CN114480234A - Strain for producing L-alanine by high-efficiency fermentation and construction method and application thereof - Google Patents

Abstract

The invention discloses a strain for producing L-alanine by high-efficiency fermentation, which is characterized in that the strain is a mutant escherichia coli recombinant engineering strain 05-DE, is preserved in China general microbiological culture Collection center in 2020, 10 and 23 months, and has the preservation number as follows: CGMCC No. 20946. The strain constructed by the invention can efficiently ferment and produce the L-alanine. The invention continuously optimizes and upgrades the strains and the manufacturing process, so that the product has better quality and lower cost, and has great market prospect.

Description

Strain for producing L-alanine by high-efficiency fermentation and construction method and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a bacterial strain for producing L-alanine by high-efficiency fermentation, and a construction method and application thereof.

Background

L-alanine is a non-essential amino acid, is also the amino acid with the highest content in human blood, is an excellent transportation tool for nitrogen in blood, is an effective glycogenic amino acid and has important physiological functions. L-alanine also has wide application in the fields of industry, daily chemicals, food, medicine and the like.

In the field of daily chemicals, L-alanine is a main raw material for producing a phosphorus-free detergent, namely methylglycine diacetic acid (MGDA), and along with the restriction of the countries in Europe and America on the use of the phosphorus-containing detergent, the phosphorus-free detergent is rapidly developed in recent years, and the demand on the MGDA raw material, namely L-alanine, is increasingly increased; N-lauroyl-L-alanine synthesized by taking L-alanine and coconut oil as raw materials is one of the most commonly used amino acid surfactants, and is used for a plurality of washing and skin care products such as toothpaste, shampoo, facial cleanser and the like; in the field of food, L-alanine can be used as a natural sweetener to improve the flavor of food, can improve the utilization rate of protein in food and beverage, and can quickly eliminate fatigue and recover physical strength after being eaten; in the field of medicine, L-alanine is a main raw material for synthesizing vitamin B6 and amino propanol, is a main component of a nutrient agent for sugar-supplementing amino acid nutrient infusion, and is also a good diuretic. The wide application of the industries such as daily chemicals, food, medicine and the like brings infinitely increased commercial opportunity for the market demand of the raw material L-alanine.

At present, a plurality of L-alanine production and sale factories are sold in the market. There are also many existing L-alanine production processes, including chemical, enzymatic conversion and biological fermentation. 1) The chemical method comprises the following steps: mainly comprises a propionic acid chloridizing ammoniation method, a bromopropionic acid chloridizing method and a cyanohydrin method. These processes all require petroleum-based raw materials such as propionic acid, -bromopropionic acid, acetaldehyde, hydrocyanic acid, etc., and thus the cost is affected by the price of crude oil. In addition, the methods are completed through complicated chemical catalysis, have heavy pollution and high separation and extraction cost, and are not suitable for the requirement of sustainable development. 2) An enzymatic conversion method: taking L-aspartic acid as a raw material, and carrying out decarboxylation reaction under the catalysis of L-aspartic acid-decarboxylase to generate L-alanine. The method is a production technology mainly used by domestic L-alanine manufacturers at present. However, since the raw material aspartic acid in this method is produced from maleic anhydride, the cost of the production route still depends on the price of petroleum. 3) A biological fermentation method: there have been many reports in recent years that synthesis of L-alanine in e. The obtained engineering bacteria can synthesize the L-alanine by taking glucose as a raw material through an anaerobic microbial fermentation method. At present, the manufacturers adopting the method in China are mainly Anhui Hua constant biological science and technology, and related patents mainly comprise CN 103602623B, CN 103898089B, CN 109055451A, CN 108642041A, CN 102603548B, CN 106748847B, CN 108484423A and the like. The constructed genetically engineered bacterium is obtained by integrating an L-alanine dehydrogenase gene on a Geobacillus stearothermophilus chromosome at a lactate dehydrogenase of an Escherichia coli ATCC8739 chromosome, sequentially knocking out a pyruvate formate lyase gene, an ethanol dehydrogenase gene, an acetate kinase gene, a fumarate reductase gene and an alanine racemase gene of the Escherichia coli chromosome, and continuously subculturing in a fermentation tank, wherein the yield can reach 115g/L after fermentation for 48 hours, the conversion rate is 0.96g/g, and the production intensity is about 2.4 g/L/h. Because the anaerobic fermentation process is adopted, the obtained cell density is low and is only about 5g/L, and the production intensity is low. And the yeast extract powder is added into the seed culture medium, so that the raw material cost is high.

Based on the above, the strain capable of efficiently producing the L-alanine by fermentation and the construction method thereof have important significance for improving the yield of the L-alanine and reducing the production cost.

Disclosure of Invention

The main purposes of the invention are as follows: aiming at the problems, the bacterial strain for producing the L-alanine by high-efficiency fermentation, the construction method and the application thereof are provided. The invention constructs the strain with high L-alanine yield through genetic engineering, can obviously improve the density and production strength of the strain, and simultaneously adopts an inorganic salt culture medium for fermentation culture, thereby further reducing the production cost, ensuring that the production process is more environment-friendly and the market competition advantage is obvious.

The purpose of the invention and the technical problem to be solved are realized by adopting the following technical scheme.

One aspect of the invention provides a strain for producing L-alanine by high-efficiency fermentation, the strain is Escherichia coli (Escherichia coli), the strain number is 05-DE, the strain is preserved in China general microbiological culture Collection center in 23.10.2020, the preservation number is: CGMCC No. 20946.

The address of the depository: the institute of microbiology, national academy of sciences No.3, Xilu No.1, Beijing, Chaoyang, Beijing.

The Escherichia coli mutant HAa05 is obtained by replacing the lactate dehydrogenase Gene (ldhA) of Escherichia coli K12 MG1655 with the glyceraldehyde-3-phosphate dehydrogenase Gene (gapA) shown in Gene ID 947679, the pyruvate oxidase Gene (poxB) with the glucose-6-phosphate isomerase Gene (pgi) shown in Gene ID 948535, the pyruvate formate lyase Gene (pflB) with the 6-phosphofructokinase II Gene (pfkB) shown in Gene ID 946230, the alanine racemase Gene (dadX) with the fructose diphosphate aldolase I Gene (fbaB) shown in Gene ID 946632, and the lipid A biosynthesis myristoyl transferase Gene (lpxM) with the pyruvate kinase I Gene (pykF) shown in Gene ID 946179 (HAa 05).

The invention also provides a construction method of the strain for producing the L-alanine by high-efficiency fermentation, which comprises the following steps:

construction of host strains: the mutant E.coli was named HAa05 by biosynthesizing a myristoyltransferase gene (lpxM) by replacing the lactate dehydrogenase gene (ldhA) in the genome of wild-type E.coli with the glyceraldehyde-3-phosphate dehydrogenase gene (gapA), the pyruvate oxidase gene (poxB) in the genome of wild-type E.coli with the glucose-6-phosphate isomerase gene (pgi), the pyruvate formate lyase gene (pfB) in the genome of wild-type E.coli with the 6-phosphofructokinase II gene (pfkB), the alanine racemase gene (dadX) in the genome of wild-type E.coli with the fructose bisphosphate aldolase I gene (fbaB), and the lipid A in the genome of wild-type E.coli with the pyruvate kinase I gene (pykF);

construction of plasmid: inserting an L-alanine dehydrogenase gene (alaD) and an L-alanine transporter gene (alaE) into a plasmid vector pSB1s between NcoI and EcoRI sites in a tandem manner to obtain a recombinant vector plasmid, which is named as pDE;

construction of engineering strains: introducing the recombinant vector plasmid pDE into the mutant escherichia coli HAa05 to obtain a recombinant engineering strain named 05-DE;

wherein the wild type escherichia coli is escherichia coli K12 MG 1655;

the genotype of the mutant Escherichia coli is E.coli BW25113 delta ldhA, gapA, delta poxB, pgi, delta pflB, pfkB, delta dadX, fbaB, delta lpxM, pykF;

the L-alanine dehydrogenase gene (alaD) is derived from Geobacillus stearothermophilus;

the L-alanine transporter gene (alaE) is derived from Escherichia coli K-12MG 1655.

Preferably, the recombinant vector plasmid pDE is constructed by the following steps:

taking the genome DNA of Geobacillus stearothermophilus as a template, and carrying out PCR amplification by using primers alaD-F and alaD-R to obtain an L-alanine dehydrogenase gene alaD; the nucleotide sequence of the forward primer alaD-F is shown as SEQ ID NO.4, and the nucleotide sequence of the reverse primer alaD-R is shown as SEQ ID NO. 5;

taking the genome DNA of Escherichia coli K12 as a template, and carrying out PCR amplification by using primers alaE-F and alaE-R to obtain an L-alanine transporter gene alaE; the nucleotide sequence of the forward primer alaE-F is shown as SEQ ID NO.6, and the nucleotide sequence of the reverse primer alaE-R is shown as SEQ ID NO. 7;

carrying out double enzyme digestion on a pSB1s vector by NcoI and EcoRI, recovering a large vector fragment, connecting the obtained alaD gene fragment, the alaE gene fragment and the large vector fragment by a Gibson method, transforming a product into competent cells, coating an LB solid plate containing streptomycin, staying overnight at 37 ℃, selecting a single clone to extract a plasmid, designing a pair of primers pBAD-F and pBAD-R for PCR verification, and screening out a correctly constructed recombinant vector plasmid pDE; the nucleotide sequence of the forward primer pBAD-F is shown as SEQ ID NO.8, and the nucleotide sequence of the reverse primer pBAD-R is shown as SEQ ID NO. 9.

Preferably, the recombinant vector plasmid pDE is obtained by replacing the fragment between NcoI and EcoRI sites of the pSB1s vector with the L-alanine dehydrogenase gene and the L-alanine transporter gene; the nucleotide sequence of the pSB1s vector is shown as SEQ ID NO.1, the nucleotide sequence of the L-alanine dehydrogenase gene is shown as SEQ ID NO.2, and the nucleotide sequence of the L-alanine transporter gene is shown as SEQ ID NO. 3.

Preferably, the mutant Escherichia coli HAa05 is constructed by the following steps:

(1) using pTargetF as a template, PCR amplifying pTarget-ldhA-F and pTarget-ldhA-R, pTarget-poxB-F and pTarget-poxB-R, pTarget-pflB-F and pTarget-pflB-R, pTarget-dadX-F and pTarget-dadX-R, pTarget-ipxM-F and pTarget-ipxM-R using primers, digesting the amplified fragment with DpnI methylase, transforming E.coli Fast-T1 competent state, screening sequencing positive clones on LB plate containing streptomycin, verifying with primers pTarget-cexu-F, and respectively naming pTarget-ldhA, pTarget-poxB, pTarget-pflB, pTarget-pxlB and pTarget-dap-R after correct;

(2) PCR-amplifying ldhA-up500-F and ldhA-up500-R, gapA-F and gapA-R, ldhA-down500-F and ldhA-down500-R with primers, respectively, to obtain three fragments, and PCR-amplifying ldhA-up500-F and ldhA-down500-R with a mixture of the three fragments as a template, to obtain ldhA-up500-F and ldhA-down500-R, and gapA targeting fragment; carrying out PCR amplification on poxB-up500-F, poxB-up500-R, pgi-F, pgi-R, poxB-down500-F and poxB-down500-R by using primers respectively to obtain three fragments, and carrying out PCR amplification on poxB-up500-F and poxB-down500-R by using a mixture of the three fragments as a template to obtain poxB, wherein pgi is a targeting fragment; respectively carrying out PCR amplification on pflB-up500-F, pflB-up500-R, pfkB-F, pfkB-R, pflB-down500-F and pflB-down500-R by using primer pairs to respectively obtain three fragments, and respectively carrying out PCR amplification on pflB-up500-F and pflB-down500-R by using a primer pair to obtain a pflB target fragment by using a mixture of the three fragments as a template; respectively carrying out PCR amplification on dadX-up500-F, dadX-up500-R, fbaB-F, fbaB-R, dadX-down500-F and dadX-down500-R by using primers to respectively obtain three fragments, and respectively carrying out PCR amplification on dadX-up500-F and dadX-down500-R by using a mixture of the three fragments as a template to obtain dadX, wherein fbaB targeting fragments; respectively carrying out PCR amplification on lpxM-up500-F, lpxM-up500-R, pykF-F, pykF-R, lpxM-down500-F and lpxM-down500-R by using primer pairs to respectively obtain three fragments, and carrying out PCR amplification on the lpxM-up500-F and lpxM-down500-R by using a mixture of the three fragments as a template to obtain an lpxM: (pykF) targeting fragment; recovering the target fragment ldhA, gapA, poxB, pgi, pflB, pfkB, dadX, fbaB and lpxM, pykF respectively;

(3) mixing the pTarget-ldhA plasmid and the targeting fragment ldhA, placing the mixture into an electric rotating cup for electric shock, adding LB liquid culture medium for resurgence at 30 ℃, coating the mixture on an LB plate containing kanamycin and streptomycin, culturing at 30 ℃, screening positive clones, carrying out PCR amplification on ldhA-up800-F and ldhA-down800-R by using primers, and sequencing and verifying the amplified fragment to screen the positive clones;

(4) the positive clones obtained above were inoculated in LB liquid medium containing IPTG and kanamycin and cultured overnight at 30 ℃ to eliminate pTarget plasmid, the overnight cultured strains were streaked on LB solid plate containing kanamycin and cultured overnight at 30 ℃ to obtain Escherichia coli mutant BW 25113. delta. ldhA containing pCas plasmid: gapA, which was named HAa 01;

(5) preparing electroporation competent cells from Escherichia coli mutant HAa01 containing pCas plasmid, mixing with pTarget-poxB plasmid and poxB:: pgi targeting fragment, repeating the steps (3) - (4), and sequencing and verifying poxB-up800-F and poxB-down800-R by using primer pair, to obtain Escherichia coli mutant BW25113 delta ldhA:: gapA delta poxB:: pgi containing pCas plasmid, which is named HAa 02;

(6) preparing an electric transfer competent cell from an Escherichia coli mutant HAa02 containing a pCas plasmid, mixing the electric transfer competent cell with pTarget-pflB plasmid and pflB, wherein pfkB targeting fragments are mixed, repeating the steps (3) to (4), and sequencing and verifying the pflB-up800-F and pflB-down800-R by using primers to obtain an Escherichia coli mutant BW25113 delta ldhA, gapA delta poxB, pgi delta pflB, pfkB and HAa03, wherein the Escherichia coli mutant contains the pCas plasmid is named as BW25113 delta ldhA;

(7) escherichia coli mutant HAa03 containing pCas plasmid was prepared as electroporation competent cells, mixed with pTarget-dadX plasmid and dadX:: fbaB targeting fragment, the above steps (3) - (4) were repeated, and sequencing of dadX-up800-F and dadX-down800-R with primers verified to give Escherichia coli mutant BW 25113. delta. ldhA:: gapA. delta. poxB:: pgi. delta. pflB:: pfkB. delta. dadX:: fbaB containing pCas plasmid, designated HAa 04;

(8) e.coli mutant HAa04 containing pCas plasmid was prepared into electroporation competent cells, mixed with pTarget-lpxM plasmid and lpxM targeting fragment, the above steps (3) - (4) were repeated, and sequencing of lpxM-up800-F and lpxM-down800-R was verified with primers to give E.coli mutant BW 25113. delta. ldhA:: gapA. delta. poxB:: pgi. delta. pflB:: pfkB. delta. dadX: fbaB. delta. lpxM:: pykF, named HAa05, containing pCas plasmid;

(9) coli mutant BW25113 delta ldhA with correct pCas plasmid, gapA delta poxB, pgi delta pflB, pfkB delta dadX, fbaB delta lpxM, pykF (HAa05), inoculated in LB liquid medium, cultured overnight at 37 ℃ to eliminate pCas plasmid, streaked overnight culture on LB solid plate, cultured overnight at 37 ℃ to obtain plasmid-free E.coli mutant BW25113 delta ldhA, gapA delta poxB, i delta pflB, pfkB delta dadX, fbaB delta lpxM, pykF, HAa 05.

Preferably, the method further comprises the step of preparing the electroporation competent cell: transforming Escherichia coli K12 with pCas plasmid by chemical transformation, culturing and screening positive clone on LB plate containing kanamycin at 30 deg.C, inoculating positive clone in LB liquid culture medium containing 2g/L arabinose, culturing at 30 deg.C to OD₆₀₀After about 0.6, electroporation competent cells were obtained.

Preferably, the nucleotide sequence of the forward primer pTarget-ldhA-F in step (1) is shown as SEQ ID NO.10, and the nucleotide sequence of the reverse primer pTarget-ldhA-R is shown as SEQ ID NO. 11; the nucleotide sequence of the forward primer pTarget-poxB-F is shown as SEQ ID NO.12, and the nucleotide sequence of the reverse primer pTarget-poxB-R is shown as SEQ ID NO. 13; the nucleotide sequence of the forward primer pTarget-pflB-F is shown as SEQ ID NO.14, and the nucleotide sequence of the reverse primer pTarget-pflB-R is shown as SEQ ID NO. 15; the nucleotide sequence of the forward primer pTarget-dadX-F is shown as SEQ ID NO.16, and the nucleotide sequence of the reverse primer pTarget-dadX-R is shown as SEQ ID NO. 17; the nucleotide sequence of the forward primer pTarget-lpxM-F is shown as SEQ ID NO.18, and the nucleotide sequence of the reverse primer pTarget-lpxM-R is shown as SEQ ID NO. 19; the nucleotide sequence of the forward primer pTarget-cexu-F is shown as SEQ ID NO. 20;

the PCR amplification system is as follows: 5X SF Buffer 10ul, dNTP Mix (10mM each)1ul, template pTargetF 20ng, primers (10uM) each 2ul, Phanta Super-Fidelity DNA Polymerase 1ul, and distilled water 34ul, the total volume is 50 ul;

the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 2 min (1 cycle); denaturation at 95 ℃ for 10 seconds, annealing at 55 ℃ for 20 seconds, and extension at 72 ℃ for 1.5 minutes (30 cycles); extension at 72 ℃ for 10 min (1 cycle).

Preferably, the nucleotide sequence of the forward primer ldhA-up500-F in the step (2) is shown as SEQ ID NO.21, and the nucleotide sequence of the reverse primer ldhA-up500-R is shown as SEQ ID NO. 22; the nucleotide sequence of the forward primer gapA-F is shown as SEQ ID NO.23, and the nucleotide sequence of the reverse primer gapA-R is shown as SEQ ID NO. 24; the nucleotide sequence of the forward primer ldhA-down500-F is shown in SEQ ID NO.25, and the nucleotide sequence of the reverse primer ldhA-down500-R is shown in SEQ ID NO. 26; the nucleotide sequence of the forward primer poxB-up500-F is shown as SEQ ID NO.27, and the nucleotide sequence of the reverse primer poxB-up500-R is shown as SEQ ID NO. 28; the nucleotide sequence of the forward primer pgi-F is shown as SEQ ID NO.29, and the nucleotide sequence of the reverse primer pgi-R is shown as SEQ ID NO. 30; the nucleotide sequence of the forward primer poxB-down500-F is shown in SEQ ID NO.31, and the nucleotide sequence of the reverse primer poxB-down500-R is shown in SEQ ID NO. 32; the nucleotide sequence of the forward primer pflB-up500-F is shown as SEQ ID NO.33, and the nucleotide sequence of the reverse primer pflB-up500-R is shown as SEQ ID NO. 34; the nucleotide sequence of the forward primer pfkB-F is shown as SEQ ID NO.35, and the nucleotide sequence of the reverse primer pfkB-R is shown as SEQ ID NO. 36; the nucleotide sequence of the forward primer pflB-down500-F is shown in SEQ ID NO.37, and the nucleotide sequence of the reverse primer pflB-down500-R is shown in SEQ ID NO. 38; the nucleotide sequence of the forward primer dadX-up500-F is shown as SEQ ID NO.39, and the nucleotide sequence of the reverse primer dadX-up500-R is shown as SEQ ID NO. 40; the nucleotide sequence of the forward primer fbaB-F is shown as SEQ ID NO.41, and the nucleotide sequence of the reverse primer fbaB-R is shown as SEQ ID NO. 42; the nucleotide sequence of the forward primer dadX-down500-F is shown as SEQ ID NO.43, and the nucleotide sequence of the reverse primer dadX-down500-R is shown as SEQ ID NO. 44; the nucleotide sequence of the forward primer lpxM-up500-F is shown as SEQ ID NO.45, and the nucleotide sequence of the reverse primer lpxM-up500-R is shown as SEQ ID NO. 46; the nucleotide sequence of the forward primer pykF-F is shown as SEQ ID NO.47, and the nucleotide sequence of the reverse primer pykF-R is shown as SEQ ID NO. 48; the nucleotide sequence of the forward primer lpxM-down500-F is shown in SEQ ID NO.49, and the nucleotide sequence of the reverse primer lpxM-down500-R is shown in SEQ ID NO. 50;

the PCR amplification system is as follows: 5X SF Buffer 10ul, dNTP Mix (10mM each)1ul, template 5-20ng, primers (10uM) each 2ul, Phanta Super-Fidelity DNA Polymerase (Nanjing Novodka Biotech Co., Ltd., catalog P501)1ul, distilled water 34ul, total volume 50 ul;

the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 2 min (1 cycle); denaturation at 95 ℃ for 10 seconds, annealing at 55 ℃ for 20 seconds, extension at 72 ℃ for 0.5-2 minutes (30 seconds/kb) (30 cycles); extension at 72 ℃ for 10 min (1 cycle).

Preferably, the nucleotide sequence of the forward primer ldhA-up800-F in the step (3) is shown as SEQ ID NO.51, and the nucleotide sequence of the reverse primer ldhA-up800-R is shown as SEQ ID NO. 52;

the nucleotide sequence of the forward primer poxB-up800-F in the step (5) is shown as SEQ ID NO.53, and the nucleotide sequence of the reverse primer poxB-up800-R is shown as SEQ ID NO. 54;

the nucleotide sequence of the forward primer pflB-up800-F in the step (6) is shown as SEQ ID No.55, and the nucleotide sequence of the reverse primer pflB-up800-R is shown as SEQ ID No. 56;

the nucleotide sequence of the forward primer dadX-up800-F in the step (7) is shown as SEQ ID NO.57, and the nucleotide sequence of the reverse primer dadX-up800-R is shown as SEQ ID NO. 58;

the nucleotide sequence of the forward primer lpxM-up800-F in the step (8) is shown as SEQ ID NO.59, and the nucleotide sequence of the reverse primer lpxM-up800-R is shown as SEQ ID NO. 60.

The object of the present invention and the technical problem to be solved can also be achieved by adopting the following technical solutions.

The invention also provides application of the strain for producing the L-alanine by high-efficiency fermentation, and the strain is used for preparing the L-alanine.

Preferably, the application is to inoculate the activated high-efficiency fermentation L-alanine production strain into a fermentation medium and prepare L-alanine by adopting a biological fermentation method, wherein the method comprises the following steps:

aerobic stage culture: the temperature is 37 ℃, the initial air flux is 2vvm, the stirring speed is 300rpm, the dissolved oxygen concentration is set as 100 percent, the air flux is adjusted to 3vvm in the thallus growth process, the stirring speed is related to the DO value to control the dissolved oxygen concentration to be always more than 30 percent, when the initial glucose consumption is finished, the feed medium is started, the pH is controlled to be 7.0 by adopting ammonia water in the fermentation process, and when the thallus density reaches the absorbance (OD) of 600nm₆₀₀) When the concentration is 30-40, adding L-arabinose with the final concentration of 1g/L to induce protein expression;

and (3) oxygen-limited stage culture: when the density of the cells reached 600nm absorbance (OD)₆₀₀) When the fermentation temperature is 60-70 ℃, entering an acid production stage of oxygen-limited fermentation, stopping ventilation, controlling the rotation speed of a stirring paddle to be 200rpm, controlling the temperature to be 37 ℃, adopting ammonia water to control the pH value to be 7.0, feeding a supplemented medium at the speed of 6g/L/h, and ending the fermentation when the supplemented medium is exhausted.

Glucose is used as the sole carbon source in the fermentation process.

In the above fermentation process, the fermentation medium consists of: 1-5g/L of citric acid, 1-20g/L of potassium dihydrogen phosphate, 1-5g/L of nitrogen source, 150 mu L/L of polyether defoamer, 5-30g/L of glucose and MgSO₄·7H₂0.3-1g/L of O, 10mg/L of VB 15, 1-10mL/L of trace inorganic salt I, and 7.0 +/-0.5 of pH. The trace inorganic salt I comprises the following components: EDTA840 mg/L, CoCl₂·6H₂O 250mg/L，MnCl₂·4H₂O 1500mg/L，CuCl₂·2H₂O 150mg/L，H₃BO₃ 300mg/L， Na₂MoO₄·2H₂O 250mg/L，Zn(CH₃COO)₂·2H₂1300mg/L of O and 10g/L of ferric citrate.

In the fermentation process, the nitrogen source is an inorganic nitrogen-containing compound, and can be one or more selected from ammonium chloride, ammonium acetate, ammonium sulfate and ammonium phosphate.

In the fermentation process, the method also comprises a feed-batch culture medium, and the feed-batch culture medium comprises the following components: glucose 100-₄·7H₂O1-5 g/L, and trace inorganic salt II 1-10 mL/L. The trace inorganic salt II comprises: EDTA 1300mg/L, CoCl₂·6H₂O 400mg/L，MnCl₂·4H₂O 2350mg/L，CuCl₂·2H₂O 250mg/L，H₃BO₃ 500 mg/L，Na₂MoO₄·2H₂O 400mg/L，Zn(CH₃COO)₂·2H₂O1600 mg/L and ferric citrate 4 g/L.

By the technical scheme, the invention at least has the following advantages: the invention constructs Escherichia coli (Escherichia coli) with strain number 05-DE, which can efficiently produce L-alanine. The invention has the advantages that through the genetic engineering transformation, the strain and the production process are greatly improved, the strain density and the production strength are improved, meanwhile, the production cost is further reduced by adopting the inorganic salt culture medium for fermentation culture, the production process is more environment-friendly, and the market competition advantage is obvious. The biological production process adopted by the invention replaces the traditional petrochemical process, and the renewable bio-based raw material replaces the non-renewable petrochemical raw material, thereby realizing the large industries of energy conservation, emission reduction, clean production, environmental protection and circular economy. Through continuous optimization and upgrade of strains and a manufacturing process, the product has better quality and lower cost, and has great market prospect.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given of preferred embodiments of the present invention with reference to the accompanying drawings.

Drawings

FIG. 1 is a physical map of pSB1 s;

FIG. 2 is a curve showing the change of L-alanine production with time during the fermentation of Escherichia coli CGMCC No. 20946.

Detailed Description

In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

The materials, reagents and the like used in the examples of the present invention are commercially available unless otherwise specified.

In the quantitative experiments in the embodiments of the present invention, three repeated experiments are set, and the results are averaged.

In the embodiment of the present invention, unless otherwise specified, the sequence verification process involved is performed by a third-party detection institution, which is national institute of Kingzhi Biotechnology, Inc.

In the examples of the present invention, Escherichia coli K12 is described in "Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H: Construction of Escherichia coli K-12in-frame, single-gene knockout variants: the Keio collection. mol Syst Biol 2006,2: 2006.0008", as a non-pathogenic bacterium, with clear genetic background, short generation time, easy cultivation and low cost of culture medium raw materials. The GenBank Accession of the whole genome sequence of Escherichia coli K12 is U00096.3 (GI: 545778205, update date is AUG 01, 2014, version is 3), which is publicly available from the institute of microbiology of Chinese academy of sciences, and the biomaterial is only used for repeating the related experiments of the invention, and cannot be used for other purposes.

In the embodiment of the invention, the coding sequence of the L-alanine dehydrogenase gene is shown as SEQ ID NO. 2; the coding sequence of the L-alanine transport protein gene is shown in SEQ ID NO. 3. The coding sequence of the glyceraldehyde-3-phosphate dehydrogenase Gene (gapA) is shown as Gene ID:947679 (consisting of 996 nucleotides), and codes glyceraldehyde-3-phosphate dehydrogenase shown as Acession number NP-416293.1 (consisting of 331 amino acid residues); the coding sequence of the glucose-6-phosphate isomerase Gene (pgi) is shown as Gene ID:948535 (consisting of 1650 nucleotides), and the coding sequence of the glucose-6-phosphate isomerase Gene (pgi) is shown as Acession NP-418449.1 (consisting of 549 amino acid residues); the coding sequence of the 6-phosphofructokinase II Gene (pfkB) is shown as Gene ID:946230 (consisting of 930 nucleotides), and codes 6-phosphofructokinase II shown as Acession number NP-416237.3 (consisting of 309 amino acid residues); the coding sequence of the fructose diphosphate aldolase I Gene (fbaB) is shown as Gene ID:946632 (consisting of 1053 nucleotides), and codes fructose diphosphate aldolase I shown as Acession No. NP-416600.4 (consisting of 350 amino acid residues); the coding sequence of the pyruvate kinase I Gene (pykF) is shown as Gene ID:946179 (consisting of 1413 nucleotides), and codes for pyruvate kinase I shown as Acession number NP-416191.1 (consisting of 470 amino acid residues); the coding sequence of the lactate dehydrogenase Gene (ldhA) is shown as Gene ID:946315 (consisting of 990 nucleotides), and codes the lactate dehydrogenase shown as Acession number NP-415898.1 (consisting of 329 amino acid residues); the coding sequence of pyruvate oxidase Gene (poxB) is shown as Gene ID:946132 (composed of 1719 nucleotides), and codes pyruvate oxidase shown as accession number NP-415392.1 (composed of 572 amino acid residues); the coding sequence of the pyruvate formate lyase Gene (pflB) is shown as Gene ID:945514 (consisting of 2283 nucleotides), and codes the pyruvate formate lyase with the accession number NP-415423.1 (consisting of 760 amino acid residues); the coding sequence of the alanine racemase Gene (dadX) is shown as Gene ID:945754 (consisting of 1071 nucleotides), and codes for alanine racemase shown as accession number NP-415708.1 (consisting of 356 amino acid residues); the coding sequence of the Gene (lpxM) for biosynthesis of lipid A shows that the Gene ID 945143 (consisting of 972 nucleotides) encodes the lipid A biosynthesis myristoyl transferase of Acession No. NP-416369.1 (consisting of 323 amino acid residues).

In the embodiment of the invention, the nucleotide sequence of the pSB1s vector is shown in SEQ ID NO.1 and comprises the following fragments: (1) araC-araBAD-MCS fragment (containing arabinose inducible promoter, multiple cloning site); (2) MCS-TrrnB fragment (containing multiple cloning site, terminator TrrnB); (3) a pSC101 replication initiation site fragment; (4) a streptomycin resistance gene Str fragment. The map of the pSB1s vector is shown in FIG. 1.

SEQ ID NO.1：

aatgtgcctgtcaaatggacgaagcagggattctgcaaaccctatgctactccgtcaagccgtcaattgtctgattcgttaccaatt atgacaacttgacggctacatcattcactttttcttcacaaccggcacggaactcgctcgggctggccccggtgcattttttaaatacccgc gagaaatagagttgatcgtcaaaaccaacattgcgaccgacggtggcgataggcatccgggtggtgctcaaaagcagcttcgcctgg ctgatacgttggtcctcgcgccagcttaagacgctaatccctaactgctggcggaaaagatgtgacagacgcgacggcgacaagcaa acatgctgtgcgacgctggcgatatcaaaattgctgtctgccaggtgatcgctgatgtactgacaagcctcgcgtacccgattatccatc ggtggatggagcgactcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttatcgccagcagctccgaatagcgccc ttccccttgcccggcgttaatgatttgcccaaacaggtcgctgaaatgcggctggtgcgcttcatccgggcgaaagaaccccgtattgg caaatattgacggccagttaagccattcatgccagtaggcgcgcggacgaaagtaaacccactggtgataccattcgcgagcctccgg atgacgaccgtagtgatgaatctctcctggcgggaacagcaaaatatcacccggtcggcaaacaaattctcgtccctgatttttcaccac cccctgaccgcgaatggtgagattgagaatataacctttcattcccagcggtcggtcgataaaaaaatcgagataaccgttggcctcaat cggcgttaaacccgccaccagatgggcattaaacgagtatcccggcagcaggggatcattttgcgcttcagccatacttttcatactccc gccattcagagaagaaaccaattgtccatattgcatcagacattgccgtcactgcgtcttttactggctcttctcgctaaccaaaccggtaa ccccgcttattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgcgtaacaaaagtgtctataatcacggcagaaa agtccacattgattatttgcacggcgtcacactttgctatgccatagcatttttatccataagattagcggatcctacctgacgctttttatcgc aactctctactgtttctccatacccgttttttgggctaacaggaggaattaaccatgggtacctctcatcatcatcatcatcacagcagcgg cctggtgccgcgcggcagcctcgagggtagatctggtactagtggtgaattcggtgagctcggtctgcagctggtgccgcgcggca gccaccaccaccaccaccactaatacagattaaatcagaacgcagaagcggtctgataaaacagaatttgcctggcggcagtagcgc ggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtggggtctccccatgcgagagtagg gaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgtcgaccagacccgccataaaacgccctgagaa gcccgtgacgggcttttcttgtattatgggtagtttccttgcatgaatccataaaaggcgcctgtagtgccatttacccccattcactgccag agccgtgagcgcagcgaactgaatgtcacgaaaaagacagcgactcaggtgcctgatggtcggagacaaaaggaatattcagcgatt tgcccgagcttgcgagggtgctacttaagcctttagggttttaaggtctgttttgtagaggagcaaacagcgtttgcgacatccttttgtaat actgcggaactgactaaagtagtgagttatacacagggctgggatctattctttttatctttttttattctttctttattctataaattataaccactt gaatataaacaaaaaaaacacacaaaggtctagcggaatttacagagggtctagcagaatttacaagttttccagcaaaggtctagcag aatttacagatacccacaactcaaaggaaaaggtctagtaattatcattgactagcccatctcaattggtatagtgattaaaatcacctaga ccaattgagatgtatgtctgaattagttgttttcaaagcaaatgaactagcgattagtcgctatgacttaacggagcatgaaaccaagctaa ttttatgctgtgtggcactactcaaccccacgattgaaaaccctacaaggaaagaacggacggtatcgttcacttataaccaatacgctca gatgatgaacatcagtagggaaaatgcttatggtgtattagctaaagcaaccagagagctgatgacgagaactgtggaaatcaggaatc ctttggttaaaggctttgagattttccagtggacaaactatgccaagttctcaagcgaaaaattagaattagtttttagtgaagagatattgcc ttatcttttccagttaaaaaaattcataaaatataatctggaacatgttaagtcttttgaaaacaaatactctatgaggatttatgagtggttatta aaagaactaacacaaaagaaaactcacaaggcaaatatagagattagccttgatgaatttaagttcatgttaatgcttgaaaataactacc atgagtttaaaaggcttaaccaatgggttttgaaaccaataagtaaagatttaaacacttacagcaatatgaaattggtggttgataagcga ggccgcccgactgatacgttgattttccaagttgaactagatagacaaatggatctcgtaaccgaacttgagaacaaccagataaaaatg aatggtgacaaaataccaacaaccattacatcagattcctacctacgtaacggactaagaaaaacactacacgatgctttaactgcaaaa attcagctcaccagttttgaggcaaaatttttgagtgacatgcaaagtaagcatgatctcaatggttcgttctcatggctcacgcaaaaaca acgaaccacactagagaacatactggctaaatacggaaggatctgaggttcttatggctcttgtatctatcagtgaagcatcaagactaa caaacaaaagtagaacaactgttcaccgttagatatcaaagggaaaactgtcgatatgcacagatgaaaacggtgtaaaaaagatagat acatcagagcttttacgagtttttggtgcatttaaagctgttcaccatgaacagatcgacaatgtaacagatgaacagcatgtaacacctaa tagaacaggtgaaaccagtaaaacaaagcaactagaacatgaaattgaacacctgagacaacttgttacagctcaacagtcacacata gacagcctgaaacaggcgatgctgcttatcgaatcaaagctgccgacaacacgggagccagtgacgcctcccgtggggaaaaaatc atggcaattctggaagaaatagcgctttcagccggcaaacctgaagccggatctgcgattctgataacaaactagcaacaccagaaca gcccgtttgcgggcagcaaaacccgcggccgcctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctg ataaatgcttcaataatattgaaaaaggaagagtatgagggaagcggtgatcgccgaagtatcgactcaactatcagaggtagttggcg tcatcgagcgccatctcgaaccgacgttgctggccgtacatttgtacggctccgcagtggatggcggcctgaagccacacagtgatatt gatttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctttgatcaacgaccttttggaaacttcggcttcccctgga gagagcgagattctccgcgctgtagaagtcaccattgttgtgcacgacgacatcattccgtggcgttatccagctaagcgcgaactgca atttggagaatggcagcgcaatgacattcttgcaggtatcttcgagccagccacgatcgacattgatctggctatcttgctgacaaaagc aagagaacatagcgttgccttggtaggtccagcggcggaggaactctttgatccggttcctgaacaggatctatttgaggcgctaaatg aaaccttaacgctatggaactcgccgcccgactgggctggcgatgagcgaaatgtagtgcttacgttgtcccgcatttggtacagcgca gtaaccggcaaaatcgcgccgaaggatgtcgctgccgactgggcaatggagcgcctgccggcccagtatcagcccgtcatacttgaa gctagacaggcttatcttggacaagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttgtccactacgtgaaaggcg agatcaccaaggtagtcggcaaataatgtctaacaattcgttcaagccgaggggccgcaagatccggccacgatgacccggtcgtcg gttcagggcagggtcgttaaatagccgcttatgtctattgctggtttaccggtttattgactaccggaagcagtgtgaccgtgtgcttctcaa atgcctgaggtttcaggcatgc

SEQ ID NO.2：

atgaagatcggcattccaaaagaaatcaaaaacaatgaaaaccgcgtcgccatcactccggcaggcgtgatgacgctcgtcaa agcggggcatgacgtgtatgtggagacggaagccggcgctgggtcgggtttttccgattccgagtatgaaaaagccggggcagtgat cgtgacgaaagcggaagatgcctgggcggcggagatggtgttgaaagtgaaagaaccgctggctgaggagttccgctattttcgccc cggattgattttgtttacgtatttgcatttagccgcggccgaagcgctcacgaaagcgctcgtcgagcaaaaagtggtcggcatcgctta cgagacggtgcagcttgcgaacggctcgctgccgctgttgacgccgatgagtgaagtcgccggccgcatgtcggtgcaagtcggcg cccagtttctcgagaagccgcacggcgggaaaggcattttgcttggcggcgtgcccggggtgcggcgcggcaaagtgacgatcatc ggcggcggcacagcggggacgaacgcggcgaaaatcgcggtcggcctcggggcggacgtgacgattttggacattaacgccgag cggctgcgcgagctcgatgatttgttcggcgaccaagtgacgacgttgatgtccaactcgtatcatatcgccgagtgcgtgcgcgaatc cgatttggtcgtcggcgccgtcttgatcccgggggcgaaagcgccgaagcttgtgacggaagagatggtgcgctcgatgacgccag gctcggtgttggtcgacgtcgccattgaccaaggcggcatttttgaaacgaccgaccgcgtcacgacgcacgacgatccgacatacgt caagcacggcgtcgtccattacgccgtcgcgaacatgccgggcgctgtgccgcgtacgtcaacattcgcgcttacgaacgtcacgatc ccatacgccttgcaaatcgccaacaaaggctaccgcgccgcttgcctcgacaatccggcgctgttaaaagggatcaacacgctcgac gggcacatcgtgtacgaagcggtcgcggcggcgcacaacatgccgtatacggatgttcattcgttgctgcagggatga

SEQ ID NO.3

atgttctcaccgcagtcacgcttgcgtcatgcagttgcagatacgttcgcgatggttgtttactgttctgtcgtgaacatgtgtattga agttttcctctccggaatgagcttcgaacagtctttttattccagattggtagcgattccggtgaacatcttaattgcatggccatacggtatg taccgtgatctgtttatgcgcgcggcacgcaaagttagcccgtcgggctggataaaaaatctggcggatatcctggcttatgtgacgttc cagtcaccggtgtatgtggcgatcttgttagtggtgggcgcagactggcatcagattatggcggcggtcagttcaaacatcgttgtttcga tgttgatgggggcggtttatggctacttcctcgattattgccgccgactgtttaaagtcagccgttaccagcaggtaaaagcctga

Example 1 construction of recombinant plasmid pDE overexpressing L-alanine dehydrogenase and L-alanine transporter

Taking the genome DNA of Geobacillus stearothermophilus as a template, and carrying out PCR amplification on alaD-F and alaD-R by using primers to obtain an L-alanine dehydrogenase gene alaD. Taking the genome DNA of Escherichia coli K12 as a template, and carrying out PCR amplification on alaE-F and alaE-R by using primers to obtain an L-alanine transporter gene alaE.

After digesting the pSB1s vector with NcoI and EcoRI, recovering a vector large fragment of about 4200bp, ligating the recovered alaD gene fragment, alaE gene fragment and vector large fragment by a Gibson method (Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA,3rd, Smith HO: enzymic analysis of DNA molecules up to segmented human cloned plasmids 2009,6: 343-. A recombinant vector obtained by replacing the fragment between NcoI and EcoRI sites of the pSB1s vector with the L-alanine dehydrogenase gene shown in SEQ ID NO.2 and the L-alanine transporter gene shown in SEQ ID NO.3 was named pDE.

The primer sequences are as follows:

in the L-alanine dehydrogenase gene and L-alanine transporter gene expression cassettes, the promoter that initiates transcription of the L-alanine dehydrogenase gene and L-alanine transporter gene is the pBAD promoter.

EXAMPLE 2 construction of E.coli mutant HAa05

Coli mutant HAa05 is a mutant of E.coli K12 obtained by replacing the lactate dehydrogenase gene (ldhA) of E.coli K12 MG1655 with the glyceraldehyde-3-phosphate dehydrogenase gene (gapA), the pyruvate oxidase gene (poxB) with the glucose-6-phosphate isomerase gene (pgi), the pyruvate formate lyase gene (pflB) with the 6-phosphofructokinase II gene (pfkB), the alanine racemase gene (dadX) with the fructose bisphosphate aldolase I gene (gaB), the lipid A biosynthetic myristoyl transferase gene (fbxM) with the pyruvate kinase I gene (pkF) using CRISPR technology (Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene in the Escherichia coli genome via the CRISPR-Cas9 system, Appl Environ Microbiol 2015 81: 2506-2514), abbreviated HAa05 in this application. The genotype of HAa05 was E.coli BW 25113. delta. ldhA: gapA,. delta. poxB: pgi,. delta. pflB: pfkB,. delta. dadX: fbaB,. delta. lpxM: pykF.

The specific construction steps of the Escherichia coli mutant HAa05 are as follows:

(1) preparing electroporation competent cells: the pCas plasmid (Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. apple Environ Microbiol 2015,81:2506-₆₀₀After about 0.6, electroporation competent cells were prepared.

(2) Construction of pTarget plasmid: the site https:// crispy. second plasmid. org was used to select N20 at the knock-out site and design primers to construct pTarget plasmid. pTarget-ldhA-F and pTarget-ldhA-R, pTarget-dadX-F and pTarget-dadX-R, pTarget-ipxM-F and pTarget-ipxM-R are PCR amplified with primers using pTargetF (Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene encoding in the Escherichia coli via the CRISPR-Cas9 system, Appl Environ Microbiol 2015,81:2506-2514.) to obtain 2100bp fragments.

The PCR amplification system is as follows: 5X SF Buffer 10ul, dNTP Mix (10mM each)1ul, template pTargetF 20ng, primers (10uM) each 2ul, Phanta Super-Fidelity DNA Polymerase 1ul (Nanjing Novodka Biotech Co., Ltd., catalog P501), and distilled water 34ul, the total volume being 50 ul.

The amplification conditions were: pre-denaturation at 95 ℃ for 2 min (1 cycle); denaturation at 95 ℃ for 10 seconds, annealing at 55 ℃ for 20 seconds, and extension at 72 ℃ for 1.5 minutes (30 cycles); extension at 72 ℃ for 10 min (1 cycle).

After digestion with DpnI methylase for about 3h, Escherichia coli Fast-T1 was transformed directly by chemical transformation, positive clones were selected on a streptomycin-containing LB plate (streptomycin concentration 50. mu.g/ml) and verified by sequencing with the primer pTarget-cexu-F. The sequences were designated pTarget-ldhA, pTarget-poxB, pTarget-pflB, pTarget-dadX and pTarget-lpxM, respectively, after they were sequenced correctly.

The primer sequences used were as follows (the sequence of N20 is underlined):

(3) amplifying the target fragment: PCR amplification was performed using primer pairs ldhA-up500-F and ldhA-up500-R, gapA-F and gapA-R, ldhA-down500-F and ldhA-down500-R, respectively, to obtain fragments having sizes of about 500bp, 1500bp and 500bp, respectively. Using the mixture of the three fragments as a template, ldhA-up500-F and ldhA-down500-R were PCR-amplified using primers to obtain a gapA targeting fragment of ldhA:, which was about 2500bp in size. PCR amplification is carried out on poxB-up500-F, poxB-up500-R, pgi-F, pgi-R, poxB-down500-F and poxB-down500-R by using primers respectively to obtain fragments with the sizes of about 500bp, 2200bp and 500bp respectively. Using the mixture of the three fragments as a template, using primers to perform PCR amplification on poxB-up500-F and poxB-down500-R to obtain a poxB-up target fragment of about 3200bp in size. PCR amplification is carried out on pflB-up500-F, pflB-up500-R, pfkB-F, pfkB-R, pflB-down500-F and pflB-down500-R by using primers respectively, and fragments with the sizes of about 500bp, 1500bp and 500bp are obtained respectively. Taking the mixture of the three fragments as a template, and carrying out PCR amplification on pflB-up500-F and pflB-down500-R by using primers to obtain a pflB targeting fragment with the size of about 2500 bp. PCR amplification was performed with primers dadX-up500-F and dadX-up500-R, fbaB-F and fbaB-R, dadX-down500-F and dadX-down500-R, respectively, to obtain fragments of about 500bp, 1600bp and 500bp, respectively. Using the mixture of the three fragments as a template, dadX-up500-F and dadX-down500-R were PCR amplified using primers to obtain a target dadX of about 2600bp in size, fbaB. PCR amplification is carried out on lpxM-up500-F, lpxM-up500-R, pykF-F, pykF-R, lpxM-down500-F and lpxM-down500-R by using primers respectively to obtain fragments with the sizes of about 500bp, 2000bp and 500bp respectively. Using the mixture of the three fragments as a template, PCR amplification is carried out on lpxM-up500-F and lpxM-down500-R by using primers to obtain lpxM: (pykF) targeting fragments with the size of about 3000 bp.

The PCR amplification system is as follows: 5X SF Buffer 10ul, dNTP Mix (10mM each)1ul, template 5-20ng, primers (10uM) each 2ul, Phanta Super-Fidelity DNA Polymerase 1ul (Nanjing Novodka Biotech Co., Ltd., catalog P501), and distilled water 34ul, the total volume is 50 ul. The amplification conditions were: pre-denaturation at 95 ℃ for 2 min (1 cycle); denaturation at 95 ℃ for 10 seconds, annealing at 55 ℃ for 20 seconds, extension at 72 ℃ for 0.5-2 minutes (30 seconds/kb) (30 cycles); extension at 72 ℃ for 10 min (1 cycle).

The targeting fragments ldhA:: gapA, poxB:: pgi, pflB:: pfkB, dadX:: fbaB and lpxM:: pykF were recovered separately. The targeting fragment comprises a 500bp upstream homology arm, a replacement gene expression cassette and a 500bp downstream homology arm from upstream to downstream in sequence.

The primer sequences used were as follows:

(4) and (3) electric conversion: gapA was mixed with 100ml of the electroporation competent cells prepared in step (1), placed in a 2mm electroporation cuvette, subjected to 2.5kV electric shock, and added with 1ml of LB liquid medium to resuscitate at 30 ℃ and spread on LB plates containing kanamycin and streptomycin (kanamycin concentration: 50ug/ml, streptomycin concentration: 50. mu.g/ml), cultured at 30 ℃ and positive clones were selected. The amplified fragments were verified by sequencing by PCR amplification using the primer pairs ldhA-up800-F and ldhA-down 800-R.

The PCR amplification system is as follows: green Taq Mix 10ul (Nanjing Novovisan Biotechnology Co., Ltd., product catalog P131), primers (10uM) 0.8ul each, distilled water 8.4ul, template bacterial liquid 0.2ul, total volume of 20 ul;

the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 3 min (1 cycle); denaturation at 95 ℃ for 15 seconds, annealing at 55 ℃ for 15 seconds, extension at 72 ℃ for 1-5 minutes (60 seconds/kb) (30 cycles); extension at 72 ℃ for 5 min (1 cycle).

(5) Elimination of pTarget plasmid: positive clones that were correctly sequenced were inoculated in LB liquid medium containing 0.1mM IPTG and kanamycin and cultured overnight at 30 ℃ to eliminate pTarget plasmid. The overnight cultured strain was streaked on LB solid plate containing kanamycin and cultured overnight at 30 ℃ to give Escherichia coli mutant BW 25113. delta. ldhA:: gapA, which was designated as HAa01, containing pCas plasmid.

(6) Single clones were picked from the plate of step (5), electroporation competent cells were prepared, mixed with pTarget-poxB plasmid and poxB:: pgi targeting fragment, the steps of steps (4) - (5) were repeated, and sequencing of poxB-up800-F and poxB-down800-R was verified with primers to give Escherichia coli mutant BW 25113. delta. ldhA:: gapA. delta. poxB:: pgi containing pCas plasmid, which was designated HAa 02.

(7) The E.coli mutant HAa02 containing pCas plasmid was made into electroporation competent cells, mixed with pTarget-pflB plasmid and pflB:: pfkB targeting fragment, the steps of steps (4) - (5) were repeated, and sequence verification was performed on pflB-up800-F and pflB-down800-R with primers to obtain the E.coli mutant BW 25113. delta. ldhA:: gapA. delta. poxB:: pgi. delta. pflB:: pfkB containing pCas plasmid, which was named HAa 03.

(8) Escherichia coli mutant HAa03 containing pCas plasmid was prepared as electroporation competent cells, mixed with pTarget-dadX plasmid and dadX:: fbaB targeting fragment, the steps of steps (4) to (5) were repeated, and sequencing of dadX-up800-F and dadX-down800-R with primers was verified to give Escherichia coli mutant BW 25113. delta. ldhA:: gapA. delta. poxB:: pgi. delta. pflB:: pfkB. delta. dadX:: fbaB containing pCas plasmid, which was designated HAa 04.

(9) The E.coli mutant HAa04 containing pCas plasmid was prepared into electroporation competent cells, mixed with pTarget-lpxM plasmid and lpxM:: pykF targeting fragment, the steps of steps (4) - (5) were repeated, and sequencing of lpxM-up800-F and lpxM-down800-R with primers verified to give an E.coli mutant BW 25113. delta. ldhA:: gapA. delta. poxB:: pgi. delta. pflB:: pfkB. delta. dadX:: fbaB. delta. lpxM:: pykF containing pCas plasmid, which was designated HAa 05.

(10) Elimination of the pCas plasmid: coli mutant BW 25113. delta. ldhA:: gapA. delta. poxB:: pgi. delta. pflB:: pfkB. delta. dadX:: fbaB. delta. lpxM:: pykF (HAa05) containing pCas plasmid, which was confirmed by sequencing, was inoculated in LB liquid medium and cultured overnight at 37 ℃ to eliminate pCas plasmid. The overnight cultured strain was streaked on LB solid plate and cultured overnight at 37 ℃ to give plasmid-free E.coli mutant BW 25113. delta. ldhA:: gapA. delta. poxB:: pgi. delta. pflB:: pfkB. delta. dadX:: fbaB. delta. lpxM:: pykF, abbreviated as HAa 05.

The primer sequences used for validation and sequencing were as follows:

EXAMPLE 3 construction of an engineered Strain 05-DE producing L-alanine at high yield

The expression vector pDE constructed in example 1 was transformed into E.coli mutant HAa05 by chemical transformation, and positive clones were selected on an LB plate containing streptomycin (the concentration of streptomycin was 50. mu.g/ml), and the resulting clone strain was named 05-DE.

EXAMPLE 4 high Density fermentation of Strain 05-DE

The fermentation medium comprises the following components: 1.7g/L of citric acid, 14g/L of potassium dihydrogen phosphate, 4g/L of diammonium hydrogen phosphate, 150uL/L of polyether antifoaming agent, 20g/L of glucose and MgSO₄·7H₂O0.6 g/L, VB 19 mg/L, trace inorganic salt I10 mL/L, and pH 7.0.

The trace inorganic salt I comprises the following components: EDTA840 mg/L, CoCl₂·6H₂O 250mg/L，MnCl₂·4H₂O 1500 mg/L，CuCl₂·2H₂O 150mg/L，H₃BO₃ 300mg/L，Na₂MoO₄·2H₂O 250mg/L，Zn(CH₃COO)₂·2H₂1300mg/L of O and 10g/L of ferric citrate.

The feed-batch culture medium comprises the following components: glucose 600g/L, MgSO₄·7H₂O2 g/L and trace inorganic salt II 10 mL/L.

The trace inorganic salt II comprises: EDTA 1300mg/L, CoCl₂·6H₂O 400mg/L，MnCl₂·4H₂O 2350 mg/L，CuCl₂·2H₂O 250mg/L，H₃BO₃ 500mg/L，Na₂MoO₄·2H₂O 400mg/L， Zn(CH₃COO)₂·2H₂O1600 mg/L and ferric citrate 4 g/L.

As for the culture medium, a person skilled in the art can make certain adjustments to the above components according to actual conditions, and this example only provides a specific implementation scheme, and is an alternative embodiment of this example. The fermentation medium may include components in amounts that are replaceable by any value within the following ranges: 1-5g/L of citric acid, 1-20g/L of potassium dihydrogen phosphate, 1-5g/L of nitrogen source, 5-30g/L of glucose and MgSO₄·7H₂0.3-1g/L of O, 10mg/L of VB 15, 1-10mL/L of trace inorganic salt I, and 7.0 +/-0.5 of pH. The nitrogen source is an inorganic nitrogen-containing compound, and can be one or more selected from ammonium chloride, ammonium acetate, ammonium sulfate and ammonium phosphate. The trace inorganic salt is selected from one or more of soluble iron salt, cobalt salt, copper salt, zinc salt, manganese salt and molybdate. The feed medium may include components in amounts that are replaceable by any value within the following ranges: glucose 100-₄·7H₂O1-5 g/L and trace inorganic salt II 1-10 mL/L.

Seed liquid culture: the LB in a 250mL triangular flask is 100mL, and sterilized at 121 ℃ for 20 min. Cooling, inoculating glycerol strain 05-DE preserved at-80 deg.C, culturing at 37 deg.C and shaking table rotation speed of 200rpm for 6-8 hr, and inoculating to fermentation culture medium. Those skilled in the art can adjust the above conditions to a certain extent according to actual conditions, and the achievement of the object of the present invention is not affected. This example provides only one specific implementation, and as an alternative embodiment of this example, the culture conditions may be replaced by any value within the following ranges: the cultivation temperature is 25-42 ℃, the rotation speed of the shaking table is 100-.

Inoculating in a fermentation tank: as a preferred embodiment of this example, the volume of the fermentation medium in the 5L fermentor was 2.5L, and the seed solution was inoculated in an amount of 4% (V/V) and at an initial glucose concentration of 20g/L after sterilization.

Aerobic stage culture: in the aerobic growth stage of the thalli, the temperature is 37 ℃, the initial air flux is 2vvm, the stirring rotating speed is 300rpm, the dissolved oxygen concentration is set to be 100 percent, the air flux is adjusted to be 3vvm in the growth process of the thalli, and the stirring rotating speed is related to a DO value to control the dissolved oxygen concentration to be always more than 30 percent. When the initial glucose was consumed, the feed was started. The pH value is controlled to be 7.0 by adopting ammonia water in the fermentation process. When the density of the cells reached 600nm absorbance (OD)₆₀₀) When the concentration is 30-40, adding L-arabinose with final concentration of 1g/L to induce protein expression, and when the thallus density reaches 600nm absorbance (OD)₆₀₀) When the fermentation temperature is 60-70 ℃, entering an acid production stage of oxygen-limited fermentation.

And (3) oxygen-limited stage culture: and in the second stage, stopping aeration, controlling the rotation speed of a stirring paddle to be 200rpm and the temperature to be 37 ℃, controlling the pH to be 7.0 by adopting ammonia water, feeding the fed-batch culture medium at the speed of 6g/L/h, and ending the fermentation when the fed-batch culture medium is exhausted. Those skilled in the art can adjust the above conditions to a certain extent according to actual conditions, without affecting the achievement of the object of the present invention.

The analysis method comprises the following steps: the components in the fermentation broth were measured using an Agilent (Agilent-1200) high performance liquid chromatograph. The detection method of the L-alanine comprises the following steps: the sample was appropriately diluted and derivatized with 2, 4-Dinitrofluorobenzene (DNFB), and 50uL of 10g/L DNFB acetonitrile and 100uL of 0.5M NaHCO were added to 100uL of the sample₃Mixing the solution, reacting at 60 deg.C in dark for 1 hr, cooling, and adding 750uL 0.01M KH₂PO₄Mixing the solution, filtering with 0.22um filter membrane, and detecting by high performance liquid chromatography. The chromatographic column is ZORBAX Eclipse XDB-C18 column (4.6X 150mm, 5 um; Agilent), the column temperature is 30 ℃, the mobile phase is 35% acetonitrile formic acid (thousandth) water solution, the flow rate is 1 mL/min, and the detection wavelength is 360 nm. Residual in fermentation brothGlucose and organic acid detection were performed using an Aminex HPX-87H sugar analysis column from Biorad.

As a result: as shown in FIG. 2, the yield of L-alanine in the fermentation broth reaches 140.8g/L, the conversion rate of L-alanine in the oxygen-limited fermentation stage can reach 91.2g/100g glucose, and the conversion rate of L-alanine in the whole fermentation stage can reach 73.3g/100g glucose. The contents of lactic acid, formic acid and acetic acid are all lower than 0.1 g/L.

Comparative example 1

The gene 201310325533.2 is a genetically engineered bacterium XZ-A26, which is obtained by integrating an L-alanine dehydrogenase gene on a Geobacillus stearothermophilus chromosome at a lactate dehydrogenase of an Escherichia coli ATCC8739 chromosome, sequentially knocking out a pyruvate formate lyase gene, an ethanol dehydrogenase gene, an acetate kinase gene, a fumarate reductase gene and an alanine racemase gene of the Escherichia coli chromosome, and continuously subculturing in a fermentation tank, wherein the preservation number is CGMCC No. 4036. The yield of L-alanine produced by the strain through fermentation reaches 115 g/L. Is significantly lower than the strain of the invention in producing L-alanine.

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Nanjing Shengde Biotechnology research institute Co., Ltd

<120> bacterial strain for producing L-alanine by high-efficiency fermentation and construction method and application thereof

<160> 58

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4792

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac tccgtcaagc 60

cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca ttcacttttt 120

cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta aatacccgcg 180

agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata ggcatccggg 240

tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag cttaagacgc 300

taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag caaacatgct 360

gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg tactgacaag 420

cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct tccatgcgcc 480

gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc ccttcccctt 540

gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg 600

ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca tgccagtagg 660

cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga tgacgaccgt 720

agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa acaaattctc 780

gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata taacctttca 840

ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac 900

ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt tgcgcttcag 960

ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat tgcatcagac 1020

attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta accccgctta 1080

ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt aacaaaagtg 1140

tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca ctttgctatg 1200

ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta tcgcaactct 1260

ctactgtttc tccatacccg ttttttgggc taacaggagg aattaaccat gggtacctct 1320

catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagcctcga gggtagatct 1380

ggtactagtg gtgaattcgg tgagctcggt ctgcagctgg tgccgcgcgg cagccaccac 1440

caccaccacc actaatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 1500

tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 1560

gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat 1620

caaataaaac gaaaggctca gtcgaaagac tgggcctttc gtcgaccaga cccgccataa 1680

aacgccctga gaagcccgtg acgggctttt cttgtattat gggtagtttc cttgcatgaa 1740

tccataaaag gcgcctgtag tgccatttac ccccattcac tgccagagcc gtgagcgcag 1800

cgaactgaat gtcacgaaaa agacagcgac tcaggtgcct gatggtcgga gacaaaagga 1860

atattcagcg atttgcccga gcttgcgagg gtgctactta agcctttagg gttttaaggt 1920

ctgttttgta gaggagcaaa cagcgtttgc gacatccttt tgtaatactg cggaactgac 1980

taaagtagtg agttatacac agggctggga tctattcttt ttatcttttt ttattctttc 2040

tttattctat aaattataac cacttgaata taaacaaaaa aaacacacaa aggtctagcg 2100

gaatttacag agggtctagc agaatttaca agttttccag caaaggtcta gcagaattta 2160

cagataccca caactcaaag gaaaaggtct agtaattatc attgactagc ccatctcaat 2220

tggtatagtg attaaaatca cctagaccaa ttgagatgta tgtctgaatt agttgttttc 2280

aaagcaaatg aactagcgat tagtcgctat gacttaacgg agcatgaaac caagctaatt 2340

ttatgctgtg tggcactact caaccccacg attgaaaacc ctacaaggaa agaacggacg 2400

gtatcgttca cttataacca atacgctcag atgatgaaca tcagtaggga aaatgcttat 2460

ggtgtattag ctaaagcaac cagagagctg atgacgagaa ctgtggaaat caggaatcct 2520

ttggttaaag gctttgagat tttccagtgg acaaactatg ccaagttctc aagcgaaaaa 2580

ttagaattag tttttagtga agagatattg ccttatcttt tccagttaaa aaaattcata 2640

aaatataatc tggaacatgt taagtctttt gaaaacaaat actctatgag gatttatgag 2700

tggttattaa aagaactaac acaaaagaaa actcacaagg caaatataga gattagcctt 2760

gatgaattta agttcatgtt aatgcttgaa aataactacc atgagtttaa aaggcttaac 2820

caatgggttt tgaaaccaat aagtaaagat ttaaacactt acagcaatat gaaattggtg 2880

gttgataagc gaggccgccc gactgatacg ttgattttcc aagttgaact agatagacaa 2940

atggatctcg taaccgaact tgagaacaac cagataaaaa tgaatggtga caaaatacca 3000

acaaccatta catcagattc ctacctacgt aacggactaa gaaaaacact acacgatgct 3060

ttaactgcaa aaattcagct caccagtttt gaggcaaaat ttttgagtga catgcaaagt 3120

aagcatgatc tcaatggttc gttctcatgg ctcacgcaaa aacaacgaac cacactagag 3180

aacatactgg ctaaatacgg aaggatctga ggttcttatg gctcttgtat ctatcagtga 3240

agcatcaaga ctaacaaaca aaagtagaac aactgttcac cgttagatat caaagggaaa 3300

actgtcgata tgcacagatg aaaacggtgt aaaaaagata gatacatcag agcttttacg 3360

agtttttggt gcatttaaag ctgttcacca tgaacagatc gacaatgtaa cagatgaaca 3420

gcatgtaaca cctaatagaa caggtgaaac cagtaaaaca aagcaactag aacatgaaat 3480

tgaacacctg agacaacttg ttacagctca acagtcacac atagacagcc tgaaacaggc 3540

gatgctgctt atcgaatcaa agctgccgac aacacgggag ccagtgacgc ctcccgtggg 3600

gaaaaaatca tggcaattct ggaagaaata gcgctttcag ccggcaaacc tgaagccgga 3660

tctgcgattc tgataacaaa ctagcaacac cagaacagcc cgtttgcggg cagcaaaacc 3720

cgcggccgcc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 3780

aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagg gaagcggtga 3840

tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc catctcgaac 3900

cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg aagccacaca 3960

gtgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg cggcgagctt 4020

tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt ctccgcgctg 4080

tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca gctaagcgcg 4140

aactgcaatt tggagaatgg cagcgcaatg acattcttgc aggtatcttc gagccagcca 4200

cgatcgacat tgatctggct atcttgctga caaaagcaag agaacatagc gttgccttgg 4260

taggtccagc ggcggaggaa ctctttgatc cggttcctga acaggatcta tttgaggcgc 4320

taaatgaaac cttaacgcta tggaactcgc cgcccgactg ggctggcgat gagcgaaatg 4380

tagtgcttac gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg 4440

atgtcgctgc cgactgggca atggagcgcc tgccggccca gtatcagccc gtcatacttg 4500

aagctagaca ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt 4560

tggaagaatt tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc aaataatgtc 4620

taacaattcg ttcaagccga ggggccgcaa gatccggcca cgatgacccg gtcgtcggtt 4680

cagggcaggg tcgttaaata gccgcttatg tctattgctg gtttaccggt ttattgacta 4740

ccggaagcag tgtgaccgtg tgcttctcaa atgcctgagg tttcaggcat gc 4792

<210> 2

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gggctaacag gaggaattaa ccatgaagat cggcattcca aaag 44

<210> 3

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cattatatct ccttctcgag tcatccctgc agcaacgaat g 41

<210> 4

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ctcgagaagg agatataatg ttctcaccgc agtcacgc 38

<210> 5

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gctgcagacc gagctcaccg aattctcagg cttttacctg ctggt 45

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cggcgtcaca ctttgctatg 20

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cgtttcactt ctgagttcgg c 21

<210> 8

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cgatccgtat ccaagtgcag gttttagagc tagaaatagc 40

<210> 9

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ctgcacttgg atacggatcg actagtatta tacctaggac 40

<210> 10

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cagcaaggtg gatatggcac gttttagagc tagaaatagc 40

<210> 11

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gtgccatatc caccttgctg actagtatta tacctaggac 40

<210> 12

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gatcaccgaa caagaagcgc gttttagagc tagaaatagc 40

<210> 13

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gcgcttcttg ttcggtgatc actagtatta tacctaggac 40

<210> 14

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gcgttcgttg tccaattcgg gttttagagc tagaaatagc 40

<210> 15

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ccgaattgga caacgaacgc actagtatta tacctaggac 40

<210> 16

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tctttctggt gccgcacggt gttttagagc tagaaatagc 40

<210> 17

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

accgtgcggc accagaaaga actagtatta tacctaggac 40

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ctttcctgcg ttatcccctg 20

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gccagacaag cagaatcaag 20

<210> 20

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gacgacgtgg tgttagctgt gcataagact ttctccagtg atg 43

<210> 21

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gaaagtctta tgcacagcta acaccacgtc gtc 33

<210> 22

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

cgttcgggca agtactcacc tgcgatatcg 30

<210> 23

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

cgatatcgca ggtgagtact tgcccgaacg aactggttta atc 43

<210> 24

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

tcgccagcgt taactggttc 20

<210> 25

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

tcacgtaccg tgatgacctg 20

<210> 26

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

gacgacgtgg tgttagctgt gcatggttct ccatctcctg aatg 44

<210> 27

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

gagaaccatg cacagctaac accacgtcgt c 31

<210> 28

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

agtttgtttt agtactcacc tgcgatatcg 30

<210> 29

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

cgatatcgca ggtgagtact aaaacaaact ggctaaggta 40

<210> 30

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ctcgcgggta aattcccatg 20

<210> 31

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ttccggcgag tatatgaccg 20

<210> 32

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gacgacgtgg tgttagctgt gcatgtaaca cctaccttct taag 44

<210> 33

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

gtgttacatg cacagctaac accacgtcgt c 31

<210> 34

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

gagtgaaggt agtactcacc tgcgatatcg 30

<210> 35

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

cgatatcgca ggtgagtact accttcactc aatctatgta 40

<210> 36

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

aaccgttggt gtccagacag 20

<210> 37

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

tccggtgtat ccgctgaaag 20

<210> 38

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

gacgacgtgg tgttagctgt gcatctcgtt tccttagctg tg 42

<210> 39

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

aaacgagatg cacagctaac accacgtcgt c 31

<210> 40

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

caaccgggac agtactcacc tgcgatatcg 30

<210> 41

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

cgatatcgca ggtgagtact gtcccggttg tgacggtgta 40

<210> 42

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

ttgtcggtga ccaggtagag 20

<210> 43

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

tgcaccacac agaggtgttg 20

<210> 44

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

acgacgtggt gttagctgtg catgcttttc cagtttcgga 40

<210> 45

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

gaaaagcatg cacagctaac accacgtcgt c 31

<210> 46

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

gataaagatc agtactcacc tgcgatatcg 30

<210> 47

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

cgatatcgca ggtgagtact gatctttatc ccatcaaata 40

<210> 48

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

ttctaaacac cgtctggacg 20

<210> 49

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

ttacaggaag tccatccggc 20

<210> 50

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

agcgttttgg gtagggtgtg 20

<210> 51

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

tacctgcgac gataaagcag 20

<210> 52

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

tgtcgtggtc ttcctgcaag 20

<210> 53

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

accgccggtg ttttcatctc 20

<210> 54

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

tcaacgttgc ccatatcacg 20

<210> 55

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

cagaagtggc gcacaaactg 20

<210> 56

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

cgaaggcgat gtgttgtgtg 20

<210> 57

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

agccatgcag tggcaaatgg 20

<210> 58

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

gtcatggacg tagcaaacgc 20

Claims (13)

1. The strain is a mutant escherichia coli recombinant engineering strain 05-DE, is preserved in China general microbiological culture Collection center (CGMCC) at 10-23 days in 2020, and has the preservation number as follows: CGMCC No. 20946.

2. A method for constructing a strain for the efficient fermentative production of L-alanine according to claim 1, comprising:

construction of host strains: the mutant E.coli was named HAa05 by biosynthesizing a myristoyltransferase gene (lpxM) by replacing the lactate dehydrogenase gene (ldhA) in the genome of wild-type E.coli with the glyceraldehyde-3-phosphate dehydrogenase gene (gapA), the pyruvate oxidase gene (poxB) in the genome of wild-type E.coli with the glucose-6-phosphate isomerase gene (pgi), the pyruvate formate lyase gene (pfB) in the genome of wild-type E.coli with the 6-phosphofructokinase II gene (pfkB), the alanine racemase gene (dadX) in the genome of wild-type E.coli with the fructose bisphosphate aldolase I gene (fbaB), and the lipid A in the genome of wild-type E.coli with the pyruvate kinase I gene (pykF);

construction of plasmid: inserting an L-alanine dehydrogenase gene (alaD) and an L-alanine transporter gene (alaE) into a plasmid vector pSB1s between NcoI and EcoRI sites in a tandem manner to obtain a recombinant vector plasmid, which is named as pDE;

construction of engineering strains: introducing the recombinant vector plasmid pDE into the mutant escherichia coli HAa05 to obtain a recombinant engineering strain named 05-DE;

wherein the wild type escherichia coli is escherichia coli K12 MG 1655;

the genotype of the mutant Escherichia coli is E.coli BW25113 delta ldhA, gapA, delta poxB, pgi, delta pflB, pfkB, delta dadX, fbaB, delta lpxM, pykF;

the L-alanine dehydrogenase gene (alaD) is derived from Geobacillus stearothermophilus;

the L-alanine transporter gene (alaE) is derived from Escherichia coli K-12MG 1655.

3. The method for constructing the strain for efficiently producing L-alanine by fermentation as claimed in claim 2, wherein the recombinant vector plasmid pDE is constructed by the following steps:

taking the genome DNA of Geobacillus stearothermophilus as a template, and carrying out PCR amplification by using primers alaD-F and alaD-R to obtain an L-alanine dehydrogenase gene alaD; the nucleotide sequence of the forward primer alaD-F is shown as SEQ ID NO.4, and the nucleotide sequence of the reverse primer alaD-R is shown as SEQ ID NO. 5;

taking the genome DNA of Escherichia coli K12 as a template, and carrying out PCR amplification by using primers alaE-F and alaE-R to obtain an L-alanine transporter gene alaE; the nucleotide sequence of the forward primer alaE-F is shown as SEQ ID NO.6, and the nucleotide sequence of the reverse primer alaE-R is shown as SEQ ID NO. 7;

carrying out double enzyme digestion on a pSB1s vector by NcoI and EcoRI, recovering a large vector fragment, connecting the obtained alaD gene fragment, the alaE gene fragment and the large vector fragment by a Gibson method, transforming a product into competent cells, coating an LB solid plate containing streptomycin, staying overnight at 37 ℃, selecting a single clone to extract a plasmid, designing a pair of primers pBAD-F and pBAD-R for PCR verification, and screening out a correctly constructed recombinant vector plasmid pDE; the nucleotide sequence of the forward primer pBAD-F is shown as SEQ ID NO.8, and the nucleotide sequence of the reverse primer pBAD-R is shown as SEQ ID NO. 9.

4. The method for constructing a strain for efficient fermentative production of L-alanine according to claim 3, wherein said recombinant vector plasmid pDE is obtained by replacing the fragment of pSB1s vector between the NcoI and EcoRI sites with the gene for L-alanine dehydrogenase and the gene for L-alanine transporter; the nucleotide sequence of the pSB1s vector is shown as SEQ ID NO.1, the nucleotide sequence of the L-alanine dehydrogenase gene is shown as SEQ ID NO.2, and the nucleotide sequence of the L-alanine transporter gene is shown as SEQ ID NO. 3.

5. The method for constructing the strain for efficiently producing L-alanine by fermentation according to claim 2, wherein the mutant Escherichia coli HAa05 is constructed by the following steps:

(1) PCR amplification of pTarget-pflB-F and pTarget-pflB-R, pTarget-dadX-F and pTarget-poxB-R, pTarget-poxB-F and pTarget-poxB-R, pTarget-pflB-F and pTarget-pflB-R, pTarget-dadX-F and pTarget-dadX-R, pTarget-ipxM-F and pTarget-ipxM-R, respectively, using pTargetF as a template, digestion of the amplified fragment with DpnI methylase followed by transformation of E.coli Fastt-T1 competence, screening of positive clones on a streptomycin-containing LB plate and verification of the sequencing with pTarget-cexu-F, and after correctness, naming pTarget-ldhaT-dE, pTarget-poxB, pTarget-ceft-and pTarxM, respectively;

(2) PCR-amplifying ldhA-up500-F and ldhA-up500-R, gapA-F and gapA-R, ldhA-down500-F and ldhA-down500-R with primers to obtain three fragments, respectively, and PCR-amplifying ldhA-up500-F and ldhA-down500-R with primers to obtain ldhA:: gapA targeting fragment, using a mixture of the three fragments as a template; carrying out PCR amplification on poxB-up500-F, poxB-up500-R, pgi-F, pgi-R, poxB-down500-F and poxB-down500-R by using primers respectively to obtain three fragments, and carrying out PCR amplification on poxB-up500-F and poxB-down500-R by using a mixture of the three fragments as a template to obtain poxB, wherein pgi is a targeting fragment; respectively carrying out PCR amplification on pflB-up500-F, pflB-up500-R, pfkB-F, pfkB-R, pflB-down500-F and pflB-down500-R by using primer pairs to respectively obtain three fragments, and respectively carrying out PCR amplification on pflB-up500-F and pflB-down500-R by using a primer pair to obtain a pflB target fragment by using a mixture of the three fragments as a template; respectively carrying out PCR amplification on dadX-up500-F, dadX-up500-R, fbaB-F, fbaB-R, dadX-down500-F and dadX-down500-R by using primers to respectively obtain three fragments, and respectively carrying out PCR amplification on dadX-up500-F and dadX-down500-R by using a mixture of the three fragments as a template to obtain dadX, wherein fbaB targeting fragments; respectively carrying out PCR amplification on lpxM-up500-F and lpxM-up500-R, pykF-F and pykF-R, and lpxM-down500-F and lpxM-down500-R by using a primer pair to respectively obtain three fragments, and carrying out PCR amplification on lpxM-up500-F and lpxM-down500-R by using a primer pair to obtain lpxM and pykF targeting fragments by using a mixture of the three fragments as a template; recovering the target fragment ldhA, gapA, poxB, pgi, pflB, pfkB, dadX, fbaB and lpxM, pykF respectively;

(3) mixing the pTarget-ldhA plasmid and the targeting fragment ldhA, placing the mixture into an electric rotating cup for electric shock, adding LB liquid culture medium for resurgence at 30 ℃, coating the mixture on an LB plate containing kanamycin and streptomycin, culturing at 30 ℃, screening positive clones, carrying out PCR amplification on ldhA-up800-F and ldhA-down800-R by using primers, and sequencing and verifying the amplified fragment to screen the positive clones;

(4) the positive clones obtained above were inoculated in LB liquid medium containing IPTG and kanamycin and cultured overnight at 30 ℃ to eliminate pTarget plasmid, the overnight cultured strains were streaked on LB solid plate containing kanamycin and cultured overnight at 30 ℃ to obtain Escherichia coli mutant BW 25113. delta. ldhA containing pCas plasmid: gapA, which was named HAa 01;

(5) preparing electroporation competent cells from Escherichia coli mutant HAa01 containing pCas plasmid, mixing with pTarget-poxB plasmid and poxB:: pgi targeting fragment, repeating the steps (3) - (4), and sequencing and verifying poxB-up800-F and poxB-down800-R by using primer pair, to obtain Escherichia coli mutant BW25113 delta ldhA:: gapA delta poxB:: pgi containing pCas plasmid, which is named HAa 02;

(6) preparing an electric transfer competent cell from an Escherichia coli mutant HAa02 containing a pCas plasmid, mixing the electric transfer competent cell with pTarget-pflB plasmid and pflB, wherein pfkB targeting fragments are mixed, repeating the steps (3) to (4), and sequencing and verifying the pflB-up800-F and pflB-down800-R by using primers to obtain an Escherichia coli mutant BW25113 delta ldhA, gapA delta poxB, pgi delta pflB, pfkB and HAa03, wherein the Escherichia coli mutant contains the pCas plasmid is named as BW25113 delta ldhA;

(7) escherichia coli mutant HAa03 containing pCas plasmid was prepared as electroporation competent cells, mixed with pTarget-dadX plasmid and dadX:: fbaB targeting fragment, the above steps (3) - (4) were repeated, and sequencing of dadX-up800-F and dadX-down800-R with primers verified to give Escherichia coli mutant BW 25113. delta. ldhA:: gapA. delta. poxB:: pgi. delta. pflB:: pfkB. delta. dadX:: fbaB containing pCas plasmid, designated HAa 04;

(8) e.coli mutant HAa04 containing pCas plasmid was prepared into electroporation competent cells, mixed with pTarget-lpxM plasmid and lpxM targeting fragment, the above steps (3) - (4) were repeated, and sequencing of lpxM-up800-F and lpxM-down800-R was verified with primers to give E.coli mutant BW 25113. delta. ldhA:: gapA. delta. poxB:: pgi. delta. pflB:: pfkB. delta. dadX: fbaB. delta. lpxM:: pykF, named HAa05, containing pCas plasmid;

(9) coli mutant BW25113 delta ldhA with correct pCas plasmid, gapA delta poxB, pgi delta pflB, pfkB delta dadX, fbaB delta lpxM, pykF (HAa05), inoculated in LB liquid medium, cultured overnight at 37 ℃ to eliminate pCas plasmid, streaked overnight culture on LB solid plate, cultured overnight at 37 ℃ to obtain plasmid-free E.coli mutant BW25113 delta ldhA, gapA delta poxB, i delta pflB, pfkB delta dadX, fbaB delta lpxM, pykF, HAa 05.

6. The method for constructing a strain for efficient fermentative production of L-alanine according to claim 5, further comprising the step of preparing electroporation competent cells: transforming Escherichia coli K12 with pCas plasmid by chemical transformation, culturing and screening positive clone on LB plate containing kanamycin at 30 deg.C, inoculating positive clone in LB liquid culture medium containing 2g/L arabinose, culturing at 30 deg.C to OD₆₀₀After about 0.6, electroporation competent cells were obtained.

7. The method for constructing a strain capable of efficiently producing L-alanine by fermentation as claimed in claim 5, wherein the nucleotide sequence of the forward primer pTarget-ldhA-F in step (1) is shown in SEQ ID NO.10, and the nucleotide sequence of the reverse primer pTarget-ldhA-R is shown in SEQ ID NO. 11; the nucleotide sequence of the forward primer pTarget-poxB-F is shown as SEQ ID NO.12, and the nucleotide sequence of the reverse primer pTarget-poxB-R is shown as SEQ ID NO. 13; the nucleotide sequence of the forward primer pTarget-pflB-F is shown as SEQ ID NO.14, and the nucleotide sequence of the reverse primer pTarget-pflB-R is shown as SEQ ID NO. 15; the nucleotide sequence of the forward primer pTarget-dadX-F is shown as SEQ ID NO.16, and the nucleotide sequence of the reverse primer pTarget-dadX-R is shown as SEQ ID NO. 17; the nucleotide sequence of the forward primer pTarget-lpxM-F is shown as SEQ ID NO.18, and the nucleotide sequence of the reverse primer pTarget-lpxM-R is shown as SEQ ID NO. 19; the nucleotide sequence of the forward primer pTarget-cexu-F is shown as SEQ ID NO. 20;

the PCR amplification system is as follows: 5X SF Buffer 10ul, dNTP Mix (10mM each)1ul, template pTargetF 20ng, primers (10uM) each 2ul, Phanta Super-Fidelity DNA Polymerase 1ul, and distilled water 34ul, the total volume is 50 ul;

the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 2 min (1 cycle); denaturation at 95 ℃ for 10 seconds, annealing at 55 ℃ for 20 seconds, and extension at 72 ℃ for 1.5 minutes (30 cycles); extension at 72 ℃ for 10 min (1 cycle).

8. The method for constructing a strain capable of efficiently producing L-alanine by fermentation as claimed in claim 5, wherein in the step (2), the nucleotide sequence of the forward primer ldhA-up500-F is shown in SEQ ID NO.21, and the nucleotide sequence of the reverse primer ldhA-up500-R is shown in SEQ ID NO. 22; the nucleotide sequence of the forward primer gapA-F is shown as SEQ ID NO.23, and the nucleotide sequence of the reverse primer gapA-R is shown as SEQ ID NO. 24; the nucleotide sequence of the forward primer ldhA-down500-F is shown in SEQ ID NO.25, and the nucleotide sequence of the reverse primer ldhA-down500-R is shown in SEQ ID NO. 26; the nucleotide sequence of the forward primer poxB-up500-F is shown as SEQ ID NO.27, and the nucleotide sequence of the reverse primer poxB-up500-R is shown as SEQ ID NO. 28; the nucleotide sequence of the forward primer pgi-F is shown as SEQ ID NO.29, and the nucleotide sequence of the reverse primer pgi-R is shown as SEQ ID NO. 30; the nucleotide sequence of the forward primer poxB-down500-F is shown in SEQ ID NO.31, and the nucleotide sequence of the reverse primer poxB-down500-R is shown in SEQ ID NO. 32; the nucleotide sequence of the forward primer pflB-up500-F is shown as SEQ ID NO.33, and the nucleotide sequence of the reverse primer pflB-up500-R is shown as SEQ ID NO. 34; the nucleotide sequence of the forward primer pfkB-F is shown as SEQ ID NO.35, and the nucleotide sequence of the reverse primer pfkB-R is shown as SEQ ID NO. 36; the nucleotide sequence of the forward primer pflB-down500-F is shown in SEQ ID NO.37, and the nucleotide sequence of the reverse primer pflB-down500-R is shown in SEQ ID NO. 38; the nucleotide sequence of the forward primer dadX-up500-F is shown as SEQ ID NO.39, and the nucleotide sequence of the reverse primer dadX-up500-R is shown as SEQ ID NO. 40; the nucleotide sequence of the forward primer fbaB-F is shown as SEQ ID NO.41, and the nucleotide sequence of the reverse primer fbaB-R is shown as SEQ ID NO. 42; the nucleotide sequence of the forward primer dadX-down500-F is shown as SEQ ID NO.43, and the nucleotide sequence of the reverse primer dadX-down500-R is shown as SEQ ID NO. 44; the nucleotide sequence of the forward primer lpxM-up500-F is shown as SEQ ID NO.45, and the nucleotide sequence of the reverse primer lpxM-up500-R is shown as SEQ ID NO. 46; the nucleotide sequence of the forward primer pykF-F is shown as SEQ ID NO.47, and the nucleotide sequence of the reverse primer pykF-R is shown as SEQ ID NO. 48; the nucleotide sequence of the forward primer lpxM-down500-F is shown in SEQ ID NO.49, and the nucleotide sequence of the reverse primer lpxM-down500-R is shown in SEQ ID NO. 50;

the PCR amplification system is as follows: 5X SF Buffer 10ul, dNTP Mix (10mM each)1ul, template 5-20ng, primers (10uM) each 2ul, Phanta Super-Fidelity DNA Polymerase 1ul, distilled water 34ul, total volume 50 ul;

the PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 2 min (1 cycle); denaturation at 95 ℃ for 10 seconds, annealing at 55 ℃ for 20 seconds, extension at 72 ℃ for 0.5-2 minutes (30 seconds/kb) (30 cycles); extension at 72 ℃ for 10 min (1 cycle).

9. The method for constructing a strain capable of efficiently producing L-alanine by fermentation as claimed in claim 5, wherein the nucleotide sequence of said forward primer ldhA-up800-F in step (3) is shown in SEQ ID NO.51, and the nucleotide sequence of said reverse primer ldhA-up800-R is shown in SEQ ID NO. 52;

the nucleotide sequence of the forward primer poxB-up800-F in the step (5) is shown as SEQ ID NO.53, and the nucleotide sequence of the reverse primer poxB-up800-R is shown as SEQ ID NO. 54;

the nucleotide sequence of the forward primer pflB-up800-F in the step (6) is shown as SEQ ID NO.55, and the nucleotide sequence of the reverse primer pflB-up800-R is shown as SEQ ID NO. 56;

the nucleotide sequence of the forward primer dadX-up800-F in the step (7) is shown as SEQ ID NO.57, and the nucleotide sequence of the reverse primer dadX-up800-R is shown as SEQ ID NO. 58;

the nucleotide sequence of the forward primer lpxM-up800-F in the step (8) is shown as SEQ ID NO.59, and the nucleotide sequence of the reverse primer lpxM-up800-R is shown as SEQ ID NO. 60.

10. Use of a strain for the efficient fermentative production of L-alanine according to claim 1, for the preparation of L-alanine.

11. The use of the strain for high efficiency fermentative production of L-alanine according to claim 10, wherein the activated strain for high efficiency fermentative production of L-alanine is inoculated into a fermentation medium and used for the production of L-alanine by a biological fermentation method, said method comprising:

aerobic stage culture: the temperature is 37 ℃, the initial air flux is 2vvm, the stirring speed is 300rpm, the dissolved oxygen concentration is set as 100 percent, the air flux is adjusted to 3vvm in the thallus growth process, the stirring speed is related to a DO value to control the dissolved oxygen concentration to be always more than 30 percent, when the initial glucose is consumed, the feed medium is started, the pH is controlled to be 7.0 by ammonia water in the fermentation process, and when the thallus density reaches the absorbance (OD) of 600nm₆₀₀) When the concentration is 30-40, adding L-arabinose with the final concentration of 1g/L to induce protein expression;

and (3) oxygen-limited stage culture: when the density of the cells reached an absorbance (OD) of 600nm₆₀₀) And when the concentration is 60-70 ℃, entering an oxygen-limited fermentation acid production stage, stopping introducing the gas, controlling the rotating speed of a stirring paddle to be 200rpm and the temperature to be 37 ℃, adopting ammonia water to control the pH to be 7.0, feeding the feed culture medium at the speed of 6g/L/h, and ending the fermentation when the feed culture medium is exhausted.

12. The use of a strain for the efficient fermentative production of L-alanine according to claim 11, characterized in that the fermentation medium consists of: 1-5g/L of citric acid, 1-20g/L of potassium dihydrogen phosphate, 1-5g/L of nitrogen source, 150 mu L/L of polyether defoamer, 5-30g/L of glucose and MgSO₄·7H₂O0.3-1 g/L, VB 15-10 mg/L, trace inorganic salt I1-10mL/L, and pH 7.0 +/-0.5;

the feed medium comprises the following components: glucose 100-₄·7H₂O1-5 g/L and trace inorganic salt II 1-10 mL/L.

13. The use of a strain for the efficient fermentative production of L-alanine according to claim 12, characterized in that said trace amount of inorganic salts I consists of: EDTA840 mg/L, CoCl₂·6H₂O 250mg/L，MnCl₂·4H₂O 1500mg/L，CuCl₂·2H₂O 150mg/L，H₃BO₃ 300mg/L，Na₂MoO₄·2H₂O 250mg/L，Zn(CH₃COO)₂·2H₂1300mg/L of O, 10g/L of ferric citrate; the nitrogen source is selected from one or more of ammonium chloride, ammonium acetate, ammonium sulfate and ammonium phosphate;

the trace inorganic salt II comprises the following components: EDTA 1300mg/L, CoCl₂·6H₂O 400mg/L，MnCl₂·4H₂O 2350mg/L，CuCl₂·2H₂O 250mg/L，H₃BO₃ 500mg/L，Na₂MoO₄·2H₂O 400mg/L，Zn(CH₃COO)₂·2H₂O1600 mg/L, ferric citrate 4 g/L.

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