Method for producing pyruvic acid by using non-NADP-dependent lactate dehydrogenase

Abstract

Provided is a method for producing pyruvic acid by using non-NADP-dependent lactate dehydrogenase. The method comprises: S1: constructing a recombinant Escherichia coli strain expressing lactate dehydrogenase; S2: obtaining lactate dehydrogenase-producing whole cells from the recombinant Escherichia coli strain expressing lactate dehydrogenase; and S3: obtaining pyruvic acid by means of a whole-cell catalytic reaction of the lactate dehydrogenase-producing whole cells. By means of constructing a genetically engineered Escherichia coli strain, the addition of NAD +／NADP + as electron acceptors is not required during the conversion to produce the pyruvic acid. Furthermore, the method achieves a higher pyruvic acid yield, simpler operation and reduced raw material consumption. The selected substrate, sodium lactate, is inexpensive and readily available, and the production process for the pyruvic acid is simple. The method has good prospects for industrial application.

Description

A method for producing pyruvate using non-NADP-dependent lactate dehydrogenase Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, relates to a method for producing pyruvate using a non-NADP-dependent lactate dehydrogenase. Background Technology

[0002] Pyruvate is a product of glycolysis and plays a crucial role in biological metabolism. As an important pharmaceutical and chemical product, many amino acid biosyntheses require pyruvate as a starting material, such as the production of L-leucine, L-tryptophan, and L-tyrosine. In addition, pyruvate is an effective starting material for the synthesis of some pesticides and plant protection products, and it is also an indispensable additive in animal cell culture media.

[0003] Pyruvic acid is finding increasingly wider applications and is in greater demand; however, its global production scale has not increased accordingly, resulting in its currently very high price. Therefore, research into the large-scale production of pyruvic acid has become a major focus in the fields of chemical engineering and biotechnology.

[0004] The traditional method for producing pyruvate is tartaric acid dehydration and decarboxylation. While simple to operate, this method suffers from severe pollution, low yield, and high raw material consumption. In recent years, biological methods for converting lactate to pyruvate have become the most competitive and attractive method due to their advantages of mild conditions, low environmental pollution, low cost, and high yield. Conventional bioconversion methods utilize NAD-dependent lactate dehydrogenases to catalyze the conversion of lactate to pyruvate, but this enzyme requires NAD+. ＋ / NADP ＋ It acts as an electron acceptor. NAD ＋ / NADP ＋ The high price and difficulty in reusing lactate dehydrogenase have limited its widespread use, which in turn limits the large-scale production of pyruvate. Summary of the Invention

[0005] The primary objective of this invention is to provide a method for producing pyruvate using a non-NADP-dependent lactate dehydrogenase, thereby enabling large-scale production of pyruvate and reducing production costs.

[0006] Therefore, the present invention provides the following technical solution.

[0007] One aspect of the present invention provides a method for producing pyruvate using a non-NADP-dependent lactate dehydrogenase, the method comprising the following steps:

[0008] S1: Constructing recombinant Escherichia coli with lactate dehydrogenase;

[0009] S2: Obtaining lactate dehydrogenase-producing whole cells from recombinant Escherichia coli using lactate dehydrogenase;

[0010] S3: Pyruvate is obtained through a whole-cell catalytic reaction of lactate dehydrogenase-producing whole cells.

[0011] In some preferred embodiments, step S1 includes:

[0012] S11: The lactate dehydrogenase gene was inserted into the expression plasmid using gene recombination technology to construct the lactate dehydrogenase recombinant expression plasmid.

[0013] S12: Transform the obtained lactate dehydrogenase recombinant expression plasmid into Escherichia coli to obtain lactate dehydrogenase recombinant Escherichia coli.

[0014] In some preferred embodiments, in step S11, the lactate dehydrogenase gene is selected from one or more of Escherichia coli BW25113, Klebsiella pneumoniae strain BH2511, and Pseudomonas thermotolerans.

[0015] In some preferred embodiments, in step S11, the amino acid sequence of the lactate dehydrogenase is the sequence shown in NCBI with accession NOs WP_000586962, WP_087874898.1, and WP_017940236.

[0016] In some preferred embodiments, in step S11, the Escherichia coli is Escherichia coli BW25113.

[0017] In some preferred embodiments, step S2 includes:

[0018] S21: The obtained recombinant lactate dehydrogenase Escherichia coli was cultured on LB plates for the first time and on LB liquid medium for the second time to obtain seed culture;

[0019] S22: The obtained seed culture was transferred to ZYM-5052 self-induction medium and cultured for the third time to obtain whole cells that produce lactate dehydrogenase.

[0020] In some preferred embodiments, in step S21, the first culture conditions are: overnight culture at 35~38°C.

[0021] In some preferred embodiments, in step S21, the second culture conditions are: temperature: 35~38℃, shaking speed: 200~220rpm, time: 6~8h.

[0022] In some preferred embodiments, in step S22, the inoculation amount of the seed liquid is 5-10% by volume.

[0023] In some preferred embodiments, in step S22, the third culture conditions are: temperature: 25~30℃, shaking speed: 200~220rpm, time: 16~18h.

[0024] In some preferred embodiments, step S3 includes:

[0025] S31: A whole-cell catalytic reaction system is constructed using sodium lactate as a substrate and whole cells that produce lactate dehydrogenase as enzymes;

[0026] S32: Performs a whole-cell catalytic reaction to obtain pyruvate.

[0027] In some preferred embodiments, in step S31, the whole-cell catalytic reaction system consists of: a phosphate buffer solution with a pH of 7.5 to 8.0, a whole-cell concentration of lactate dehydrogenase of 20 to 40 OD, and sodium lactate of 0.35 to 0.45 M.

[0028] In some preferred embodiments, in step S32, the conditions for the whole-cell catalytic reaction are: temperature 35~38℃, shaking speed 200~220rpm, and time 1~22h.

[0029] By employing the above technical solution, the present invention has at least the following advantages:

[0030] This invention constructs a novel genetically engineered *Escherichia coli* strain that overexpresses lactate dehydrogenase, thereby enabling the production of pyruvate. Compared to conventional enzymatic conversion methods, the *E. coli* strain constructed in this invention does not require the addition of NAD+ when producing pyruvate. ＋ / NADP ＋ As an electron acceptor, it circumvents NAD. ＋ / NADP ＋ This invention overcomes cost limitations and offers advantages such as higher pyruvate yield, simpler operation, and less raw material loss. The substrate sodium lactate used in this invention is inexpensive and readily available, and the pyruvate production process is simple, demonstrating promising prospects for industrial application.

[0031] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0032] Figure 1 shows the recombinant plasmid pattern constructed in Example 1. Detailed Implementation

[0033] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0034] Unless otherwise specified, the percentage content mentioned in this invention refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0035] Unless otherwise specified, all percentage concentrations mentioned in this invention refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0036] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows temperature fluctuations within the precision range controlled by the instrument.

[0037] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0038] Unless otherwise specified, the ZYM-5052 self-induction culture medium formulation used in the following examples is 100ml A + 2ml B + 2ml C + 200μl D + 100μl E (all are mass percentage concentrations); A. ZY: 1% tryptone, 0.5% yeast extract; B. 50×M: 1.25M Na2HPO4, 1.25M KH2PO4, 2.5M NH4Cl and 0.25M Na2SO4; C. 50×5052: 25% glycerol, 2.5% glucose, 10% arabinose; D. 1M MgSO4; E. 1000× micronutrients: 50mM FeCl3, 20mM CaCl2, 10mM MnCl2, 2mM each of CoCl2, NiCl2, Na2MoO4, Na2SeO3 and H3BO3; F. 20% arabinose; other materials and reagents are available commercially.

[0039] Example 1: Construction of recombinant Escherichia coli

[0040] 1. Constructing a recombinant expression plasmid for lactate dehydrogenase

[0041] The synthesized gene fragments of Ec-lld (Escherichia coli BW25113), Kp-lld (Klebsiella pneumoniae strain BH2511), and Pth-lld (Pseudomonas thermotolerans) were used as templates for PCR amplification. The primers used are shown in Table 1. The amplification program was as follows: denaturation at 95℃ for 5 minutes, followed by 30 cycles of denaturation at 95℃ for 30 seconds, annealing at 60℃ for 30 seconds, and polymerization at 72℃ for 60 seconds, and then polymerization at 72℃ for 5 minutes to obtain the PCR amplification product. The obtained PCR product was electrophoresed on a 1.0% agarose gel, and the DNA band of approximately 1200 bp was then purified. The recovered DNA fragments were seamlessly cloned with the pBAD-hisB vector digested with XhoI and NcoI to obtain recombinant expression plasmids corresponding to the three lactate dehydrogenase genes. The plasmid map is shown in Figure 1. The genes in the plasmids can refer to Ec-lld, Kp-lld, and Pth-lld.

[0042] The obtained recombinant expression plasmids were transformed into Escherichia coli BW25113, and then plated on LB agar plates containing ampicillin. Recombinant Escherichia coli overexpressing the corresponding lactate dehydrogenases were obtained by screening and were denoted as Ec-lld / Bw, Kp-lld / Bw and Pth-lld / Bw, respectively.

[0043] Table 1 Primer Sequences

[0044]

[0045] Example 2: Comparison of catalytic effects of lactate dehydrogenases from different sources

[0046] The three recombinant *E. coli* strains Ec-lld / Bw, Kp-lld / Bw, and Pth-lld / Bw constructed in Example 1 were induced and cultured according to the following method: First, the recombinant *E. coli* were transferred to LB medium at a volume ratio of 2% and cultured at 37°C and 220 rpm for 16 h to obtain a seed culture. Then, the obtained seed culture was transferred to self-induction medium at a volume ratio of 2% and cultured at 30°C and 220 rpm for 16 h. After the induction culture was completed, the obtained product was centrifuged at 4°C and 6000 rpm for 10 min, and the cell pellet was collected, which was the whole cell producing lactate dehydrogenase.

[0047] The obtained whole-cell lactate dehydrogenase-producing cells were subjected to whole-cell catalysis to produce pyruvate. The whole-cell catalytic system consisted of: pH 8.0 phosphate buffer, 20 OD whole-cell lactate dehydrogenase concentration, and 0.4 M sodium lactate. The obtained whole-cell catalytic system was incubated at 37℃ and shaken at 220 rpm for 22 h. Finally, the pyruvate content in the product was determined by high-performance liquid chromatography (HPLC): the column was Bio-rad Aminex HPX-87H, the mobile phase was 5 mM sulfuric acid, the column temperature was 35℃, the flow rate was 0.5 mL / min, and the detection wavelength was 210 nm. The results are shown in Table 2.

[0048] Table 2 Comparison of pyruvate production catalyzed by lactate dehydrogenases from different sources

[0049]

[0050] As shown in Table 2 above, the pyruvate conversion rate of recombinant Escherichia coli Kp-lld / Bw is the highest among the three enzymes, with a pyruvate content of 357.7 mM at 22 h, and a calculated pyruvate conversion rate of 89.4%. Therefore, recombinant Escherichia coli Kp-lld / Bw was selected for subsequent transformation condition optimization experiments.

[0051] Example 3: Optimization of whole-cell catalytic reaction system

[0052] 1. Determination of the optimal whole-cell concentration of lactate dehydrogenase

[0053] Recombinant Escherichia coli Kp-lld / Bw was induced and cultured according to the induction culture method described in Example 2. After induction culture, the cells were collected by centrifugation at 4°C and 6000 rpm for 10 min, yielding whole cells that produce lactate dehydrogenase.

[0054] The obtained whole-cell lactate dehydrogenase-producing cells were subjected to whole-cell catalysis to produce pyruvate. The whole-cell catalytic system used consisted of: pH 8.0 phosphate buffer, whole-cell lactate dehydrogenase concentrations of 20 OD, 30 OD, 40 OD, and 50 OD, and 0.4 M sodium lactate. The obtained whole-cell catalytic system was incubated at 37°C and shaken at 220 rpm for 6 h. Finally, the pyruvate content was determined by high-performance liquid chromatography (HPLC), and the results are shown in Table 3.

[0055] Table 3 Comparison of pyruvate production at different whole-cell concentrations

[0056]

[0057] As shown in Table 3 above, within the concentration range of 20–40 OD, the pyruvate content and conversion rate of the product increase with increasing whole-cell concentration. However, when the concentration is increased to 50 OD, the substrate reaction is essentially complete, and further increasing the OD has no significant effect on increasing pyruvate production. Therefore, the pyruvate content produced by recombinant *E. coli* Kp-lld / Bw at a cell concentration of 40 OD is the highest, reaching 387.9 mM. Calculations show that the highest pyruvate conversion rate at this concentration is 97.0%. Further increasing the cell concentration to 50 OD does not significantly increase pyruvate production and will increase the cost of the bacterial culture. Therefore, the optimal whole-cell concentration is determined to be 40 OD.

[0058] 2. Determination of the optimal substrate concentration

[0059] Recombinant Escherichia coli Kp-lld / Bw was induced and cultured according to the induction culture method described in Example 2. After induction culture, the cells were collected by centrifugation at 4°C and 6000 rpm for 10 min, yielding whole cells that produce lactate dehydrogenase.

[0060] The obtained whole-cell lactate dehydrogenase-producing cells were subjected to whole-cell catalysis to produce pyruvate. The whole-cell catalytic system used consisted of: pH 8.0 phosphate buffer, whole-cell lactate dehydrogenase concentration of 40 OD, and sodium lactate concentrations of 0.3 M, 0.4 M, 0.5 M, 0.6 M, and 0.8 M, respectively. The obtained whole-cell catalytic system was incubated at 37 °C and shaken at 220 rpm for 6 h. Finally, the pyruvate content was determined by high-performance liquid chromatography (HPLC), and the results are shown in Table 4.

[0061] Table 4 Comparison of pyruvate production at different substrate concentrations

[0062]

[0063] The results in Table 4 show that: ① When the substrate concentration is 0.3M and 0.4M, the pyruvate conversion rate is ≥95%, indicating that the substrate reaction is basically complete, but the pyruvate content is higher at 0.4M; ② When the substrate concentration is greater than 0.4M, the yield of pyruvate in the catalytic reaction does not change significantly and may even decrease, leading to a corresponding decrease in conversion rate. Considering all factors, the substrate concentration of 0.4M is the most effective. Calculations show that the pyruvate conversion rate under this condition is 97.3% after 6 hours.

[0064] Therefore, the optimal whole-cell catalytic reaction system was determined to be: pH 8.0 phosphate buffer, whole-cell concentration of lactate dehydrogenase 40 OD, and sodium lactate concentration of 0.4 M.

[0065] Example 4: Large-scale production of lactate dehydrogenase in whole cells

[0066] In another aspect of the present invention, a method for large-scale production of lactate dehydrogenase in whole cells is also provided, specifically comprising:

[0067] Seed culture: The glycerol recombinant Escherichia coli engineered strain preserved at -80℃ obtained in Example 1 was inoculated into LB medium and cultured at 25~42℃ and 100~300rpm for 6~8h to obtain seed culture;

[0068] Fermentation culture: The obtained seed liquid is inoculated into the fermentation medium at an inoculation rate of 5~10% (V / V) for fermentation culture. When the initial glucose in the fermentation medium is consumed, the feed medium is turned on. When the cell density reaches 600nm absorbance OD600 of 30, an inducer is added to induce protein expression. When the feed medium is exhausted, the fermentation is stopped, centrifuged, and the cell precipitate is collected.

[0069] In some preferred embodiments, the fermentation medium consists of: citric acid 2-5 g / L, potassium dihydrogen phosphate 10-20 g / L, diammonium hydrogen phosphate 2-5 g / L, polyether defoamer 0.1-1 mL / L, glucose 5-30 g / L, MgSO4·7H2O 0.3-1 g / L, VB1 5-15 mg / L, trace inorganic salt I 1-10 mL / L, and pH 7.0±0.5.

[0070] In some preferred embodiments, the composition of the trace inorganic salt I is: EDTA 820~840 mg / L, CoCl2·6H2O 240~250 mg / L, MnCl2·4H2O 1450~1500 mg / L, CuCl2·2H2O 140~150 mg / L, H3BO3 290~300 mg / L, Na2MoO4·2H2O 240~250 mg / L, Zn(CH3COO)2·2H2O 1250~1300 mg / L, and ferric citrate 9~10 g / L.

[0071] In some preferred embodiments, the fed-batch culture medium consists of: glucose 600-650 g / L, MgSO4·7H2O 2.0-2.5 g / L, and trace inorganic salt II 10.0-10.5 mL / L.

[0072] In some preferred embodiments, the trace inorganic salt II comprises: EDTA 1300~1350 mg / L, CoCl2·6H2O 400~450 mg / L, MnCl2·4H2O 2350~2400 mg / L, CuCl2·2H2O 250~300 mg / L, H3BO3 500~550 mg / L, Na2MoO4·2H2O 400~450 mg / L, Zn(CH3COO)2·2H2O 1600~1650 mg / L, and ferric citrate 4.0~4.5 g / L.

[0073] In some preferred embodiments, the inducing agent consists of: L-arabinose 1.0~1.2 g / L and MgSO4·7H2O 1.0~1.2 g / L.

[0074] In some preferred embodiments, the fermentation culture conditions are as follows:

[0075] Initial conditions: glucose concentration of 20-25 g / L, temperature of 35-37℃, air flow rate of 2-3 vvm, stirring speed of 300-320 rpm, and dissolved oxygen concentration of 99%-100%.

[0076] Fermentation process conditions: Adjust the air flow rate to 2~3 vvm, control the dissolved oxygen concentration to 30%~35% at all times, and use ammonia water to control the pH to 7.0~7.5 during the fermentation process.

[0077] In the above process, the initial conditions refer to the period from the start of cultivation until the initial glucose is consumed; the fermentation process refers to the period from the start of feeding culture medium to the end of fermentation.

[0078] Example 5: Production of pyruvate by fermentation

[0079] The fermentation medium consisted of: citric acid 1.7 g / L, potassium dihydrogen phosphate 14 g / L, diammonium hydrogen phosphate 4 g / L, polyether defoamer 0.5 mL / L, glucose 20 g / L, MgSO4·7H2O 0.6 g / L, vitamin B1 9 mg / L, trace inorganic salt I 10 mL / L, and pH 7.0. The trace inorganic salt I consisted of: EDTA 840 mg / L, CoCl2·6H2O 250 mg / L, MnCl2·4H2O 1500 mg / L, CuCl2·2H2O 150 mg / L, H3BO3 300 mg / L, Na2MoO4·2H2O 250 mg / L, Zn(CH3COO)2·2H2O 1300 mg / L, and ferric citrate 10 g / L.

[0080] The fed-batch culture medium consisted of: glucose 600 g / L, MgSO4·7H2O 2 g / L, polyether defoamer 2 mL / L, and trace inorganic salt II 10 mL / L. Trace inorganic salt II consisted of: EDTA 1300 mg / L, CoCl2·6H2O 400 mg / L, MnCl2·4H2O 2350 mg / L, CuCl2·2H2O 250 mg / L, H3BO3 500 mg / L, Na2MoO4·2H2O 400 mg / L, Zn(CH3COO)2·2H2O 1600 mg / L, and ferric citrate 4 g / L.

[0081] Seed culture: 100 mL of LB solution was placed in a 250 mL Erlenmeyer flask and sterilized at 121 °C for 20 min. After cooling, the glycerol-recombinant Escherichia coli engineered strain Kp-lld / Bw, stored at -80 °C, was inoculated. The culture temperature was 37 °C, the shaking speed was 200 rpm, and the culture time was 6-8 h to obtain the seed culture, which was used for inoculation of fermentation medium.

[0082] Fermentation Tank Inoculation: In a preferred embodiment of this study, the fermentation medium volume in a 5L fermenter is 2.0L. After sterilization, the above-mentioned seed culture is inoculated at an inoculation rate of 5% (V / V), with an initial glucose concentration of 20g / L. The temperature is 37℃, the initial airflow is 2vvm, the stirring speed is 300rpm, and the dissolved oxygen concentration is set to 100%. During cell growth, the airflow is adjusted up to 3vvm, while the stirring speed is correlated with the DO value to ensure that the dissolved oxygen concentration is always greater than 30%. When the initial glucose is depleted, feeding is initiated. Ammonia is used to control the pH at 7.0±0.5 during fermentation. After 16 hours of cultivation, an inducer (1g / L L-arabinose, 1g / L MgSO4·7H2O) is added to induce protein expression. Fermentation is terminated when the feeding medium is exhausted. After fermentation, the cells are centrifuged at 4℃ and 6000rpm for 10min, and the cell pellet is collected. Those skilled in the art can adjust the above conditions to a certain extent according to actual conditions without affecting the achievement of the purpose of this invention.

[0083] Fermentation continued under the above conditions until the feed medium was exhausted, at which point fermentation ended. The total fermentation time was 40 hours, and the bacterial concentration reached 140 OD. Samples were taken, centrifuged, and whole cells producing lactate dehydrogenase were collected and subjected to whole-cell catalysis.

[0084] The obtained whole-cell lactate dehydrogenase was used to produce pyruvate via whole-cell catalysis. The whole-cell catalytic system consisted of: pH 8.0 phosphate buffer, a cell concentration of 40 OD, and 0.4 M sodium lactate. The whole-cell catalytic system was incubated at 37°C and a shaking speed of 220 rpm for 6 h. Finally, the pyruvate content was determined by high-performance liquid chromatography (HPLC), and the results are shown in Table 5.

[0085] Table 5 Comparison of pyruvate production from different fermentation methods

[0086]

[0087] As shown in Table 5 above, when the bacterial sample obtained by fermentation culture of recombinant Escherichia coli strain Kp-lld / Bw for 40 h was used for whole-cell catalysis, the yield of pyruvate was 398.4 mM after 6 h of reaction, with a conversion rate of 99.6%, which was significantly higher than the yield and conversion rate of pyruvate obtained by whole-cell catalysis of the bacterial sample obtained by fermentation culture for 36 h.

[0088] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, various changes in form and detail can still be made by those skilled in the art within the scope of protection of the present invention.

[0089] The construction of genetically engineered bacteria, the composition of the culture medium, the culture method, and the transformation described above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Theoretically, other bacteria, filamentous fungi, actinomycetes, and animal cells can also undergo genome modification and be used to transform and produce pyruvate. Any modifications made within the principles and spirit of the present invention are equivalent to substitutions.

[0090] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for producing pyruvate using a non-NADP-dependent lactate dehydrogenase, characterized in that, The method includes the following steps: S1: Constructing recombinant Escherichia coli with lactate dehydrogenase; S2: Obtaining lactate dehydrogenase-producing whole cells from recombinant Escherichia coli using lactate dehydrogenase; S3: Pyruvate is obtained through a whole-cell catalytic reaction of lactate dehydrogenase-producing whole cells.

2. The method according to claim 1, characterized in that, Step S1 includes: S11: The lactate dehydrogenase gene was inserted into the expression plasmid using gene recombination technology to construct the lactate dehydrogenase recombinant expression plasmid. S12: Transform the obtained lactate dehydrogenase recombinant expression plasmid into Escherichia coli to obtain lactate dehydrogenase recombinant Escherichia coli.

3. The method according to claim 2, characterized in that, In step S11, the lactate dehydrogenase gene is selected from one or more of Escherichia coli BW25113, Klebsiella pneumoniae strain BH2511, and Pseudomonas thermotolerans.

4. The method according to claim 1, characterized in that, Step S2 includes: S21: The obtained recombinant lactate dehydrogenase Escherichia coli was cultured on LB plates for the first time and on LB liquid medium for the second time to obtain seed culture; S22: The obtained seed culture was transferred to ZYM-5052 self-induction medium and cultured for the third time to obtain whole cells that produce lactate dehydrogenase.

5. The method according to claim 4, characterized in that, In step S21, the first culture conditions are: overnight culture at 35~38℃.

6. The method according to claim 4, characterized in that, In step S21, the second culture conditions are: temperature: 35~38℃, shaking speed: 200~220rpm, time: 6~8h.

7. The method according to claim 4, characterized in that, In step S22, the inoculation amount of the seed liquid is 5-10% by volume. The conditions for the third culture were: temperature: 25~30℃, shaking speed: 200~220rpm, and time: 16~18h.

8. The method according to claim 1, characterized in that, Step S3 includes: S31: A whole-cell catalytic reaction system is constructed using sodium lactate as a substrate and whole cells that produce lactate dehydrogenase as enzymes; S32: Performs a whole-cell catalytic reaction to obtain pyruvate.

9. The method according to claim 8, characterized in that, In step S31, the whole-cell catalytic reaction system consists of: pH 7.5~8.0 phosphate buffer, whole-cell concentration of lactate dehydrogenase 20~40 OD, and sodium lactate 0.35~0.45 M.

10. The method according to claim 8, characterized in that, In step S32, the conditions for the whole-cell catalytic reaction are: temperature 35~38℃, shaking speed 200~220rpm, and time 1~22h.

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