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CN-121975711-A - L-alanine production strain and construction method and application thereof

CN121975711ACN 121975711 ACN121975711 ACN 121975711ACN-121975711-A

Abstract

The invention provides an L-alanine production strain, a construction method and application thereof, wherein the strain takes vibrio natriegens as an original strain, and the dns gene, the vnp1 gene and the vnp2 gene in the genome are sequentially knocked out through genetic modification, so that a series of key competitive pathway genes pflB, lldh, dldh, pta, ackA and adhE are knocked out for blocking the organic acid fermentation pathway of the vibrio natriegens, the production of L-alanine is increased, the accumulation of organic acid is reduced, then pXtuf-alaD plasmid is transferred to increase the gene copy number, the ultra-fast growth characteristic of the vibrio natriegens is cooperated with the original strain, the constructed strain has clear genetic background, high-efficiency transformation capacity is shown in the subsequent anaerobic production stage, the L-alanine can be efficiently produced, and the strain has good industrial application prospect.

Inventors

  • LI YANJUN
  • Yang Zunshuo
  • JIANG YAN
  • ZHAO GUIHONG

Assignees

  • 天津科技大学

Dates

Publication Date
20260505
Application Date
20260409

Claims (10)

  1. 1. An L-alanine production strain is characterized in that wild type vibrio natrii is used as an original strain for genetic modification, a dns gene, a pnp 1 gene and a pnp 2 gene in the genome are sequentially knocked out, a pyruvate formate lyase gene pflB, an L-lactate dehydrogenase gene lldh, a D-lactate dehydrogenase gene dldh, phosphotransacetylase genes pta1 and pta2, acetate kinase genes ackA1 and ackA2 and alcohol dehydrogenase genes adhE1 and adhE2 are knocked out, a strain VNA9 is obtained, a Hind III and BamH I are used for linearization pXMJ plasmid, a P tuf -alaD gene fragment is connected with the strain to obtain pXtuf-alaD, and the pXtuf-alaD plasmid is electrotransferred into a VNA9 competent.
  2. 2. The L-alanine production strain of claim 1, wherein the P tuf -alaD gene fragment is obtained by amplifying the alaD gene fragment using the Vibrio natrii genome as a template, ligating the gene fragment to P tuf , and then ligating the P tuf -alaD gene fragment to the pXMJ plasmid by homologous recombination using ClonExpress II One Step cloning kit, thereby obtaining the pXtuf-alaD plasmid.
  3. 3. The L-alanine producing strain of claim 1, wherein the wild-type Vibrio natrii is wild-type Vibrio natrii Vibrio NATRIEGENS ATCC 14048.
  4. 4. The L-alanine producer strain according to claim 1, wherein the nucleotide sequence of pyruvate formate lyase gene pflB is shown in SEQ ID NO.1, the nucleotide sequence of L-lactate dehydrogenase gene lldh is shown in SEQ ID NO.2, the nucleotide sequence of D-lactate dehydrogenase gene dldh is shown in SEQ ID NO.3, the nucleotide sequence of phosphotransacetylase gene pta1 is shown in SEQ ID NO.4, the nucleotide sequence of phosphotransacetylase gene pta2 is shown in SEQ ID NO.5, the nucleotide sequence of acetate kinase gene ackA1 is shown in SEQ ID NO.6, the nucleotide sequence of acetate kinase gene ackA2 is shown in SEQ ID NO.7, the nucleotide sequence of alcohol dehydrogenase gene adhE1 is shown in SEQ ID NO.8, the nucleotide sequence of alcohol dehydrogenase gene adhE2 is shown in SEQ ID NO.9, and the nucleotide sequence of alaD gene is shown in SEQ ID NO. 10.
  5. 5. The method for constructing L-alanine production strain as claimed in any one of claims 1 to 4, wherein the gene modification is performed by using a naturally transformed gene editing technique, and the dns gene, the vnp1 gene and the vnp2 gene in the wild-type Vibrio natrii genome are sequentially knocked out, and the subsequent gene modification is performed based on the genes, wherein the following steps are performed: (1) Knocking out a pyruvate formate lyase gene pflB; (2) Knocking out the L-lactate dehydrogenase gene lldh; (3) Knocking out the D-lactate dehydrogenase gene dldh; (4) Knocking out phosphotransacetylase genes pta1 and pta2; (5) Knocking out acetate kinase genes ackA1 and ackA2; (6) Knocking out ethanol dehydrogenase genes adhE1 and adhE2; (7) Linearizing pXMJ plasmid with HindIII and BamH I, ligating Ptuf with the alaD gene fragment amplified in Vibrio natrii Vibrio NATRIEGENS ATCC 14048 genome by homologous recombination, ligating end-to-end P tuf -alaD fragment to pXMJ linearizing vector to obtain pXtuf-alaD plasmid, electrotransferring pXtuf-alaD plasmid into the strain competence obtained in step (6) to obtain final strain.
  6. 6. Use of the L-alanine producer strain according to any one of claims 1 to 4 for producing L-alanine.
  7. 7. The method according to claim 6, wherein L-alanine is produced by fermentation.
  8. 8. The method according to claim 6 or 7, wherein the fermentation tank is used for culturing, and the method comprises the following specific steps: (1) Strain activation, namely inoculating the strain into an LB shake tube added with chloramphenicol for culture; (2) Seed culture, namely adding bacterial liquid, glucose solution and chloramphenicol which are cultivated by LB shaking tube into a seed culture medium, wherein the pH is maintained at 7.0+/-0.1 in the cultivation process, the temperature is maintained at 32+/-0.2 ℃, fermenting the seed culture by using the seed culture medium, and transferring the fermentation culture medium to ferment and cultivate when the OD 600 is reached to 15; (3) The fermentation culture comprises inoculating seed solution into fermentation medium, fermenting at pH 7.0+ -0.1, maintaining temperature at 32+ -0.2deg.C, and maintaining dissolved oxygen at 25% -30%, and maintaining carbon source required by thallus during fermentation by feeding nutrient solution during the culture process, and adjusting pH to 7.0+ -0.1 by adding ammonia water.
  9. 9. The method according to claim 8, wherein the seed culture medium and the fermentation culture medium are 20g/L glucose, 7g/L NaCl, 5g/L yeast powder and 10g/L,Na 2 HPO 4 ×2H 2 O 5g/L,KH 2 PO 4 3g/L,MgSO 4 ×7H 2 O 0.74g/L,(NH 4 ) 2 SO 4 5g/L,NH 4 Cl 1g/L,% peptone trace elements.
  10. 10. The method according to claim 8, wherein the fed-batch nutrient solution comprises glucose 600g/L,NaCl 15g/L,(NH 4 ) 2 SO 4 20g/L,KH 2 PO 4 20g/L-1,K 2 HPO 4 20g/L,MgSO 4 5g/L and 1% trace elements.

Description

L-alanine production strain and construction method and application thereof Technical Field The invention relates to the technical field of genetic engineering, in particular to an L-alanine production strain, a construction method and application thereof. Background L-alanine plays a key role in a variety of fields as an important organic compound. Currently, the production of L-alanine is mainly dependent on chemical synthesis or microbial fermentation. The chemical synthesis method still has a plurality of limitations that on one hand, depending on non-renewable resources, the production process often needs severe conditions such as high temperature, high pressure and the like, reactants possibly having toxicity are used, harmful byproducts are possibly generated, and environmental burden is brought, on the other hand, the route often needs expensive catalysts and specific raw materials, the subsequent purification steps are complex, and the overall cost is high. With the development of biotechnology, the use of whole cell catalysts to effect bioconversion of renewable substrates to L-alanine has become an important research direction. At present, most chassis strains commonly used for producing amino acids are escherichia coli or corynebacterium glutamicum, and the strains are mature in genetic operation, but generally have the problems of slow cell growth and long fermentation period, so that the equipment utilization rate in unit time is low, and the production cost is increased. Vibrio natrii (Vibrio natriegens) is required as one of the free living microorganisms with the highest known growth rate, and the generation time is less than 10min in rich culture medium and far faster than that of escherichia coli. This feature enables it to greatly shorten the strain construction period and fermentation culture time, and to show great potential in improving production efficiency. Disclosure of Invention The invention aims to provide an L-alanine production strain. Another technical problem to be solved by the present invention is to provide a method for constructing the strain for producing L-alanine. Another technical problem to be solved by the present invention is to provide the use of the strain for producing L-alanine. In order to solve the technical problems, the technical scheme of the invention is as follows: An L-alanine producing strain is obtained by taking wild type vibrio natrii as an initial strain for genetic modification, namely, the dns gene, the vnp1 gene and the vnp2 gene in the genome are sequentially knocked out, the pyruvate formate lyase gene pflB, the L-lactate dehydrogenase gene lldh, the D-lactate dehydrogenase gene dldh, the phosphotransacetylase genes pta1 and pta2, the acetate kinase genes ackA1 and ackA2 and the alcohol dehydrogenase genes adhE1 and adhE2 are knocked out, the strain VNA9 is obtained, a HindIII and BamH I are used for linearization pXMJ19 plasmid (derived from GenBank: AJ 133195.1), a P tuf -alaD gene fragment is connected with the genes to obtain pXtuf-alaD, and the pXtuf-alaD plasmid is electrically transferred into VNA9 competence. Preferably, in the L-alanine producer strain, the P tuf -alaD gene fragment is obtained by amplifying the alaD gene fragment by using the vibrio natrii genome as a template, connecting the gene fragment with P tuf, and then connecting the P tuf -alaD gene fragment with pXMJ19 plasmid by homologous recombination by using ClonExpress II One Step cloning kit (Nanjing Vazyme Biotech Co.), thus obtaining pXtuf-alaD plasmid. Preferably, the L-alanine producing strain mentioned above, the wild-type Vibrio natrii is wild-type Vibrio natrii Vibrio natriegensATCC 14048. Preferably, in the above L-alanine producing strain, the nucleotide sequence of pyruvate formate lyase gene pflB is shown in SEQ ID NO.1, the nucleotide sequence of L-lactate dehydrogenase gene lldh is shown in SEQ ID NO.2, the nucleotide sequence of D-lactate dehydrogenase gene dldh is shown in SEQ ID NO.3, the nucleotide sequence of phosphotransacetylase gene pta1 is shown in SEQ ID NO.4, the nucleotide sequence of phosphotransacetylase gene pta2 is shown in SEQ ID NO.5, the nucleotide sequence of acetate kinase gene ackA1 is shown in SEQ ID NO.6, the nucleotide sequence of acetate kinase gene ackA2 is shown in SEQ ID NO.7, the nucleotide sequence of alcohol dehydrogenase gene adhE1 is shown in SEQ ID NO.8, the nucleotide sequence of alcohol dehydrogenase gene adhE2 is shown in SEQ ID NO.9, and the nucleotide sequence of alaD gene is shown in SEQ ID NO. 10. The plasmids of the above-mentioned L-alanine producer strain, see the sequence of the dns gene Weinstock MT, Hesek ED, Wilson CM,gibson DG. Vibrio natriegens as a fast-growing host for molecular biology. Nat Methods.2016 Oct;13(10):849-51.doi: 10.1038/nmeth.3970. Epub 2016 Aug 29. PMID: 27571549.;vnp1 and the sequence of the vnp2, see the Pfeifer E, Michniewski S,gätgens C, Münch E, Müller F, Polen T, Millard A, Blomb