CN111748535B

CN111748535B - Alanine dehydrogenase mutant and application thereof in fermentation production of L-alanine

Info

Publication number: CN111748535B
Application number: CN201910243105.2A
Authority: CN
Inventors: 张学礼; 郭恒华; 马延和; 张冬竹; 刘萍萍
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS; Anhui Huaheng Biotechnology Co Ltd
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS; Anhui Huaheng Biotechnology Co Ltd
Priority date: 2019-03-28
Filing date: 2019-03-28
Publication date: 2022-07-05
Anticipated expiration: 2039-03-28
Also published as: CN111748535A

Abstract

The invention discloses an alanine dehydrogenase mutant and application thereof in fermentation production of L-alanine. The alanine dehydrogenase mutant of the present invention is a protein obtained by mutating the 327 th lysine of an amino acid sequence of alanine dehydrogenase to asparagine. The alanine dehydrogenase mutant with obviously improved activity is obtained based on a screening technology of metabolic domestication, and is used for the construction of an L-alanine engineering strain and the high-efficiency fermentation of L-alanine. Experiments prove that: the L-alanine yield and the enzyme activity of the L-alanine dehydrogenase of the strain provided by the invention are both greatly improved by 88.2 percent compared with the strain expressing wild-type alanine dehydrogenase, the fermentation capacity and the L-alanine yield of the L-alanine engineering strain can be effectively improved, and the strain has great application value.

Description

Alanine dehydrogenase mutant and application thereof in fermentation production of L-alanine

Technical Field

The invention relates to the field of biotechnology, in particular to an alanine dehydrogenase mutant and application thereof in fermentation production of L-alanine, and particularly relates to an alanine dehydrogenase mutant with obviously improved activity and application thereof in construction of an L-alanine engineering strain and efficient fermentation production of L-alanine.

Background

L-alanine is a non-essential amino acid for human body, and has wide application in the fields of food and pharmaceutical industry. The L-alanine can improve the nutritive value of food in the field of food industry, and can obviously improve the utilization rate of protein in food and beverage after being added. L-alanine can improve the taste of synthetic sweeteners, making them like natural sweeteners. In addition, L-alanine can also improve the sourness of organic acids, making them closer to natural taste. L-alanine is commonly used as an amino acid based nutritional agent in the medical field. Meanwhile, L-alanine is also a key precursor for synthesizing vitamin B6 and an important raw material for synthesizing calcium pantothenate and other organic compounds. With the further exploitation of the potential of L-alanine applications, the global demand for L-alanine is increasing.

The traditional biological technology for producing L-alanine mainly takes L-aspartic acid as a raw material, and the L-alanine is produced by decarboxylation reaction under the catalysis of aspartate-decarboxylase. The method is a production technology mainly used by domestic L-alanine manufacturers at present. However, in this method, the raw material aspartic acid is produced from maleic anhydride, and therefore the production cost of L-alanine is limited by the price of petroleum. Along with the shortage of petroleum resources and the improvement of prices, the supply of aspartic acid has huge hidden danger due to the shortage of maleic anhydride resources and the rise of prices, thereby influencing the production and the cost of L-alanine.

Through microbial fermentation, the L-alanine is produced by taking renewable lignocellulose as a raw material, so that the dependence on petroleum-based raw materials can be eliminated, the emission of carbon dioxide is reduced, and the low-cost and environment-friendly production of the L-alanine is realized. Smith et al, starting from E.coli, constructed an E.coli ALS929(pTrc99A-alaD) in which an alanine dehydrogenase (alaD) expressed in a plasmid was able to convert intracellular pyruvate into L-alanine. The strain can produce 88 g/L-alanine after 48 hours of fermentation. Lee et al constructed an E.coli ALA887(pTrc99A-alaD) strain that was able to produce 32 g/L-alanine within 27 hours of fermentation production. The applicant of the invention realizes the high-level synthesis of L-alanine in Escherichia coli by introducing alanine dehydrogenase from Geobacillus stearothermophilus XL-65-6 into Escherichia coli and inactivating competitive metabolic pathway of pyruvic acid, and realizes industrial production for the first time in the world. Since alanine dehydrogenase is a key step in the production of L-alanine, the specificity and catalytic activity of the enzyme directly limit the L-alanine production capacity of the engineered strain. Therefore, the modification of the activity of the alanine dehydrogenase has important significance on the construction of an L-alanine engineering strain and the high-efficiency fermentation of L-alanine.

Disclosure of Invention

The invention aims to provide an alanine dehydrogenase mutant and application thereof in fermentation production of L-alanine.

In a first aspect, the invention features a protein.

The protein protected by the invention is obtained by mutating the 327 th lysine of an alanine dehydrogenase (AlaD protein) amino acid sequence into asparagine.

The amino acid sequence of the alanine dehydrogenase (AlaD protein) is shown as a sequence 4 in a sequence table.

The protein protected by the invention can be any one of the following (a1) - (a 3):

(a1) a protein consisting of an amino acid sequence shown in a sequence 6 in a sequence table;

(a2) a protein having the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues other than the 327 th amino acid residue of the amino acid sequence shown in (a 1);

(a3) and (b) a fusion protein obtained by attaching a tag to the N-terminus or/and C-terminus of the amino acid sequence shown in (a1) or (a 2).

In order to facilitate the purification of the protein of (a1), a tag as shown in Table 1 may be attached to the amino terminus or the carboxy terminus of the protein represented by sequence 6 in the sequence listing.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein of (a2), wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein of (a2) or (a3) may be artificially synthesized, or may be obtained by synthesizing a gene encoding the protein and then performing biological expression.

In any of the above proteins, the protein has an alanine dehydrogenase activity higher than that of the alanine dehydrogenase (AlaD protein).

In a second aspect, the invention features nucleic acid molecules encoding the above-described proteins.

The nucleic acid molecule may be a DNA molecule as described in any one of (b1) to (b4) below:

(b1) the coding region is a DNA molecule shown as a sequence 5 in a sequence table;

(b2) the nucleotide sequence is a DNA molecule shown as a sequence 5 in a sequence table;

(b3) a DNA molecule having 75% or more identity to the nucleotide sequence defined in (b1) or (b2) and encoding the protein;

(b4) a DNA molecule which hybridizes with the nucleotide sequence defined in (b1) or (b2) under stringent conditions and encodes the protein.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc. The nucleic acid molecule may be a nucleic acid molecule formed by a gene encoding the protein and its regulatory sequences.

The nucleotide sequence encoding the protein of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have a homology of 75% or more with the nucleotide sequence of the protein of the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein. The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, 80% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of the present invention that encodes the protein.

The above stringent conditions are hybridization and washing of the membrane 2 times 5min at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of the membrane 2 times 15min at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

In a third aspect, the present invention also protects any one of the following biomaterials (c1) to (c 3):

(c1) an expression cassette comprising the nucleic acid molecule;

(c2) a recombinant vector comprising the nucleic acid molecule;

(c3) a recombinant microorganism comprising the above nucleic acid molecule.

Further, the recombinant vector may be a recombinant plasmid obtained by inserting the nucleic acid molecule into an expression vector or a cloning vector.

The recombinant microorganism may be a starting microorganism containing the recombinant plasmid. The starting microorganism may be a yeast, bacterium, algae or fungus. The bacterium may be escherichia coli.

Further, the recombinant plasmid may be a Paladmut plasmid. The Palamout plasmid is obtained by inserting the nucleic acid molecule into an M1-46 artificial promoter in a Palamut-1 plasmid.

The Escherichia coli may be the XZ-A51 strain or the XZ-A53 strain mentioned in examples.

In a fourth aspect, the present invention protects the use of any one of the following (d1) to (d 5):

(d1) the use of the above protein as an alanine dehydrogenase;

(d2) the application of the nucleic acid molecule or the biological material in the preparation of alanine dehydrogenase;

(d3) the application of the protein or nucleic acid molecule or biological material in the construction of alanine engineering bacteria;

(d4) the application of the protein or nucleic acid molecule or biological material in the synthesis or preparation of alanine;

(d5) the use of the above protein or nucleic acid molecule or biomaterial for increasing the production of alanine.

In a fifth aspect, the invention also protects a recombinant bacterium B capable of producing alanine.

The alanine dehydrogenase involved in the pathway for producing alanine by metabolizing the recombinant bacterium B protected by the invention can comprise any one of the proteins.

The "alanine dehydrogenase may include any of the proteins" described above means that the alanine dehydrogenase involved therein may be all of any of the proteins described above, or may be a part of any of the proteins described above.

When all of the above proteins are present, all of the alanine dehydrogenases in the recombinant strain B are the mutated alanine dehydrogenases of the present invention (i.e., any of the above proteins).

When a part is any of the above-mentioned proteins, it means that a part of the bacterial cells is the mutated alanine dehydrogenase of the present invention (i.e., any of the above-mentioned proteins), and another part is the non-mutated alanine dehydrogenase (the AlaD protein shown in SEQ ID NO: 4).

The recombinant bacterium capable of producing alanine may be any bacterium known to those skilled in the art, which has alanine-producing ability and is involved in alanine dehydrogenase in the alanine-producing pathway.

As for conventional recombinant bacteria capable of producing alanine, one example of the present invention is a recombinant bacterium obtained by knocking out pyruvate formate lyase gene (pflB gene), fumarate reductase gene (frd gene), alcohol dehydrogenase gene (adhE gene), acetate kinase (ackA gene), methylglyoxal synthase (mgsA gene) and alanine racemase gene (dadX gene) in Escherichia coli. Wherein the Escherichia coli may be wild type or any mutant type of Escherichia coli which does not affect the purpose of producing alanine.

As to the conventional recombinant bacterium capable of producing alanine, another example of the present invention is a recombinant bacterium obtained by knocking out pyruvate formate lyase gene (pflB gene), fumarate reductase gene (frd gene), alcohol dehydrogenase gene (adhE gene), acetate kinase (ackA gene), methylglyoxal synthase (mgsA gene) and alanine racemase gene (dadX gene) in E.coli and then introducing alaD gene into the bacterium. Also, E.coli may be a wild type or any mutant type which does not affect the purpose of producing alanine.

The skilled in the art can change the alanine dehydrogenase and the coding gene thereof into the alanine dehydrogenase and the coding gene thereof of the invention on the basis of any of the existing recombinant bacteria capable of producing alanine by using the conventional technical means in the field. Such as homologous recombination, site-directed mutagenesis, and the like. An example of the present invention is a recombinant bacterium capable of producing alanine by introducing a gene encoding the mutated alanine dehydrogenase of the present invention (i.e., any of the proteins described above) into the bacterium.

In a sixth aspect, the invention also provides a method for producing alanine.

The method for producing alanine protected by the invention sequentially comprises the following steps:

(e1) improving the expression level and/or activity of any protein in the starting bacterium to obtain recombinant bacterium A; the recombinant bacterium A has an increased ability to produce alanine compared to the starting bacterium;

(e2) fermenting and culturing the recombinant bacterium A to obtain alanine.

In the above method, the recombinant bacterium A may be the recombinant bacterium B.

In the above method, the "increasing the expression level and/or activity of any of the proteins in the starting bacterium" may be carried out by introducing a nucleic acid molecule encoding the protein into the starting bacterium.

The initiating bacterium can be escherichia coli.

In the above method, the solvent of the culture medium used for the fermentation may be tap water or distilled water.

Any one of the recombinant bacterium A or the recombinant bacterium B can be XZ-A51(Paladmut) or XZ-A53(Paladmut) mentioned in the examples.

Any recombinant bacterium A described above also belongs to the protection scope of the invention.

Any of the XZ-A51 strains described above can be obtained by knocking out the alaD gene on the chromosome of the XZ-A12 strain. The XZ-A12 strain can be a genetic engineering bacterium obtained by integrating an alanine dehydrogenase gene on a Geobacillus stearothermophilus XL-65-6 chromosome in a lactate dehydrogenase of an escherichia coli (E.coli) ATCC 8739 chromosome, sequentially knocking out a pyruvate formate lyase gene (pflB gene), a fumarate reductase gene (frd gene), an ethanol dehydrogenase gene (adhE gene), an acetate kinase (ackA gene), a methylglyoxal synthase (mgsA gene) and an alanine racemase gene (dadX gene) of the obtained escherichia coli chromosome, and then continuously subculturing in a fermentation tank.

Any of the XZ-A53 strains described above can be obtained by knocking out the alaD gene on the chromosome of the XZ-A47 strain. The strain XZ-A47 is disclosed in Chinese patent publication CN 103898089B, and is named as XZ-A47. The XZ-A47 strain is an engineering strain which can tolerate tap water and can efficiently produce L-alanine in a culture medium prepared by the tap water.

The XZ-A51(Paladmut) can be a recombinant strain obtained by introducing the plasmid Paladmut into the XZ-A51 strain.

The XZ-A53(Paladmut) can be a recombinant strain obtained by introducing the plasmid Paladmut into the XZ-A53 strain.

The nucleotide sequence of any one of the alaD genes can be shown as a sequence 3 in a sequence table.

The amino acid sequence of any AlaD protein can be shown as a sequence 4 in a sequence table.

Any one of the above alanines may specifically be L-alanine.

The alanine dehydrogenase mutant with obviously improved activity is obtained based on a screening technology of metabolic acclimation and is used for the construction of an L-alanine engineering strain and the efficient fermentation of L-alanine. Experiments prove that: the L-alanine yield and the enzyme activity of the L-alanine dehydrogenase of the strain provided by the invention are both greatly improved by 88.2 percent compared with the strain expressing wild-type alanine dehydrogenase, the fermentation capacity and the L-alanine yield of the L-alanine engineering strain can be effectively improved, and the strain has great application value.

Drawings

FIG. 1 is a map of the Palamut-1 plasmid.

FIG. 2 is a schematic diagram of a plasmid expressing the alaD gene.

Detailed Description

The present invention is further illustrated by the following examples, but any examples or combination thereof should not be construed as limiting the scope or embodiment of the present invention. The scope of the invention is defined by the appended claims, and the scope defined by the claims will be clearly understood by those skilled in the art from this description and the common general knowledge in the art. Those skilled in the art can make any modification or change to the technical solution of the present invention without departing from the spirit and scope of the present invention, and such modifications and changes are also included in the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The methods for preparing competent cells in the following examples are described in the following references: dower et al, 1988, Nucleic Acids Res 16: 6127-6145.

The pKD46 plasmid (Datsenko and Wanner 2000, Proc Natl Acad Sci USA 97: 6640-. The MicroPulser electroporator is a product of Bio-Rad. pEASY Blunt simple vector is a product of Beijing Quanyujin Biotechnology, Inc. Phusion 5 Xbuffer is a product of NEB corporation.

Coli (E.coli) ATCC 8739 in the examples described below was deposited in the American Type Culture Collection (ATCC, address: American Type Culture Collection (ATCC)10801 University Boulevard Manassas, VA 20110 USA), and the strain was publicly available from the American Type Culture Collection. Escherichia coli (e.coli) ATCC 8739 is hereinafter abbreviated ATCC 8739 strain.

TABLE 1 Escherichia coli strains used in the present invention

TABLE 2 plasmids used in the present invention

TABLE 3 primers used in the present invention

The strain XZ-A47 in the following examples is described in patent document CN103898089A entitled "a strain which produces L-alanine at a high yield and is resistant to tap water and a method for constructing the same", and is publicly available to the applicant, and the biomaterial is used only for repeating the experiments related to the present invention and is not used for other purposes.

The nucleotide sequence of the alaD gene of alanine dehydrogenase derived from Geobacillus stearothermophilus XL-65-6 in the following examples is shown as sequence 3 in the sequence listing. The amino acid sequence of the protein coded by the alaD gene (namely the alaD protein) is shown as a sequence 4 in a sequence table.

Example 1 construction of XZ-A51 Strain

From XZ-A12, the alanine dehydrogenase alaD gene from Geobacillus stearothermophilus XL-65-6 integrated at the ldhA site was knocked out by a two-step homologous recombination method. The method comprises the following specific steps:

construction of XZ-A50 Strain

1. A DNA fragment I for homologous recombination was obtained by PCR amplification using a plasmid pXZ-CS (Tan et al, apple Environ Microbiol.2013, 9:4838-4844) containing a chloramphenicol resistance gene cat and a fructan sucrose transferase gene sacB as a template and using primers ldhA-cat-up/ldhA-sacB-down, and the DNA fragment I was 2719bp in total (sequence 1 in the sequence listing). The DNA fragment I includes 50 bases upstream of the lactate dehydrogenase gene ldhA, a cat-sacB DNA fragment, and 50 bases downstream of the ldhA gene.

The amplification system is as follows: new England Biolabs Phusion 5 Xbuffer 10U L, dNTP (10 mM each dNTP) 1U L, DNA template 20ng, primers (10U M) 2.5U L, Phusion High-Fidelity DNA polymerase (2.5U/. mu.L) 0.5U L, distilled water 32.5U L, total volume 50U L.

The amplification conditions were: pre-denaturation at 98 ℃ for 2 min (1 cycle); denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 10 seconds, and extension at 72 ℃ for 30 seconds (30 cycles); extension at 72 ℃ for 5min (1 cycle).

2. After completion of step 1, the pKD46 plasmid was transformed into XZ-A12 strain by calcium chloride transformation, and then the DNA fragment I was electrically transferred into XZ-A12 strain carrying pKD46, to obtain XZ-A50 strain.

The method comprises the following specific steps: firstly, preparing competent cells of XZ-A12 strain carrying pKD46 plasmid; 50 μ L of competent cells were placed on ice, 50ng of DNA fragment I was added, placed on ice for 2 minutes, and transferred to a 0.2cm Bio-Rad cuvette. A MicroPulser (Bio-Rad) electroporator was used with a shock parameter of 2.5 kv. After the electric shock, 1mL of LB medium was quickly transferred to a cuvette, and after 5 strokes, transferred to a tube, and incubated at 75rpm for 2 hours at 30 ℃. After 200. mu.L of the suspension was applied to LB plate containing chloramphenicol (final concentration: 34. mu.g/mL) and cultured overnight at 37 ℃, 5 single colonies were selected and verified by PCR using the primers XZ-ldhA-up/XZ-ldhA-down, and one correct single colony (correct single colony PCR amplification product: 3807bp DNA fragment) was selected and named as XZ-A50.

Construction of the second, XZ-A51 Strain

1. A DNA fragment II for homologous recombination was obtained by PCR amplification using ATCC 8739 genomic DNA as a template and the primers ldhA-del-up/XZ-ldhA-down, and the total length was 453bp (shown in sequence 2 in the sequence listing).

2. After completion of step 1, the pKD46 plasmid was transformed into XZ-A50 strain by calcium chloride transformation, and then the DNA fragment II was electrotransferred into XZ-A50 strain carrying pKD46, to obtain XZ-A51 strain.

The method comprises the following specific steps: firstly, preparing competent cells of XZ-A50 strain with pKD46 plasmid; 50. mu.L of competent cells were placed on ice, 50ng of DNA fragment II was added, and the mixture was placed on ice for 2 minutes and transferred to a 0.2cm Bio-Rad cuvette. A MicroPulser (Bio-Rad) electroporator was used with a shock parameter of 2.5 kv. After the electric shock, 1mL of LB medium was quickly transferred to a cuvette, and after 5 strokes, transferred to a tube, and incubated at 75rpm for 4 hours at 30 ℃. The culture broth was transferred to LB liquid medium without sodium chloride containing 10% sucrose (50 mL medium in a 250mL Erlenmeyer flask), and after culturing for 16-24 hours, streaked on LB solid medium without sodium chloride containing 6% sucrose. A correct single colony (a DNA fragment of 847bp which was a PCR-amplified product of the correct single colony) was selected by PCR verification using XZ-ldhA-up/XZ-ldhA-down primers and designated as XZ-A51.

Example 2 construction of AlaD mutant library of alanine dehydrogenase-encoding Gene

An artificial promoter M1-46 (this promoter M1-46 is described in the following document: construction of a stringent promoter on the chromosome of E.coli) was ligated to a commercial pACYC184 plasmid to obtain plasmid Palamut-1 (FIG. 1) for gene expression. The expression is carried out by connecting the alaD gene mutation library DNA fragment with an artificial promoter M1-46. Wherein the M1-46 artificial promoter is a weaker promoter, which is convenient for screening alanine dehydrogenase mutants with higher activity and can not increase the activity of intracellular enzymes due to the influence of too high transcription level. The method comprises the following specific steps:

construction of Palamut-2 plasmid for alaD gene mutation

1. PCR amplification was performed using the genomic DNA of XZ-A12 as a template and primers alaD-184up/alaD-184down to obtain an alaD-containing fragment of 1159bp in size.

2. The fragments containing alaD obtained by amplification were ligated into pEASY Blunt simple vector plasmid (all-in-one gold) to obtain the Palamut-2 plasmid.

The method comprises the following specific steps: mu.L of pEASY Blunt simple vector and 3. mu.L of PCR reaction solution containing alaD were gently mixed and reacted at room temperature for 5 minutes to obtain a ligation product. mu.L of the ligation product was added to 50. mu.L of TransT-T1 competent cells and gently mixed. The ice bath was carried out for 30 minutes, and the mixture was heat-shocked at 42 ℃ for 30 seconds and immediately placed on ice for 2 minutes. 500. mu.L of LB medium was added and incubated at 37 ℃ for 1 hour at 200 rpm. After incubation was complete, plates containing ampicillin were plated. PCR validation and sequencing was performed using M13F and M13R primers, and the correct clone stored the extracted plasmid, Palamut-2.

Mutation of the Dia, alaD Gene

The mutation of the alaD gene was accomplished using the GeneMorph II EZClone Domain Mutagenesis Kit (Agilent Technologies, Catalog # 200552).

The PCR amplification system is as follows: mu.L of 10 XMutazyme II buffer solution, 1. mu.L of each primer (alaD-184up/alaD-184down, 125 ng/. mu.L), 200ng of 40mM dNTP mixture, and water to make up to a total volume of 50. mu.L. mu.L of Mutazyme II DNA polymerase (2.5U/. mu.L) was added and mixed well.

The PCR amplification conditions were: pre-denaturation at 95 ℃ for 2 min (1 cycle); denaturation at 95 ℃ for 30 seconds, annealing at 53 ℃ for 30 seconds, and extension at 72 ℃ for 80 seconds (25 cycles); extension for 10 min at 72 ℃ (1 cycle).

Construction of three, alaD mutant library plasmid

And (3) recovering and purifying the mutant fragments of alaD obtained by PCR in the step two by cutting gel, and constructing the mutant fragments into a Palamut-1 plasmid by a CPEC method. The method comprises the following specific steps:

1. construction of the Palamut-1 plasmid backbone

The linearized plasmid backbone of Palamut-1 was obtained by PCR amplification using the Palamut-1 plasmid as template and primers 184-alaDup/184-alaDdown. Both ends of the backbone contain 20bp bases homologous to alaD. The PCR amplification system and conditions were as described in step two, except that the extension time was adjusted to 2 minutes.

After the completion of PCR, 1. mu.L of DpnI (NEB) was directly added and mixed, and reacted at 37 ℃ for 2 hours to eliminate the original Palamut-1 plasmid template, to obtain a single plasmid backbone fragment for subsequent reactions.

2. Construction of AlaD plasmid mutant library

The Palamut-1 plasmid backbone and the alaD mutant fragment were ligated into a library of plasmids containing the nick by the method of CPEC.

The PCR reaction system is as follows: new England Biolabs Phusion 5 Xbuffer 10U L, dNTP (10 mM each dNTP) 1U L, Palamut-1 plasmid backbone 200ng, alaD mutant 58ng, Phusion High-Fidelity DNA polymerase (2.5U/. mu.L) 0.5U L, DMSO (100%) 1.5U L, water to make up the total volume of 50U L.

The PCR amplification conditions were: pre-denaturation at 98 ℃ for 2 min (1 cycle); denaturation at 98 ℃ for 15 seconds, annealing at 55 ℃ for 15 seconds, and extension at 72 ℃ for 5 minutes (30 cycles); extension at 72 ℃ for 10 min (1 cycle).

Construction of four, alaD mutation library

Transferring the alaD mutant library plasmid containing the nick obtained in the third step into XZ-A51 strain by electric transformation to obtain the mutant library of the alaD gene. A schematic representation of the plasmid for the expression of the alaD gene is shown in FIG. 2.

The method comprises the following specific steps: 50 μ L of competent cells were placed on ice, 8 μ L of the alaD mutant library plasmid obtained in step three was added, placed on ice for 2 minutes, and transferred to a 0.2cm Bio-Rad cuvette. A Micro Pulser (Bio-Rad) electroporator was used, and the electric shock parameter was 2.5 kv. After the electric shock, 1mL of LB medium was quickly transferred to a cuvette, and after 5 strokes, transferred to a test tube, and incubated at 37 ℃ for 1 hour at 200 rpm. The incubated broth was spread on LB plates containing chloramphenicol (final concentration: 34. mu.g/mL) and cultured overnight at 37 ℃.20 clones are randomly picked and sequenced by using primers alamut-YZ-up/alamut-YZ-down, and the mutation rate is controlled to be 1-4.5 basic groups/kb, thereby meeting the requirement of library establishment.

Example 3 screening of the alaD mutant library

First, use metabolic domestication to carry on the primary screen

Since the growth rate of cells in the XZ-A51 strain containing the alaD gene and the alaD gene mutation library is proportional to the L-alanine producing ability, the higher the activity of L-alanine dehydrogenase, the stronger the L-alanine producing ability, and the better the cell growth. Thus, strains with higher enzymatic activity after the alaD mutation grow at a faster rate and are more easily screened for enrichment during serial passage. Based on this, the alaD gene mutant library clone obtained in example 2 was collected from the plate, washed 2 times with a seed medium, transferred to a fermentation medium, and cultured at 30 ℃ and 250rpm for 24 hours. The cells were transferred to a new medium at an initial OD550 of 0.1 and cultured continuously, and then transferred continuously for subculture. After continuous passage for 10-15 times, the fermentation liquor is diluted and coated on LB plate containing chloramphenicol.

The seed culture medium comprises the following components (solvent is distilled water): glucose 20g/L, ammonium chloride 5g/L, NaH₂PO₄ 5g/L，Na₂HPO₄ 5g/L，MgSO₄·7H₂O 1g/L，CaCl₂·2H₂O0.1 g/L, trace inorganic salt 5ml/L, and culture medium pH 6.5.

The trace inorganic salt comprises the following components: FeCl₃·6H₂O 1.5mg，CoCl₂·6H₂O 0.1mg，CuCl₂·2H₂O 0.1mg，ZnCl₂ 0.1mg，Na₂MoO₄·2H₂O 0.1mg，MnCl₂·4H₂O0.2 mg, distilled water to a constant volume of 1L, and filtering for sterilization.

The fermentation medium comprises the following components: the same seed medium, except that the glucose concentration was 50 g/L.

The fermentation conditions were as follows: 250mL of fermentation medium was added to the 500mL fermentor. The initial OD550 of the transition was 0.1. The culture was carried out at 30 ℃ and 250 rpm. 5M aqueous ammonia was added dropwise to maintain the pH at 7.0.

Second, double screen of alaD mutation storehouse

The clones diluted and coated after the initial screening of the metabolic acclimatization are inoculated into a 96-hole deep-well plate, 1.5mL of LB medium (containing 34g/mL of chloramphenicol) is added into 2mL of deep-well plate, and the culture is carried out by shaking. The cells were collected by centrifugation at 4000rpm at 4 ℃. After washing twice with 500. mu.L of 100mM Tris-HCl (pH7.5) buffer, cells were lysed by addition of Lysozyme and DNase I and centrifuged, and the supernatant was collected for enzyme activity measurement.

The enzyme activity determination method comprises the following steps: a new 96-well plate was used, and 200. mu.L of an enzyme reaction solution (100mM Tris-HCl (pH7.5) buffer, 100mM sodium pyruvate, 100mM ammonium chloride, 0.3mM NADH) was added to each well. The reaction was initiated by adding the supernatant. The change of NADH absorbance at 340nm was detected by a microplate reader, and the enzyme activity was calculated.

Comparison of enzyme Activity and L-alanine production of alaD mutant

1. Evaluation of L-alanine fermentation Performance

And (3) fermenting the 10 alaD mutants with the most remarkable enzyme activity change after screening in the second step in a 500mL fermentation tank respectively and detecting the yield of the L-alanine. Meanwhile, taking fermentation broth in the logarithmic phase of fermentation for enzyme activity determination. Plasmid Palad-184 containing the wild-type alaD gene was used as a control. The method comprises the following specific steps:

the compositions of the seed culture medium and the fermentation culture medium are the same as those in the step one.

10 clones were inoculated in seed medium, respectively, 50mL of the seed medium was contained in a 250mL triangular flask, and cultured at 37 ℃ and 250rpm until logarithmic phase, to obtain seed solutions.

According to initial OD₅₅₀The cultured seed solution was inoculated into a fermentation medium at 0.1, and 250mL of the fermentation medium was contained in a 500mL fermentor. Fermenting at 30 deg.C and 250rpm for 72h (adding 5M ammonia water dropwise during fermentation to maintain pH of 7.0) to obtain fermentation broth.

The components of the fermentation broth were determined by high performance liquid chromatography using Agilent 1200. The concentration of glucose and organic acid in the fermentation broth was measured by using an Aminex HPX-87H organic acid analytical column from Biorad. L-alanine quantification and chiral determination using a ligand exchange type chiral isomer liquid chromatography separation column (Chiralpak MA (+)) from Daciel.

2. AlaD enzyme Activity evaluation

30mL of the fermentation broth was taken at the logarithmic phase of the fermentation in step 1, centrifuged at 5000rpm at 4 ℃ and the cells were collected. Then, the cells were washed twice with 30mL of 100mM Tris-HCl buffer (pH7.5), and then suspended by adding 1mL of 100mM Tris-HCl buffer (pH7.5) to give a cell suspension. After the cell suspension was sonicated (35%, 1s/3s, 6min), it was centrifuged at 12000rpm at 4 ℃ for 40 minutes to obtain a crude extract of the enzyme, which was used for enzyme activity assay.

The enzyme activity determination system is as follows: 1mL of the reaction mixture contained 100mM Tris-HCl (pH7.5) buffer, 100mM sodium pyruvate, 100mM ammonium chloride, and 20. mu.L of the crude enzyme extract. NADH was added to the reaction solution to initiate the reaction (NADH concentration in the reaction system was 0.3 mM). The change in absorbance at 340nm of NADH was measured.

Through the evaluation of L-alanine fermentation performance and enzyme activity, a clone extraction plasmid (named Paladmut) with the most significant improvement in both L-alanine fermentation performance and enzyme activity is selected and sequenced. The sequencing primer is alamut-YZ-up/alamut-YZ-down.

The sequencing result shows that: the Paladmut plasmid contains a mutant gene shown as a sequence 5 in a sequence table, and the mutant gene is different from an alaD gene in that: the mutant gene is obtained by mutating the 981 th position of the alaD gene shown in the sequence 3 from a base A to a base C.

The protein encoded by the mutant gene was designated as L-alanine dehydrogenase mutant (AlaD mutant). The amino acid sequence of the L-alanine dehydrogenase mutant is shown as a sequence 6, and is obtained by mutating lysine (Lys) at the 327 th position of the amino acid sequence of the L-alanine dehydrogenase shown as a sequence 4 into asparagine (Asn).

The results of the enzyme activity and L-alanine production assay for AlaD are shown in Table 4. The results show that: compared with the XZ-A51 strain containing the Palad-184 plasmid, the XZ-A51 strain containing the Paladmut plasmid has 90 percent of L-alanine dehydrogenase activity and 88.2 percent of L-alanine yield.

TABLE 4 evaluation of alaD mutants

Example 4 application of AlaD mutants in Industrial L-alanine fermentation

The production cost of the L-alanine can be greatly reduced by using the culture medium prepared by tap water to produce the L-alanine by fermentation. The strain XZ-A47 in the embodiment is disclosed in Chinese patent publication CN 103898089B, and is named as XZ-A47. The XZ-A47 strain is an engineering strain which can tolerate tap water and can efficiently produce L-alanine in a culture medium prepared by the tap water.

Construction of L-alanine engineering strain

1. The alaD gene integrated at the ldhA site in the XZ-A47 strain is knocked out to obtain an engineering strain XZ-A53. The specific procedure was the same as in example 1.

2. The Palad-184 plasmid and the Paladmut plasmid were electroporated into XZ-A53 competent cells, respectively, to obtain the engineered strains XZ-A53(Palad-184) and XZ-A53(Paladmut), respectively.

The Palad-184 plasmid is obtained by inserting the alaD gene sequence shown in sequence 3 into the M1-46 artificial promoter in the Palamut-1 plasmid.

The Paladmut plasmid is obtained by replacing the alaD gene sequence in the Palad-184 plasmid with the alaD mutant gene sequence shown in the sequence 5 and keeping other sequences unchanged.

Preparation of di, L-alanine

1. Preparation of culture Medium

The seed culture medium comprises the following components (solvent is tap water): glucose 20g/L, ammonium chloride 5g/L, NaH₂PO₄ 5g/L，Na₂HPO₄ 5g/L，MgSO₄·7H₂O 1g/L，CaCl₂·2H₂O0.1 g/L, trace inorganic salt 5ml/L, and culture medium pH 6.5.

The fermentation medium consists of (the solvent is tap water): 170g/L glucose, 5g/L ammonium chloride, NaH₂PO₄ 5g/L，Na₂HPO₄ 5g/L，MgSO₄·7H₂O 1g/L，CaCl₂·2H₂O0.1 g/L, trace inorganic salt 5ml/L, and culture medium pH 6.5.

2. Fermentation culture

1) The engineered strains XZ-A47, XZ-A53(Palad-184) and XZ-A53(Paladmut) were inoculated into a flask (250mL standard) containing 50mL of seed medium, and cultured at 37 ℃ and 250rpm until logarithmic phase to obtain seed solutions.

2) Inoculating the seed solution into a fermentation tank (3L) filled with 2.4L fermentation medium, wherein the initial inoculation amount is 0.1% (V/V); then, the mixture was fermented at 30 ℃ and 100rpm for 48 hours (ammonia water as a neutralizing agent during the fermentation, pH in the fermentor was controlled at 6.5) to obtain a fermentation broth.

3) The components of the fermentation broth were determined by high performance liquid chromatography using Agilent 1200. The glucose and organic acid concentrations in the fermentation broth were analyzed using an Aminex HPX-87H organic acid analytical column from Biorad. L-alanine quantification and chiral determination were performed using a ligand exchange type chiral isomer liquid chromatography separation column (Chiralpak MA (+)) from Daciel corporation.

The results are shown in Table 5. The results show that: the Paladmut mutant also shows good activity in engineering strains for industrial production. The L-alanine production of strain XZ-A53, which contained the Paladmut plasmid, was increased by 40.2% compared to strain XZ-A53, which contained the Palad-184 plasmid.

TABLE 5 application of alaD mutants in commercial fermentation strains

	L-alanine yield (g/L)	Mutation site of alaD gene
			XZ-A47	114	Wild type
XZ-A53(PalaD-184)	122	Wild type
			XZ-A53(PalaDmut)	171	Lys327Asn

Sequence listing

<110> institute of Tianjin Industrial Biotechnology of China academy of sciences, Inc. of Anhui Hua Heng Biotechnology

<120> alanine dehydrogenase mutant and application thereof in fermentation production of L-alanine

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 2719

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 1

agcggcaaga ttaaaccagt tcgttcgggc aggtttcgcc tttttccaga ttatttgtta 60

actgttaatt gtccttgttc aaggatgctg tctttgacaa cagatgtttt cttgcctttg 120

atgttcagca ggaagcttgg cgcaaacgtt gattgtttgt ctgcgtagaa tcctctgttt 180

gtcatatagc ttgtaatcac gacattgttt cctttcgctt gaggtacagc gaagtgtgag 240

taagtaaagg ttacatcgtt aggatcaaga tccattttta acacaaggcc agttttgttc 300

agcggcttgt atgggccagt taaagaatta gaaacataac caagcatgta aatatcgtta 360

gacgtaatgc cgtcaatcgt catttttgat ccgcgggagt cagtgaacag gtaccatttg 420

ccgttcattt taaagacgtt cgcgcgttca atttcatctg ttactgtgtt agatgcaatc 480

agcggtttca tcactttttt cagtgtgtaa tcatcgttta gctcaatcat accgagagcg 540

ccgtttgcta actcagccgt gcgtttttta tcgctttgca gaagtttttg actttcttga 600

cggaagaatg atgtgctttt gccatagtat gctttgttaa ataaagattc ttcgccttgg 660

tagccatctt cagttccagt gtttgcttca aatactaagt atttgtggcc tttatcttct 720

acgtagtgag gatctctcag cgtatggttg tcgcctgagc tgtagttgcc ttcatcgatg 780

aactgctgta cattttgata cgtttttccg tcaccgtcaa agattgattt ataatcctct 840

acaccgttga tgttcaaaga gctgtctgat gctgatacgt taacttgtgc agttgtcagt 900

gtttgtttgc cgtaatgttt accggagaaa tcagtgtaga ataaacggat ttttccgtca 960

gatgtaaatg tggctgaacc tgaccattct tgtgtttggt cttttaggat agaatcattt 1020

gcatcgaatt tgtcgctgtc tttaaagacg cggccagcgt ttttccagct gtcaatagaa 1080

gtttcgccga ctttttgata gaacatgtaa atcgatgtgt catccgcatt tttaggatct 1140

ccggctaatg caaagacgat gtggtagccg tgatagtttg cgacagtgcc gtcagcgttt 1200

tgtaatggcc agctgtccca aacgtccagg ccttttgcag aagagatatt tttaattgtg 1260

gacgaatcga attcaggaac ttgatatttt tcattttttt gctgttcagg gatttgcagc 1320

atatcatggc gtgtaatatg ggaaatgccg tatgtttcct tatatggctt ttggttcgtt 1380

tctttcgcaa acgcttgagt tgcgcctcct gccagcagtg cggtagtaaa ggttaatact 1440

gttgcttgtt ttgcaaactt tttgatgttc atcgttcatg tctccttttt tatgtactgt 1500

gttagcggtc tgcttcttcc agccctcctg tttgaagatg gcaagttagt tacgcacaat 1560

aaaaaaagac ctaaaatatg taaggggtga cgccaaagta tacactttgc cctttacaca 1620

ttttaggtct tgcctgcttt atcagtaaca aacccgcgcg atttacttag atctagcggc 1680

tatttaacga ccctgccctg aaccgacgac cgggtcgaat ttgctttcga atttctgcca 1740

ttcatccgct tattatcact tattcaggcg tagcaccagg cgtttaaggg caccaataac 1800

tgccttaaaa aaattacgcc ccgccctgcc actcatcgca gtactgttgt aattcattaa 1860

gcattctgcc gacatggaag ccatcacaaa cggcatgatg aacctgaatc gccagcggca 1920

tcagcacctt gtcgccttgc gtataatatt tgcccatggt gaaaacgggg gcgaagaagt 1980

tgtccatatt ggccacgttt aaatcaaaac tggtgaaact cacccaggga ttggctgaga 2040

cgaaaaacat attctcaata aaccctttag ggaaataggc caggttttca ccgtaacacg 2100

ccacatcttg cgaatatatg tgtagaaact gccggaaatc gtcgtggtat tcactccaga 2160

gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta acaagggtga acactatccc 2220

atatcaccag ctcaccgtct ttcattgcca tacggaattc cggatgagca ttcatcaggc 2280

gggcaagaat gtgaataaag gccggataaa acttgtgctt atttttcttt acggtcttta 2340

aaaaggccgt aatatccagc tgaacggtct ggttataggt acattgagca actgactgaa 2400

atgcctcaaa atgttcttta cgatgccatt gggatatatc aacggtggta tatccagtga 2460

tttttttctc cattttagct tccttagctc ctgaaaatct cgataactca aaaaatacgc 2520

ccggtagtga tcttatttca ttatggtgaa agttggaacc tcttacgtgc cgatcaacgt 2580

ctcattttcg ccaaaagttg gcccagggct tcccggtatc aacagggaca ccaggattta 2640

tttattctgc gaagtgatct tccgtcacat ttgtgctata aacggcgagt ttcataagac 2700

tttctccagt gatgttgaa 2719

<210> 2

<211> 453

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 2

ttcaacatca ctggagaaag tcttatgaaa ctcgccgttt atagcacaaa tctggaaaaa 60

ggcgaaacct gcccgaacga actggtttaa tcttgccgct cccctgcatt ccaggggagc 120

tgattcagat aatccccaat gacctttcat cctctattct taaaatagtc ctgagtcaga 180

aactgtaatt gagaaccaca atgaagaaag tagccgcgtt tgttgcgcta agcctgctga 240

tggcgggatg tgtaagtaat gacaaaattg ctgttacgcc agaacagcta cagcatcatc 300

gctttgtgct ggaaagcgta aacggtaagc ccgtgaccag cgataaaaat ccgccagaaa 360

tcagctttgg tgaaaaaatg atgatttccg gcagcatgtg taaccgcttt agcggtgaag 420

gcaaactgtc taatggtgaa ctgacagcca aag 453

<210> 3

<211> 1119

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 3

atgaagatcg gcattccaaa agaaatcaaa aacaatgaaa accgcgtcgc catcactccg 60

gcaggcgtga tgacgctcgt caaagcgggg catgacgtgt atgtggagac ggaagccggc 120

gctgggtcgg gtttttccga ttccgagtat gaaaaagccg gggcagtgat cgtgacgaaa 180

gcggaagatg cctgggcggc ggagatggtg ttgaaagtga aagaaccgct ggctgaggag 240

ttccgctatt ttcgccccgg attgattttg tttacgtatt tgcatttagc cgcggccgaa 300

gcgctcacga aagcgctcgt cgagcaaaaa gtggtcggca tcgcttacga gacggtgcag 360

cttgcgaacg gctcgctgcc gctgttgacg ccgatgagtg aagtcgccgg ccgcatgtcg 420

gtgcaagtcg gcgcccagtt tctcgagaag ccgcacggcg ggaaaggcat tttgcttggc 480

ggcgtgcccg gggtgcggcg cggcaaagtg acgatcatcg gcggcggcac agcggggacg 540

aacgcggcga aaatcgcggt cggcctcggg gcggacgtga cgattttgga cattaacgcc 600

gagcggctgc gcgagctcga tgatttgttc ggcgaccaag tgacgacgtt gatgtccaac 660

tcgtatcata tcgccgagtg cgtgcgcgaa tccgatttgg tcgtcggcgc cgtcttgatc 720

ccgggggcga aagcgccgaa gcttgtgacg gaagagatgg tgcgctcgat gacgccaggc 780

tcggtgttgg tcgacgtcgc cattgaccaa ggcggcattt ttgaaacgac cgaccgcgtc 840

acgacgcacg acgatccgac atacgtcaag cacggcgtcg tccattacgc cgtcgcgaac 900

atgccgggcg ctgtgccgcg tacgtcaaca ttcgcgctta cgaacgtcac gatcccatac 960

gccttgcaaa tcgccaacaa aggctaccgc gccgcttgcc tcgacaatcc ggcgctgtta 1020

aaagggatca acacgctcga cgggcacatc gtgtacgaag cggtcgcggc ggcgcacaac 1080

atgccgtata cggatgttca ttcgttgctg cagggatga 1119

<210> 4

<211> 372

<212> PRT

<213> Artificial sequence

<220>

<223>

<400> 4

Met Lys Ile Gly Ile Pro Lys Glu Ile Lys Asn Asn Glu Asn Arg Val

1 5 10 15

Ala Ile Thr Pro Ala Gly Val Met Thr Leu Val Lys Ala Gly His Asp

20 25 30

Val Tyr Val Glu Thr Glu Ala Gly Ala Gly Ser Gly Phe Ser Asp Ser

35 40 45

Glu Tyr Glu Lys Ala Gly Ala Val Ile Val Thr Lys Ala Glu Asp Ala

50 55 60

Trp Ala Ala Glu Met Val Leu Lys Val Lys Glu Pro Leu Ala Glu Glu

65 70 75 80

Phe Arg Tyr Phe Arg Pro Gly Leu Ile Leu Phe Thr Tyr Leu His Leu

85 90 95

Ala Ala Ala Glu Ala Leu Thr Lys Ala Leu Val Glu Gln Lys Val Val

100 105 110

Gly Ile Ala Tyr Glu Thr Val Gln Leu Ala Asn Gly Ser Leu Pro Leu

115 120 125

Leu Thr Pro Met Ser Glu Val Ala Gly Arg Met Ser Val Gln Val Gly

130 135 140

Ala Gln Phe Leu Glu Lys Pro His Gly Gly Lys Gly Ile Leu Leu Gly

145 150 155 160

Gly Val Pro Gly Val Arg Arg Gly Lys Val Thr Ile Ile Gly Gly Gly

165 170 175

Thr Ala Gly Thr Asn Ala Ala Lys Ile Ala Val Gly Leu Gly Ala Asp

180 185 190

Val Thr Ile Leu Asp Ile Asn Ala Glu Arg Leu Arg Glu Leu Asp Asp

195 200 205

Leu Phe Gly Asp Gln Val Thr Thr Leu Met Ser Asn Ser Tyr His Ile

210 215 220

Ala Glu Cys Val Arg Glu Ser Asp Leu Val Val Gly Ala Val Leu Ile

225 230 235 240

Pro Gly Ala Lys Ala Pro Lys Leu Val Thr Glu Glu Met Val Arg Ser

245 250 255

Met Thr Pro Gly Ser Val Leu Val Asp Val Ala Ile Asp Gln Gly Gly

260 265 270

Ile Phe Glu Thr Thr Asp Arg Val Thr Thr His Asp Asp Pro Thr Tyr

275 280 285

Val Lys His Gly Val Val His Tyr Ala Val Ala Asn Met Pro Gly Ala

290 295 300

Val Pro Arg Thr Ser Thr Phe Ala Leu Thr Asn Val Thr Ile Pro Tyr

305 310 315 320

Ala Leu Gln Ile Ala Asn Lys Gly Tyr Arg Ala Ala Cys Leu Asp Asn

325 330 335

Pro Ala Leu Leu Lys Gly Ile Asn Thr Leu Asp Gly His Ile Val Tyr

340 345 350

Glu Ala Val Ala Ala Ala His Asn Met Pro Tyr Thr Asp Val His Ser

355 360 365

Leu Leu Gln Gly

370

<210> 5

<211> 1119

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgaagatcg gcattccaaa agaaatcaaa aacaatgaaa accgcgtcgc catcactccg 60

gcaggcgtga tgacgctcgt caaagcgggg catgacgtgt atgtggagac ggaagccggc 120

gctgggtcgg gtttttccga ttccgagtat gaaaaagccg gggcagtgat cgtgacgaaa 180

gcggaagatg cctgggcggc ggagatggtg ttgaaagtga aagaaccgct ggctgaggag 240

ttccgctatt ttcgccccgg attgattttg tttacgtatt tgcatttagc cgcggccgaa 300

gcgctcacga aagcgctcgt cgagcaaaaa gtggtcggca tcgcttacga gacggtgcag 360

cttgcgaacg gctcgctgcc gctgttgacg ccgatgagtg aagtcgccgg ccgcatgtcg 420

gtgcaagtcg gcgcccagtt tctcgagaag ccgcacggcg ggaaaggcat tttgcttggc 480

ggcgtgcccg gggtgcggcg cggcaaagtg acgatcatcg gcggcggcac agcggggacg 540

aacgcggcga aaatcgcggt cggcctcggg gcggacgtga cgattttgga cattaacgcc 600

gagcggctgc gcgagctcga tgatttgttc ggcgaccaag tgacgacgtt gatgtccaac 660

tcgtatcata tcgccgagtg cgtgcgcgaa tccgatttgg tcgtcggcgc cgtcttgatc 720

ccgggggcga aagcgccgaa gcttgtgacg gaagagatgg tgcgctcgat gacgccaggc 780

tcggtgttgg tcgacgtcgc cattgaccaa ggcggcattt ttgaaacgac cgaccgcgtc 840

acgacgcacg acgatccgac atacgtcaag cacggcgtcg tccattacgc cgtcgcgaac 900

atgccgggcg ctgtgccgcg tacgtcaaca ttcgcgctta cgaacgtcac gatcccatac 960

gccttgcaaa tcgccaacaa cggctaccgc gccgcttgcc tcgacaatcc ggcgctgtta 1020

aaagggatca acacgctcga cgggcacatc gtgtacgaag cggtcgcggc ggcgcacaac 1080

atgccgtata cggatgttca ttcgttgctg cagggatga 1119

<210> 6

<211> 372

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Lys Ile Gly Ile Pro Lys Glu Ile Lys Asn Asn Glu Asn Arg Val

1 5 10 15

Ala Ile Thr Pro Ala Gly Val Met Thr Leu Val Lys Ala Gly His Asp

20 25 30

Val Tyr Val Glu Thr Glu Ala Gly Ala Gly Ser Gly Phe Ser Asp Ser

35 40 45

Glu Tyr Glu Lys Ala Gly Ala Val Ile Val Thr Lys Ala Glu Asp Ala

50 55 60

Trp Ala Ala Glu Met Val Leu Lys Val Lys Glu Pro Leu Ala Glu Glu

65 70 75 80

Phe Arg Tyr Phe Arg Pro Gly Leu Ile Leu Phe Thr Tyr Leu His Leu

85 90 95

Ala Ala Ala Glu Ala Leu Thr Lys Ala Leu Val Glu Gln Lys Val Val

100 105 110

Gly Ile Ala Tyr Glu Thr Val Gln Leu Ala Asn Gly Ser Leu Pro Leu

115 120 125

Leu Thr Pro Met Ser Glu Val Ala Gly Arg Met Ser Val Gln Val Gly

130 135 140

Ala Gln Phe Leu Glu Lys Pro His Gly Gly Lys Gly Ile Leu Leu Gly

145 150 155 160

Gly Val Pro Gly Val Arg Arg Gly Lys Val Thr Ile Ile Gly Gly Gly

165 170 175

Thr Ala Gly Thr Asn Ala Ala Lys Ile Ala Val Gly Leu Gly Ala Asp

180 185 190

Val Thr Ile Leu Asp Ile Asn Ala Glu Arg Leu Arg Glu Leu Asp Asp

195 200 205

Leu Phe Gly Asp Gln Val Thr Thr Leu Met Ser Asn Ser Tyr His Ile

210 215 220

Ala Glu Cys Val Arg Glu Ser Asp Leu Val Val Gly Ala Val Leu Ile

225 230 235 240

Pro Gly Ala Lys Ala Pro Lys Leu Val Thr Glu Glu Met Val Arg Ser

245 250 255

Met Thr Pro Gly Ser Val Leu Val Asp Val Ala Ile Asp Gln Gly Gly

260 265 270

Ile Phe Glu Thr Thr Asp Arg Val Thr Thr His Asp Asp Pro Thr Tyr

275 280 285

Val Lys His Gly Val Val His Tyr Ala Val Ala Asn Met Pro Gly Ala

290 295 300

Val Pro Arg Thr Ser Thr Phe Ala Leu Thr Asn Val Thr Ile Pro Tyr

305 310 315 320

Ala Leu Gln Ile Ala Asn Asn Gly Tyr Arg Ala Ala Cys Leu Asp Asn

325 330 335

Pro Ala Leu Leu Lys Gly Ile Asn Thr Leu Asp Gly His Ile Val Tyr

340 345 350

Glu Ala Val Ala Ala Ala His Asn Met Pro Tyr Thr Asp Val His Ser

355 360 365

Leu Leu Gln Gly

370

Claims

1. A protein obtained by mutating the 327 th lysine of an alanine dehydrogenase amino acid sequence into asparagine; the amino acid sequence of the alanine dehydrogenase is shown as a sequence 4 in a sequence table.

2. The protein of claim 1, wherein: the protein is (a1) or (a 2):

(a2) and (b) a fusion protein obtained by attaching a tag to the N-terminus or/and C-terminus of the amino acid sequence shown in (a 1).

3. A nucleic acid molecule encoding the protein of claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein: the nucleic acid molecule is a DNA molecule shown in a sequence 5 in a sequence table.

5. Any one of the following biomaterials (c1) to (c 3):

(c1) an expression cassette comprising the nucleic acid molecule of claim 3 or 4;

(c2) a recombinant vector comprising the nucleic acid molecule of claim 3 or 4;

(c3) a recombinant microorganism comprising the nucleic acid molecule of claim 3 or 4.

6. Any one of the following (d1) to (d5) is used:

(d1) use of the protein of claim 1 or 2 as an alanine dehydrogenase;

(d2) use of the nucleic acid molecule of claim 3 or 4 or the biomaterial of claim 5 for the preparation of an alanine dehydrogenase;

(d3) use of the protein of claim 1 or 2 or the nucleic acid molecule of claim 3 or 4 or the biomaterial of claim 5 for the construction of engineered alanine bacteria;

(d4) use of a protein according to claim 1 or 2 or a nucleic acid molecule according to claim 3 or 4 or a biological material according to claim 5 for the production or preparation of alanine;

(d5) use of a protein according to claim 1 or 2 or a nucleic acid molecule according to claim 3 or 4 or a biological material according to claim 5 for increasing the production of alanine.

7. A recombinant bacterium capable of producing alanine is characterized in that: an alanine dehydrogenase involved in a pathway for producing alanine by its metabolism, comprising the protein of claim 1 or 2.

8. A method for producing alanine, comprising the following steps in sequence:

(e1) increasing the expression level and/or activity of the protein of claim 1 or 2 in a starting bacterium to obtain a recombinant bacterium A; the recombinant bacterium A has an increased ability to produce alanine compared to the starting bacterium;

(e2) fermenting and culturing the recombinant bacterium A to obtain alanine.

9. The method of claim 8, wherein: the method for improving the expression level and/or activity of the protein in the starting bacteria is achieved by introducing a nucleic acid molecule encoding the protein into the starting bacteria.

10. The method according to claim 8 or 9, characterized in that: the spawn is escherichia coli.