Method for producing L-proline by fermenting recombinant corynebacterium glutamicum

Abstract

The invention provides a method for producing L-proline by fermenting recombinant corynebacterium glutamicum, which comprises the following steps: on the basis of a previously researched strain C.glutamicum SO40, inserting plasmid pXMJ19-ppocd to convert intracellular L-ornithine into L-proline, randomly selecting corynebacterium glutamicum into which the plasmid pXMJ19-ppocd is successfully inserted, and carrying out shake-flask fermentation, so as to obtain L-proline. Corynebacterium glutamicum with high L-proline production capacity is obtained and named as C. glutamicum PO1, and then optimization of the adding time and the adding concentration of an inducer IPTG is carried out on PO1. Then, the aerobic fermentation yield of the L-proline is further improved through a method of modifying gene target genes, namely knocking out a putA gene and a cgocd gene and double knockout, the recombinant corynebacterium glutamicum PO5 with relatively high L-proline yield is finally obtained, the shake-flask fermentation yield can reach 25.4 g / L, the yield of the proline can be improved to 99 g / L through fed-batch feeding of a 5L fermentation tank, and the yield of the L-proline can be increased to 99 g / L. The yield of relative glucose is 0.54 g / g, and the method has good industrial application value.

Description

Technical Field

[0001] The present invention relates to the field of microorganism or enzyme technology, and specifically, the present invention relates to a method for producing L-proline by fermentation using recombinant Corynebacterium glutamicum. Technical Background

[0002] L-proline is one of the important amino acids for synthesizing human protein and is an important raw material for amino acid infusion; L-proline is also widely used in the fields of medicine, industry and agriculture. In the field of medicine, it can be used to prepare drugs for hypertension and congestive heart failure, becoming the most promising substitute for small molecule and macromolecular drugs. In the industrial field, it is widely used in various catalytic reactions due to its strong activity, less environmental pollution, good stereospecificity, simple operation and low cost. In the agricultural field, it can play an important role in the growth stages of different plants, for example, it plays an important role in photosynthesis, biological nitrogen fixation and relief of salt stress. The industrial production of L-proline currently mainly includes chemical synthesis, protein hydrolysis and microbial fermentation. Microbial fermentation is currently the main method used in industrial production. The chemical synthesis method has simple synthesis steps but it has a large environmental pollution and is difficult to achieve industrial production. The protein hydrolysis process requires a large amount of strong acid and the quality of the raw materials obtained is unstable. Therefore, these two methods are generally not used in industrial production. The microbial fermentation method mainly relies on traditional mutagenesis, cell fusion and genetic engineering to obtain high-yield strains for synthesizing L-proline. This method has little environmental pollution and low production cost. It is currently the main method for industrial production of L-proline. Corynebacterium glutamicum is currently an important strain for industrial microbial fermentation to produce amino acids. C. glutamicum ATCC 13032 uses biological editing tools to perform metabolic engineering on target genes, thereby achieving the release of feedback inhibition of key enzymes in Corynebacterium glutamicum, knocking out metabolic branches in the amino acid synthesis pathway, efficiently expressing key genes, and knocking in exogenous genes, ultimately obtaining an efficient amino acid production strain; Microbial fermentation is a very promising method for producing L-proline. Currently, there are few reports on the construction of high-yield strains of L-proline. Therefore, it is necessary to further construct a recombinant high-yield strain of Corynebacterium glutamicum that produces L-proline. Summary of the invention

[0003] In view of the current technical status of L-proline at home and abroad, the present invention aims to provide a method for producing L-proline by fermentation using recombinant Corynebacterium glutamicum, and the specific steps are as follows: (1) Based on previous research C.glutamicumBased on SO40, randomly selected plasmids that had been successfully inserted into pXMJ19- ppocd The Corynebacterium glutamicum was fermented in a shake flask to obtain a Corynebacterium glutamicum with a high ability to produce L-proline, which was named C. glutamicum PO1, and then optimize the addition time and concentration of inducer IPTG to PO1; (2) The PO1 selected in step (1) above is genetically modified again, i.e., knocked out putA Genes get PO2, knockout cgocd The genes were knocked out respectively. putA , cgocd It is beneficial to increase the production of L-proline, and then C.glutamicum Superimposed knockout gene on SO40 putA , cgocd PO4 was obtained; the results showed that the double knockout gene putA , cgocd The effect of producing L-proline was higher than that of single knockout. Finally, the plasmid pXMJ19- ppocd After integration into PO4 to obtain PO5, shake flask fermentation was carried out to verify the maximum production of L-proline by feeding the fermenter.

[0004] According to the transformation result in step (1), the method of synthesizing L-proline by metabolizing glucose using recombinant Corynebacterium glutamicum, the content of each component in the fermentation medium is fermentation medium (g / L): yeast powder 6 g / L, (NH 4 ) 2 SO 4 50 g / L, MgSO 4 2.5 g / L, KH 2 PO 4 1 g / L, K 2 HPO 4 0.5 g / L, Na 2 HPO 4 0.5 g / L, FeSO 4 0.02g / L, MnSO 4 0.02 g / L, 100 g glucose, pH 6.8. By implementing the specific content of the present invention, the following effects can be achieved: The present invention adopts the strain based on previous research C. glutamicum SO40, inserted into plasmid pXMJ19- ppocd And knockout putA Gene, cgocdThe recombinant strain PO5 of Corynebacterium glutamicum was obtained by the expression of the gene, and then shake flask fermentation and 5 L fermentation tank feeding were carried out to explore the highest production of L-proline, providing a basis for the subsequent transformation of Corynebacterium glutamicum. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1 for C. glutamicum SO40-pXMJ19- ppocd PCR verification of the recombinant strain PO1.

[0006] Figure 2 for C. glutamicum SO40-pXMJ19- ppocd Optimization diagram of the addition time of the inducer IPTG.

[0007] Figure 3 for C. glutamicum SO40-pXMJ19- ppocd Optimization diagram of the addition concentration of the inducer IPTG.

[0008] Figure 4 This is the standard curve of L-proline.

[0009] Figure 5 for C. glutamicum SO40-pXMJ19- ppocd - cgocd Verification diagram of recombinant strain PO2 PCR.

[0010] Figure 6 for C. glutamicum SO40-pXMJ19- ppocd - putA Verification diagram of recombinant strain PO3 PCR.

[0011] Figure 7 for C. glutamicum SO40- ppocd - putA - cgocd Verification diagram of recombinant strain PO4 PCR.

[0012] Figure 8 This is a diagram of the shake flask fermentation test of the PO1-PO3 recombinant strains.

[0013] Figure 9 For C glutamicum SO40- cgocd- putA -pXMJ19- ppocd PCR verification of the recombinant strain PO5.

[0014] Figure 10This is a shake flask fermentation test diagram of the recombinant strains PO1, PO2, PO3, and PO5.

[0015] Figure 11 This is the feeding test diagram of PO5 5 L fermenter. DETAILED DESCRIPTION

[0016] The present invention will be described below with reference to examples; however, the present invention is not limited to the following examples.

[0017] In addition, in the following description, unless otherwise specified, % refers to m / m mass percentage. All reagents, raw materials and instruments used in the present invention are well known in the art and can be purchased from the market, but do not limit the implementation of the present invention. Other reagents and equipment well known in the art can be applied to the implementation of the following embodiments of the present invention. Example

[0018] Screening a recombinant strain capable of producing L-proline; Based on strain C.glutamicum Based on SO40, randomly selected plasmids that had been successfully inserted into pXMJ19- ppocd The Corynebacterium glutamicum was fermented in a shake flask to obtain a Corynebacterium glutamicum with a high ability to produce L-proline, which was named C. glutamicum PO1, please refer to the instruction manual Figure 1 shown. Example

[0019] Optimization of the addition time and concentration of the inducer IPTG; Optimize the addition time and concentration of IPTG for the selected strains. Figure 2 and 3 As shown, 1 mM IPTG was added at 0 h, 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h. C. glutamicum PO1 for fermentation, such as Figure 2 As shown, the addition of IPTG at various time periods had no significant effect on the growth of its bacteria; When IPTG was added at 8 h to induce expression, the yield of L-proline was the highest, reaching 21.2 g / L. Then, as the addition time was delayed, the yield of L-proline gradually decreased. When IPTG was added at 0 h, 4 h, and 8 h, L-ornithine was not detected in the fermentation broth, indicating that all L-ornithine was converted into L-proline. When IPTG was added at 12 h, 16 h, 20 h, and 24 h, the concentration of L-ornithine gradually increased with the delay of IPTG addition time. In summary, 8 h is the best IPTG induction time. C. glutamicumDuring the PO1 fermentation process, 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6 mM IPTG were added at 8 h to seek the optimal inducer concentration. The results are shown in Figure 3 As shown in the figure, the amount of PO1 bacteria was slightly more when IPTG was not added than when IPTG was added, indicating that the gene ppocd The expression of may have a certain effect on bacterial growth, but with the increase of IPTG concentration, there is no obvious difference in bacterial growth. The results showed that when 0.8 mM IPTG was added, the L-proline production was the highest, which was 19.5 g / L. Example

[0020] Construction of recombinant Corynebacterium glutamicum PO2-PO5; Plasmid pK18-T- cgocd Transform into PO1 competent cells by electroporation, and verify whether the T terminator is inserted after two homologous recombination. cgocd The gene coding region, the selected strains were named C. glutamicum PO2, please refer to the instruction manual Figure 5 As shown; Plasmid pK18-T- putA Transform into PO1 competent cells by electroporation, and verify whether the T terminator is inserted after two homologous recombination. putA The gene coding region, the selected strains were named C. glutamicum PO3, please refer to the instruction manual Figure 6 As shown; Plasmid pK18-T- cgocd Transform into PO1 competent cells by electroporation, and verify whether the T terminator is inserted after two homologous recombination. cgocd After the gene coding region, the plasmid pK18-T- putA Electroporation into competent cells of the obtained strain, after two homologous recombination, verify whether the T terminator is inserted cgocd as well as putA The gene coding region, the selected strains were named C. glutamicum PO4, please refer to the instruction manual Figure 7 shown. Example

[0021] The shake flask fermentation results showed that PO2 had the highest yield among PO1, PO2 and PO3. The reason for this was genetic cgocd knockout blocked the conversion of L-proline to L-ornithine; putA The knockout of blocked the catabolism of L-proline, and its L-proline accumulation was increased. Please refer to the attached manual for details. Figure 8 As shown; LB medium (g / L): 10 g tryptone, 5 g yeast powder, 10 g NaCl; solid medium is supplemented with 2% agar powder; LBG medium (g / L): 10 g tryptone, 5 g yeast powder, 10 g NaCl, 2% glucose; Seed medium (g / L): 10 g yeast powder, 10 g corn steep liquor, 15 g (NH 4 ) 2 SO 4 , 2.5 g MgSO 4 , 1 gKH 2 PO 4 , 0.5 g K 2 HPO 4 , 0.5 g Na 2 HPO 4 , 10 g CaCO 3 , 3% glucose, pH 6.8; The seed culture solution was inoculated into 20 mL of fermentation medium at a 10% inoculation rate. The fermentation was carried out in a 250 mL Erlenmeyer flask at a temperature of 32°C and a rotation speed of 250 rpm. Fermentation medium (g / L): 6 g yeast powder, 50 g (NH 4 ) 2 SO 4 , 2.5 g MgSO 4 , 1 g KH 2 PO 4 , 0.5 gK 2 HPO 4 , 0.5 g Na 2 HPO 4 , 0.02 g FeSO 4 , 0.02 g MnSO 4 , 100 g glucose, pH 6.8. Example

[0022] Screening of recombinant strains C. glutamicum PO5; Plasmid pXMJ19- ppocd Electroporation into PO4 competent cells, after two homologous recombination, was used to verify whether the plasmid was inserted into the gene coding region. The selected strain was named C. glutamicum PO5, please refer to the instruction manual Figure 9 shown. Example

[0023] Shake flask fermentation test of PO1, PO2, PO3, PO5 recombinant strains and fed-batch performance test of PO5 5 L fermenter; To verify the recombinant strain C. glutamicum The fermentation performance of PO1, PO2, PO3, and PO5 was tested by shake flask fermentation using glucose and sucrose as mixed carbon sources. The recombinant strains were cultured overnight on LB plates, and a single colony was streaked from the fresh plate into a 100 mL triangular shake flask containing 10 mL seed medium and cultured at 32 °C and 220 rpm for 12 h. LB medium (g / L): 10 g tryptone, 5 g yeast powder, 10 g NaCl; solid medium is supplemented with 2% agar powder; LBG medium (g / L): 10 g tryptone, 5 g yeast powder, 10 g NaCl, 2% glucose; Seed medium (g / L): 10 g yeast powder, 10 g corn steep liquor, 15 g (NH 4 ) 2 SO 4 , 2.5 g MgSO 4 , 1 gKH 2 PO 4 , 0.5 g K 2 HPO 4 , 0.5 g Na 2 HPO 4 , 10 g CaCO 3 , 3% glucose, pH 6.8; The seed culture solution was inoculated into 20 mL of fermentation medium at a 10% inoculation rate. The fermentation was carried out in a 250 mL Erlenmeyer flask at a temperature of 32°C and a rotation speed of 250 rpm. Fermentation medium (g / L): 6 g yeast powder, 50 g (NH 4 ) 2 SO 4 , 2.5 g MgSO 4 , 1 g KH 2 PO 4 , 0.5 gK 2 HPO 4 , 0.5 g Na 2 HPO 4 , 0.02 g FeSO 4 , 0.02 g MnSO 4 , 100 g glucose, pH 6.8; Inoculate the seed culture into 2 L fermentation tank medium at 10% inoculation rate, the temperature is 32°C, and the initial speed is controlled at 650 rpm; Fermentation tank medium (g / L): 10 g yeast extract, 15 g peptone, 20 g (NH 4 ) 2 SO 4 , 25 g MgSO 4 , 1g KH 2 PO 4 , 0.5 g K 2 HPO 4 , 0.5 g Na 2 HPO 4 , 0.02 g FeSO 4 , 0.02 g MnSO 4 , 30 g glucose, pH 6.8; Feed medium (g / L): 15 g yeast powder, 15 g (NH 4 ) 2 SO 4 , 800 g glucose.

[0024] After 72 h of shake flask fermentation, samples were taken to detect the accumulation of L-proline in the fermentation broth. The accumulation of L-proline in the fermentation broth reached 25.4 g / L after 72 h. Figure 10 As shown, the strains based on previous studies C.glutamicum SO40, inserted into plasmid pXMJ19- ppocd Genes and knockouts putA Gene, cgocd The strain with the gene has a high production capacity of L-proline; the production of L-proline can be increased to 99 g / L by sampling and measuring the production in a 5 L fermenter with feeding for 72 h, and the yield relative to glucose is 0.54 g / g. The conversion rate is the highest level so far. Please refer to the attached manual for details. Figure 11 shown.

[0025] As described above, the present invention is better implemented. The above embodiments are only descriptions of the preferred implementation methods of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the present invention.

Claims

1. A method for producing L-proline by fermentation using recombinant Corynebacterium glutamicum, characterized in that: (1) Based on previous research C.glutamicum Based on SO40, randomly selected plasmids that had been successfully inserted into pXMJ19- ppocd The Corynebacterium glutamicum was fermented in a shake flask to obtain a Corynebacterium glutamicum with a high ability to produce L-proline, which was named C. glutamicum PO1, and then optimize the addition time and concentration of inducer IPTG to PO1; (2) The PO1 selected through the above steps is genetically modified again, i.e., knocked out putA Genes get PO2, knockout cgocd Gene gets PO3, then in C.glutamicum Superimposed knockout gene on SO40 putA , cgocd Get PO4; The results showed that the double knockout gene putA , cgocd The effect of producing L-proline was better. Finally, the plasmid pXMJ19- ppocd After integration into PO4 to obtain PO5, shake flask fermentation was carried out to verify the maximum production of L-proline by feeding in a 5 L fermenter.

2. The method for producing L-proline by fermentation using recombinant Corynebacterium glutamicum according to claim 1, characterized in that: The screening C.glutamicum SO40 was inserted into plasmid pXMJ19- ppocd strains, and conducted shake flask fermentation to select a high-yield strain with a high ability to produce L-proline, which was named PO1.

3. The method for producing L-proline by fermentation using recombinant Corynebacterium glutamicum according to claim 1, characterized in that: The contents of the components in the fermentation medium are as follows (g / L): yeast powder 6 g / L, (NH4)2SO4 50 g / L, MgSO4 2.5 g / L, KH2PO4 1 g / L, K2HPO4 0.5 g / L, Na2HPO4 0.5 g / L, FeSO4 0.02 g / L, MnSO4 0.02 g / L, 100 g glucose, pH 6.8.

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