System for producing valerolactam through whole-cell catalysis and application of system

Abstract

The invention relates to the technical field of bioengineering, in particular to a system for producing valerolactam through whole-cell catalysis and application of the system. The system provided by the invention comprises L-lysine, and also comprises halomonas with the functions of synthesizing 5-aminovaleric acid, inhibiting GabT protein and enhancing beta-alanine coenzyme A transferase. According to the method, L-lysine is taken as a substrate, halomonas is utilized, 5-aminovaleric acid is synthesized firstly, degradation of the 5-aminovaleric acid is reduced by inhibiting the function of GabT protein in the halomonas, the function of beta-alanine coenzyme A transferase is enhanced, the metabolic flux from the 5-aminovaleric acid to the valerolactam is increased, and finally the valerolactam is efficiently produced.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a system for producing valerolactam by whole-cell catalysis and application thereof. Background Art

[0002] Lactam is a cyclic form of ω-amino acid and an important precursor for the production of polyamide (nylon). Among them, valerolactam (VL) is a five-carbon lactam that can be used in the synthesis of nylon 5 and nylon 65. At present, valerolactam is mainly produced through petrochemical derivatives, but this route has disadvantages such as harsh reaction conditions and the generation of toxic waste.

[0003] Existing research discloses a method for producing valerolactam using a biosynthetic pathway. This method uses Corynebacterium glutamicum GA16 (C. glutamicum GA16) as the base bacteria. By introducing L-lysine monooxygenase (davB) and 5-aminovaleramide amidohydrolase (davA) from Pseudomonas putida into the base bacteria, as well as β-alanine coenzyme A transferase (AcT) from Clostridium propionicum, valerolactam is synthesized using L-lysine as a substrate. Corynebacterium glutamicum GA16 has a strong ability to synthesize L-lysine. It is derived from Corynebacterium glutamicum BE. Corynebacterium glutamicum BE has a high tolerance to valerolactam and can tolerate 60g / L valerolactam. However, Corynebacterium glutamicum is easily contaminated with foreign bacteria during the fermentation process and requires strict hygiene control, which brings many inconveniences to industrial production.

[0004] In view of this, the present invention is proposed In view of this, the present invention is proposed. Summary of the invention

[0005] To solve the above technical problems, the present invention provides a system for producing valerolactam based on L-lysine and whole-cell catalysis of Halomonas and its application, which provides a new idea for the production of valerolactam.

[0006] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a whole-cell catalytic system for producing valerolactam, including L-lysine, and also includes Halomonas with the function of synthesizing 5-aminovaleric acid, inhibiting GabT protein function and enhancing β-alanine CoA transferase function.

[0007] The present invention finds that in the process of synthesizing valerolactam using L-lysine as a substrate, the intermediate product 5-aminovaleric acid is first synthesized. 5-aminovaleric acid can be used as a nitrogen source by Halomonas to improve the tricarboxylic acid cycle (TCA). Although this metabolic step improves the growth and reproduction rate of the fermentation bacteria, it reduces the metabolic flow from 5-aminovaleric acid to valerolactam, thereby causing a waste of resources. In addition, in the process of converting 5-aminovaleric acid into valerolactam using β-alanine coenzyme A transferase Act, the enzyme activity of β-alanine coenzyme A transferase Act is generally low, and the conversion rate is also low, thereby causing a low yield. The whole-cell catalytic production system of valerolactam provided by the present invention uses L-lysine as a substrate, and uses Halomonas to first synthesize 5-aminovaleric acid, and then inhibits the GabT protein function in Halomonas to reduce the degradation of 5-aminovaleric acid and improve the metabolic flow of 5-aminovaleric acid to valerolactam; at the same time, the whole-cell catalytic production system of valerolactam provided by the present invention uses Halomonas with enhanced β-alanine coenzyme A transferase function to produce valerolactam, improves the efficiency of producing valerolactam with 5-aminovaleric acid and L-lysine as substrates, and finally efficiently produces valerolactam. The yield and output of valerolactam synthesized with L-lysine as a substrate in the present invention are higher than those of conventional methods. And Halomonas can be used as a fermentation bacterium for open fermentation without additional sterilization, and various air filter membranes and complex aseptic operations are not required during the fermentation process to reduce the possibility of bacterial contamination, which greatly saves production costs and manpower and material resources.

[0008] Preferably, the GabT includes at least one of GabT1, GabT2, GabT3, and GabT4.

[0009] More preferably, the GabT comprises GabT1 and GabT2.

[0010] More preferably, the GabT includes GabT1, GabT2 and GabT4.

[0011] In the present invention, the method of inhibiting the function of GabT protein is preferably at least one of the following: knocking out GabT gene, silencing GabT gene, reducing GabT gene expression, and inhibiting GabT protein activity.

[0012] Among them, the amino acid sequence of GabT1 is preferably as shown in SEQ ID NO.01.

[0013] And / or, the amino acid sequence of GabT2 is preferably as shown in SEQ ID NO.02.

[0014] And / or, the amino acid sequence of GabT3 is preferably as shown in SEQ ID NO.03.

[0015] And / or, the amino acid sequence of GabT4 is preferably as shown in SEQ ID NO.04.

[0016] And / or, the nucleotide sequence of GabT1 is preferably as shown in SEQ ID NO.05.

[0017] And / or, the nucleotide sequence of GabT2 is preferably as shown in SEQ ID NO.06.

[0018] And / or, the nucleotide sequence of GabT3 is preferably as shown in SEQ ID NO.07.

[0019] And / or, the nucleotide sequence of GabT4 is preferably as shown in SEQ ID NO.08.

[0020] Furthermore, in the present invention, the method for enhancing the function of β-alanine CoA transferase is selected from at least one of the following: overexpressing β-alanine CoA transferase, enhancing the enzyme activity of β-alanine CoA transferase.

[0021] Among them, the amino acid sequence of β-alanine CoA transferase is preferably as shown in SEQ ID NO.09 or SEQ ID NO.11.

[0022] And / or, the nucleotide sequence of β-alanine CoA transferase is preferably as shown in SEQ ID NO.10 or SEQ ID NO.12.

[0023] Table 1 Amino acid sequences of SEQ ID NO. 01-04, 09, 11 Table 2 Nucleotide sequences of SEQ ID NO. 5-8, 10, 12 In one of the more specific embodiments provided by the present invention, the Halomonas is based on Halomonas having the function of synthesizing 5-aminovaleric acid and is obtained by knocking out the GabT gene.

[0024] More specifically, the Halomonas can be selected as a Halomonas having the function of synthesizing 5-aminovaleric acid and having GabT1 and GabT2 knocked out, such as Halomonas bluephagenesis F (reference: Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas bluephagenesis. ACS Synth. Biol. 2018, 7, 8, 1897–1906); or, it can be selected as a Halomonas bluephagenesis M having the function of synthesizing 5-aminovaleric acid and having GabT1, GabT2 and GabT4 knocked out, which is based on Halomonas bluephagenesis F and obtained by knocking out the GabT4 gene).

[0025] Preferably, in the system of the present invention, the concentration of L-lysine is 5±3 g / L; and / or the cell mass of Halomonas is 10±2 g / L).

[0026] The system provided by the present invention realizes the synthesis of valerolactam by taking L-lysine as a substrate in Halomonas through whole-cell catalysis, and improves the yield of valerolactam by inhibiting the function of GabT protein and enhancing the function of β-alanine coenzyme A transferase.

[0027] In a second aspect, the present invention provides application of the system in preparing valerolactam or downstream products containing valerolactam.

[0028] In a third aspect, the present invention provides a method for preparing valerolactam, which utilizes the aforementioned system, takes L-lysine as a substrate, and produces valerolactam by whole-cell catalysis (i.e., takes L-lysine as a substrate, first synthesizes 5-aminovaleric acid, and then undergoes cyclization to form valerolactam).

[0029] Preferably, the reaction temperature of whole-cell catalysis is 37±2°C, the stirring speed is 200±50rpm, the pH value is 7-8.5, and the reaction time is 48-72h.

[0030] The preparation method provided by the invention has the advantages of simple production process, low cost and broad application prospect.

[0031] Beneficial effects: The present invention provides a system for whole-cell catalytic production of valerolactam and its application, the system includes L-lysine, and also includes Halomonas with the function of synthesizing 5-aminovaleric acid, inhibiting GabT protein function and enhancing β-alanine coenzyme A transferase function. The present invention uses L-lysine as a substrate, uses Halomonas, first synthesizes 5-aminovaleric acid, then inhibits the GabT protein function in Halomonas, reduces the degradation of 5-aminovaleric acid, and then enhances the β-alanine coenzyme A transferase function to increase the metabolic flow of 5-aminovaleric acid to valerolactam, and finally efficiently produces valerolactam. The yield and output of valerolactam synthesized using L-lysine as a substrate in the present invention are higher than those of conventional methods; and Halomonas as a fermentation bacterium can be open fermented without additional sterilization, and various air filter membranes and complex aseptic operations are not required during the fermentation process to reduce the possibility of bacterial contamination, which greatly saves production costs and manpower and material resources. DETAILED DESCRIPTION

[0032] The invention provides a system for producing valerolactam based on L-lysine and whole-cell catalysis of Halomonas and its application. The system comprises L-lysine and Halomonas which can synthesize 5-aminovaleric acid, inhibit the function of GabT protein and enhance the function of β-alanine coenzyme A transferase.

[0033] 5-aminovaleric acid can be degraded by GabT protein. The present invention preferably reduces the degradation of the intermediate product 5-aminovaleric acid by knocking out the endogenous GabT protein gene in Halomonas. The present invention found 4 endogenous GabT proteins (i.e., 5-aminovaleric acid transaminase, the amino acid sequence is shown in SEQ ID NO.01-04) in Halomonas. In the early stage of the present invention, 5-aminovaleric acid transaminase GabT (GabT1, GabT2, GabT3) was knocked out in combination in Halomonas H. bluephagenesis, and it was determined that the strain with GabT1+2 knocked out had the best effect (the highest accumulation of 5-aminovaleric acid). In further research, the present invention further knocked out GabT4 on the basis of knocking out GabT1+2 strains, and the results showed that the knockout of GabT4 can further reduce the degradation of 5-aminovaleric acid and improve the synthesis of valerolactam.

[0034] The conversion rate of β-alanine CoA transferase Act, which converts 5-aminovaleric acid into valerolactam, is low. Enhancing the function of β-alanine CoA transferase helps to improve the efficiency of producing valerolactam using 5-aminovaleric acid and L-lysine as substrates. Specifically, the present invention achieves the synthesis of valerolactam from 0 to 1 by overexpressing β-alanine CoA transferase AcT in Halomonas H. bluephagenesis F (a strain used to synthesize 5-aminovaleric acid), using 10 g / L L-lysine as a substrate and initially adding L-lysine. Among them, the expression plasmid of β-alanine CoA transferase AcT uses a quorum sensing cell expression system, namely pSEVA321-plux-acT. The plasmid is overexpressed in the strain Halomonas H. bluephagenesis F, and the expression of the enzyme AcT is induced by the inducer AHL. The inducer is 5×10 -7 , 5×10 -6 , 5×10 -5 , 5×10 -4 , initially added 10g / L L-lysine as substrate, and the production of valerolactam under different inducer conditions was detected. Specific results: Under AHL, 5×10 -7 , 5×10 -6 , 5×10 -5 , 5×10 -4 When 50 MM-YU medium was used, the yields of valerolactam were 0.11, 0.23, 0.29, and 0.41 g / L, respectively. Among them, 50 MM-YU medium is Yeast 5g / L, urea 5g / L, and other culture conditions are the 50 MM conditions used in the laboratory (except for the different substrates, the experimental operation process is referenced: Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonasbluephagenesis. ACS Synth. Biol. 2018, 7, 8, 1897–1906).

[0035] In addition to the method of overexpressing Act, the present invention further improves the enzymatic activity of Act by constructing a mutant library of acT, thereby improving the conversion rate of valerolactam. Specifically, the present invention uses an error-prone PCR kit to perform random mutation of in situ error-prone PCR between 200-400 bp from the start codon of Act (the operation of random mutation is referenced: Shao WL, Ma KS, Le YL, et al. Development and use of a novel random mutagenesis method: in situerror-prone PCR (is-epPCR) / / Reeves A, Ed. In vitro Mutagenesis. New York, NY: Humana Press, 2017: 497-506), after multiple rounds of PCR, the mutants were transferred into Escherichia coli s17-1 through ligation reaction to construct a mutant library; the mutants were further overexpressed in Halomonas, the production of valerolactam was detected, and a high concentration of L-lysine substrate was added (Halomonas is intolerant to high concentrations of L-lysine. If the strain can grow well under the condition of high concentrations of L-lysine, it means that the enzyme activity of acT is good and the content of L-lysine converted to valerolactam is increased). Finally, the present invention screened a gene with two mutation points between act 200-400 bp, and overexpressed it in Halomonas H. bluephagenesis F. Compared with the overexpression of non-mutated act strain, the production of valerolactam was increased by 1.5 times, and the production of valerolactam reached nearly 1 g / L, indicating that the act gene screened by the present invention can significantly increase the production of valerolactam.

[0036] In summary, the present invention discovered a new 5-aminovaleric acid transaminase GabT4 in Halomonas H. bluephagenesis, and after knocking out the enzyme, the accumulation of 5-aminovaleric acid can be improved; the present invention realizes the synthesis of valerolactam in Halomonas H. bluephagenesis for the first time; the present invention improves the activity of the enzyme β-alanine coenzyme A transferase Act for synthesizing valerolactam, and the yield of valerolactam is increased by 1.5 times; the present invention has low cost and broad application prospects.

[0037] The technical solutions provided by the present invention are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used can be obtained from commercial sources.

[0038] Example 1 In this example, GabT4 (ie, H. bluephagenesis M strain) was further knocked out based on the knockout of GabT1+2 strain (ie, H. bluephagenesis F strain). The results showed that the knockout of GabT4 could further reduce the degradation of 5-aminovaleric acid and increase the synthesis of valerolactam.

[0039] During the shake flask experiment, 5 g / L of 5-aminovaleric acid was initially added, 50 MM-YU medium was used, the fermentation temperature was 37 ° C, 200 rpm, and the fermentation time was 48 h. After shaking the flask, 5 mL of bacterial liquid was taken, diluted 10 times, and passed through a high-pressure homogenizer at 800-1000 mpa for 1.5 min, then offline at 4 degrees and 12000 rpm for 10 min, and the residual 5-aminovaleric acid was detected by HPLC. The experimental results show that when the strain H. bluephagenesis F (i.e., GabT1 and GabT2 were knocked out), the residual 5-aminovaleric acid was 2.5 g / L when the flask was shaken, and the cell dry weight was 12.1 g / L. After GabT was knocked out, the residual 5-aminovaleric acid was 3.1 g / L and the cell dry weight was 11.8 g / L. Then, H. bluephagenesis F and H. bluephagenesis M were overexpressed with pSEVA321-plux-act, and the AHL was 5×10 -4 When the degradation rate of 5-aminovaleric acid was 4.34%, the yield of valerolactam was 0.4 g / L and 0.64 g / L, respectively. This indicates that the knockout of GabT4 can reduce the degradation of 5-aminovaleric acid and increase the yield of valerolactam.

[0040] Example 2 In this example, the synthesis of valerolactam from 0 to 1 was achieved by overexpressing β-alanine coenzyme A transferase Act in Halomonas bluephagenesis M, that is, overexpressing pSEVA321-plux-act, using L-lysine as a substrate.

[0041] Initially add 5 g / L L-lysine, in 50MM-YU medium, fermentation temperature is 37 ° C, 200 rpm, fermentation time is 48 h, after shaking the bottle, take 5mL of bacterial solution, dilute 10 times, pass through a high pressure homogenizer, 800-1000 mpa, 1.5 min, 4 degrees, 12000 rpm offline for 10 min, and pass through HPLC Specific results: In AHL, they were 5×10 -7 , 5×10 -6 , 5×10 -5 , 5×10 -4When 50 MM-YU medium was used, the production of valerolactam was 0.11, 0.23, 0.29, and 0.41 g / L, and the remaining L-lysine was 2.0, 1.8, 1.7, and 1.5 g / L, respectively.

[0042] Example 3 In this example, the Act enzyme activity is further improved by constructing an Act mutant library, thereby increasing the conversion rate of valerolactam.

[0043] Firstly, an act mutant plasmid, i.e., pSEVA321-plux-act (mutant), was constructed, and the act mutant gene screened by the present invention was overexpressed into Halomonas H. bluephagenesis M. 5 g / L of 5-aminovaleric acid was initially added, 50 MM-YU medium was used, the fermentation temperature was 37°C, 200 rpm, and the fermentation time was 48 h. After shaking the flask, 5 mL of bacterial solution was taken, diluted 10 times, and passed through a high-pressure homogenizer at 800-1000 mpa for 1.5 min, and then offline at 4°C and 12000 rpm for 10 min. HPLC detection showed that the yield of valerolactam was increased by 1.5 times compared with the overexpression of non-mutated Act strain, the yield of valerolactam reached nearly 1 g / L, and the remaining 5-aminovaleric acid was 0.5 g / L.

[0044] The constructed act mutant plasmid, i.e., pSEVA321-plux-act (mutant), was overexpressed in H. bluephagenesis M. Initially, 5 g / L of 5-L-lysine was added, 50 MM-YU medium was used, the fermentation temperature was 37°C, 200 rpm, and the fermentation time was 48 h. After shaking the flask, 5 mL of the bacterial solution was taken and diluted 10 times. After passing through a high-pressure homogenizer at 800-1000 mpa for 1.5 min, it was offline at 4°C and 12,000 rpm for 10 min. HPLC detection showed that the production of valerolactam was 0.7 g / L and the remaining L-lysine was 1.2 g / L compared with the overexpression of the unmutated Act strain.

[0045] The above-described embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solution of the present invention in detail, but cannot be understood as limiting the scope of protection of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the scope of protection of the present invention.

Claims

1. A system for whole-cell catalytic production of valerolactam, characterized in that: The invention includes L-lysine and also includes Halomonas which has the function of synthesizing 5-aminovaleric acid, inhibiting GabT protein function and enhancing β-alanine coenzyme A transferase function.

2. The system according to claim 1, characterized in that: The GabT includes at least one of GabT1, GabT2, GabT3, and GabT4; preferably includes GabT1 and GabT2.

3. The system according to claim 2, characterized in that: The GabT includes GabT1, GabT2 and GabT4.

4. The system according to any one of claims 1 to 3, characterized in that: The method for inhibiting the function of GabT protein is selected from at least one of the following: knocking out GabT gene, silencing GabT gene, reducing GabT gene expression, and inhibiting GabT protein activity.

5. The system according to any one of claims 1 to 4, characterized in that: The amino acid sequence of GabT1 is shown in SEQ ID NO.01; and / or, the amino acid sequence of GabT2 is shown in SEQ ID NO.02; and / or, the amino acid sequence of GabT3 is shown in SEQ ID NO.03; and / or, the amino acid sequence of GabT4 is shown in SEQ ID NO.04; and / or, the nucleotide sequence of GabT1 is shown in SEQ ID NO.05; and / or, the nucleotide sequence of GabT2 is shown in SEQ ID NO.06; and / or, the nucleotide sequence of GabT3 is shown in SEQ ID NO.07; And / or, the nucleotide sequence of GabT4 is shown in SEQ ID NO.

08.

6. The system according to any one of claims 1 to 5, characterized in that: The method for enhancing the function of β-alanine CoA transferase is selected from at least one of the following: overexpressing β-alanine CoA transferase and enhancing the enzyme activity of β-alanine CoA transferase.

7. The system according to any one of claims 1 to 6, characterized in that: The amino acid sequence of β-alanine CoA transferase is shown in SEQ ID NO.09 or SEQ ID NO.11; And / or, the nucleotide sequence of β-alanine CoA transferase is shown as SEQ ID NO.10 or SEQ ID NO.

12.

8. Use of the system according to any one of claims 1 to 7 in the preparation of valerolactam or downstream products containing valerolactam.

9. A method for preparing valerolactam, characterized in that: The system according to claims 1 to 7 is used to produce valerolactam by whole-cell catalysis using L-lysine as a substrate.

10. The preparation method according to claim 9, characterized in that: The reaction temperature of whole-cell catalysis is 37±2°C, the stirring speed is 200±50rpm, the pH value is 7-8.5, and the reaction time is 48-72h.

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