-3g/L MgSO 4·7H 2O，1-5g/L柠檬酸或柠檬酸盐，10-30mg/L FeSO 4·7H 2O，10-20mg/L MnSO 4·H 2O，0.2-2mg/L V H和0.3-1mg/L V B1，加或不加甜菜碱，优选地，加1-3g/L甜菜碱。
进一步地，发酵培养时，流加或不流加培养基，所述培养基的组成为：1-10g/L酵母粉，1-10g/L蛋白胨，1-10g/L KH 2PO 4，1-5g/L MgSO 4·7H 2O，1-5g/L柠檬酸，5-15mg/L FeSO 4·7H 2O，5-15mg/L MnSO 4·H 2O，0.1-1mg/L V H和0.1-1mg/L V B1。
进一步地，在发酵14-20h期间流加培养基。
借由上述方案，本发明至少具有以下优点：
本发明提供了一种生产L-高丝氨酸的基因工程菌株，基因工程菌株以大肠杆菌为出发菌株，敲除了lacI基因，弱化了高丝氨酸激酶编码基因thrB，强化了天冬氨酸激酶I/高丝氨酸脱氢酶I编码基因thrA、磷酸烯醇式丙酮酸羧化酶编码基因ppc、天冬氨酸转氨酶编码基因aspC和天冬氨酸氨裂合酶编码基因aspA，引入了来源于C.glutamicum的天冬氨酸激酶编码基因lysC cgl，过表达苏氨酸和高丝氨酸外排系统编码基因rhtA和吡啶核苷酸转氢酶编码基因pntAB，并引入来源于T.mobilis的天冬氨酸半醛脱氢酶编码基因asd tmo和来源于P.aeruginosa的天冬氨酸脱氢酶编码基因aspdh pae，在发酵生产L-高丝氨酸中表现良好，摇瓶发酵36h产物浓度高达42g/L，发酵罐发酵48h产物浓度高达120g/L，糖酸转化率达到60％，具有良好的工业应用前景。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
为了使本发明的内容更容易被清楚的理解，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。
图1为L-高丝氨酸基因工程菌株摇瓶发酵结果；
图2为菌株E.coli HOM10在5L发酵罐中分批补料发酵过程曲线。
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。
实施例1 基因工程菌E.coli W3110 HOM10的构建
1.敲除E.coli W3110的lacI基因：
以E.coli W3110基因组为模板，根据其lacI基因的上下游序列设计上游同源臂引物(lacI-1、lacI-2)和下游同源臂引物(lacI-3、lacI-4)，并通过PCR扩增其上下游同源臂片段。然后，通过重叠PCR的方法将上述片段融合，获得lacI基因的敲除片段。将引物gRNA-lacI-1和gRNA-lacI-2退火后得到的DNA片段与质粒pGRB连接，构建质粒pGRB-lacI。将质粒pGRB-lacI和lacI基因的敲除片段同时电转化至含有pREDCas9的E.coli W3110的电转感受态细胞中，获得阳性转化子，消除质粒后获得E.coli W3110 ΔlacI菌株。
2.将thrB基因的原启动子替换为P fliC
以E.coli W3110基因组为模板，根据其thrB基因启动子区域的上下游序列设计上游同源臂引物(P thrB-1、P thrB-2)和下游同源臂引物(P thrB-3、P thrB-4)，将P fliC序列(SEQ ID NO.1)设计在引物P thrB-2和P thrB-3上，并通过PCR扩增其上下游同源臂片段，再以其为模板，进行融合PCR，获得P thrB基因的替换片段。将引物gRNA-P thrB-1和gRNA-P thrB-2退火后得到的DNA片段与质粒pGRB连接，构建质粒pGRB-P thrB。将质粒pGRB-P thrB和P thrB基因的替换片段同时电转化至含有pREDCas9的E.coli W3110 ΔlacI的电转感受态细胞中，获得阳性转化子，消除质粒后获得菌株HOM1。
3.将P trc-thrA基因分别整合至ycgH、ydeU、yjhE和tfaD位点
(1)将P trc-thrA基因整合至ycgH位点
以E.coli W3110基因组为模板，根据ycgH基因序列设计上游同源臂引物(ycgH-1、ycgH-2)和下游同源臂引物(ycgH-5、ycgH-6)，根据thrA基因序列设计引物ycgH-3和ycgH-4，其中，将P trc启动子序列(SEQ ID NO.2)设计在引物ycgH-2和ycgH-3上，通过PCR扩增获得各个片段，再以其为模板，进行融合PCR，获得P trc-thrA基因的整合片段。将引物gRNA-ycgH-1和gRNA-ycgH-2退火后得到的DNA片段与质粒pGRB连接，构建质粒pGRB-ycgH。将质粒pGRB-ycgH和P trc-thrA基因的整合片段同时电转化至含有pREDCas9的HOM1的电转感受态细胞中，获得阳性转化子，消除质粒后获得菌株HOM2-1。
(2)将P trc-thrA基因整合至ydeU位点
以E.coli W3110基因组为模板，根据ydeU基因序列设计上游同源臂引物(ydeU-1、ydeU-2)和下游同源臂引物(ydeU-5、ydeU-6)，根据thrA基因序列设计引物ydeU-3和ydeU-4，其中，将P trc启动子序列设计在引物ydeU-2和ydeU-3(引物ydeU-3与上述ycgH-3序列一致)上，通过PCR扩增获得各个片段，再以其为模板，进行融合PCR，获得P trc-thrA基因的整合片段。将引物gRNA-ydeU-1和gRNA-ydeU-2退火后得到的DNA片段与质粒pGRB连接，构建质粒pGRB-ydeU。将质粒pGRB-ydeU和P trc-thrA基因的整合片段同时电转化至含有pREDCas9的HOM2-1的电转感受态细胞中，获得阳性转化子，消除质粒后获得菌株HOM2-2。
(3)将P trc-thrA基因整合至yjhE位点
以E.coli W3110基因组为模板，根据yjhE基因序列设计上游同源臂引物(yjhE-1、yjhE-2)和下游同源臂引物(yjhE-5、yjhE-6)，根据thrA基因序列设计引物yjhE-3和yjhE-4，其中，将P trc启动子序列设计在引物yjhE-2和yjhE-3(引物yjhE-3与上述ycgH-3序列一致)上，通过PCR扩增获得各个片段，再以其为模板，进行融合PCR，获得P trc-thrA基因的整合片段。将引物gRNA-yjhE-1和gRNA-yjhE-2退火后得到的DNA片段与质粒pGRB连 接，构建质粒pGRB-yjhE。将质粒pGRB-yjhE和P trc-thrA基因的整合片段同时电转化至含有pREDCas9的HOM2-2的电转感受态细胞中，获得阳性转化子，消除质粒后获得菌株HOM2-3。
(4)将P trc-thrA基因整合至tfaD位点
以E.coli W3110基因组为模板，根据tfaD基因序列设计上游同源臂引物(tfaD-1、tfaD-2)和下游同源臂引物(tfaD-5、tfaD-6)，根据thrA基因序列设计引物tfaD-3和tfaD-4，其中，将P trc启动子序列设计在引物tfaD-2和tfaD-3(引物tfaD-3与上述ycgH-3序列一致)上，通过PCR扩增获得各个片段，再以其为模板，进行融合PCR，获得P trc-thrA基因的整合片段。将引物gRNA-tfaD-1和gRNA-tfaD-2退火后得到的DNA片段与质粒pGRB连接，构建质粒pGRB-tfaD。将质粒pGRB-tfaD和P trc-thrA基因的整合片段同时电转化至含有pREDCas9的HOM2-3的电转感受态细胞中，获得阳性转化子，消除质粒后获得菌株HOM2-4。
4.将ppc基因的原启动子替换为P trc，并将P trc-ppc基因整合至yeeL位点
(1)将ppc基因的原启动子替换为P trc
以E.coli W3110基因组为模板，根据其ppc基因启动子区域的上下游序列设计上游同源臂引物(P ppc-1、P ppc-2)和下游同源臂引物(P ppc-3、P ppc-4)，将P trc序列设计在引物P ppc-2和P ppc-3上，并通过PCR扩增其上下游同源臂片段，再以其为模板，进行融合PCR，获得P ppc基因的替换片段。将引物gRNA-P ppc-1和gRNA-P ppc-2退火后得到的DNA片段与质粒pGRB连接，构建质粒pGRB-P ppc。将质粒pGRB-P ppc和P ppc基因的替换片段同时电转化至含有pREDCas9的HOM2-4的电转感受态细胞中，获得阳性转化子，消除质粒后获得菌株HOM3-1。
(2)将P trc-ppc基因整合至yeeL位点
以E.coli W3110基因组为模板，根据yeeL基因序列设计上游同源臂引物(yeeL-1、yeeL-2)和下游同源臂引物(yeeL-5、yeeL-6)，根据ppc基因 序列设计引物yeeL-3和yeeL-4，其中，将P trc启动子序列设计在引物yeeL-2和yeeL-3上，通过PCR扩增获得各个片段，再以其为模板，进行融合PCR，获得P trc-ppc基因的整合片段。将引物gRNA-yeeL-1和gRNA-yeeL-2退火后得到的DNA片段与质粒pGRB连接，构建质粒pGRB-yeeL。将质粒pGRB-yeeL和P trc-ppc基因的整合片段同时电转化至含有pREDCas9的HOM3-1的电转感受态细胞中，获得阳性转化子，消除质粒后获得菌株HOM3-2。
5.将P trc-aspC基因整合至ylbE位点并将P trc-aspA基因整合至ycdN位点
(1)将P trc-aspC基因整合至ylbE位点
以E.coli W3110基因组为模板，根据ylbE基因序列设计上游同源臂引物(ylbE-1、ylbE-2)和下游同源臂引物(ylbE-5、ylbE-6)，根据aspC基因序列设计引物ylbE-3和ylbE-4，其中，将P trc启动子序列设计在引物ylbE-2和ylbE-3上，通过PCR扩增获得各个片段，再以其为模板，进行融合PCR，获得P trc-aspC基因的整合片段。将引物gRNA-ylbE-1和gRNA-ylbE-2退火后得到的DNA片段与质粒pGRB连接，构建质粒pGRB-ylbE。将质粒pGRB-ylbE和P trc-aspC基因的整合片段同时电转化至含有pREDCas9的HOM3-2的电转感受态细胞中，获得阳性转化子，消除质粒后获得菌株HOM4。
(2)将P trc-aspA基因整合至ycdN位点
以E.coli W3110基因组为模板，根据ycdN基因序列设计上游同源臂引物(ycdN-1、ycdN-2)和下游同源臂引物(ycdN-5、ycdN-6)，根据aspA基因序列设计引物ycdN-3和ycdN-4，其中，将P trc启动子序列设计在引物ycdN-2和ycdN-3上，通过PCR扩增获得各个片段，再以其为模板，进行融合PCR，获得P trc-aspA基因的整合片段。将引物gRNA-ycdN-1和gRNA-ycdN-2退火后得到的DNA片段与质粒pGRB连接，构建质粒pGRB-ycdN。将质粒pGRB-ycdN和P trc-aspA基因的整合片段同时电转化至含有pREDCas9的 HOM4的电转感受态细胞中，获得阳性转化子，消除质粒后获得菌株HOM5。
6.将来源于C.glutamicum的lysC cgl基因整合至ycjV位点
以E.coli W3110基因组为模板，根据ycjV基因序列设计上游同源臂引物(ycjV-1、ycjV-2)和下游同源臂引物(ycjV-5、ycjV-6)，然后，以C.glutamicum13032基因组为模板，根据lysC cgl基因序列设计引物ycjV-3和ycjV-4，其中，将P trc启动子序列设计在引物ycjV-2和ycjV-3上，通过PCR扩增获得各个片段，再以其为模板，进行融合PCR，获得P trc-lysC cgl基因的整合片段。将引物gRNA-ycjV-1和gRNA-ycjV-2退火后得到的DNA片段与质粒pGRB连接，构建质粒pGRB-ycjV。将质粒pGRB-ycjV和P trc-lysC cgl基因的整合片段同时电转化至含有pREDCas9的HOM5的电转感受态细胞中，获得阳性转化子，消除质粒后获得菌株HOM6。
7.将P lpp-rhtA基因整合至yjiP位点
以E.coli W3110基因组为模板，根据yjiP基因序列设计上游同源臂引物(yjiP-1、yjiP-2)和下游同源臂引物(yjiP-7、yjiP-8)，根据P lpp基因序列(SEQ ID NO.3)设计引物yjiP-3和yjiP-4，根据rhtA基因序列设计引物yjiP-5和yjiP-6，通过PCR扩增获得各个片段，再以其为模板，进行融合PCR，获得P lpp-rhtA基因的整合片段。将引物gRNA-yjiP-1和gRNA-yjiP-2退火后得到的DNA片段与质粒pGRB连接，构建质粒pGRB-yjiP。将质粒pGRB-yjiP和P lpp-rhtA基因的整合片段同时电转化至含有pREDCas9的HOM6的电转感受态细胞中，获得阳性转化子，消除质粒后获得菌株HOM7。
8.将P trc-pntAB基因分别整合至ilvG和ygaY位点
(1)将P trc-pntAB基因整合至ilvG位点
以E.coli W3110基因组为模板，根据ilvG基因序列设计上游同源臂引物(ilvG-1、ilvG-2)和下游同源臂引物(ilvG-5、ilvG-6)，根据pntAB基因序列设计引物ilvG-3和ilvG-4，其中，将P trc启动子序列设计在引物ilvG-2和ilvG-3上，通过PCR扩增获得各个片段，再以其为模板，进行融合PCR，获得P trc-pntAB基因的整合片段。将引物gRNA-ilvG-1和gRNA-ilvG-2退火 后得到的DNA片段与质粒pGRB连接，构建质粒pGRB-ilvG。将质粒pGRB-ilvG和P trc-pntAB基因的整合片段同时电转化至含有pREDCas9的HOM7的电转感受态细胞中，获得阳性转化子，消除质粒后获得菌株HOM8-1。
(2)将P trc-pntAB基因整合至ygaY位点
以E.coli W3110基因组为模板，根据ygaY基因序列设计上游同源臂引物(ygaY-1、ygaY-2)和下游同源臂引物(ygaY-5、ygaY-6)，其中，将P trc启动子序列设计在引物ygaY-2和ilvG-3上，通过PCR扩增获得各个片段，再以其为模板，进行融合PCR，获得P trc-pntAB基因的整合片段。将引物gRNA-ygaY-1和gRNA-ygaY-2退火后得到的DNA片段与质粒pGRB连接，构建质粒pGRB-ygaY。将质粒pGRB-ygaY和P trc-pntAB基因的整合片段同时电转化至含有pREDCas9的HOM8-1的电转感受态细胞中，获得阳性转化子，消除质粒后获得菌株HOM8-2。
9.将来源于T.mobilis的天冬氨酸半醛脱氢酶编码基因整合至yeeP位点
以E.coli W3110基因组为模板，根据yeeP基因序列设计上游同源臂引物(yeeP-1、yeeP-2)和下游同源臂引物(yeeP-5、yeeP-6)，来源于T.mobilis的天冬氨酸半醛脱氢酶编码基因asd tmo经密码子优化后由公司合成，根据其序列设计引物yeeP-3和yeeP-4，其中，将P trc启动子序列设计在引物yeeP-2和yeeP-3上，通过PCR扩增获得各个片段，再以其为模板，进行融合PCR，获得P trc-asd tmo基因的整合片段。将引物gRNA-yeeP-1和gRNA-yeeP-2退火后得到的DNA片段与质粒pGRB连接，构建质粒pGRB-yeeP。将质粒pGRB-yeeP和P trc-asd tmo基因的整合片段同时电转化至含有pREDCas9的HOM8-2的电转感受态细胞中，获得阳性转化子，消除质粒后获得菌株HOM9。
10.来源于P.aeruginos的aspdh pae基因整合至yghX位点
以E.coli W3110基因组为模板，根据yghX基因序列设计上游同源臂引物(yghX-1、yghX-2)和下游同源臂引物(yghX-5、yghX-6)，来源于P.aeruginos 的天冬氨酸脱氢酶编码基因aspdh pae经密码子优化后由公司合成，根据其序列设计引物yghX-3和yghX-4，其中，将P trc启动子序列设计在引物yghX-2和yghX-3上，通过PCR扩增获得各个片段，再以其为模板，进行融合PCR，获得P trc-aspdh pae基因的整合片段。将引物gRNA-yghX-1和gRNA-yghX-2退火后得到的DNA片段与质粒pGRB连接，构建质粒pGRB-yghX。将质粒pGRB-yghX和P trc-aspdh pae基因的整合片段同时电转化至含有pREDCas9的HOM9的电转感受态细胞中，获得阳性转化子，消除质粒后获得菌株HOM10。
上述实验过程所用引物见下表：
Figure PCTCN2022114482-appb-000001
Figure PCTCN2022114482-appb-000002
Figure PCTCN2022114482-appb-000003
Figure PCTCN2022114482-appb-000004
Figure PCTCN2022114482-appb-000005
实施例2 利用基因工程菌HOM10摇瓶发酵生产L-高丝氨酸
(1)种子培养：取-80℃保藏菌种划线接种于斜面培养基中，37℃培养12h，并传代一次，然后，用接种环刮取一环斜面种子接种至装有30mL种子培养基的500mL圆底三角瓶中，九层纱布封口，37℃、220rmp培养8-10h。
种子培养基组成为：30g/L葡萄糖，10g/L酵母粉，4g/L(NH 4) 2SO 4，3g/L KH 2PO 4，2g/L MgSO 4·7H 2O，2g/L柠檬酸钠，5mg/L FeSO 4·7H 2O，0.5mg/L V H和0.5mg/L V B1，其余为水，pH 7.0-7.2。
(2)发酵培养：按15％的接种量将种子液接种至装有30mL发酵培养基的500mL挡板三角瓶中，九层纱布封口，37℃、240r/min培养，发酵过程中以苯酚红作指示剂，通过补加25％的氨水控制pH在7.0-7.2，当培养基中葡萄糖耗尽时，通过补加60％(m/v)的葡萄糖溶液维持发酵进行；发酵周期36h。
发酵培养基组成为：10g/L葡萄糖，5g/L酵母粉，5g/L(NH 4) 2SO 4，3g/L KH 2PO 4，2g/L MgSO 4·7H 2O，2g/L柠檬酸钠，30mg/L FeSO 4·7H 2O，0.5mg/L V H，0.5mg/L V B1和8mg/L苯酚红，其余为水，pH 7.0-7.2。
经过36h的摇瓶发酵，各L-高丝氨酸基因工程菌株摇瓶发酵结果见图1。其中，HOM10菌株发酵液中L-高丝氨酸的产量为42g/L。未检测到其它氨基酸和有机酸副产物。
实施例3 利用基因工程菌HOM10在5L发酵罐中发酵生产L-高丝氨酸
(1)种子培养：将适量无菌水倒入斜面中，用接种环将菌体悬浮，然后，将菌悬液接种于种子培养基中进行培养。培养温度为37℃，初始通气量为2L/min，初始搅拌转速为200r/min，通过自动流加25％的氨水控制培养基pH为7.0-7.2，通过搅拌和通风控制溶氧在25-30％，当OD 600达到15-20时，准备接入发酵培养基。
种子培养基组成为：30g/L葡萄糖，5g/L酵母粉，3g/L蛋白胨，1.5g/L KH 2PO 4，0.5g/L MgSO 4·7H 2O，1g/L柠檬酸钠，10mg/L FeSO 4·7H 2O，10mg/L MnSO 4·H 2O，1mg/L V H和0.5mg/L V B1，其余为水，pH 7.0-7.2。
(2)发酵培养：按15％的接种量将种子液接种至发酵培养基中，培养温度为37℃，通过自动流加25％的氨水控制培养基pH为7.0-7.2，通过搅拌和通风控制溶氧在25-30％，当培养基中葡萄糖耗尽时，自动流加80％的葡萄糖溶液，控制发酵液中葡萄糖残糖浓度为0.05-5g/L。
发酵培养基组成为：10g/L葡萄糖，10g/L玉米浆，4g/L酵母粉，3g/L蛋白胨，4g/L KH 2PO 4，1g/L MgSO 4·7H 2O，2g/L柠檬酸钠，10mg/L FeSO 4·7H 2O，10mg/L MnSO 4·H 2O，0.2mg/L V H和0.3mg/L V B1，其余为水，pH 7.0-7.2。
在5L发酵罐中发酵48h，L-高丝氨酸产量达到85g/L。未检测到其它氨基酸和有机酸副产物。
实施例4 基因工程菌HOM10在5L发酵罐中发酵生产L-高丝氨酸的条件优化
(1)种子培养：将适量无菌水倒入斜面中，用接种环将菌体悬浮，然后，将菌悬液接种于种子培养基中进行培养。培养温度为37℃，初始通气量为2L/min，初始搅拌转速为200r/min，通过自动流加25％的氨水控制培养基pH为7.0-7.2，通过搅拌和通风控制溶氧在25-30％，当OD 600达到15-20时，准备接入发酵培养基。
种子培养基组成为：35g/L葡萄糖，5g/L酵母粉，3g/L蛋白胨，1.5g/L KH 2PO 4，0.5g/L MgSO 4·7H 2O，1g/L柠檬酸，10mg/L FeSO 4·7H 2O， 10mg/L MnSO 4·H 2O，1mg/L V H和0.5mg/L V B1，其余为水，pH 7.0-7.2。
(2)发酵培养：按20％的接种量将种子液接种至发酵培养基中，培养温度为37℃，通过自动流加25％的氨水控制培养基pH为7.0-7.2，通过搅拌和通风控制溶氧在25-30％，当培养基中葡萄糖耗尽时，自动流加80％的葡萄糖溶液控制发酵液中葡萄糖残糖浓度为0.05-5g/L。
发酵培养基组成为：10g/L葡萄糖，12g/L玉米浆，4g/L酵母粉，3g/L蛋白胨，4g/L KH 2PO 4，1g/L MgSO 4·7H 2O，2g/L柠檬酸，10mg/L FeSO 4·7H 2O，10mg/L MnSO 4·H 2O，0.2mg/L V H和0.3mg/L V B1，其余为水，pH 7.0-7.2，并随糖流加1g/L甜菜碱。
在5L发酵罐中发酵48h，L-高丝氨酸产量最高达到96g/L。未检测到其它氨基酸和有机酸副产物。
实施例5 基因工程菌HOM10在5L发酵罐中发酵生产L-高丝氨酸的条件优化
(1)种子培养：将适量无菌水倒入斜面中，用接种环将菌体悬浮，然后，将菌悬液接种于种子培养基中进行培养。培养温度为37℃，初始通气量为2L/min，初始搅拌转速为200r/min，通过自动流加25％的氨水控制培养基pH为7.0-7.2，通过搅拌和通风控制溶氧在25-30％，当OD 600达到15-20时，准备接入发酵培养基。
种子培养基组成为：35g/L葡萄糖，5g/L酵母粉，3g/L蛋白胨，1.5g/L KH 2PO 4，0.5g/L MgSO 4·7H 2O，1g/L柠檬酸，10mg/L FeSO 4·7H 2O，10mg/L MnSO 4·H 2O，1mg/L V H和0.5mg/L V B1，其余为水，pH 7.0-7.2。
(2)发酵培养：按20％的接种量将种子液接种至发酵培养基中，培养温度为37℃，通过自动流加25％的氨水控制培养基pH为7.0-7.2，通过搅拌和通风控制溶氧在25-30％，当培养基中葡萄糖耗尽时，自动流加80％的葡萄糖溶液控制发酵液中葡萄糖残糖浓度为0.05-5g/L，在发酵14-20h之间自动流加培养基。
发酵培养基组成为：10g/L葡萄糖，12g/L玉米浆，4g/L酵母粉，3g/L蛋白胨，4g/L KH 2PO 4，1g/L MgSO 4·7H 2O，2g/L柠檬酸，10mg/L FeSO 4·7H 2O，10mg/L MnSO 4·H 2O，0.2mg/L V H和0.3mg/L V B1，其余为水，pH 7.0-7.2，并随糖流加1g/L甜菜碱。
流加培养基组成为：4g/L酵母粉，3g/L蛋白胨，4g/L KH 2PO 4，1g/L MgSO 4·7H 2O，2g/L柠檬酸，10mg/L FeSO 4·7H 2O，10mg/L MnSO 4·H 2O，0.5mg/L V H和0.5mg/L V B1。
在5L发酵罐中发酵48h，发酵过程曲线见图2，其中，L-高丝氨酸产量最高达到120g/L，糖酸转化率最高达到60％。未检测到其它氨基酸和有机酸副产物。
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 (12)

  1. 一种生产L-高丝氨酸的重组大肠杆菌，其特征在于：所述生产L-高丝氨酸的重组大肠杆菌以大肠杆菌为出发菌株，敲除乳糖操纵子阻遏蛋白编码基因lacI，弱化高丝氨酸激酶编码基因thrB的表达，过表达天冬氨酸激酶I/高丝氨酸脱氢酶I编码基因thrA、磷酸烯醇式丙酮酸羧化酶编码基因ppc、天冬氨酸转氨酶编码基因aspC、天冬氨酸氨裂合酶编码基因aspA、苏氨酸和高丝氨酸外排系统编码基因rhtA和吡啶核苷酸转氢酶编码基因pntAB，引入异源天冬氨酸激酶编码基因lysC、天冬氨酸半醛脱氢酶编码基因asd和天冬氨酸脱氢酶编码基因aspdh；
    其中，
    通过将thrB的原启动子替换为启动子P fliC弱化高丝氨酸激酶编码基因thrB的表达；
    所述天冬氨酸激酶I/高丝氨酸脱氢酶I编码基因thrA、磷酸烯醇式丙酮酸羧化酶编码基因ppc、天冬氨酸转氨酶编码基因aspC、天冬氨酸氨裂合酶编码基因aspA、天冬氨酸激酶编码基因lysC、吡啶核苷酸转氢酶编码基因pntAB、天冬氨酸半醛脱氢酶编码基因asd和天冬氨酸脱氢酶编码基因aspdh通过启动子P trc调控表达；
    所述苏氨酸和高丝氨酸外排系统编码基因rhtA通过启动子P lpp调控表达。
  2. 根据权利要求1所述的重组大肠杆菌，其特征在于：将thrA分别整合至ycgH、ydeU、yjhE和tfaD基因位点；将ppc整合至yeeL基因位点；将aspC整合至ylbE基因位点；将aspA整合至ycdN基因位点；将lysC整合至ycjV基因位点；将pntAB分别整合至ilvG和ygaY基因位点；将rhtA整合至yjiP基因位点；将asd整合至yeeP基因位点；将aspdh整合至yghX基因位点。
  3. 根据权利要求1所述的重组大肠杆菌，其特征在于：所述启动子P fliC的核苷酸序列如SEQ ID NO.1所示，启动子P trc的核苷酸序列如SEQ ID NO.2 所示，启动子P lpp的核苷酸序列如SEQ ID NO.3所示。
  4. 根据权利要求1所述的重组大肠杆菌，其特征在于：高丝氨酸激酶编码基因thrB的核苷酸序列如SEQ ID NO.4所示，天冬氨酸激酶I/高丝氨酸脱氢酶I编码基因thrA的核苷酸序列如SEQ ID NO.5所示，磷酸烯醇式丙酮酸羧化酶编码基因ppc的核苷酸序列如SEQ ID NO.6所示，天冬氨酸转氨酶编码基因aspC的核苷酸序列如SEQ ID NO.7所示，天冬氨酸氨裂合酶编码基因aspA的核苷酸序列如SEQ ID NO.8所示，天冬氨酸激酶编码基因lysC的核苷酸序列如SEQ ID NO.9所示，苏氨酸和高丝氨酸外排系统编码基因rhtA的核苷酸序列如SEQ ID NO.10所示，吡啶核苷酸转氢酶编码基因pntAB的核苷酸序列如SEQ ID NO.11所示，天冬氨酸半醛脱氢酶编码基因asd的核苷酸序列如SEQ ID NO.12所示，天冬氨酸脱氢酶编码基因aspdh的核苷酸序列如SEQ ID NO.13所示。
  5. 根据权利要求1所述的重组大肠杆菌，其特征在于：所述出发菌株为Escherichia coli W3110。
  6. 权利要求1-5任一项所述的重组大肠杆菌在制备L-高丝氨酸中的应用。
  7. 根据权利要求6所述的应用，其特征在于：所述的应用是将权利要求1-5任一项所述的重组大肠杆菌活化后在种子培养基中培养得到种子液，再将种子液接种至发酵培养基中进行发酵培养制备L-高丝氨酸。
  8. 根据权利要求7所述的应用，其特征在于：在种子培养基中培养时，培养温度为35-37℃，pH为7.0-7.2，控制溶氧为25-30％。
  9. 根据权利要求7所述的应用，其特征在于：发酵培养过程中，培养温度为35-37℃，pH为7.0-7.2，葡萄糖浓度控制在0.05-5g/L。
  10. 根据权利要求7所述的应用，其特征在于，发酵培养基的组成为：10-20g/L葡萄糖，10-15g/L玉米浆，1-5g/L酵母粉，1-5g/L蛋白胨，1-5g/L KH 2PO 4，0.5-3g/L MgSO 4·7H 2O，1-5g/L柠檬酸或柠檬酸盐，10-30mg/L FeSO 4·7H 2O，10-20mg/L MnSO 4·H 2O，0.2-2mg/L V H和0.3-1mg/L V B1，加 或不加甜菜碱。
  11. 根据权利要求7所述的应用，其特征在于，发酵培养过程中流加培养基，所述流加的培养基组成为：1-10g/L酵母粉，1-10g/L蛋白胨，1-10g/L KH 2PO 4，1-5g/L MgSO 4·7H 2O，1-5g/L柠檬酸，5-15mg/L FeSO 4·7H 2O，5-15mg/L MnSO 4·H 2O，0.1-1mg/L V H和0.1-1mg/L V B1。
  12. 根据权利要求11所述的应用，其特征在于：在发酵14-20h之间流加培养基。
PCT/CN2022/114482 2022-06-15 2022-08-24 一种生产l-高丝氨酸的重组大肠杆菌及其应用 WO2023240794A1 (zh)

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Families Citing this family (1)

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Publication number Priority date Publication date Assignee Title
CN108504613B (zh) * 2017-02-27 2021-03-30 四川利尔生物科技有限公司 一种l-高丝氨酸生产菌株及其构建方法和应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018152918A1 (zh) * 2017-02-27 2018-08-30 苏州引航生物科技有限公司 一种l-高丝氨酸生产菌株及其构建方法和应用
CN109055290A (zh) * 2018-07-27 2018-12-21 浙江工业大学 一种高产l-高丝氨酸的重组大肠杆菌及其应用
CN109666617A (zh) * 2017-10-13 2019-04-23 四川利尔生物科技有限公司 一种l-高丝氨酸的生产菌株及其构建方法和应用
CN112592875A (zh) * 2020-12-08 2021-04-02 鲁东大学 一株高丝氨酸生产菌及其构建方法和应用
CN112779204A (zh) * 2021-01-26 2021-05-11 天津科技大学 一种生产l-高丝氨酸的基因工程菌及其应用
CN113122487A (zh) * 2020-01-10 2021-07-16 中国科学院微生物研究所 一种高产l-高丝氨酸的重组菌及其制备方法与应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113956992B (zh) * 2021-03-18 2023-09-01 中国科学院微生物研究所 一株耐受l-高丝氨酸的大肠杆菌及其应用

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018152918A1 (zh) * 2017-02-27 2018-08-30 苏州引航生物科技有限公司 一种l-高丝氨酸生产菌株及其构建方法和应用
CN108504613A (zh) * 2017-02-27 2018-09-07 苏州引航生物科技有限公司 一种l-高丝氨酸生产菌株及其构建方法和应用
CN113151127A (zh) * 2017-02-27 2021-07-23 四川利尔生物科技有限公司 一种l-高丝氨酸生产菌株及其构建方法和应用
CN109666617A (zh) * 2017-10-13 2019-04-23 四川利尔生物科技有限公司 一种l-高丝氨酸的生产菌株及其构建方法和应用
CN109055290A (zh) * 2018-07-27 2018-12-21 浙江工业大学 一种高产l-高丝氨酸的重组大肠杆菌及其应用
CN113122487A (zh) * 2020-01-10 2021-07-16 中国科学院微生物研究所 一种高产l-高丝氨酸的重组菌及其制备方法与应用
CN112592875A (zh) * 2020-12-08 2021-04-02 鲁东大学 一株高丝氨酸生产菌及其构建方法和应用
CN112779204A (zh) * 2021-01-26 2021-05-11 天津科技大学 一种生产l-高丝氨酸的基因工程菌及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HUANG JIAN-FENG, ZHANG BO, SHEN ZHEN-YANG, LIU ZHI-QIANG, ZHENG YU-GUO: "Metabolic engineering of E. coli for the production of O-succinyl-l-homoserine with high yield", 3 BIOTECH, SPRINGEROPEN, DE, vol. 8, no. 7, 1 July 2018 (2018-07-01), DE , XP093118013, ISSN: 2190-572X, DOI: 10.1007/s13205-018-1332-x *
LIU PENG, ZHANG BO, YAO ZHEN-HAO, LIU ZHI-QIANG, ZHENG YU-GUO: "Multiplex Design of the Metabolic Network for Production of l -Homoserine in Escherichia coli", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 86, no. 20, 1 October 2020 (2020-10-01), US , XP093118014, ISSN: 0099-2240, DOI: 10.1128/AEM.01477-20 *

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