Methods for increasing lysine and pentamethylene diamine fermentation yield

Abstract

The application provides a method for improving the fermentation yield of lysine and pentanediamine, which is a method for dynamically regulating the content of NADH and NADPH in cells, the expression of a gapA gene in a corynebacterium glutamicum is controlled by using a growth-regulated promoter Pald, and the expression of an additionally introduced gapN gene is controlled by using a Psod promoter, so that the expression of gapA is strong in the early growth stage, thereby maintaining the normal NADH concentration in the cells and the cell growth rate, and in the middle and late exponential growth stage and the cell growth stable stage, due to the sharp decrease in the expression strength of Pald and the sharp increase in the expression strength of Psod, gapN is highly expressed, so that the NADPH concentration in the cells is significantly increased, thereby significantly improving the yield and the rate of lysine, and the strategy is also applicable to significantly improving the yield of pentanediamine.

Description

Technical Field

[0001] This invention belongs to the field of biochemical technology, specifically, it relates to a method for increasing the fermentation yield of lysine and pentanediamine. Background Technology

[0002] Lysine is an important essential amino acid with wide applications in feed additives, pharmaceuticals, and health products. Industrial production of lysine primarily relies on microbial fermentation, with commonly used industrial strains including *Corynebacterium glutamicum* and *Escherichia coli*. During lysine biosynthesis, four molecules of NADPH are required to synthesize one molecule of lysine. Therefore, enhancing NADPH supply is crucial for increasing lysine production. Reported methods include enhancing the expression of key genes in the pentose phosphate cycle to increase its metabolic flux, expressing transhydrogenase genes to achieve the conversion from NADH to NADPH, and replacing NADPH-dependent amino acid dehydrogenases in the lysine synthesis pathway with NADH-dependent dehydrogenases. The biosynthesis of pentanediamine is also highly dependent on NADPH supply; it is obtained by expressing lysine decarboxylases in lysine-producing strains.

[0003] NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase catalyzes the irreversible oxidation of glyceraldehyde-3-phosphate to produce NADPH. Previous studies have found that replacing the NADH-dependent glyceraldehyde-3-phosphate dehydrogenase gene gapA with the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase gene gapN in Corynebacterium glutamicum can improve the yield of lysine synthesis in Corynebacterium glutamicum. However, excessively high intracellular NADPH concentrations in the early stages of fermentation significantly inhibit the growth of Corynebacterium glutamicum, thus reducing lysine production intensity. Therefore, dynamically regulating intracellular NADPH levels to improve lysine yield and production without affecting cell growth and lysine production intensity has become a pressing technical challenge. Summary of the Invention

[0004] The purpose of this invention is to provide a method for increasing the fermentation yield of lysine and pentanediamine.

[0005] The present invention is conceived as follows: This invention provides a method for dynamically regulating intracellular NADH and NADPH levels. By utilizing the growth-regulated promoter Pald to control the expression of the gapA gene in Corynebacterium glutamicum, and by utilizing the Psod promoter to control the expression of an additionally introduced gapN gene, the expression of gapA is strong in the early stages of growth, thereby maintaining normal intracellular NADH concentration and cell growth rate. In the mid-to-late stages of exponential growth and the stationary phase of cell growth, due to the sharp decrease in the expression intensity of Pald and the sharp increase in the expression intensity of Psod, gapN is highly expressed, resulting in a significant increase in intracellular NADPH concentration, thereby significantly increasing the yield and production of lysine. This strategy is also applicable to significantly increasing the production of pentanediamine.

[0006] To achieve the objectives of this invention, in a first aspect, this invention provides recombinant Corynebacterium glutamicum, wherein the recombinant Corynebacterium glutamicum is constructed by replacing the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene gapA of Corynebacterium glutamicum with the promoter Pald of the endogenous acetaldehyde dehydrogenase gene of Corynebacterium glutamicum, and by codon optimization of the exogenous NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase gene, either by introducing it into Corynebacterium glutamicum via plasmid or by integrating it into the chromosome of Corynebacterium glutamicum through genetic engineering.

[0007] Preferably, the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase gene is derived from Streptococcus mutans.

[0008] Furthermore, the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase gene is driven by the Psod promoter.

[0009] In this invention, the starting strain can be Corynebacterium glutamicum ATCC 21543.

[0010] Secondly, the present invention provides a method for constructing recombinant Corynebacterium glutamicum, the method comprising: replacing the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene gapA of Corynebacterium glutamicum with the promoter of the endogenous acetaldehyde dehydrogenase gene Pald of Corynebacterium glutamicum using genetic engineering methods, and overexpressing the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase gene gapN derived from Streptococcus mutans driven by the Psod promoter in Corynebacterium glutamicum.

[0011] Specifically, the method for constructing the recombinant Corynebacterium glutamicum includes the following steps:

[0012] A. Construction of recombinant strain C. glutamicum-ald

[0013] a1. Using the genome of Corynebacterium glutamicum ATCC 21543 as a template, PCR was performed using ald-up-F and ald-up-R primers to obtain the gene fragment ald-up, and the PCR product was purified. Using the genome of Corynebacterium glutamicum ATCC 21543 as a template, PCR was performed using ald-F and ald-R primers to obtain the gene fragment ald, and the PCR product was purified. Using the genome of Corynebacterium glutamicum ATCC 21543 as a template, PCR was performed using ald-down-F and ald-down-R primers to obtain the gene fragment ald-down, and the PCR product was purified.

[0014] a2. The suicide plasmid pK18mobsacB of Corynebacterium glutamicum was double-digested with EcoRI / XbaI. The gene fragments ald-up, ald, and ald-down were ligated into pK18mobsacB in one step using the Gibson Assembly kit. The resulting recombinant plasmid was named pK18-ald.

[0015] a3. pK18-ald was transformed into Corynebacterium glutamicum ATCC 21543, and the recombinant strain obtained was named C. glutamicum-ald;

[0016] The recombinant bacteria C. glutamicum-ald contains the gapA gene, driven by the Pald promoter, with the sequence shown in SEQ ID NO:1.

[0017] B. Construction of recombinant strain C.glutamicum-ald / pXMJ-gapN

[0018] b1. The NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase gene gapN from Streptococcus mutans containing the Psod promoter and codon optimization was artificially synthesized. The sequence is shown in SEQ ID NO:3. This fragment was ligated into the shuttle vector pXMJ19 of Corynebacterium glutamicum, and the resulting recombinant plasmid was named pXMJ-gapN.

[0019] b2. Transform the recombinant plasmid pXMJ-gapN into the recombinant bacterium C. glutamicum-ald, and the resulting recombinant bacterium is named C. glutamicum-ald / pXMJ-gapN.

[0020] The primer sequences are as follows:

[0021] ald-up-F: 5′-cacagaattcgcaattttaaggtttgctggtg-3′;

[0022] ald-up-R: 5′-agtggcgtcgacaagcatgttttttgcagggaacgaacctg-3′;

[0023] ald-F: 5′-caggttcgttccctgcaaaaaacatgcttgtcgacgccact-3′;

[0024] ald-R: 5′-ataccaacacgaatggtcattgggtctcctttgggccacc-3′;

[0025] ald-down-F: 5′-ggtggcccaaaggagacccaatgaccattcgtgttggtat-3′;

[0026] ald-down-R: 5′-cacatctagattagagcttggaagctacgag-3′.

[0027] Preferably, steps a3 and b2 employ an electro-conversion method, with the following electro-shock conditions: voltage 2.5KV, resistance 200Ω, and capacitance 25μF (electro-shock cup width is 2mm).

[0028] Thirdly, the present invention provides the application of the recombinant Corynebacterium glutamicum or the recombinant Corynebacterium glutamicum constructed according to the method in the production of lysine by fermentation.

[0029] Fourthly, the present invention provides a method for increasing lysine fermentation yield, the method comprising: fermenting and culturing the recombinant Corynebacterium glutamicum or the recombinant Corynebacterium glutamicum constructed according to the method, and collecting lysine from the fermentation product.

[0030] Fifthly, the present invention provides an engineered bacterium that produces pentamethylenediamine, wherein the engineered bacterium is constructed by expressing a lysine decarboxylase gene in the recombinant Corynebacterium glutamicum or the recombinant Corynebacterium glutamicum constructed according to the method.

[0031] Preferably, the lysine decarboxylase gene is derived from *Escherichia coli*. The codon-optimized sequence of the *E. coli* lysine decarboxylase gene ldcC is shown in SEQ ID NO:4.

[0032] In a sixth aspect, the present invention provides a method for increasing the fermentation yield of pentanediamine, the method comprising: fermenting the engineered bacteria and collecting pentanediamine from the fermentation product.

[0033] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0034] This invention provides a method for dynamically regulating intracellular NADH and NADPH levels. By using the growth-regulated promoter Pald to control the expression of the gapA gene in Corynebacterium glutamicum and using the Psod promoter to control the expression of an additionally introduced gapN gene, the production of NADH and NADPH in cells at different fermentation cycles is balanced, thereby significantly improving the production and efficiency of lysine and pentanediamine. Detailed Implementation

[0035] This invention addresses the technical problem mentioned above, which requires dynamic control of intracellular NADH and NADPH levels to balance cell growth and the synthesis of lysine or pentanediamine. It utilizes two different promoters, Pald and Psod, to control the expression of gapA and gapN genes, respectively, thereby achieving switching of NADH and NADPH synthesis at different fermentation stages. Specifically, the Pald promoter exhibits high expression intensity in the early stages of fermentation, but its expression intensity rapidly decreases in the middle and later stages; conversely, the Psod promoter shows low expression intensity in the early stages of fermentation, but its expression intensity rapidly increases in the middle and later stages.

[0036] This invention provides a method for increasing the production of lysine and pentanediamine by dynamically regulating the supply of NADPH in Corynebacterium glutamicum.

[0037] The present invention adopts the following technical solution:

[0038] (1) The original promoter (SEQ ID NO:2) of the NADH-dependent glyceraldehyde-3-phosphate dehydrogenase gene gapA in Corynebacterium glutamicum was replaced with the Pald promoter; (2) The gapN gene containing the Psod promoter was expressed in the above strain; (3) The growth of the cells and the yield of lysine were detected by fermentation culture of the above strain; (4) Lysine decarboxylase ldcC was expressed in the above strain, and the yield of pentanediamine was detected by fermentation culture.

[0039] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.

[0040] The plasmids pEC-K18mob, pK18mobsacB, and vector pXMJ19 used in the following examples were purchased from Addgene.

[0041] Example 1: The original gapA promoter in Corynebacterium glutamicum was replaced with the Pald promoter.

[0042] Corynebacterium glutamicum ATCC 21543 is a lysine-producing bacterium. In this embodiment, the original promoter of gapA (SEQ ID NO:2) is replaced with the Pald promoter.

[0043] Using the genome of *Corynebacterium glutamicum* ATCC 21543 as a template, PCR was performed using primers ald-up-F (5′-cacagaattcgcaattttaaggtttgctggtg-3′) and ald-up-R (5′-agtggcgtcgacaagcatgttttttgcagggaacgaacctg-3′) to obtain the gene fragment ald-up of approximately 1.0 kb, which was then purified. Using the genome of *Corynebacterium glutamicum* ATCC 21543 as a template, PCR was performed using primers ald-F (5′-caggttcgttccctgcaaaaaacatgcttgtcgacgccact-3′) and ald-R (5′-ataccaacacgaatggtcattgggtctcctttgggccacc-3′) to obtain the gene fragment ald of approximately 0.36 kb, which was then purified. Using the genome of Corynebacterium glutamicum ATCC 21543 as a template, PCR was performed using ald-down-F (5′-ggtggcccaaaggagacccaatgaccattcgtgttggtat-3′) and ald-down-R (5′-cacatctagattagagcttggaagctacgag-3′) as primers to obtain the gene fragment ald-down of approximately 1.0 kb, and the PCR product was purified. The Corynebacterium glutamicum suicide plasmid pK18mobsacB (Journal of Biotechnology 104(2003)287-299) was double-digested with EcoRI / XbaI, and the gene fragments ald-up, ald, and ald-down were ligated into pK18mobsacB in one step using the Gibson Assembly kit (NEB). The resulting recombinant plasmid was named pK18-ald. pK18-ald was electroporated into *Corynebacterium glutamicum* ATCC21543 using an electroporator (Bio-Rad). The electroporation conditions were 2.5 kV, 200 Ω, and 25 μF (electroporation cuvette width 2 mm). Recombinant bacteria were obtained through two selection processes and named *C. glutamicum-ald*. The key characteristic of this recombinant bacteria is the gapA gene combination containing the ald promoter, the sequence of which is shown in SEQ ID NO:1.

[0044] As a control, this embodiment also constructed a control strain with direct knockout of the gapA gene. Using the genome of Corynebacterium glutamicum ATCC 21543 as a template, PCR was performed using gap-up-F (5′-cacagaattccgtgcgagcaggtcggtgca-3′) and gap-up-R (5′-atctttagaggagacacaacttagttcacatcgctaacgtgg-3′) primers to obtain a gene fragment gap-up of approximately 1.0 kb, and the PCR product was purified. Using the genome of Corynebacterium glutamicum ATCC 21543 as a template, PCR was performed using gap-down-F (5′-ccacgttagcgatgtgaactaagttgtgtctcctctaaagat-3′) and gap-down-R (5′-cacatctagagctgcatggcgcggtgcgtt-3′) as primers to obtain a gene fragment gap-down of approximately 1.0 kb, and the PCR product was purified. The Corynebacterium glutamicum suicide plasmid pK18mobsacB (Journal of Biotechnology 104(2003)287-299) was double-digested with EcoRI / XbaI, and the gene fragment gap-up and gap-down were ligated into pK18mobsacB in one step using the Gibson Assembly kit (NEB). The resulting recombinant plasmid was named pK18-gap. pK18-ald was transferred into Corynebacterium glutamicum ATCC 21543 via electroporation using an electroporator (Bio-Rad). The electroporation conditions were 2.5 kV, 200 Ω, and 25 μF (electroporation cup width was 2 mm). Recombinant bacteria were obtained through two screenings and named C. glutamicum-Δgap.

[0045] Example 2: Expression of the gapN gene with the Psod promoter

[0046] A gapN gene from *Streptococcus mutans*, containing the Psod promoter and codon optimization, was artificially synthesized. The sequence is shown in SEQ ID NO:3. This fragment was ligated into the shuttle vector pXMJ19 of *Corynebacterium glutamicum*, and the resulting recombinant plasmid was named pXMJ-gapN. The recombinant plasmid pXMJ-gapN was transformed into *Corynebacterium glutamicum* C. glutamicum-ald via electroporation (under the same conditions as in Example 1), and the resulting recombinant strain was named C. glutamicum-ald / pXMJ-gapN.

[0047] As a control, pXMJ-gapN was also transformed into strain ATCC 21543 and strain C. glutamicum-△gap by electroporation. The resulting recombinant strains were named C. glutamicum / pXMJ-gapN and C. glutamicum-△gap / pXMJ-gapN, respectively.

[0048] Example 3: Fermentation culture of recombinant Corynebacterium glutamicum to produce lysine

[0049] Corynebacterium glutamicum ATCC 21543 and recombinant strains C. glutamicum-ald / pXMJ-gapN, C. glutamicum / pXMJ-gapN, and C. glutamicum-Δgap / pXMJ-gapN were cultured overnight on LB agar plates. Single colonies from these fresh agar plates were inoculated into 250 ml shake flasks with baffles containing 30 ml of seed culture medium and incubated at 32°C and 200 rpm for 12 hours.

[0050] The seed culture medium formula includes (g / L): glucose 40, (NH4)2SO4 5.0, K2HPO4 1.5, MgSO4 1.0, MnSO4 0.005, FeSO4 0.005, and corn steep liquor 30.

[0051] The seed culture was inoculated into a 500ml shake flask with baffles containing 30ml of fermentation medium at a 10% inoculation rate and cultured at 32℃ and 200rpm for 72h.

[0052] The fermentation medium formula includes: glucose 80g / L, corn steep liquor 10g / L, urea 4.5g / L, ammonium sulfate 45g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate heptahydrate 0.5g / L, ferrous sulfate heptahydrate 10mg / L, manganese sulfate tetrahydrate 10mg / L, β-alanine 5mg / L, nicotinic acid 5mg / L, thiamine-hydrochloric acid 5mg / L, biotin 0.3mg / L, and calcium carbonate 30g / L.

[0053] During fermentation, product concentration and strain growth were detected by liquid chromatography, and the results are shown in Tables 1 and 2. As can be seen from Tables 1 and 2, by introducing Palm to dynamically control gapA expression and Psod to control gapN expression, *C. glutamicum-ald / pXMJ-gapN* significantly improved lysine yield and production efficiency without affecting the cell structure, and was significantly higher than the control strains *C. glutamicum / pXMJ-gapN* and *C. glutamicum-Δgap / pXMJ-gapN*, which directly knocked out the gapA gene or did not change gapA expression. Therefore, the dynamic control strategy adopted in this invention can significantly improve lysine yield.

[0054] Table 1. Growth of different strains (OD) 600 )

[0055]

[0056] Table 2. Lysine production (g / L) of different strains

[0057]

[0058] Example 4: Production of pentamethylenediamine by fermentation culture of recombinant Corynebacterium glutamicum

[0059] Using the genome of *Escherichia coli* MG1655 as a template, PCR was performed using ldcC-F (5′-cacagaattccatgaacatcatcgcaatcat-3′) and ldcC-R (5′-cacatctagactagccagccatcttcagga-3′) as primers to obtain the gene fragment ldcC of approximately 2.1 kb, and the PCR product was purified. The *Corynebacterium glutamicum* shuttle plasmid pEC-K18mob (Journal of Biotechnology 104(2003)287-299) was double-digested with EcoRI / XbaI, and the ldcC fragment was ligated to pEC-K18mob using the Gibson Assembly kit (NEB). The resulting recombinant plasmid was named pEC-ldcC. The recombinant plasmid was electroporated into Corynebacterium glutamicum ATCC 21543 and strains C.glutamicum-ald / pXMJ-gapN, C.glutamicum / pXMJ-gapN, and C.glutamicum-△gap / pXMJ-gapN. The resulting recombinant strains were named C.glutamicum / pEC-ldcC, C.glutamicum-ald / pXMJ-gapN / pEC-ldcC, C.glutamicum / pXMJ-gapN / pEC-ldcC, and C.glutamicum-△gap / pXMJ-gapN / pEC-ldcC, respectively.

[0060] Corynebacterium glutamicum / pEC-ldcC, C. glutamicum-ald / pXMJ-gapN / pEC-ldcC, C. glutamicum / pXMJ-gapN / pEC-ldcC, and C. glutamicum-Δgap / pXMJ-gapN / pEC-ldcC were cultured overnight on LB agar plates. Single colonies from these fresh agar plates were inoculated into 250 ml shake flasks with baffles containing 30 ml of seed culture medium and incubated at 32°C and 200 rpm for 12 hours.

[0061] The seed culture medium formula includes (g / L): glucose 40, (NH4)2SO4 5.0, K2HPO4 1.5, MgSO4 1.0, MnSO4 0.005, FeSO4 0.005, and corn steep liquor 30.

[0062] The seed culture was inoculated into a 500ml shake flask with baffles containing 30ml of fermentation medium at a 10% inoculation rate and cultured at 32℃ and 200rpm for 72h.

[0063] The fermentation medium formula includes: glucose 80g / L, corn steep liquor 10g / L, urea 4.5g / L, ammonium sulfate 45g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate heptahydrate 0.5g / L, ferrous sulfate heptahydrate 10mg / L, manganese sulfate tetrahydrate 10mg / L, β-alanine 5mg / L, nicotinic acid 5mg / L, thiamine-hydrochloric acid 5mg / L, biotin 0.3mg / L, and calcium carbonate 30g / L.

[0064] During fermentation, product concentration and strain growth were detected by liquid chromatography, and the results are shown in Tables 3 and 4. As can be seen from Tables 3 and 4, by introducing Pald to dynamically control gapA expression and Psod to control gapN expression, *C. glutamicum-ald / pXMJ-gapN / pEC-ldcC* significantly improved the yield and efficiency of pentanediamine without affecting the cell structure. These results were significantly higher than those of the control strains *C. glutamicum / pXMJ-gapN / pEC-ldcC* and *C. glutamicum-Δgap / pXMJ-gapN / pEC-ldcC*, which either directly knocked out the gapA gene or did not alter gapA expression. Therefore, the dynamic control strategy employed in this invention can also significantly improve the yield of pentanediamine.

[0065] Table 3. Growth of different strains (OD) 600 )

[0066]

[0067] Table 4. Pentanediamine production (g / L) by different strains

[0068]

[0069] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Recombinant Corynebacterium glutamicum, characterized in that, The recombinant Corynebacterium glutamicum is derived from the Corynebacterium glutamicum glyceraldehyde-3-phosphate dehydrogenase gene. gapA The promoter was replaced with the Pald promoter of the endogenous acetaldehyde dehydrogenase gene of Corynebacterium glutamicum, and the exogenous NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase gene gapN was codon optimized and then introduced into Corynebacterium glutamicum by plasmid or integrated into the chromosome of Corynebacterium glutamicum by genetic engineering. The NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase gene gapN is derived from Streptococcus mutans ( Streptococcus mutans ); The NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase gene gapN is driven by the Psod promoter. The recombinant Corynebacterium glutamicum contains the gapA gene, driven by the Pald promoter, with the sequence shown in SEQ ID NO:

1. The sequence of gapN, a NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase gene derived from Streptococcus mutans containing the Psod promoter and codon optimization, is shown in SEQ ID NO:

3.

2. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that, The starting strain was Corynebacterium glutamicum ATCC 21543.

3. A method for constructing recombinant Corynebacterium glutamicum, characterized in that, The method includes: using genetic engineering techniques to modify the glyceraldehyde-3-phosphate dehydrogenase gene of Corynebacterium glutamicum. gapA The promoter was replaced with the Pald promoter of the endogenous acetaldehyde dehydrogenase gene from Corynebacterium glutamicum, and the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase gene gapN, driven by the Psod promoter, from Streptococcus mutans, was overexpressed in Corynebacterium glutamicum. Includes the following steps: A. Recombinant bacteria C. glutamicum-ald Construction a1. Using the genome of Corynebacterium glutamicum ATCC 21543 as a template, PCR was performed using ald-up-F and ald-up-R primers to obtain the gene fragment ald-up, and the PCR product was purified. Using the genome of Corynebacterium glutamicum ATCC 21543 as a template, PCR was performed using ald-F and ald-R primers to obtain the gene fragment ald, and the PCR product was purified. Using the genome of Corynebacterium glutamicum ATCC 21543 as a template, PCR was performed using ald-down-F and ald-down-R primers to obtain the gene fragment ald-down, and the PCR product was purified. a2. The Corynebacterium glutamate suicide plasmid pK18mobsacB was double-digested with EcoRI / XbaI. The gene fragments ald-up, ald, and ald-down were ligated into pK18mobsacB in one step using the Gibson Assembly kit. The resulting recombinant plasmid was named pK18-ald. a3. pK18-ald was transformed into Corynebacterium glutamicum ATCC 21543, and the resulting recombinant bacteria was named C. glutamicum-ald ; The recombinant bacteria C. glutamicum-ald It contains the gapA gene, a glyceraldehyde-3-phosphate dehydrogenase driven by the Pald promoter, with the sequence shown in SEQ ID NO:1; B. Recombinant bacteria C. glutamicum-ald / Construction of pXMJ-gapN b1. The NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase gene gapN from Streptococcus mutans containing the Psod promoter and codon optimization was artificially synthesized. The sequence is shown in SEQ ID NO:

3. This fragment was ligated into the shuttle vector pXMJ19 of Corynebacterium glutamicum, and the resulting recombinant plasmid was named pXMJ-gapN. b2. Transform the recombinant plasmid pXMJ-gapN into the recombinant bacteria. C. glutamicum-ald The recombinant bacteria obtained were named C. glutamicum-ald / pXMJ-gapN; The primer sequences are as follows: ald-up-F: 5′-cacagaattcgcaattttaaggtttgctggtg-3′; ald-up-R: 5′-agtggcgtcgacaagcatgttttttgcagggaacgaacctg-3′; ald-F: 5′-caggttcgttccctgcaaaaaacatgcttgtcgacgccact-3′; ald-R: 5′-ataccaacacgaatggtcattgggtctcctttgggccacc-3′; ald-down-F: 5′-ggtggcccaaaggagacccaatgaccattcgtgttggtat-3′; ald-down-R: 5′-cacatctagattagagcttggaagctacgag-3′.

4. The application of the recombinant Corynebacterium glutamicum according to claim 1 or 2 or the recombinant Corynebacterium glutamicum constructed according to the method of claim 3 in the fermentation production of lysine.

5. A method for increasing lysine fermentation yield, characterized in that, The method includes: fermenting and culturing the recombinant Corynebacterium glutamicum as described in claim 1 or 2 or the recombinant Corynebacterium glutamicum constructed according to the method described in claim 3, and collecting lysine from the fermentation product.

6. An engineered bacterium that produces pentamethylenediamine, characterized in that, The engineered bacteria are constructed by expressing the lysine decarboxylase gene in the recombinant Corynebacterium glutamicum as described in claim 1 or 2 or the recombinant Corynebacterium glutamicum constructed according to the method described in claim 3.

7. The engineered bacteria according to claim 6, characterized in that, The lysine decarboxylase gene is derived from Escherichia coli (E. coli) Escherichia coli ).

8. A method for increasing the fermentation yield of pentamethylenediamine, characterized in that, The method includes: fermenting the engineered bacteria of claim 6 or 7, and collecting pentanediamine from the fermentation product.

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