Recombinant bacterium for synthesizing phenyllactic acid as well as construction method and application of recombinant bacterium

Abstract

The invention discloses a recombinant bacterium for synthesizing phenyllactic acid as well as a construction method and application thereof, and relates to the technical field of microorganisms. The recombinant strain expresses lactic dehydrogenase, and overexpresses YjbB, MdtF, LapA and / or OrtT. According to the invention, a strain for producing the phenyllactic acid is modified by replacing promoters of yjbB, mdtF, lapA and / or ortT genes with strong promoters and the like, so that the strain overexpresses YjbB, MdtF, LapA and / or OrtT, and the yield of the phenyllactic acid can be obviously improved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to recombinant bacteria for synthesizing phenyllactic acid, their construction methods, and applications. Background Technology

[0002] Phenylated acid, also known as 2-hydroxy-3-phenylpropionic acid, is a monomer for synthesizing polyphenyllactic acid (PPA). Because PPA monomers contain phenyl groups, they possess stronger thermal stability, mechanical strength, and UV absorption properties than PPA, making them a promising new biodegradable bio-based polymer material following PPA. Besides being a monomer for PPA synthesis, PPA is also a broad-spectrum natural preservative, exhibiting significant antibacterial effects against most Gram-positive and Gram-negative bacteria and fungi. In medicine, it can be used as a substitute for tanshinone in the treatment of coronary heart disease, possessing hemostatic and analgesic effects, antiplatelet aggregation, and coronary artery dilation functions. In the cosmetics industry, it has anti-wrinkle and skin-brightening effects. As a novel antibacterial agent, it exhibits particularly good inhibitory effects against intestinal pathogens. PPA compounds have been successfully applied in livestock and poultry farming, replacing antibiotic components and improving chicken quality, enhancing the immunity of laying hens and the quality of eggs, reducing intestinal pathogens in pigs, and improving production performance.

[0003] Currently, phenyllactic acid (PLA) is mainly synthesized through chemical and biological methods. Chemical synthesis is complex, requires organic solvents, and causes significant environmental pollution. Biological methods include microbial fermentation, enzymatic methods, and whole-cell catalysis. Enzymatic methods require coenzyme I, are costly, and are unsuitable for industrial production. Whole-cell catalysis involves heterologous expression of PLA-related enzyme genes in chassis cells, catalyzing the synthesis of the target product by adding precursors such as phenylalanine and phenylpyruvic acid. However, these precursors are expensive, resulting in low profit margins for PLA and making it unsuitable for industrial production. Microbial fermentation includes natural strains and engineered microbial fermentation. Natural strain fermentation has low yields, making it difficult to meet industrial production demands. With the development of synthetic biology and metabolic engineering technologies, engineered microbial fermentation has become the most promising method for PLA synthesis. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a recombinant bacterium capable of efficiently producing phenyllactic acid.

[0005] The present invention also provides a method for constructing the above-mentioned recombinant bacteria.

[0006] The present invention also provides applications of the above-mentioned recombinant bacteria.

[0007] The present invention also provides a method for producing phenyl lactic acid.

[0008] According to a first aspect of the present invention, a recombinant bacterium expresses lactate dehydrogenase and overexpresses yjbB, mdtF, lapA and / or ortT.

[0009] The recombinant bacteria according to embodiments of the present invention have at least the following beneficial effects:

[0010] By modifying strains that produce phenyllactic acid by replacing the promoters of the yjbB, mdtF, lapA, and / or ortT genes with strong promoters (such as promoter P37), the overexpression of yjbB, mdtF, lapA, and / or ortT can significantly increase the yield of phenyllactic acid.

[0011] According to some embodiments of the present invention, the gene encoding lactate dehydrogenase is one of the following: CnldhA gene of Hookworm Copper-Greek, LdhdhA gene of Lactobacillus delbrueckii, LpD-ldh gene of Sugar-Free Lactobacillus, WfpprA gene of Wickham's yeast, PaldhA gene of Micrococcus acidophilus, Lpldh gene of Lactobacillus plantarum, and ppr gene of Lactobacillus.

[0012] According to some embodiments of the present invention, the genes encoding lactate dehydrogenase are *Cupriavidus necator H16* CnldhA (GenBank: CAJ91827.1), *Lactobacillus delbrueckii* LdhdhA (GenBank: CR954253.1), *Lactobacillus pentosus* LpD-ldh (GenBank: FR874854.1), *Wickerhamia fluorescens* TK-1 WfpprA (GenBank: AB621792.1), *Pediococcus acidilactici* PaldhA (GenBank: AB776697), *Lactobacillus plantarum* Lpldh (GenBank: ACGZ02000027.1), and *Lactobacillus* One of the sp.CGMCC 9967)ppr(GenBank:KP735960.1).

[0013] According to some embodiments of the present invention, the overexpression is achieved by any one of A1) to A5):

[0014] A1) Introduce a vector containing the coding genes of YjbB, MdtF, LapA and / or OrtT into the starting bacteria;

[0015] A2) Increase the copy number of the coding genes for YjbB, MdtF, LapA and / or OrtT on the chromosome;

[0016] A3) Alter the promoter sequence of the gene encoding YjbB, MdtF, LapA and / or OrtT on the chromosome;

[0017] A4) The strong promoter is operatively linked to the coding genes of YjbB, MdtF, LapA and / or OrtT;

[0018] A5) Modify the nucleotide sequences encoding yjbB, mdtF, lapA and / or ortT.

[0019] According to some embodiments of the present invention, the originating bacteria of the recombinant bacteria include Escherichia coli.

[0020] According to some embodiments of the present invention, the Escherichia coli is a common Escherichia coli, such as K12 derivatives (BW25113, MG1665, W3110, DH10B, BW2952, MDS42 and their derivatives), BL21(DE3) and its derivatives, BREL606, W, DH1, etc., or it may be a phenylalanine-producing Escherichia coli.

[0021] According to some embodiments of the present invention, the phenylalanine-producing Escherichia coli includes E. coli PHE03.

[0022] According to some embodiments of the present invention, the vector in A1) is an expression vector. The expression vector can replicate and be expressed normally in host cells.

[0023] According to some embodiments of the present invention, the vectors include, but are not limited to, pETDuet series expression vectors, pACYCDuet series expression vectors, pRSFDuet series expression vectors, pCOLADuet series expression vectors, or pCDFDuet series expression vectors.

[0024] According to some embodiments of the present invention, A3) includes replacing the original promoter of the coding gene for yjbB, mdtF, lapA and / or ortT on the chromosome with a strong promoter.

[0025] According to some embodiments of the present invention, the strong promoter includes at least one of the P37 promoter, P8 promoter, J23100 promoter, J23119 promoter, P21285 promoter, M1-93 promoter, and PL1118 promoter. The strong promoter can be recognized by the recombinant bacteria.

[0026] According to some embodiments of the present invention, the changes in A5) include codon optimization.

[0027] The method for constructing the recombinant bacteria described in the first aspect embodiment or the method for increasing phenyl lactic acid production according to a second aspect embodiment of the present invention includes the following steps:

[0028] Genetic engineering techniques were used to overexpress YjbB, MdtF, LapA, and / or OrtT in the starting strain;

[0029] The starting bacteria express lactate dehydrogenase.

[0030] According to some embodiments of the present invention, the genetic engineering methods include, but are not limited to, gene editing technology.

[0031] According to some embodiments of the present invention, the method includes: replacing the original promoters of the yjbB, mdtF, lapA and / or ortT genes of the originating strain with strong promoters using gene editing technology.

[0032] According to some embodiments of the present invention, the strong promoter includes at least one of the P37 promoter, P8 promoter, J23100 promoter, J23119 promoter, P21285 promoter, M1-93 promoter, and PL1118 promoter. The strong promoter can be recognized by the recombinant bacteria.

[0033] According to some embodiments of the present invention, the gene editing technology includes, but is not limited to, gene replacement. For example, gene replacement can be performed using the method described in the reference (Applied and Environmental Microbiology 2015, 81(7):2506-2514).

[0034] According to some embodiments of the present invention, the gene replacement method includes, but is not limited to, the CRISPR / Cas method.

[0035] Application of the recombinant bacteria according to the first aspect embodiment of the third aspect of the present invention in any one of B1) to B3):

[0036] B1) Production of phenyllactic acid or its derivatives;

[0037] B2) Prepare products for the production of phenyllactic acid or its derivatives;

[0038] B3) Increase the yield of phenyllactic acid or its derivatives.

[0039] According to some embodiments of the present invention, the product includes at least one of reagents and kits.

[0040] A method for producing phenyl lactic acid according to a fourth aspect of the present invention includes: culturing the recombinant bacteria described in the first aspect of the present invention.

[0041] According to some embodiments of the present invention, the initial OD of the culture 600 It is between 0.01 and 0.2. For example, it can be 0.05.

[0042] According to some embodiments of the present invention, the culture temperature is 30-37°C. For example, it can be 37°C.

[0043] According to some embodiments of the present invention, the culture time is 48-96 hours. For example, it can be 72 hours.

[0044] According to some embodiments of the present invention, the culture rotation speed is 150-300 rpm. For example, it can be 200 rpm.

[0045] According to some embodiments of the present invention, the method further includes the step of separating and purifying phenyllactic acid from the fermentation broth.

[0046] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the biosynthetic pathway of phenyllactic acid. Detailed Implementation

[0048] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0049] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0050] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0051] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0052] Using CRISPRa activation screening technology (Advanced Biotechnology 2024, 2:15), all endogenous stress protein (123) and transport protein (396) genes of Escherichia coli were activated and screened. Some target genes that could improve the robustness of the strain were identified, and the following promoter replacements were performed to enhance expression.

[0053] Promoter replacement was performed using CRISPR gene editing technology (Applied Environmental Microbiology, 2015, 81:2506-2514), and the plasmids used (pCas*, pTargetB) were the modified plasmids used in this experiment (Fronters Microbiology 2018, 9:1623).

[0054] Example 1 PHE03 (P yjbB Construction of strain ::P37)

[0055] In this embodiment, the phenylalanine-producing bacterium E. coli PHE03 (AdvancedBiotechnology (2024) 2:15), previously constructed in the laboratory, was used as the starting bacterium for modification. The natural promoter of the yjbB gene (Uniprot P0AF43) was replaced with promoter P37 to construct PHE03 (P yjbB strain ::P37). The specific experimental methods are as follows:

[0056] (1) Construction of the shooting segment:

[0057] Using the E. coli W3110 genome as a template, primer P was used... yjbB -up-F、P yjbB -up-R (sequence shown in Table 1), the upstream homologous arm of the yjbB gene promoter was amplified, and then amplified using primer P. yjbB -dn-F、P yjbB -dn-R (sequence shown in Table 1) was used to amplify the downstream homologous arm of the yjbB gene promoter.

[0058] Using plasmid pZBK (Biotechnology Biofuels 2019, 12:94) as a template, the P37 promoter sequence was amplified using primers P37-F and P37-R (sequences shown in Table 1). The amplified target fragment P37-UTR (sequence shown in SEQ ID NO:1) was recovered using a DNA recovery kit.

[0059] Using the upstream homologous arm of the recovered yjbB gene promoter and the P37 promoter (P37-UTR) as templates, primer P was used. yjbB Overlap PCR was performed using -up-F and P37-R to obtain P yjbB -up-P37. With recycled P yjbB Using -up-P37 and downstream homologous arms as templates, primer P yjbB -up-F and P yjbB Overlap PCR was performed again with -dn-R to obtain PCR products. The PCR products were verified by agarose gel electrophoresis, and the target fragment with the correct band was recovered from the gel to obtain the targeting fragment P. yjbB -up-P37-dn.

[0060] (2)pTargetB-P yjbB plasmid construction

[0061] Using pTargetB plasmid as a template, primer P was used. yjbB -N20-F, P yjbB The template was amplified by reverse PCR using -N20-R (sequence shown in Table 1) to obtain the PCR product. The PCR product was then treated with DpnI to remove the template plasmid. The treated product was immediately transformed into E. coli DH5α competent cells, plated on Spe resistant plates, and incubated upside down at 37°C. One or two single colonies were randomly selected for testing. For colonies with correct sequencing results, the corresponding single colonies were inoculated into 5 mL of LB broth and cultured overnight to preserve the culture and extract the plasmid to obtain pTargetB-P. yjbB .

[0062] (3) Preparation and transformation of E. coli PHE03 electrocompetent cells containing pCas* plasmid

[0063] The pCas* plasmid was transformed into the originating strain, phenylalanine-producing E. coli PHE03, plated, and incubated upside down. A single colony was picked and transferred to 50 mL of liquid LB medium, and incubated in a shaker at 30°C and 200 rpm until OD500 was achieved. 600When the pH reaches approximately 0.4, add 500 μL of arabinose and continue culturing for about 1 hour. Then, prepare electrocompetent cells and aliquot them into 1.5 mL EP tubes. Add 500 ng of pTargetF-P. yjbB The plasmid and 500 ng of the targeting fragment were electrotransformed. After recovery at 30℃ and 200 rpm for 1 h, 10 μL of arabinose was added for another 1 h of recovery. The mixture was centrifuged at 4500 rpm for 10 min and plated on Kan+Spe dual-antibody plates. The plates were incubated upside down at 30℃ for 36 h. After single bacteria grew, primer P was used... yjbB -P37-UTR-F and P37-R were used for PCR verification. If there were obvious and correctly sized bands on agarose gel electrophoresis, it indicated that the promoter replacement was successful. The correct colonies were selected for preservation and sequencing for further verification.

[0064] (4) Plasmid pTargetB-P yjbB removal

[0065] The successfully replaced promoter strain was inoculated into 5 mL of liquid LB (Kan) medium, and 25 μL of 0.5 mM / mL IPTG solution was added. After overnight incubation at 30°C and 200 rpm on a shaker, the culture was streaked onto Kan resistant plates and incubated upside down at 30°C. Once single colonies appeared, they were spotted onto LB (Spe+Kan) solid medium and LB (Kan) solid medium, respectively, and incubated upside down at 30°C to verify the removal of the pTargetB-PX plasmid. If colonies grew only on LB (Kan) solid plates and not on LB (Spe+Kan) solid plates, it indicated that the pTargetB-PX plasmid removal was ineffective. yjbB Plasmid removal was successful.

[0066] (5) Removal of plasmid pCas*

[0067] Successfully removed pTargetB-P yjbB Colonies containing the plasmid were inoculated into 5 mL of liquid LB medium and incubated overnight at 37°C and 200 rpm on a shaker. The inoculum was then streaked onto LB antibiotic-free plates. After single colonies grew, they were selected and spotted onto LB solid plates and LB (Kan) plates, respectively, and incubated upside down at 37°C to verify pCas plasmid removal. If colonies grew only on LB plates and not on LB (Kan) solid plates, the pCas* plasmid removal was successful. The resulting strain, PHE03(P), with promoter replacement completed and plasmid successfully removed, was obtained. yjbB ::P37).

[0068] The sequences of the primers used are shown in Table 1.

[0069] Table 1

[0070]

[0071] Example 2 PHE03 (P mdtF Construction of strain ::P37)

[0072] In this embodiment, the phenylalanine-producing bacterium E. coli PHE03 (AdvancedBiotechnology (2024) 2:15), previously constructed in the laboratory, was used as the starting bacterium for modification. The natural promoter of the mdtF gene (Uniprot P37637) was replaced with promoter P37 to construct PHE03 (P mdtF strain ::P37). The specific experimental methods are as follows:

[0073] (1) Construction of the shooting segment:

[0074] Using the E. coli W3110 genome as a template, primer P was used... mdtF -up-F、P mdtF -up-R (sequence shown in Table 2), the upstream homologous arm of the mdtF gene promoter was amplified, and primer P was used to amplify it. mdtF -dn-F、P mdtF -dn-R (sequence shown in Table 2) was amplified to obtain the downstream homologous arm of the mdtF gene promoter.

[0075] Using plasmid pZBK (Biotechnology Biofuels 2019, 12:94) as a template, the P37 promoter sequence was amplified using primers P37-F and P37-R (sequences shown in Table 2). The amplified target fragment P37-UTR (sequence shown in SEQ ID NO:1) was recovered using a DNA recovery kit.

[0076] Using the upstream homologous arm of the recovered mdtF gene promoter and the P37 promoter (P37-UTR) as templates, primer P was used. mdtF Overlap PCR was performed using -up-F and P37-R to obtain P mdtF -up-P37. With recycled P mdtF Using -up-P37 and downstream homologous arms as templates, primer P mdtF -up-F and P mdtF Overlap PCR was performed again with -dn-R to obtain PCR products. The PCR products were verified by agarose gel electrophoresis, and the target fragment with the correct band was recovered from the gel to obtain the targeting fragment P. mdtF -up-P37-dn.

[0077] (2)pTargetB-P mdtF plasmid construction

[0078] Using pTargetB plasmid as a template, primer P was used. mdtF -N20-F, P mdtF The template was amplified by reverse PCR using -N20-R (sequence shown in Table 2) to obtain the PCR product. The PCR product was then treated with DpnI to remove the template plasmid. The treated product was immediately transformed into E. coli DH5α competent cells, plated on Spe resistant plates, and incubated upside down at 37°C. One or two single colonies were randomly selected for testing. For colonies with correct sequencing results, the corresponding single colonies were inoculated into 5 mL of LB broth and cultured overnight to preserve the culture and extract the plasmid to obtain pTargetB-P. mdtF .

[0079] (3) Preparation and transformation of E. coli PHE03 electrocompetent cells containing pCas* plasmid

[0080] The pCas* plasmid was transformed into the originating strain, phenylalanine-producing E. coli PHE03, plated, and incubated upside down. A single colony was picked and transferred to 50 mL of liquid LB medium, and incubated in a shaker at 30°C and 200 rpm until OD500 was achieved. 600 When the pH reaches approximately 0.4, add 500 μL of arabinose and continue culturing for about 1 hour. Then, prepare electrocompetent cells and aliquot them into 1.5 mL EP tubes. Add 500 ng of pTargetF-P. mdtF The plasmid and 500 ng of the targeting fragment were electrotransformed. After recovery at 30℃ and 200 rpm for 1 h, 10 μL of arabinose was added for another 1 h of recovery. The mixture was centrifuged at 4500 rpm for 10 min and plated on Kan+Spe dual-antibody plates. The plates were incubated upside down at 30℃ for 36 h. After single bacteria grew, primer P was used... mdtF -P37-UTR-F and P37-R (sequences shown in Table 2) were used for PCR verification. If there were obvious and correctly sized bands on agarose gel electrophoresis, it indicated that the promoter replacement was successful. The correct colonies were selected for preservation and sequencing for further verification.

[0081] (4) Plasmid pTargetB-P mdtF removal

[0082] The successfully replaced promoter strain was inoculated into 5 mL of liquid LB (Kan) medium, and 25 μL of 0.5 mM / mL IPTG solution was added. After overnight incubation at 30°C and 200 rpm on a shaker, the culture was streaked onto Kan resistant plates and incubated upside down at 30°C. Once single colonies appeared, they were spotted onto LB (Spe+Kan) solid medium and LB (Kan) solid medium, respectively, and incubated upside down at 30°C to verify the removal of the pTargetB-PX plasmid. If colonies grew only on LB (Kan) solid plates and not on LB (Spe+Kan) solid plates, it indicated that the pTargetB-PX plasmid removal was ineffective. mdtF Plasmid removal was successful.

[0083] (5) Removal of plasmid pCas*

[0084] Successfully removed pTargetB-P mdtF Colonies containing the plasmid were inoculated into 5 mL of liquid LB medium and incubated overnight at 37°C and 200 rpm on a shaker. The inoculum was then streaked onto LB antibiotic-free plates. After single colonies grew, they were selected and spotted onto LB solid plates and LB (Kan) plates, respectively, and incubated upside down at 37°C to verify pCas plasmid removal. If colonies grew only on LB plates and not on LB (Kan) solid plates, the pCas* plasmid removal was successful. The resulting strain, PHE03(P), with promoter replacement completed and plasmid successfully removed, was obtained. mdtF ::P37).

[0085] The sequences of the primers used are shown in Table 2.

[0086] Table 2

[0087]

[0088]

[0089] Example 3 PHE03 (P lapA Construction of strain ::P37)

[0090] In this embodiment, the phenylalanine-producing bacterium E. coli PHE03 (AdvancedBiotechnology (2024) 2:15), which was previously constructed in the laboratory, was used as the starting bacterium for modification. The natural promoter of the lapA gene (Uniprot P0ACV4) was replaced with promoter P37 to construct PHE03 (P lapA strain ::P37). The specific experimental methods are as follows:

[0091] (1) Construction of the shooting segment:

[0092] Using the E. coli W3110 genome as a template, primer P was used... lapA -up-F、P lapA -up-R (sequence shown in Table 3), the upstream homologous arm of the lapA gene promoter was amplified, and then amplified using primer P. lapA -dn-F、P lapA -dn-R (sequence shown in Table 3) was used to amplify the downstream homologous arm of the lapA gene promoter.

[0093] Using plasmid pZBK (Biotechnology Biofuels 2019, 12:94) as a template, the P37 promoter sequence was amplified using primers P37-F and P37-R (sequences shown in Table 3). The amplified target fragment P37-UTR (sequence shown in SEQ ID NO:1) was recovered using a DNA recovery kit.

[0094] Using the upstream homologous arm of the recovered lapA gene promoter and the P37 promoter (P37-UTR) as templates, primer P was used. lapA Overlap PCR was performed using -up-F and P37-R to obtain P lapA -up-P37. With recycled P lapA Using -up-P37 and downstream homologous arms as templates, primer P lapA -up-F and P lapA Overlap PCR was performed again with -dn-R to obtain PCR products. The PCR products were verified by agarose gel electrophoresis, and the target fragment with the correct band was recovered from the gel to obtain the targeting fragment P. lapA -up-P37-dn.

[0095] (2)pTargetB-P lapA plasmid construction

[0096] Using pTargetB plasmid as a template, primer P was used. lapA -N20-F, P lapA The template was amplified by reverse PCR using -N20-R (sequence shown in Table 3) to obtain the PCR product. The PCR product was then treated with DpnI to remove the template plasmid. The treated product was immediately transformed into E. coli DH5α competent cells, plated on Spe resistant plates, and incubated upside down at 37°C. One or two single colonies were randomly selected for testing. For colonies with correct sequencing results, the corresponding single colonies were inoculated into 5 mL of LB broth and cultured overnight to preserve the culture and extract the plasmid to obtain pTargetB-P. lapA .

[0097] (3) Preparation and transformation of E. coli PHE03 electrocompetent cells containing pCas* plasmid

[0098] The pCas* plasmid was transformed into the originating strain, phenylalanine-producing E. coli PHE03, plated, and incubated upside down. A single colony was picked and transferred to 50 mL of liquid LB medium, and incubated in a shaker at 30°C and 200 rpm until OD500 was achieved. 600 When the pH reaches approximately 0.4, add 500 μL of arabinose and continue culturing for about 1 hour. Then, prepare electrocompetent cells and aliquot them into 1.5 mL EP tubes. Add 500 ng of pTargetF-P. lapA The plasmid and 500 ng of the targeting fragment were electrotransformed. After recovery at 30℃ and 200 rpm for 1 h, 10 μL of arabinose was added for another 1 h of recovery. The mixture was centrifuged at 4500 rpm for 10 min and plated on Kan+Spe dual-antibody plates. The plates were incubated upside down at 30℃ for 36 h. After single bacteria grew, primer P was used... lapA -P37-UTR-F and P37-R (sequences shown in Table 3) were used for PCR verification. If there were obvious and correctly sized bands on agarose gel electrophoresis, it indicated that the promoter replacement was successful. The correct colonies were selected for preservation and sequencing for further verification.

[0099] (4) Plasmid pTargetB-P lapA removal

[0100] The successfully replaced promoter strain was inoculated into 5 mL of liquid LB (Kan) medium, and 25 μL of 0.5 mM / mL IPTG solution was added. After overnight incubation at 30°C and 200 rpm on a shaker, the culture was streaked onto Kan resistant plates and incubated upside down at 30°C. Once single colonies appeared, they were spotted onto LB (Spe+Kan) solid medium and LB (Kan) solid medium, respectively, and incubated upside down at 30°C to verify the removal of the pTargetB-PX plasmid. If colonies grew only on LB (Kan) solid plates and not on LB (Spe+Kan) solid plates, it indicated that the pTargetB-PX plasmid removal was ineffective. lapA Plasmid removal was successful.

[0101] (5) Removal of plasmid pCas*

[0102] Successfully removed pTargetB-P lapAColonies containing the plasmid were inoculated into 5 mL of liquid LB medium and incubated overnight at 37°C and 200 rpm on a shaker. The inoculum was then streaked onto LB antibiotic-free plates. After single colonies grew, they were selected and spotted onto LB solid plates and LB (Kan) plates, respectively, and incubated upside down at 37°C to verify pCas plasmid removal. If colonies grew only on LB plates and not on LB (Kan) solid plates, the pCas* plasmid removal was successful. The resulting strain, PHE03(P), with promoter replacement completed and plasmid successfully removed, was obtained. lapA ::P37).

[0103] The sequences of the primers used are shown in Table 3.

[0104] Table 3

[0105]

[0106]

[0107] Example 4 PHE03 (P ortT Construction of strain ::P37)

[0108] In this embodiment, the phenylalanine-producing bacterium E. coli PHE03 (AdvancedBiotechnology (2024) 2:15), which was previously constructed in the laboratory, was used as the starting bacterium for modification. The natural promoter of the ortT gene (Uniprot P64453) was replaced with promoter P37 to construct PHE03 (P ortT strain ::P37). The specific experimental methods are as follows:

[0109] (1) Construction of the shooting segment:

[0110] Using the E. coli W3110 genome as a template, primer P was used... ortT -up-F、P ortT -up-R (sequence shown in Table 4), the upstream homologous arm of the ortT gene promoter was amplified, and then amplified using primer P. ortT -dn-F、P ortT -dn-R (sequence shown in Table 4) was amplified to obtain the downstream homologous arm of the ortT gene promoter.

[0111] Using plasmid pZBK (Biotechnology Biofuels 2019, 12:94) as a template, the P37 promoter sequence was amplified using primers P37-F and P37-R (sequences shown in Table 4). The amplified target fragment P37-UTR (sequence shown in SEQ ID NO:1) was recovered using a DNA recovery kit.

[0112] Using the upstream homologous arm of the recovered ortT gene promoter and the P37 promoter (P37-UTR) as templates, primer P was used. ortT Overlap PCR was performed using -up-F and P37-R to obtain P ortT -up-P37. With recycled P ortT Using -up-P37 and downstream homologous arms as templates, primer P ortT -up-F and P ortT Overlap PCR was performed again with -dn-R to obtain PCR products. The PCR products were verified by agarose gel electrophoresis, and the target fragment with the correct band was recovered from the gel to obtain the targeting fragment P. ortT -up-P37-dn.

[0113] (2)pTargetB-P ortT plasmid construction

[0114] Using pTargetB plasmid as a template, primer P was used. ortT -N20-F, P ortT The template was amplified by reverse PCR using -N20-R (sequence shown in Table 4) to obtain the PCR product. The PCR product was then treated with DpnI to remove the template plasmid. The treated product was immediately transformed into E. coli DH5α competent cells, plated on Spe resistant plates, and incubated upside down at 37°C. One or two single colonies were randomly selected for testing. For colonies with correct sequencing results, the corresponding single colonies were inoculated into 5 mL of LB broth and cultured overnight to preserve the culture and extract the plasmid to obtain pTargetB-P. ortT .

[0115] (3) Preparation and transformation of E. coli PHE03 electrocompetent cells containing pCas* plasmid

[0116] The pCas* plasmid was transformed into the originating strain, phenylalanine-producing E. coli PHE03, plated, and incubated upside down. A single colony was picked and transferred to 50 mL of liquid LB medium, and incubated in a shaker at 30°C and 200 rpm until OD500 was achieved. 600 When the pH reaches approximately 0.4, add 500 μL of arabinose and continue culturing for about 1 hour. Then, prepare electrocompetent cells and aliquot them into 1.5 mL EP tubes. Add 500 ng of pTargetF-P. ortT The plasmid and 500 ng of the targeting fragment were electrotransformed. After recovery at 30℃ and 200 rpm for 1 h, 10 μL of arabinose was added for another 1 h of recovery. The mixture was centrifuged at 4500 rpm for 10 min and plated on Kan+Spe dual-antibody plates. The plates were incubated upside down at 30℃ for 36 h. After single bacteria grew, primer P was used... ortT-P37-UTR-F and P37-R (sequences shown in Table 4) were used for PCR verification. If there were obvious and correctly sized bands on agarose gel electrophoresis, it indicated that the promoter replacement was successful. The correct colonies were selected for preservation and sequencing for further verification.

[0117] (4) Plasmid pTargetB-P ortT removal

[0118] The successfully replaced promoter strain was inoculated into 5 mL of liquid LB (Kan) medium, and 25 μL of 0.5 mM / mL IPTG solution was added. After overnight incubation at 30°C and 200 rpm on a shaker, the culture was streaked onto Kan resistant plates and incubated upside down at 30°C. Once single colonies appeared, they were spotted onto LB (Spe+Kan) solid medium and LB (Kan) solid medium, respectively, and incubated upside down at 30°C to verify the removal of the pTargetB-PX plasmid. If colonies grew only on LB (Kan) solid plates and not on LB (Spe+Kan) solid plates, it indicated that the pTargetB-PX plasmid removal was ineffective. ortT Plasmid removal was successful.

[0119] (5) Removal of plasmid pCas*

[0120] Successfully removed pTargetB-P ortT Colonies containing the plasmid were inoculated into 5 mL of liquid LB medium and incubated overnight at 37°C and 200 rpm on a shaker. The inoculum was then streaked onto LB antibiotic-free plates. After single colonies grew, they were selected and spotted onto LB solid plates and LB (Kan) plates, respectively, and incubated upside down at 37°C to verify pCas plasmid removal. If colonies grew only on LB plates and not on LB (Kan) solid plates, the pCas* plasmid removal was successful. The resulting strain, PHE03(P), with promoter replacement completed and plasmid successfully removed, was obtained. ortT ::P37).

[0121] The sequences of the primers used are shown in Table 4.

[0122] Table 4

[0123]

[0124]

[0125] Detection example

[0126] Shake-flask fermentation of promoter-replaced bacteria: plasmid pZBK-P esaR -CnldhA(Advanced Biotechnology(2024)2:15) was transferred to the promoter replacement bacteria (PHE03(P) prepared in Examples 1-4.yjbB ::P37), PHE03(P mdtF ::P37), PHE03(P lapA ::P37), PHE03(P ortT ::P37)) and its originating bacterium, phenylalanine-producing E. coli PHE03, were used to obtain recombinant Escherichia coli producing phenyllactic acid. A schematic diagram of the biosynthetic pathway of phenyllactic acid is shown below. Figure 1 As shown.

[0127] A single colony of the above-mentioned phenyl lactic acid-producing recombinant Escherichia coli was picked and inoculated into 5 mL of liquid LB medium and cultured at 37°C and 200 rpm for about 14 h. A certain volume of seed culture was transferred to 50 mL of fermentation medium (fermentation medium (g / L): NH4Cl 0.58 g, MgSO4·7H2O 0.61 g, Na2HPO4 18.0 g, KH2PO4 9.0 g, yeast extract 4.0 g, NaCl 0.5 g, CaCl2 0.008 g, glucose 28.05 g, 2 mL / L trace element solution. Trace element solution (g / L): ZnSO4·7H2O 2.0 g, FeSO4·7H2O 0.5 g, MnCl2·4H2O 0.5 g, CuSO4·5H2O 0.16 g, CoCl2·6H2O 0.16 g, H3BO3 1.1 g, (NH4)6Mo7O 24 • 0.11 g of 4H₂O and 5.0 g of EDTA (sterilized using a 0.22 μm water-soluble filter membrane) were added to induce an initial OD of […]. 600 The concentration was 0.05. After fermentation on a shaker at 37℃ and 200rpm for 72 hours, the OD of the fermentation broth was analyzed. 600 The concentration of phenyllactic acid was determined by HPLC.

[0128] Table 5. Synthesis of Phenolic Acid by Recombinant Escherichia coli

[0129] Host bacteria <![CDATA[OD 600 ]]> Phenylated acid (g / L) PHE03 5.82±0.12 3.64±0.11 <![CDATA[PHE03(P yjbB ::P37)]]> 5.67±0.12 3.98±0.12 <![CDATA[PHE03(P mdtF ::P37)]]> 5.67±0.20 3.90±0.18 <![CDATA[PHE03(P lapA ::P37)]]> 5.91±0.04 4.01±0.03 <![CDATA[PHE03(P ortT ::P37)]]> 5.72±0.00 3.93±0.08

[0130] The results are shown in Table 5. As can be seen from the table, replacing the promoters of the yjbB, mdtF, lapA, and ortT genes with the strong P37 promoter significantly promoted the synthesis of phenyllactic acid, and the yield of phenyllactic acid increased by 7.1%-10.2%.

[0131] In addition to the expression vectors and phenylalanine-producing strains described in this embodiment, any expression vector and phenylalanine-producing strain can be used.

[0132] In addition to the methods mentioned in this embodiment, phenyl lactic acid can also be produced using a whole-cell catalytic method.

[0133] In addition to the above embodiments, the phenyllactic acid synthesis pathway enzyme gene can also be integrated into the Escherichia coli chromosome to construct the corresponding recombinant Escherichia coli.

[0134] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A recombinant bacterium, characterized in that, The recombinant bacteria express lactate dehydrogenase and overexpress YjbB, MdtF, LapA and / or OrtT.

2. The recombinant bacteria according to claim 1, characterized in that, The overexpression is achieved through any of the methods from A1) to A5): A1) Introduce a vector containing the coding genes of YjbB, MdtF, LapA and / or OrtT into the starting bacteria; A2) Increase the copy number of the coding genes for YjbB, MdtF, LapA and / or OrtT on the chromosome; A3) Alter the promoter sequence of the coding genes for YjbB, MdtF, LapA and / or OrtT on the chromosome; A4) The strong promoter is operatively linked to the coding genes of YjbB, MdtF, LapA and / or OrtT; A5) Modify the nucleotide sequences encoding yjbB, mdtF, lapA and / or ortT.

3. The recombinant bacteria according to claim 2, characterized in that, The originating bacteria of the recombinant bacteria include Escherichia coli.

4. The recombinant bacteria according to claim 2, characterized in that, A3) This includes replacing the original promoters of the genes encoding YjbB, MdtF, LapA and / or OrtT on the chromosome with strong promoters; Preferably, the strong promoter includes at least one of the following: P37 promoter, P8 promoter, J23100 promoter, J23119 promoter, P21285 promoter, M1-93 promoter, and PL1118 promoter; The changes described in (and / or, A5) include codon optimization.

5. The method for constructing the recombinant bacteria according to any one of claims 1 to 4, or a method for increasing phenyllactic acid production, characterized in that, The steps include: Genetic engineering techniques were used to overexpress YjbB, MdtF, LapA, and / or OrtT in the starting strain; The starting bacteria express lactate dehydrogenase.

6. The method according to claim 5, characterized in that, The method includes replacing the original promoters of the yjbB, mdtF, lapA and / or ortT genes of the starting strain with strong promoters using gene editing technology.

7. The method according to claim 6, characterized in that, The gene editing technology includes gene replacement.

8. The use of the recombinant bacteria according to any one of claims 1 to 4 in any one of B1) to B3): B1) Production of phenyllactic acid or its derivatives; B2) Prepare products for the production of phenyllactic acid or its derivatives; B3) Increase the yield of phenyllactic acid or its derivatives.

9. The application according to claim 8, characterized in that, The product includes at least one of reagents and reagent kits.

10. A method for producing phenyllactic acid, characterized in that, include: Cultivate the recombinant bacteria according to any one of claims 1 to 4.

Images