Alanine dehydrogenase mutant and application thereof

Abstract

According to the alanine dehydrogenase mutant and the application thereof, compared with wild alanine dehydrogenase, the enzyme activity of the alanine dehydrogenase mutant is improved, the alanine dehydrogenase mutant can be used for fermentation production of L-alanine, and the production efficiency of the L-alanine is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of genetic engineering and fermentation, and in particular, the present application provides an alanine dehydrogenase mutant and its application. BACKGROUND

[0002] L-alanine is a non-essential amino acid that can be synthesized by the human body, and is mainly used as a protein component in physiology and participates in energy circulation. L-alanine is widely used in the fields of food, medicine and daily chemicals. In the food field, L-alanine is a flavor enhancer approved for use in China, which can improve the umami taste and moderate the salty taste when added to soy sauce, and can also be used for compound sweeteners for food and beverages; in the pharmaceutical field, L-alanine is the main raw material for the synthesis of vitamin B6 and aminopropanol, and can also be used as a pharmaceutical ingredient to produce enalapril, sofosbuvir and other drugs; in the daily chemical field, L-alanine is mainly used to synthesize the new green chelating agent MGDA and amino acid surfactants.

[0003] The main production methods of L-alanine include chemical synthesis, extraction, enzyme conversion and fermentation. Among them, fermentation has become the main method for large-scale production of L-alanine due to its lower production cost and environmental friendliness. Chinese patent CN110904062B discloses a strain with high yield of L-alanine, which introduces alanine dehydrogenase from Geobacillus stearothermophilus XL-65-6 into Escherichia coli and inactivates the competitive metabolic pathway of pyruvic acid to achieve high-level synthesis of L-alanine in Escherichia coli, and it is the first time to realize industrial production. Among them, alanine dehydrogenase is an oxidoreductase that mainly catalyzes the reversible conversion between alanine and pyruvic acid. In anaerobic or high-ammonia environment, microorganisms use it to reduce and aminate pyruvic acid to produce alanine, which is a key step in the production of L-alanine. However, the catalytic activity of the existing alanine dehydrogenase directly limits the acid production capacity of the L-alanine engineering strain. Therefore, it is of great significance to develop L-alanine high-yield strains by mining enzymes with improved activity or high activity. SUMMARY

[0004] In order to solve the above problems, the present application mines a kind of alanine dehydrogenase mutant with P241L single point mutation compared with wild type by whole genome sequencing and comparison of a L-alanine production strain of Huaheng Bioengineering which is genetically modified and domesticated, the specific activity of the mutant is improved by 15.7% compared with wild type alanine dehydrogenase, and the L-alanine fermentation yield is improved by 32.8% when the mutant is introduced into the L-alanine production strain.

[0005] In the first aspect, the present application provides an alanine dehydrogenase mutant, which is any one of the following:

[0006] 1. An alanine dehydrogenase mutant, which is any one of the following:

[0007] (1) an alanine dehydrogenase mutant having a P241L mutation based on the sequence shown in SEQ ID NO: 1;

[0008] The alanine dehydrogenase mutant has alanine dehydrogenase activity, and its enzyme activity and / or specific activity is improved compared to the sequence shown in SEQ ID NO: 1 (wild-type alanine dehydrogenase);

[0009] (2) an alanine dehydrogenase variant obtained by substituting, deleting or adding one or at least two amino acid residues based on the amino acid sequence of the alanine dehydrogenase mutant of (1), and having the same or similar function as the alanine dehydrogenase mutant of (1).

[0010] In some embodiments, the specific activity of the alanine dehydrogenase mutant is improved by 15.7% compared to the wild-type alanine dehydrogenase.

[0011] In a second aspect, the present application provides a biological material, which is any one of the following A1) to A3):

[0012] A1) a nucleic acid molecule encoding the alanine dehydrogenase mutant of the first aspect;

[0013] A2) an expression cassette containing the nucleic acid molecule of A1);

[0014] A3) a recombinant vector containing the nucleic acid molecule of A1) or containing the expression cassette of A2).

[0015] In some embodiments, the nucleic acid molecule contains a DNA molecule of the coding sequence shown in SEQ ID NO: 3;

[0016] Preferably, the nucleotide sequence of the nucleic acid molecule is SEQ ID NO: 3.

[0017] In a third aspect, the present application provides a recombinant microorganism expressing the alanine dehydrogenase mutant of the first aspect, containing the nucleic acid molecule of A1) of the second aspect, containing the expression cassette of A2), or containing the recombinant vector of A3).

[0018] In some embodiments, the recombinant microorganism has the ability to produce L-alanine, and the host of the recombinant microorganism can be a strain that naturally exists or is genetically engineered to be able to produce L-alanine; it can be any strain known to those skilled in the art that has the ability to produce alanine in the prior art and is involved in alanine dehydrogenase in the pathway for producing alanine.

[0019] The L-alanine-producing engineered microorganism is a bacterial or fungal expression host; preferably a bacterium, more preferably an industrially commonly used Escherichia coli, Bacillus subtilis, or Corynebacterium glutamicum, etc.

[0020] In some embodiments, the recombinant microorganism can be further modified to attenuate the competitive metabolic pathway of pyruvate to achieve high level of L-alanine synthesis in the microorganism.

[0021] In some embodiments, the attenuation of the competitive metabolic pathway of pyruvate is achieved by at least one modification selected from the group consisting of B1) to B7) below:

[0022] B1) a modification of reduced activity of pyruvate formate lyase, by which modification the synthesis of mixed acids such as formic acid is reduced;

[0023] B2) a modification of reduced activity of alcohol dehydrogenase, by which modification the synthesis of ethanol is reduced;

[0024] B3) a modification of reduced activity of acetate kinase, by which modification the synthesis of acetate is reduced;

[0025] B4) a modification of reduced activity of methylglyoxal synthase, by which modification the synthesis of toxic by-product methylglyoxal is reduced;

[0026] B5) a modification of reduced activity of alanine racemase, by which modification the conversion of L-alanine to D-alanine is reduced;

[0027] B6) a modification of reduced activity of fumarate reductase, by which modification the metabolic flow from fumarate to succinate is reduced and the metabolic flow from oxaloacetate to phosphoenolpyruvate (PEP) to pyruvate is increased.

[0028] B7) a modification of reduced activity of lactate dehydrogenase, by which modification the synthesis of lactate is reduced.

[0029] In some embodiments, the modification described in B1) to B7) above can be achieved by the following:

[0030] B1) the activity of the pyruvate formate lyase is reduced by reducing the expression of the gene pflB encoding the pyruvate formate lyase or by disrupting the gene;

[0031] B2) the activity of the alcohol dehydrogenase is reduced by reducing the expression of the gene adhE encoding the alcohol dehydrogenase or by disrupting the gene;

[0032] B3) the activity of the acetate kinase is reduced by reducing the expression of the gene mgsA encoding the acetate kinase or by disrupting the gene;

[0033] B4) the activity of the methylglyoxal synthase is reduced by reducing the expression of the gene mgsA encoding the methylglyoxal synthase or by disrupting the gene;

[0034] B5) the activity of the alanine racemase is reduced by reducing the expression of the gene dadX encoding the alanine racemase or by disrupting the gene;

[0035] B6) the activity of the fumarate reductase is reduced by reducing the expression of the gene frd encoding the fumarate reductase or by disrupting the gene; and / or

[0036] B7) the activity of the lactate dehydrogenase is reduced by reducing the expression of the gene IdhA encoding the lactate dehydrogenase or by disrupting the gene.

[0037] In some embodiments, the nucleic acid molecule of the second aspect is integrated at the site of the lactate dehydrogenase-encoding gene IdhA.

[0038] In some embodiments, all the activities of B1) to B7) above are reduced by knocking out the genes encoding the relevant enzymes.

[0039] In a fourth aspect, the present application provides uses, at least one of the following C1) to C3):

[0040] C1) use of the alanine dehydrogenase mutant of the first aspect or the biological material of the second aspect in constructing an engineered bacterium producing L-alanine;

[0041] C2) use of the alanine dehydrogenase mutant of the first aspect, the biological material of the second aspect, or the recombinant microorganism of the third aspect in producing L-alanine;

[0042] C3) use of the alanine dehydrogenase mutant of the first aspect, the biological material of the second aspect, or the recombinant microorganism of the third aspect in improving the yield of L-alanine.

[0043] In a fifth aspect, the present application provides a method for producing L-alanine, the method comprising: culturing the recombinant microorganism of the third aspect; and collecting L-alanine from the culture.

[0044] In some embodiments, the recombinant microorganism of the third aspect produces L-alanine by fermentation, preferably an anaerobic fermentation process.

[0045] In some embodiments, the recombinant microorganism of the third aspect produces L-alanine by anaerobic fermentation, and the yield of L-alanine is improved by about 32.7%, and the sugar acid conversion rate is improved by about 4.6%.

[0046] It is to be understood that the foregoing detailed description of the application is intended to be illustrative only and not limiting. Various changes and modifications will become apparent to those skilled in the art, from the detailed description.

[0047] Technical effects: Compared with the wild-type alanine dehydrogenase from Geobacillus stearothermophilus XL-65-6, the enzyme activity of the alanine dehydrogenase mutant screened in the application is improved, and the specific enzyme activity is improved by 15.7%; when introduced into an L-alanine production chassis, the L-alanine yield is increased by about 32.7%, and the sugar acid conversion rate is increased by about 4.6%. DETAILED DESCRIPTION

[0048] The application is described in detail below.

[0049] 1. Terms

[0050] The term "weakening" of the competitive metabolic pathway of pyruvate refers to significantly reducing or completely blocking the carbon flux of the target metabolic pathway through genetic modification or environmental regulation, including but not limited to: partial or complete inhibition of the activity of related enzymes in the competitive metabolic pathway (such as gene knockout, promoter down-regulation, introduction of competitive inhibitors); expression regulation at the transcriptional or translational level (such as CRISPRi, antisense RNA interference).

[0051] The concept of the term "weakening" of an enzyme or protein includes all cases of reduced activity or absence of activity compared to the intrinsic activity or wild-type enzyme. Weakening can be used interchangeably with the terms "down-regulation", "reduction", "decrease", "weakening", "inactivation", etc.

[0052] The reduction of the activity of the enzyme or protein can be confirmed by determining the activity of the protein, or by confirming the reduction of the expression of the gene encoding the enzyme or protein, and the reduction of the expression of the gene can be confirmed by confirming the reduction of the amount of transcription of the gene or the reduction of the amount of protein expressed by the gene.

[0053] Specifically, in the present application, the weakening of the activity can be achieved by:

[0054] 1) deleting part or all of the polynucleotide encoding the protein,

[0055] 2) modifying the expression control sequence for reducing the expression of the polynucleotide,

[0056] 3) modifying the polynucleotide sequence on the chromosome to weaken the activity of the protein,

[0057] 4) selecting a method from the combination to weaken the activity of the protein.

[0058] However, the method is not limited thereto.

[0059] The term "nucleic acid molecule encoding an enzyme" refers to any naturally occurring or artificially synthesized DNA / RNA molecule capable of encoding an enzyme protein, including genes, cDNAs, synthetic fragments, etc.

[0060] The nucleic acid molecule can be introduced into a host cell in the form of an expression cassette, or can be introduced into a host cell by itself, and can be operably linked to sequences necessary for expression in a host cell.

[0061] The term "expression cassette" is a polynucleotide construct comprising all elements for expression of a gene, including a promoter operably linked to the gene, a transcription termination signal, a ribosome binding site, and a translation termination signal.

[0062] The term "vector" refers to a DNA construct containing a base sequence operably linked to suitable control sequences for expressing a target gene in a suitable host. The control sequences can include a promoter that initiates transcription, certain operator sequences that control such transcription, sequences that encode suitable ribosome binding sites on mRNA, and sequences that control transcription and translation termination. The vector used in the present application is not particularly limited and can be any vector known in the art as long as it can replicate in a host; or the vector used in the present application is a vector capable of transforming a host cell, thereby inserting a nucleic acid molecule or polynucleotide encoding a target protein into the host cell chromosome.

[0063] The term "functionally the same or similar" means that the enzyme activity and / or specific activity of the alanine dehydrogenase mutant is improved compared to wild-type alanine dehydrogenase, to the same extent as the alanine dehydrogenase mutant of the present application, to a higher extent than the alanine dehydrogenase mutant of the present application, or to a reduced extent but still improved compared to wild-type.

[0064] The term "recombinant microorganism" refers to a strain containing a nucleic acid molecule, an expression cassette, or a recombinant vector of interest, and the host cell of the recombinant microorganism can be a eukaryotic cell or a prokaryotic cell. In some embodiments, the host cell is a eukaryotic cell, and commonly used eukaryotic cells include Saccharomyces, such as S. cerevisiae. In some embodiments, the host cell is a prokaryotic cell, and commonly used prokaryotic cells include Enterobacteriaceae, Bacillaceae, or corynebacteriaceae bacteria, such as Corynebacterium, Bacillus, Escherichia bacteria, commonly used such as E. coli, B. subtilis, C. glutamicum, etc.

[0065] 2. The experimental methods in the following examples are all routine methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified.

[0066] The materials, reagents, etc. used in the following examples can be obtained from commercial sources, unless otherwise specified.

[0067] 1) The kits, solutions, media and their formulations involved in the following examples are as follows:

[0068] General Agarose Gel DNA Recovery Kit (DP209) was purchased from Tiangen Biochemical Technology Co., Ltd.

[0069] Plasmid Miniprep Kit (DP103) was purchased from Tiangen Biochemical Technology Co., Ltd.

[0070] 2x Phanta Mix High-Fidelity Enzyme was purchased from Novozyme, Cat. No. P525-01.

[0071] DH5α Competent Cells were purchased from Novozyme, Cat. No. C502-03.

[0072] 1000x Kanamycin Sulfate (Kan, 0.05 g / mL stock concentration 50 mg / ml)

[0073] 1000x Spectinomycin Dihydrochloride (SD, 0.05 g / mL stock concentration 50 mg / ml)

[0074] 100x L-Arabinose (L-Ara, 1 mol / L stock concentration 0.01 mol / L)

[0075] 100x Rhamnose (1 mol / L stock concentration 0.01 mol / L)

[0076] 5x Sucrose (50% stock concentration 2%)

[0077] Protein Purification Buffer A: NaCl 8.8 g / L, Tris-base 3 g / L, pH adjusted with NaOH / HCl

[0078] Protein Purification Buffer B: NaCl 8.8 g / L, Tris-base 3 g / L, Imidazole 32 g / L, pH adjusted with NaOH / HCl LB Broth: 10 g / L Tryptone, 5 g / L Yeast Extract, 10 g / L Sodium Chloride, pH 7.2.

[0079] LB Plate: 20 g / L Agar powder was added to the LB Broth.

[0080] Fermentation medium: glucose 50 g / L, yeast powder 1 g / L, ammonium chloride 5 g / L, NaH2PO4 5 g / L, Na2HPO4 5 g / L, MgSO4·7H2O 1 g / L, CaCl2·2H2O 0.1 g / L, trace inorganic salt 5 ml / L, medium pH 7; wherein, the composition of trace inorganic salt is: FeCl3·6H2O 1.5 mg / L, CoCl2·6H2O 0.1 mg / L, CuCl2·2H2O 0.1 mg / L, ZnCl2 0.1 mg / L, Na2MoO4·2H2O 0.1 mg / L, MnCl2·4H2O 0.2 mg / L.

[0081] 2) The strain, plasmid and primer information involved in the following examples are as follows: the primer information used in the application is shown in Table 1.

[0082] Table 1

[0083]

[0084]

[0085] The strain and plasmid information constructed in the application are shown in Table 2.

[0086] Table 2

[0087]

[0088] 3) The method involved in the following examples is as follows:

[0089] (1) PCR reaction method:

[0090] PCR reaction system (50 μL): ddH2O 20 μL, primer F (10 mM) 2 μL, primer R (10 mM) 2 μL, plasmid template 1 μL, 2×Phanta Mix high-fidelity enzyme 25 μL;

[0091] The PCR reaction program is: ① 95℃, 3min ② 95℃, 15s; Tm℃, 10s; 72℃, 30s / kb; repeat 30-35 cycles, ③ 72℃, 5min; 4℃ store the product.

[0092] (2) Overlapping PCR method:

[0093] PCR reaction system (50 μL): primer F (10 mM) 2 μL, primer R (10 mM) 2 μL, template fragment 1 μL each, 2×Phanta Mix high-fidelity enzyme 25 μL, ddH2O to 50 μL;

[0094] PCR reaction procedure: ① 95℃, 3min ② 95℃, 15s; Tm℃, 10s; 72℃, 30s / kb; repeat 30-35 cycles, ③ 72℃, 5min; 4℃ store product.

[0095] (3) Alanine dehydrogenase enzyme activity detection method:

[0096] Alanine dehydrogenase enzyme is NADH / NAD+ dependent, since NADH has maximum absorbance near 340nm, by detecting the change of NADH absorbance at 340nm, the enzyme can be determined.

[0097] Reaction system is 200μL: 100mM Tris-HCl (pH=7.4) buffer, 100mM sodium pyruvate, 100mM NH4Cl, 1mM NADH, 1μg protein, add water to 200μL.

[0098] Enzyme activity unit is defined as: the amount of enzyme required to catalyze 1μmol of NADH per minute.

[0099] Enzyme activity calculation formula:

[0100] EW is the change of absorbance at 340nm per minute, V is the reaction volume (mL), ε is the molar extinction coefficient of NADH (6.22L / mmol / cm), and ι is the optical path distance (cm).

[0101] Specific activity calculation formula:

[0102] (4) Sugar acid conversion rate (%) calculation: the ratio of the total mass of L-alanine g to the total glucose mass g consumed at the time of tank, i.e. conversion rate % = tank volume L x L-alanine concentration (g / L) / total glucose mass (g) x 100%.

[0103] (5) Alanine content detection: 1mL of fermentation broth is diluted 100 times in a 100mL volumetric flask, and after filtering the diluted solution with a 0.22μm water filter, high performance liquid chromatography is used for content detection.

[0104] HPLC determination conditions: chromatographic column HILIC Amphion II, 5μm, 4.6x150mm

[0105] Mobile phase preparation: solution A preparation: 0.05M potassium phosphate dibasic (pH adjusted to 3 with phosphoric acid, and then filtered with a 0.22μm water filter); solution B: acetonitrile (chromatographic grade). Mix A, B solutions uniformly according to the ratio A:B=1:3, ultrasonic degassing, and the mobile phase is obtained.

[0106] HPLC detection conditions: detection time 15 min; detection wavelength 250 nm; column oven temperature 35 °C; flow rate 1 mL / min; injection volume 10 uL.

[0107] (6) Glucose concentration detection: SBA-40D-100 biosensor analyzer was used to determine the glucose concentration.

[0108] Example 1: Obtaining alanine dehydrogenase mutation site

[0109] The L-alanine producing strain obtained by genetic modification and domestication from Huaheng Biotechnology was subjected to whole genome sequencing, and sequence alignment was performed with wild type alanine dehydrogenase with amino acid sequence shown as SEQ ID NO: 1. It was found that the alanine dehydrogenase mutant with P241L single point mutation compared with wild type amino acid sequence, denoted as AlaD*P241L.

[0110] The sequences of wild type AlaD (nucleotide sequence shown as SEQ ID NO: 2) and AlaD*P241L (nucleotide sequence shown as SEQ ID NO: 3) were provided to a biological company to synthesize pET28a / alaD and pET28a / alaD*P241L recombinant plasmids for subsequent enzyme activity detection and recombinant strain construction.

[0111] Example 2: Enzyme activity determination of wild type and mutant alanine dehydrogenase

[0112] 1. Protein expression and purification

[0113] The recombinant plasmids pET28a / alaD*P241L and pET28a / alaD were transformed into E. coli BL21 (DE3) respectively, and single colonies were picked and cultured overnight at 37 °C. According to 1% inoculation amount, they were transferred to 100 mL LB liquid medium and kanamycin was added. After 37 °C culture to OD 600 = 0.5-0.8, IPTG (0.2 mM) was added for low-temperature induction culture at 20 °C for 18-20 h.

[0114] The supernatant was discarded after centrifugation of the bacterial solution, and the precipitate was collected. The precipitate was resuspended with an appropriate amount of Buffer A, and was ultrasonically broken on ice for 20 min. The solution was centrifuged at 4°C and 12000 rpm for 30 min, and the supernatant was collected. The supernatant was combined with a nickel column, and eluent was prepared with Buffer A and Buffer B at different concentrations of imidazole. Gradient elution was performed, and the eluent was collected. The eluent at different gradients was added to Protein Loading Buffer, and was heated in boiling water for 8 min. The solution was centrifuged at high speed for 1 min, and the upper liquid was taken and spotted for SDS-PAGE electrophoresis detection. The voltage was 80 V for 0.5 h, and then was adjusted to 120 V for 1 h. The protein gel was dyed with Coomassie Brilliant Blue staining solution for 30 min, and was destained to observe the protein expression. According to the SDS-PAGE electrophoresis result, the protein eluent was concentrated in a protein concentration ultrafiltration tube in a 4°C refrigerated centrifuge at 4200 rpm. After the imidazole was washed away with an appropriate amount of Buffer A, the solution was concentrated to 0.5-1 mL and was sucked out. An appropriate amount of sterilized glycerol was added, and the solution was stored in a -40°C refrigerator for standby.

[0115] 2. Alanine dehydrogenase enzyme activity assay

[0116] An equal amount of AlaD and AlaD*P241L proteins purified in step 1 were taken, and were detected according to the alanine dehydrogenase enzyme activity detection method described above. The specific activity of AlaD protein was 19.7 U / mg, and the specific activity of AlaD*P241L protein was 22.8 U / mg. It can be seen that the activity of AlaD after mutation increased by 15.7%, and it can be used for biological catalysis of pyruvic acid to synthesize L-alanine, so as to improve the production efficiency of L-alanine.

[0117] Example 3: Construction of L-alanine production strains WL7 and WL8

[0118] 3.1. Construction of recombinant Escherichia coli WL7 expressing mutant alanine dehydrogenase AlaD*P241L

[0119] 1. Construction of Escherichia coli WΔpflB (WL1) strain

[0120] The endogenous gene pflB was knocked out on the basis of Escherichia coli W to obtain Escherichia coli WΔpflB (WL1). The specific steps are as follows:

[0121] S1, Preparation of E. coli W competent cells and electroporation of pEcCas plasmid: E. coli W single colony was inoculated into a 250 mL flask containing 50 mL LB liquid medium, and incubated at 37°C, 200 r / min until OD600 was about 0.5-0.6. The culture was centrifuged at 4°C, 5000 r / min for 5 min. The supernatant was discarded, and the pellet was resuspended in 20 mL 10% glycerol. The resuspended pellet was centrifuged again at 4°C, 5000 r / min for 5 min. The glycerol washing step was repeated once. The final pellet was resuspended in 1 mL 10% glycerol, and aliquoted at 100 μL per tube. The aliquots were stored at -80°C for later use. The pECas9 plasmid and the above E. coli W competent cells were added to an electroporation cup, and subjected to 2.5KV electric shock using a MicroPulser (Bio-Rad) electroporation instrument. Then, 1 mL LB liquid medium was added to the electroporation cup, and transferred to a 1.5 mL centrifuge tube. The tube was incubated at 37°C, 220 rpm for 1-2 h. The tube was plated on an LB plate containing kanamycin, and incubated at 37°C to obtain positive transformants (E. coli W / pEcCas). The positive transformants were prepared into competent cells according to the above steps.

[0122] S2, Construction of pTarget targeting plasmid: The pTarget plasmid was used as a template for PCR reaction using pflB-N20-F / R primers. The PCR product was purified using a gel recovery kit, and transformed into DH5α competent cells. The positive transformants were subjected to plasmid extraction to obtain the pTarget targeting plasmid.

[0123] S3, Construction of donor: E. coli w was used as a template for PCR using pflB-up-F / R and pflB-down-F / R primers, respectively. The PCR products were purified using a gel recovery kit after agarose gel electrophoresis to obtain pflB-up and pflB-down fragments. The two fragments were used as templates for overlap PCR using pflB-up-F and pflB-down-R primers. The donor fragment was obtained after purification using a gel recovery kit.

[0124] S4, E. coli WL1 construction: according to the above step S1 electroporation step, the appropriate amount of pTarget targeting plasmid and donor fragment are simultaneously electroporated into E. coli W / pEcCas competent cells, the mutant strain is screened using pflB-id-F / R primers, the fragment with correct band size is sequenced and verified, the correct strain is inoculated into LB liquid medium containing rhamnose, and cultured at 37°C, 220r / min for 6h, then centrifuged at 5000r / min for 5min, the supernatant is discarded, resuspended with 10mL LB liquid medium, cultured at 37°C, 220r / min for 3h, then streaked on LB plate containing sucrose, and cultured overnight, a single colony is selected and screened on LB plate containing kanamycin, spectinomycin and no antibiotic, and the strain without antibiotic is selected and named as WL1.

[0125] 2. E. coli WΔpflBΔadhE (WL2) strain construction

[0126] On the basis of E. coli WL1, the endogenous gene adhE is knocked out to obtain E. coli WΔpflBΔadhE (WL2), and the specific steps are as follows:

[0127] Preparation of E. coli WL1 / pEcCas competent cells: according to the preparation and electroporation steps of the competent cells described in S1 of step 1 above, the pEcCas plasmid is electroporated into E. coli WL1 competent cells.

[0128] Construction of pTarget plasmid: using pTarget plasmid as template, PCR reaction is carried out using adhE-N20-F / R primers, the product is purified using gel recovery kit after agarose gel electrophoresis, and the positive transformant is transformed into DH5α competent cells, and the pTarget targeting plasmid is obtained by plasmid extraction.

[0129] Donor construction: using E. coli W as template, PCR is carried out using adhE-up-F / R and adhE-down-F / R primers, respectively, the product is purified using gel recovery kit after agarose gel electrophoresis, adhE-up and adhE-down fragments are obtained; then using adhE-up-F and adhE-down-R primers, overlap PCR is carried out using the two fragments as templates, and the donor fragment is obtained after gel recovery kit purification.

[0130] E. coli WL2 construction: according to the above electroporation steps, the appropriate amount of pTarget plasmid and donor fragment were simultaneously electroporated into E. coli WL1 / pEcCas competent cells, the mutant strain was screened using adhE-id-F / R primers, the fragment with correct band size was sequenced and verified, the correct strain was inoculated in LB liquid medium containing rhamnose, and cultured at 37°C, 220r / min for 6h, then centrifuged at 5000r / min for 5min, the supernatant was discarded, resuspended with 10mL LB liquid medium, cultured at 37°C, 220r / min for 3h, then streaked on LB plate containing sucrose, and cultured overnight, a single colony was selected and screened on kanamycin, spectinomycin and antibiotic-free plates, and the antibiotic-free strain was named WL2.

[0131] 3. Construction of E. coli WΔpflBΔadhEΔackA (WL3) strain

[0132] On the basis of E. coli WL2, the endogenous gene ackA was knocked out to obtain E. coli WΔpflBΔadhEΔmgsA (WL3), and the specific steps were as follows:

[0133] Preparation of E. coli WL2 / pEcCas competent cells: according to the preparation and electroporation steps of S1 in the above step 1, the pEcCas plasmid was electroporated into E. coli WL2 competent cells.

[0134] Construction of pTarget plasmid: using pTarget plasmid as template, PCR reaction was carried out using ackA-N20-F / R primers, the product was purified using gel recovery kit after agarose gel electrophoresis, and the positive transformant was transformed into DH5α competent cells, and the pTarget targeting plasmid was obtained by plasmid extraction.

[0135] Construction of donor: using E. coli W as template, PCR was carried out using ackA-up-F / R and ackA-down-F / R primers, respectively, the product was purified using gel recovery kit after agarose gel electrophoresis, and the ackA-up and ackA-down fragments were obtained; then using ackA-up-F and ackA-down-R primers, the donor fragment was obtained by overlapping PCR using the two fragments as templates.

[0136] E. coli WL3 construction: according to the above-mentioned electroporation steps, the appropriate pTarget plasmid and donor fragment were simultaneously electroporated into E. coli WL2 / pEcCas competent cells, the ackA-id-F / R primer was used to screen the mutant strain, the fragment with correct band size was sequenced and verified, the correct strain was inoculated in LB liquid medium containing rhamnose, and cultured at 37°C, 220r / min for 6h, then centrifuged at 5000r / min for 5min, the supernatant was discarded, resuspended with 10mL LB liquid medium, cultured at 37°C, 220r / min for 3h, then streaked on LB solid plate containing sucrose, and cultured overnight, a single colony was selected and screened on kanamycin, spectinomycin and antibiotic-free plates, and the antibiotic-free strain was named WL3.

[0137] 4. Construction of E. coli WΔpflBΔadhEΔackAΔmgsA (WL4) strain

[0138] On the basis of E. coli WL3, the endogenous gene mgsA was knocked out to obtain E. coli WΔpflBΔadhEΔackAΔmgsA (WL4), and the specific steps were as follows:

[0139] Preparation of E. coli WL3 / pEcCas competent cells: according to the preparation and electroporation steps of S1 in the above-mentioned step 1, the pEcCas plasmid was electroporated into E. coli WL3 competent cells.

[0140] Construction of pTarget plasmid: using pTarget plasmid as template, mgsA-N20-F / R primers were used for PCR reaction, and the product was purified by agarose gel electrophoresis and gel recovery kit, then transformed into DH5α competent cells, and the positive transformants were subjected to plasmid extraction to obtain pTarget targeting plasmid.

[0141] Donor construction: using E. coli W as template, mgsA-up-F / R and mgsA-down-F / R primers were used for PCR, respectively, and the product was purified by agarose gel electrophoresis and gel recovery kit to obtain mgsA-up and mgsA-down fragments; then using mgsA-up-F and mgsA-down-R primers for overlap PCR with the two fragments as templates, the donor fragment was obtained after purification by gel recovery kit.

[0142] E. coli WL4 construction: according to the above-mentioned electroporation steps, the appropriate pTarget plasmid and donor fragment were simultaneously electroporated into E. coli WL3 / pEcCas competent cells, the mutant strain was screened using mgsA-id-F / R primers, the fragment with correct band size was sequenced and verified, the correct strain was inoculated into LB liquid medium containing rhamnose, and cultured at 37°C, 220r / min for 6h, then centrifuged at 5000r / min for 5min, the supernatant was discarded, resuspended with 10mL LB liquid medium, cultured at 37°C, 220r / min for 3h, then streaked on LB solid plate containing sucrose, and cultured overnight, a single colony was selected and screened on kanamycin, spectinomycin and antibiotic-free plates, and the antibiotic-free strain was named WL4.

[0143] 5. Construction of E. coli WΔpflBΔadhEΔackAΔmgsAΔdadX (WL5) strain

[0144] Knockout of endogenous gene dadX based on E. coli WL4 to obtain E. coli wΔpflBΔadhEΔackAΔmgsAΔdadX (WL5), the specific steps are as follows:

[0145] Preparation of E. coli WL4 / pEcCas competent cells: according to the preparation and electroporation steps of S1 in the above-mentioned step 1, the pEcCas plasmid was electroporated into E. coli WL4 competent cells.

[0146] Construction of pTarget plasmid: using pTarget plasmid as template, PCR reaction was carried out using dadX-N20-F / R primers, the product was purified using gel recovery kit after agarose gel electrophoresis, and the positive transformant was subjected to plasmid extraction to obtain pTarget targeting plasmid.

[0147] Construction of donor: using E. coli W as template, PCR was carried out using dadX-up-F / R and dadX-down-F / R primers, respectively, the product was purified using gel recovery kit after agarose gel electrophoresis, to obtain dadX-up and dadX-down fragments; then using dadX-up-F and dadX-down-R primers for overlap PCR, the donor fragment was obtained after gel recovery kit purification.

[0148] E. coli WL5 construction: according to the above-mentioned electroporation steps, the appropriate pTarget plasmid and donor fragment were simultaneously electroporated into E. coli WL4 / pEcCas competent cells, the mutant strain was screened using dadX-id-F / R primers, the fragment with correct band size was sequenced and verified, the correct strain was inoculated in LB liquid medium containing rhamnose, and cultured at 37°C, 220r / min for 6h, then centrifuged at 5000r / min for 5min, the supernatant was discarded, resuspended with 10mL LB liquid medium, cultured at 37°C, 220r / min for 3h, then streaked on LB solid plate containing sucrose, and cultured overnight, a single colony was selected and screened on kanamycin, spectinomycin and antibiotic-free plates, and the antibiotic-free strain was named WL5.

[0149] 6. Construction of E. coli WΔpflBΔadhEΔackAΔmgsAΔdadXΔfrd (WL6) strain

[0150] Knocking out the endogenous gene frd based on E. coli WL5 to obtain E. coli wΔpflBΔadhEΔackAΔmgsAΔdadXΔfrd (WL6), the specific steps are as follows:

[0151] Preparation of E. coli WL5 / pEcCas competent cells: according to the preparation and electroporation steps of S1 in the above-mentioned step 1, the pEcCas plasmid was electroporated into E. coli WL5 competent cells.

[0152] Construction of pTarget plasmid: using pTarget plasmid as template, PCR reaction was carried out using frd-N20-F / R primers, the product was purified using gel recovery kit after agarose gel electrophoresis, and the positive transformant was subjected to plasmid extraction to obtain pTarget targeting plasmid.

[0153] Donor construction: using E. coli W as template, PCR was carried out using frd-up-F / R and frd-down-F / R primers, respectively, the product was purified using gel recovery kit after agarose gel electrophoresis, frd-up and frd-down fragments were obtained, and then using frd-up-F and frd-down-R primers, donor fragment was obtained by overlap PCR after gel recovery kit purification.

[0154] E. coli WL6 construction: according to the above electroporation steps, the appropriate amount of pTarget plasmid and donor fragment were simultaneously electroporated into E. coli WL5 / pEcCas competent cells, the mutant strain was screened using frd-id-F / R primers, the fragment with correct band size was sequenced and verified, the correct strain was inoculated in LB liquid medium containing rhamnose, and cultured at 37°C, 220r / min for 6h, then centrifuged at 5000r / min for 5min, the supernatant was discarded, resuspended with 10mL LB liquid medium, and cultured at 37°C, 220r / min for 3h, then streaked on LB solid plate containing sucrose, and incubated overnight, and single colonies were selected and screened on kanamycin, spectinomycin and antibiotic-free plates, and the antibiotic-free strain was named WL6.

[0155] 7. E. coli E. coli WΔpflBΔadhEΔackAΔmgsAΔdadXΔfrd; ΔldhA::alaD*P241L (WL7) strain construction

[0156] On the basis of E. coli WL6, the endogenous gene ldhA was knocked out and the alaD*P241L mutant gene (nucleotide sequence is SEQ ID NO: 3) was introduced into the ldhA site to obtain E. coli wΔpflBΔadhEΔackAΔmgsAΔdadXΔfrd; ΔldhA::alaD*PL (WL7), and the specific steps are as follows:

[0157] Preparation of E. coli WL6 / pEcCas competent cells: according to the preparation and electroporation steps of the competent cells described in S1 in the above step 1, the pEcCas plasmid was electroporated into E. coli WL6 competent cells.

[0158] pTarget plasmid construction: using pTarget plasmid as template, PCR reaction was carried out using ldhA-N20-F / R primers, the product was purified using gel recovery kit after agarose gel electrophoresis, and the positive transformant was subjected to plasmid extraction to obtain pTarget targeting plasmid.

[0159] donor construction: using pET28a / alaD*P241L as template, PCR amplification was carried out using primers alaD-F / R to obtain fragment 1; then using E. coli w as template, PCR amplification was carried out using ldhA-up-F / R and ldhA-down-F / R primers to obtain fragments 2 and 3; finally, using ldhA-up-F and ldhA-down-R primers, overlapping PCR was carried out using the three fragments as template, and the donor fragment was obtained after purification by gel recovery kit.

[0160] Construction of E. coli WL7: The pTarget plasmid and the donor fragment were simultaneously electroporated into E. coli WL6 / pEcCas competent cells according to the above-mentioned electroporation procedure. The mutant strain was screened using the ldhA-id-F / R primers, and the fragment with the correct band size was sequenced and verified. The correct strain was inoculated into LB liquid medium containing rhamnose, and cultured at 37°C and 220 r / min for 6 h. Then, the supernatant was discarded by centrifugation at 5000 r / min for 5 min, and the cells were resuspended in 10 mL of LB liquid medium. The resuspended cells were cultured at 37°C and 220 r / min for 3 h, and then streaked on LB solid medium containing sucrose. After overnight culture, single colonies were selected and screened on plates containing kanamycin, spectinomycin, and no antibiotics. The strain without antibiotics was named WL7.

[0161] 3.2, Construction of recombinant E. coli WL8 expressing wild-type alanine dehydrogenase AlaD

[0162] On the basis of E. coli WL6, the endogenous gene ldhA was knocked out, and the wild-type alaD gene (nucleotide sequence: SEQ ID NO: 2) was introduced into the ldhA site to obtain E. coli wApflBAdhEAdckAAdmsAAdadXAdfrd; AldhA::alaD (WL8). The specific steps are as follows:

[0163] Preparation of E. coli WL6 / pEcCas competent cells: Refer to step 7 above.

[0164] Construction of pTarget plasmid: The pTarget plasmid was used as the template for PCR reaction with ldhA-N20-F / R primers. After agarose gel electrophoresis, the product was purified using a gel recovery kit, transformed into DH5a competent cells, and the positive transformants were subjected to plasmid extraction to obtain the pTarget targeting plasmid.

[0165] Construction of donor: The pET28a / alaD was used as the template, and the primers alaD-F / R were used for PCR amplification to obtain fragment 1. Then, the E. coli w was used as the template, and the primers ldhA-up-F / R and ldhA-down-F / R were used for PCR amplification to obtain fragments 2 and 3, respectively. Finally, the three fragments were used as the templates for overlap PCR with the primers ldhA-up-F and ldhA-down-R. After purification with a gel recovery kit, the donor fragment was obtained.

[0166] E. coli WL8 construction: according to the above-mentioned electroporation procedure, an appropriate amount of pTarget plasmid was electroporated into WL6 / pEcCas competent cells together with the donor fragment, and the mutant strain was screened using ldhA-id-F / R primers. The fragment with the correct band size was sequenced and verified, and the correct strain was inoculated into LB liquid medium containing rhamnose and cultured at 37°C and 220 r / min for 6 h. Then, the supernatant was discarded by centrifugation at 5000 r / min for 5 min, and the bacteria were resuspended in 10 mL of LB liquid medium. After being cultured at 37°C and 220 r / min for 3 h, the bacteria were streaked on LB solid medium containing sucrose and incubated overnight. Single colonies were selected and screened on kanamycin-containing, spectinomycin-containing, and antibiotic-free plates, and the antibiotic-free strain was named WL8.

[0167] Example 4: Fermentation of L-alanine by recombinant E. coli

[0168] 1. Parallel reactor fermentation

[0169] Activation of the strain: the recombinant E. coli WL7 and WL8 strains on the LB plate were inoculated into 5 mL of LB liquid medium and incubated at 37°C and 200 rpm overnight.

[0170] Seed culture: the activated bacteria were inoculated into a 250 mL conical flask containing 50 mL of LB liquid medium at a 5% (v / v) inoculation amount, and incubated at 37°C and 220 rpm for about 8 h to obtain the seed culture.

[0171] Parallel reactor fermentation: 25 mL of the seed culture was inoculated into 500 mL of fermentation medium, and incubated at 37°C and 300 rpm for 43 h. No additional aeration was performed during the fermentation, and the pH was maintained at about 7.0.

[0172] 2. Product detection

[0173] 1 mL of the fermentation broth was taken into a 100 mL volumetric flask and diluted with water to a total volume of 100 mL. After the diluted solution was filtered using a 0.22 μm water filter, the L-alanine concentration was detected according to the above-mentioned HPLC detection method.

[0174] According to the detection, the L-alanine fermentation results of the recombinant E. coli WL7 and WL8 are shown in Table 3.

[0175] Table 3

[0176]

[0177] As shown in Table 3, compared with the wild-type AlaD, the introduction of the AlaD*P241L mutant into the L-alanine chassis increased the L-alanine fermentation yield of the microorganism by about 32.8%, and the sugar acid conversion rate was increased by about 4.9%.

[0178] It can be understood that, although the application has been described above with reference to the specific forms thereof, the application is not limited to the specific form described above. It is obvious to those skilled in the art that various equivalent changes can be made to the technical features involved in the application without departing from the spirit of the application described in the application, and these changes should all belong to the scope of the application.

Claims

1. Alanine dehydrogenase mutant, any of the following: (1) an alanine dehydrogenase mutant having a P241L mutation based on the sequence shown in SEQ ID NO: 1; The alanine dehydrogenase mutant has alanine dehydrogenase activity, and its enzyme activity and / or specific activity is improved compared to the sequence shown in SEQ ID NO: 1; (2) An alanine dehydrogenase variant obtained by substituting, deleting or adding one or at least two amino acid residues based on the amino acid sequence of the alanine dehydrogenase mutant described in (1), and having the same or similar function as the alanine dehydrogenase mutant described in (1).

2. Biomaterial, any one of the following A1) to A3): A1) a nucleic acid molecule encoding the alanine dehydrogenase mutant according to claim 1; A2) an expression cassette containing the nucleic acid molecule described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1) or the expression cassette described in A2).

3. The biomaterial according to claim 2, characterized in that: The nucleic acid molecule contains a DNA molecule encoding the sequence shown in SEQ ID NO: 3; preferably, the nucleotide sequence of the nucleic acid molecule is SEQ ID NO:

3.

4. A recombinant microorganism expressing the alanine dehydrogenase mutant according to claim 1, containing the nucleic acid molecule according to claim 2 or 3, containing the expression cassette according to claim 2, or containing the recombinant vector according to claim 2.

5. The recombinant microorganism according to claim 4, characterized in that: The recombinant microorganism has the ability to produce L-alanine, and the recombinant microorganism uses bacteria or fungi as expression hosts; preferably, the expression host is bacteria, more preferably Escherichia coli, Bacillus subtilis, or Corynebacterium glutamicum.

6. The recombinant microorganism according to claim 5, characterized in that: The recombinant microorganism is further modified to weaken the competing metabolic pathway of pyruvate.

7. The recombinant microorganism according to claim 6, characterized in that: The weakening of the competitive metabolic pathway of pyruvate is achieved by at least one modification selected from the following B1) to B7): B1) modification that reduces the activity of pyruvate formate lyase; B2) modification that reduces the activity of alcohol dehydrogenase; B3) Modification that reduces the activity of acetate kinase; B4) modification that reduces the activity of methylglyoxal synthase; B5) modification that reduces alanine racemase activity; B6) Modification that reduces the activity of fumarate reductase. B7) Modification that reduces the activity of lactate dehydrogenase. Preferably, the modifications in B1) to B7) are achieved by: B1) the activity of the pyruvate formate lyase is reduced by reducing the expression of the gene pflB encoding the pyruvate formate lyase or by disrupting the gene; B2) the activity of the alcohol dehydrogenase is reduced by reducing the expression of the alcohol dehydrogenase encoding gene adhE or by destroying the gene; B3) the activity of the acetate kinase is reduced by reducing the expression of the gene encoding acetate kinase, mgsA, or by disrupting the gene; B4) the activity of the methylglyoxal synthase is reduced by reducing the expression of the gene encoding methylglyoxal synthase, mgsA, or by destroying the gene; B5) the activity of the alanine racemase is reduced by decreasing the expression of the alanine racemase encoding gene dadX or by disrupting the gene; B6) the activity of the fumarate reductase is reduced by reducing the expression of the fumarate reductase encoding gene frd or by destroying the gene; and / or B7) the activity of the lactate dehydrogenase is reduced by reducing the expression of the lactate dehydrogenase encoding gene ldhA or by disrupting the gene; Preferably, the nucleic acid molecule according to claim 3 or 4 is integrated into the ldhA site of the lactate dehydrogenase encoding gene; Preferably, all modifications in B1) to B7) are achieved by knocking out enzyme encoding genes.

8. Application, which is at least one of the following C1) to C3): C1) Use of the alanine dehydrogenase mutant according to claim 1, or the biomaterial according to claim 2 or 3, in constructing an engineered bacterium that produces L-alanine; C2) Use of the alanine dehydrogenase mutant according to claim 1, the biomaterial according to claim 2 or 3, or the recombinant microorganism according to any one of claims 4 to 7 in the production of alanine; C3) Use of the alanine dehydrogenase mutant according to claim 1, the biomaterial according to claim 2 or 3, or the recombinant microorganism according to any one of claims 4 to 7 in improving alanine production and / or conversion rate.

9. A method for producing L-alanine, comprising: Cultivating the recombinant microorganism according to any one of claims 4 to 7; L-alanine was collected from the culture.

10. The method according to claim 9, characterized in that: The recombinant microorganism according to any one of claims 4 to 7 produces L-alanine by fermentation, preferably by anaerobic fermentation.

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