Genetically engineered bacteria for producing rosmarinic acid and application thereof

Abstract

The application belongs to the technical field of microorganisms and biosynthesis, and particularly relates to a genetically engineered bacteria for producing rosmarinic acid and application thereof. Desulfatirhabdium butyrativorans In order to solve the problem that the existing microbial synthesis system is difficult to break through the cost threshold of industrialized production of rosmarinic acid, the application introduces Db4CL from Populus trichocarpa into the rosmarinic acid synthesis pathway to efficiently catalyze coffee acid and CoA to generate coffee acyl-CoA, and performs gene knockout on the chassis bacteria and overexpresses YcjP protein, so that the rosmarinic acid synthesis capacity is greatly improved. tyrR, pheA, pykF The rosmarinic acid production strain constructed by using the strategy disclosed in the application can reach a rosmarinic acid yield of 6469 mg / L in a fermenter scale production.

Description

Technical Field

[0001] This invention belongs to the field of microbial and biosynthetic technology, specifically relating to genetically engineered bacteria that produce rosmarinic acid and their applications. Background Technology

[0002] Rosmarinic acid (RA) is a natural product with an orthodiol-α,β-unsaturated carboxylic acid skeleton. It was first isolated from rosemary (Rosmarinus officinalis) and is a natural phenylpropanoid compound with good antioxidant, anti-inflammatory and neuroprotective activities. It is often used as a natural preservative, functional additive or anti-inflammatory ingredient in medicine, food and cosmetics.

[0003] Rosmarinic acid was initially obtained primarily from water extraction of plants in the Lamiaceae family, such as rosemary and perilla. However, plant extraction methods are limited by the plant's growth cycle and geographical location, and suffer from low content of active ingredients and impurity accumulation due to repeated solvent concentration. Chemical semi-synthesis solved the problem of mass production difficulties associated with traditional plant extraction, but it brought new challenges: it requires expensive or toxic reagents such as boron trifluoride diethyl ether complexes, which can easily cause environmental pollution, and the finished product often fails to meet pharmaceutical-grade quality requirements.

[0004] With the development of synthetic biology, the preparation of rosmarinic acid has shifted to microbial cell factories. Compared with traditional plant extraction and chemical semi-synthesis methods, synthetic biology preparation has many advantages, such as not being limited by natural conditions, short production cycle, high product purity, strong controllability, and environmental friendliness, providing a new feasible way for the industrial-scale production of rosmarinic acid.

[0005] Currently, the microbial synthesis of rosmarinic acid mainly relies on two technical strategies: single-strain full-pathway integration and multi-strain co-culture systems. The first strategy involves integrating all key enzymes of the complete RA biosynthetic pathway—including tyrosine aminolyase (TAL), 4-coumarate-CoA ligase (4CL), hydroxyphenylpyruvate reductase (HPPR), and rosmarinic acid synthase (RAS)—into a single *E. coli* host, and optimizing precursor supply and enzyme expression balance through metabolic engineering. The second strategy employs a modular design, breaking down the RA biosynthetic pathway into different modules / strains (such as CA, SAA, and RA modules), and redistributing metabolic flux by regulating the proportions of each module / strain. However, regardless of whether it is full-path integration or modular integration, the core bottlenecks faced by existing technologies are concentrated in the following aspects: the catalytic efficiency of key enzymes 4CL (such as Pc4CL, At4CL, etc.) for caffeic acid is low, resulting in insufficient accumulation of the intermediate caffeoyl-CoA; metabolic pathway imbalance leads to the accumulation of a large amount of byproducts such as L-DOPA, which seriously affects product purity and yield; uneven distribution of metabolic flux in the synthesis modules of caffeic acid and salvianolic acid A (SAA) causes the overall pathway efficiency to decline; and the imbalance of the cofactor regeneration system (especially FADH2 / NADH) further restricts the distribution of metabolic flux. These factors together make it difficult for existing microbial synthesis systems to break through the cost threshold for the industrial production of rosmarinic acid. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention, based on the existing synthetic route for rosmarinic acid, further utilizes... Desulfatirhabdium butyrativorans The derived Db4CL replaces the traditional 4CL to improve the catalytic efficiency for caffeic acid and efficiently synthesize caffeoyl-CoA. Overexpression of the E. coli endogenous transport protein YcjP (ABC transporter family) enhances caffeic acid efflux capacity. Knockout... tyrR, pheA, pykF Modifying the basal microorganisms to increase the metabolic flux of the shikimic acid pathway and accumulate rosmarinic acid precursors can significantly improve the current state of industrial synthesis of rosmarinic acid.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] One of the technical solutions provided by the present invention is the application of a protein as a 4-coumaric acid coenzyme A ligase, wherein the amino acid sequence of the protein is shown in SEQ ID NO.1; Furthermore, the protein is named Db4CL, and the nucleotide sequence encoding the protein is shown in SEQ ID NO.2; Furthermore, there is the application of Db4CL in the catalytic synthesis of caffeic acid into caffeoyl-CoA; and even further, there is the application of Db4CL in the synthesis of rosmarinic acid.

[0009] The second technical solution provided by this invention is a method for improving rosmarinic acid production capacity, the method comprising performing gene editing on the host in any one or more of the following (1)-(3): (1) Knockout of the gene encoding the aromatic amino acid biotransport regulator in the host genome tyrR, Phenylalanine synthase encoding gene pheA and the gene encoding pyruvate kinase I pykF ; (2) Overexpression of 4-coumarate coenzyme A ligase, lactate dehydrogenase, rosmarinic acid synthase, tyrosine ammonia-lyase, 4-hydroxyphenylacetic acid-3-hydroxylase and DAHP synthase. (3) Overexpression of YcjP; Furthermore, in (2), the expression intensity of 4-coumarate coenzyme A ligase, lactate dehydrogenase and rosmarinic acid synthase is higher than that of tyrosine ammonia-lyase, 4-hydroxyphenylacetic acid-3-hydroxylase and DAHP synthase, so as to achieve the purpose of balancing the metabolic flux of heterologous multi-enzyme complex pathway; the methods to control the expression intensity include, but are not limited to, using plasmids with different copy numbers to express different target genes. Furthermore, the 4-coumaric acid coenzyme A ligase is selected from Db4CL, At4CL, Pc4CL, Pt4CL, or Os4CL; Db4CL is a preferred embodiment. Furthermore, the nucleotide sequence of the gene encoding the lactate dehydrogenase is shown in SEQ ID NO.8; Furthermore, the rosmarinic acid synthase is from lemon balm (Lemon Balm). Melissa officinalis The MoRAS gene from which this gene originates has the GenBank accession number FR670523.1. Furthermore, the tyrosine ammonia-lyase is Flavobacterium johnsonii (Flavobacterium johnsonii). Flavobacterium johnsoniae (Source: FjTAL, NCBI Reference Sequence: WP_012023194.1) Furthermore, the 4-hydroxyphenylacetic acid-3-hydroxylase is *Escherichia coli* (E. coli). Escherichia coli The nucleotide sequence of HpaBC from this source is shown in SEQ ID NO.3; Furthermore, the gene encoding the DAHP synthase is aroG. fbr The gene, NCBI Reference Sequence is: AXN70009.1; Furthermore, the nucleotide sequence of the gene encoding the YcjP protein is shown in SEQ ID NO.4; Furthermore, the host is selected from: *Escherichia coli*, *Bacillus subtilis*, or *Saccharomyces cerevisiae*; preferably *Escherichia coli*, such as... E. coli BL21(DE3), etc.

[0010] The third technical solution provided by this invention is a strain that produces rosmarinic acid. This strain is obtained by modifying a host in the following way: modifying the gene encoding aromatic amino acid biotransport regulators on the genome. tyrR, Phenylalanine synthase encoding gene pheA and the gene encoding pyruvate kinase I pykF Knockout was performed; 4-coumarate-coenzyme A ligase, lactate dehydrogenase, and rosmarinic acid synthase were overexpressed using a first expression vector, and tyrosine ammonia-lyase, 4-hydroxyphenylacetic acid-3-hydroxylase, and DAHP synthase were overexpressed using a second expression vector to construct a heterologous rosmarinic acid synthesis pathway; wherein the copy number of the first expression vector was higher than that of the second expression vector. Furthermore, the replicon of the first expression vector is selected from... ColE1 Replicator pMB1 Replicator pUC Replicator or RSF1030 Replicon; the replicon of the second expression vector is selected from: p15A Replicator pSC101 Replicator or F Replicator; Furthermore, the first expression vector is selected from pET28a, pET21a, pET22b or pTrc99A vector; the second expression vector is selected from pACYC184, pACYCDuet-1, pCDFDuet-1 or pSC101 vector; Preferably, the first expression vector is pET28a; the second expression vector is pACYC184; Furthermore, the YcjP protein was overexpressed in the host. Furthermore, the host is selected from: *Escherichia coli*, *Bacillus subtilis*, or *Saccharomyces cerevisiae*; preferably *Escherichia coli*, such as... E. coli BL21(DE3), etc.; Furthermore, the 4-coumarate coenzyme A ligase is Db4CL, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2; the lactate dehydrogenase is Ldh, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.8; the rosmarinic acid synthase is MoRAS, and the Genbank encoding gene is FR670523.1; the tyrosine ammonia-lyase is FjTAL, NCBI Reference Sequence: WP_012023194.1; the 4-hydroxyphenylacetic acid-3-hydroxylase is HpaBC, and the nucleotide sequence is shown in SEQ ID NO.3; the encoding gene for the DAHP synthase is aroG. fbr The gene, NCBI Reference Sequence: AXN70009.1; the nucleotide sequence of the gene encoding the YcjP protein is shown in SEQ ID NO.4; Furthermore, the aforementioned tyrR GenBank accession number for the gene: QJZ03772.1; pheA GenBank accession number for the gene: QJZ04816.1; pykF GenBank accession number for the gene: QJZ04062.1.

[0011] The fourth technical solution provided by this invention is the application of the strain described in the third technical solution in the production of rosmarinic acid; The application includes the process of culturing the strain in a culture medium using glucose, glycerol, acetic acid, xylose, or a mixture of the above substances as a carbon source and obtaining rosmarinic acid from the culture.

[0012] Beneficial effects: (1) This invention is the first to determine Desulfatirhabdium butyrativorans The protein, with an amino acid sequence as shown in SEQ ID NO.1, possesses the function of a 4-coumarate-CoA ligase, which can efficiently catalyze the synthesis of caffeic acid and CoA into caffeoyl-CoA. Therefore, it can be further used for the construction of rosmarinic acid biosynthesis pathways or production strains.

[0013] Meanwhile, Db4CL consumes more caffeic acid than other microbial 4CLs. Compared with other 4CLs, the rosmarinic acid biosynthesis pathway constructed using Db4CL can significantly increase the yield of rosmarinic acid, which is 2.29 to 8.6 times that of other strains.

[0014] (2) To balance the metabolic flux of the heterologous multi-enzyme complex pathway, different target genes were expressed using plasmid backbones with different copy numbers. Among them, the enzymes encoded by FjTAL, HpaBC, and aroG have high catalytic activity; therefore, they were constructed in low-copy vectors to limit their intracellular expression and prevent the rapid transport of highly active enzymes from leading to the accumulation of intermediate metabolites and causing cytotoxicity. Conversely, Db4CL, ldh, and MoRAS are rate-limiting enzymes in the pathway, requiring high protein abundance to maintain sufficient metabolic flux; therefore, they were cloned into the medium-to-high copy vector pET28a. This strategy increased rosmarinic acid production by approximately 90%.

[0015] (3) The rosmarinic acid producing strain constructed using the strategy described in this invention can achieve a rosmarinic acid yield of 6469 mg / L in fermenter-scale production. Detailed Implementation

[0016] This invention discloses a method for producing rosmarinic acid, an engineered microorganism, and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0017] 1. The modification principles involved in this invention (1) Modification (knockout) of chassis bacteria tyrR, pheA, pykF ) By knocking out the gene encoding aromatic amino acid biotransport regulators in Chameleon bacteria tyrR, Phenylalanine synthase encoding gene pheA Increase the supply of caffeic acid precursors and knock out the gene encoding pyruvate kinase I. pykF This leads to the accumulation of PEP and a greater flow to the shikimic acid pathway, and the increased metabolic flux of the shikimic acid pathway results in an increase in aromatic compounds that are precursors to rosmarinic acid.

[0018] (2) Expression of key enzymes in the biosynthetic pathway of rosmarinic acid The aroG gene encodes DAHP synthase, a key rate-limiting enzyme in the shikimic acid pathway. In the native metabolic system of *E. coli*, the activity of DAHP synthase is strictly allosterically inhibited by downstream products, particularly L-tyrosine and L-phenylalanine. Since the synthesis of rosmarinic acid requires a large amount of L-tyrosine as a starting substrate, this invention introduces a DAHP synthase mutant to break this inhibition. In this invention, the aroG gene encodes the DAHP synthase mutant. fbr The gene's NCBI accession number is AXN70009.1.

[0019] TAL (tyrosine ammonia-lyase) catalyzes the deamination of tyrosine to produce 4-coumaric acid; HpaBC (4-hydroxyphenylacetic acid-3-hydroxylase) catalyzes the production of caffeic acid from 4-coumaric acid; 4CL (4-coumaric acid-CoA ligase) catalyzes the binding of caffeic acid to CoA (CoA) to form activated caffeoyl-CoA (RA precursor); and RAS (rosmarinic acid synthase) catalyzes the synthesis of the final product rosmarinic acid from caffeoyl-CoA and tanshinone. TAL, HpaBC, 4CL, and RAS are all key enzymes in the rosmarinic acid biosynthesis pathway. This invention enhances the rosmarinic acid biosynthesis pathway by overexpressing TAL, HpaBC, 4CL, and RAS.

[0020] Among them, Flavobacterium johnsonii ( Flavobacterium johnsoniae (Source: FjTAL, NCBI ReferenceSequence: WP_012023194.1) Among them, Escherichia coli ( Escherichia coli The nucleotide sequence of HpaBC from this source is shown in SEQ ID NO.3; Among them, the present invention for the first time Desulfatirhabdium butyrativorans The source, labeled "acyl-CoAsynthetase", is a protein with the amino acid sequence shown in SEQ ID NO.1, used as 4CL and denoted as Db4CL (nucleotide sequence shown in SEQ ID NO.2).

[0021] Among them, lemon balm ( Melissa officinalis The gene encoding the rosmarinic acid synthase MoRAS, derived from [source], has the GenBank accession number FR670523.1.

[0022] Ldh (lactate dehydrogenase) catalyzes the production of 4-hydroxyphenylpyruvate to 4-hydroxyphenyllactic acid in the SSA module. 4-hydroxyphenyllactic acid is a precursor for the synthesis of tanshinone. In this invention, the nucleotide sequence of the gene encoding the lactate dehydrogenase Ldh is shown in SEQ ID NO. 8.

[0023] YcjP is a putative inner membrane protein in *E. coli* whose function is not fully elucidated. This invention enhances caffeic acid efflux by overexpressing YcjP to balance various metabolic flows. In this invention, the gene encoding the YcjP protein has the nucleotide sequence shown in SEQ ID NO.4.

[0024] 2. The culture medium information involved in this application and its embodiments is as follows: LB liquid medium: Tryptone: 10 g, Yeast Extract: 5 g, Sodium Chloride (NaCl): 10 g, Deionized Water: bring to a final volume of 1000 mL.

[0025] M9Y liquid culture medium: Na₂HPO₄ (disodium hydrogen phosphate): 16.78 g, KH₂PO₄ (potassium dihydrogen phosphate): 3.0 g, NaCl (sodium chloride): 0.5 g, NH₄Cl (ammonium chloride): 1.0 g, yeast extract: 25 g, glucose: 20.0 g, and deionized water to a final volume of 1000 mL. The culture medium was sterilized at 121℃ for 20 min, cooled to room temperature, and then aseptically added with 0.246 g of MgSO₄·7H₂O, 0.015 g of CaCl₂·2H₂O, 0.003 g of FeSO₄·7H₂O, and 0.01 g of VB1 (thiamine hydrochloride).

[0026] 3. The strains and vectors involved in the experiments of this invention are as follows: Chassis cells, Escherichia coli BL21(DE3); pEcCas9 plasmid, purchased from General Bio; pEcgRNA plasmid, purchased from General Biotechnology; pET28a plasmid, purchased from General Bio; pACYC184, purchased from General Bio.

[0027] 4. Information regarding some of the enzymes / proteins and their encoding genes involved in the embodiments of this invention is shown in Table 1 below: Table 1. Information on the enzymes involved and their encoding genes.

[0028] 5. The primer sequences used to construct the strains in the embodiments of the present invention are shown in Table 2: Table 2 Primer Table

[0029] It should be noted that the construction methods provided in the embodiments of the present invention are exemplary and not restrictive. Those skilled in the art can use any technical means to achieve the final gene editing purpose.

[0030] The present invention will be further explained and illustrated below through specific embodiments.

[0031] Example 1: Chassis microbial modification (knockout) tyrR, pheA, pykF ) Using the CRISPR-Cas9 method with pEcCas9 and pEcgRNA plasmid systems, starting with Escherichia coli BL21(DE3), the process was completed through cyclic operations. tyrR, pheA and pykF Three genes were knocked out in succession.

[0032] First, regarding the above tyrR, pheA and pykF Specific gRNA targets were designed based on the coding sequences of the genes. Using primer circularization PCR, double-stranded oligonucleotides targeting each target were cloned upstream of the gRNA scaffold of the pEcgRNA plasmid, respectively, to construct recombinant pEcgRNA plasmids targeting the tyrR, pheA, and pykF genes. tyrR, pheA, pykF The primers for the corresponding gRNAs were designated as pEcgRNA-tyrR-F / R, pEcgRNA-pheA-F / R, and pEcgRNA-pykF-F / R (primer sequences are detailed in Table 2). Using pEcgRNA as a template, the gRNA primers were looped to obtain the pEcgRNA-tyrR, pEcgRNA-pheA, and pEcgRNA-pykF plasmids, respectively. Simultaneously, for each target gene, corresponding repair template DNA was synthesized via overlap PCR. The repair template DNA consisted of 500 bp segments above and below the gene to be knocked out, fused together via overlap PCR.

[0033] tyrR, pheA and pykF The knockout is performed sequentially; the following example uses the knockout of the tyrR gene: a) Preparation of competent cells: pEcCas9 plasmid was electroporated into Escherichia coli BL21(DE3) host bacteria to obtain engineered strains with Cas9 expression and homologous recombination ability, which were cultured at 30°C in a medium containing kanamycin.

[0034] b) First electroporation and gene editing: Using Escherichia coli BL21(DE3) genomic DNA as a template, and tyrR-UF and tyrR-UR, tyrR-DF and tyrR-DR as primers, PCR amplification was performed respectively. tyrRHomologous arms, approximately 500 bp each upstream and downstream of the gene, were spliced ​​and fused using overlap extension PCR. The resulting fragments were purified and recovered as [the desired gene]. tyrR Gene repair template DNA: tyrR500Up-tyrR500Down.

[0035] The constructed pEcgRNA-tyrR plasmid and the corresponding tyrR gene repair template DNA: tyrR500Up-tyrR500Down were electroporated into the competent cells obtained in step a).

[0036] c) Induction and Screening: After the electroporated bacterial culture was revived at 30°C, arabinose (final concentration 3 g / L) was added to induce λ-Red recombination system expression and promote homologous recombination. The culture was then plated on plates containing kanamycin and appropriate antibiotics (selected according to the repair template) and incubated at 30°C.

[0037] d) Verification and elimination of gRNA plasmid: Single clones were selected, and the correct knockout of the tyrR gene was verified by colony PCR and DNA sequencing. Positive clones were cultured in a medium containing rhamnose (final concentration 2 g / L) and kanamycin to induce gRNA expression and self-eliminate the pEcgRNA-tyrR plasmid. Elimination was verified by streaking the plasmid onto plates containing and without spectinomycin.

[0038] e) Elimination of Cas9 plasmid: Strains with eliminated gRNA plasmids were cultured in antibiotic-free LB medium at 42°C. The pEcCas9 plasmid was diluted using temperature-sensitive replicon characteristics. Subsequently, it was plated on plates containing sucrose (final concentration 20 g / L) for reverse selection. Finally, by streaking on plates containing antibiotics and kanamycin, selection was made to obtain strains containing only gRNA. tyrR Pure engineered strains with gene knockout and containing no exogenous plasmids are designated as chassis strain BL21(DE3)-ΔtyrR.

[0039] f) Cyclic operation: Using the BL21(DE3)-ΔtyrR strain with the tyrR gene knocked out in step e) as the starting strain for the new round of operation, repeat steps a) to e) to knock out the pheA and pykF genes sequentially. In each cycle, a recombinant pEcgRNA plasmid targeting the next gene and repair templates with different resistance markers are used. After the above three consecutive gene knockout cycles, the three genes (…) are finally obtained. tyrR, pheA, pykF The chassis strain BL21(DE3)-ΔtyrR- was obtained by knocking out all exogenous plasmids and producing a multiple mutant strain of E. coli BL21(DE3)-ΔtyrR-. pheA - For strain information of pykF, please refer to Table 3.

[0040] Table 3 Chassis bacteria information

[0041] Example 2: 4CL Screening from Different Biological Sources Based on the codon bias of Escherichia coli, the different biological sources in Table 1 were analyzed separately. Db4CL Gene, At4CL Genes, Pc 4CL Genes, Pt 4CL Genes and Os4CL Genes are artificially synthesized after codon optimization.

[0042] Using pET28a as a vector, restriction endonucleases were used to... Db4CL The gene was inserted downstream of the T7 promoter to construct the recombinant expression plasmid pET28a-Db4CL. The constructed recombinant plasmid pET28a-Db4CL was then transformed... E. coli Positive clones of BL21(DE3) were selected from LB agar containing 50 μg / mL kanamycin. Single colonies were picked for colony PCR and sequencing verification to confirm the positive clones. Db4CL After the gene was correctly inserted and the sequence was free of mutations, the Db4CL recombinant expression strain was obtained and named BL21(DE3) / pET28a-Db4CL.

[0043] Using the same method, expression strains BL21(DE3) / pET28a-Db4CL, BL21(DE3) / pET28a-At4CL, BL21(DE3) / pET28a-Pc4CL, BL21(DE3) / pET28a-Pt4CL, and BL21(DE3) / pET28a-Os4CL were obtained, respectively.

[0044] The four CL expression strains were inoculated at a 1:100 volume ratio into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 200 rpm until OD. 600 When the expression level reaches 0.6-0.8, add IPTG at a final concentration of 0.5-1.0 mmol / L to induce expression, and then transfer to 20℃ for low-temperature induction culture for 12-16 hours.

[0045] After fermentation, the cells were collected by centrifugation at 8000 rpm for 10 min at 4 °C. The cells were resuspended in 50 mmol / L Tris-HCl buffer (containing 1 mmol / L DTT and 1 mmol / L PMSF) at 1 / 20 of the original fermentation volume. The cells were then sonicated in an ice bath (250 W, 3 s on, 5 s off, 20 min total). The lysate was centrifuged at 12000 rpm for 20 min at 4 °C, and the supernatant was collected as crude enzyme solution for later use.

[0046] A 1 mL enzymatic reaction system was constructed, comprising: 100 mmol / L Tris-HCl buffer (pH 7.5), 5 mmol / L ATP, 5 mmol / L MgCl2, 0.5 mmol / L coenzyme A (CoA), and 80 μL crude enzyme solution (with consistent protein concentration in all groups). The reaction system was preheated in a 30°C water bath for 3 min, then caffeic acid was added to a final concentration of 0.2 mmol / L to initiate the reaction, and the reaction was continued at 30°C for 8 h.

[0047] After the reaction was completed, an equal volume of methanol was added to terminate the reaction. The precipitate was removed by centrifugation at 12,000 rpm for 10 min. The supernatant was then analyzed by HPLC to determine the consumption rate of caffeic acid substrate.

[0048] Table 4. Caffeine substrate consumption rate

[0049] The above results indicate that this invention has, for the first time, determined... Desulfatirhabdium butyrativorans The protein, with the amino acid sequence shown in SEQ ID NO.1, possesses the function of 4CL and can efficiently catalyze the reaction of caffeic acid and CoA to produce caffeoyl-CoA. This protein is designated as Db4CL in this application. Furthermore, it can be seen that Db4CL consumes more caffeic acid than 4CLs from other microbial sources, which is beneficial for the accumulation of rosmarinic acid, the product of this invention. Therefore, Db4CL will be further used in the construction of subsequent rosmarinic acid-producing strains.

[0050] Example 3 Construction of Recombinant Plasmids The enzyme or gene sequence information involved in this embodiment is detailed in Table 1.

[0051] (1) Construct a plasmid expressing the Db4CL encoding gene, LDH encoding gene, and MoRAS encoding gene on the pET28a plasmid to obtain the recombinant plasmid pET28a-Db4CL-ldh-MoRAS, denoted as P01.

[0052] Based on the codon preference of E. coli, the codons of the Db4CL, LDH, and MoRAS coding genes were optimized, and the whole genomes were synthesized by a commercial company.

[0053] Using the synthesized gene fragment or its cloning vector as a template, DNA polymerase was used to amplify gene fragments containing homologous arms. Db4CL , ldh and MoRAS The target gene fragment was extracted. Simultaneously, the pET28a empty vector was linearized by double digestion with restriction endonucleases BamHI and XhoI, and the linearized plasmid backbone was recovered by gel electrophoresis.

[0054] Purified Db4CL , ldh , MoRAS The fragments and linearized pET28a backbone were mixed at a molar ratio of 2:2:2:1 and reacted at 50°C for 30 min using a multi-fragment homologous recombinase. The reaction product was transformed into E. coli DH5α competent cells, plated on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 30°C / 37°C.

[0055] Single colonies were selected for colony PCR and sequencing verification. The recombinant plasmid with correct sequencing was named pET28a-Db4CL-ldh-MoRAS and denoted as P01.

[0056] (2) Construct expression structures for FjTAL encoding gene, HpaBC encoding gene, and aroG on pACYC184 plasmid. fbr Genes were extracted to obtain the recombinant plasmid pACYC184-FjTAL-HpaBC-aroG. fbr , denoted as P02.

[0057] Based on the codon preference of *E. coli*, the FjTAL coding gene underwent codon optimization, and the entire genome was synthesized by a commercial company. Simultaneously, [the following was obtained / obtained / etc.]. HpaBC Encoding genes and feedback inhibition resistance aroG fbr Gene fragments.

[0058] The low-copy-value plasmid pACYC184 backbone was linearized using restriction endonucleases EcoRI and BamHI. Using the same multi-fragment homologous recombination cloning strategy described above, FjTAL, HpaBC, and aroG were cloned. fbr Gene fragments were seamlessly assembled into the linearized pACYC184 backbone.

[0059] The recombinant product was transformed into DH5α competent cells and screened on LB agar plates containing 34 mg / L chloramphenicol. Colony PCR and full-length sequencing confirmed the presence of a recombinant plasmid with the correct sequence, which was named pACYC184-FjTAL-HpaBC-aroG. fbr It is abbreviated as P02.

[0060] In this invention, to balance the metabolic flux of the heterologous multi-enzyme complex pathway, different target genes are expressed using plasmid backbones with different copy numbers. The enzymes encoded by FjTAL, HpaBC, and aroG exhibit high catalytic activity; therefore, they are constructed in the low-copy vector pACYC184 (p15A replicon) to limit their intracellular expression and prevent excessively rapid enzymatic transport leading to the accumulation of intermediate metabolites and cytotoxicity. Conversely, Db4CL, ldh, and MoRAS are rate-limiting enzymes in the pathway, requiring high protein abundance to maintain sufficient metabolic flux; therefore, they are cloned into the medium-to-high copy vector pET28a.

[0061] (3) Comparative Example Construct expression on pET28a plasmid At4CL Gene, ldh Gene, MoRAS The gene was extracted to obtain the recombinant plasmid pET28a-At4CL-ldh-MoRAS, denoted as Cr1-P01-1. The construction method is the same as that of P01.

[0062] After replacing the 4CL from different sources, Crl-P01-2, Crl-P01-3 and Crl-P01-4 were constructed using the same method (see Table 5 for details).

[0063] The recombinant plasmid pACYC184-FjTAL-HpaBC, denoted as Cr1-P02, was constructed by expressing the FjTAL and HpaBC genes on the pACYC184 plasmid. The construction method is the same as that for P02.

[0064] Genes encoding Db4CL, LDH, MoRAS, FjTAL, HpaBC, and aroG were constructed on the pET28a plasmid. fbr Genes were extracted to obtain the recombinant plasmid pET28a-aroG. fbr -FjTAL-HpaBC-Db4CL-ldh-MoRAS,Crl-P03. The construction method is the same as P01.

[0065] (4) with E. coliUsing K-12 MG1655 genomic DNA as a template and YcjP-F and YcjP-R as primers, PCR amplification was performed. ycjP Gene fragments; Using pET28a(+) plasmid as template, upstream primer F / downstream primer R as primers, PCR amplification was performed to obtain the linearized pET28a(+) vector fragment; The PCR amplification obtained above ycjP Gene fragments and pET28a(+) linearized vector fragments were ligated via seamless cloning.

[0066] The ligated system was directly transformed into DH5α, mixed, and incubated on ice for 5 min. It was then heat-shocked at 42°C for 90 s, immediately followed by an ice incubation of 2 min. 600 μL of LB liquid culture was added, and the culture was restored at 37°C for 1 h. 200 μL of the restored system was then plated onto LB plates containing Kan resistance and incubated overnight at 37°C. Single colonies were picked and identified by colony PCR and sequencing to obtain the plasmid pET28a-YcjP, designated P04.

[0067] Specific information for each recombinant plasmid is shown in Table 5.

[0068] Table 5. Specific information for each recombinant plasmid

[0069] Example 4: Construction of recombinant strains and production of rosmarinic acid 1. The recombinant plasmid obtained in Example 3 was transformed into the BL21(DE3)-ΔtyrR- bacterial strain obtained in Example 1 according to the combination in Table 6. pheA - In pykF, recombinant strains RA01-RA10 were obtained for subsequent fermentation experiments.

[0070] Table 6 Detailed information on recombinant strains

[0071] 2. Fermentation verification experiment Frozen strains RA01-RA10 were removed from a -80°C freezer and streaked onto plates containing the corresponding antibiotics, then incubated at 37°C for 18 hours. Single colonies were picked from the plates and incubated overnight at 30°C and 250 rpm. Seed culture was then transferred at a 5% inoculum to 250 mL shake flasks containing 25 mL of M9Y liquid medium and fermented at 37°C and 250 rpm, with three replicates per group. The fermentation OD was measured. 600 After reaching a concentration of 0.6-0.8, IPTG was added for induction. After fermentation for 72 hours, the organic phase was collected by centrifugation at 12,000 rpm for 10 minutes. The organic phase was diluted with ethanol by the corresponding factor and then analyzed by HPLC. The specific results are shown in Table 7 below.

[0072] Table 7. Rosemary acid yield

[0073] As can be seen from the data in Table 7: (1) The difference between recombinant strain RA02 and RA01 is that RA02 has three genes knocked out on the basal bacteria. tyrR, pheA, pykF The rosmarinic acid production of RA02 was 11.36 times that of RA01, indicating that knocking out... tyrR, pheA, pykF It can significantly block the synthesis of byproducts or relieve feedback inhibition.

[0074] (2) The recombinant strain RA02 differs from RA03, RA08, RA09, and RA10 in that the source of the overexpressed 4CL is different. The rosmarinic acid yield of RA02 is 2.39 times, 2.29 times, 4.5 times, and 8.6 times that of RA03, RA08, RA09, and RA10, respectively. This indicates that the present invention is the first to confirm... Desulfatirhabdium butyrativorans The protein shown in SEQ ID NO.1 has 4-coumaric acid coenzyme A ligase function, and its catalytic effect is far superior to that of commonly used enzymes in the prior art.

[0075] (3) The difference between recombinant strains RA02 and RA04 is that the recombinant plasmid Cr1-P02 of RA04 does not contain aroG. fbr The rosmarinic acid yield of RA02 was 1.78 times that of RA04. This indicates that aroG fbr Overexpression of the gene can effectively enhance the metabolic flux of the shikimic acid pathway and has a significant positive promoting effect on the synthesis of rosmarinic acid.

[0076] (4) The difference between recombinant strains RA02 and RA06 lies in their gene expression strategies. In recombinant strain RA02, in order to balance the metabolic flux of this heterologous pathway, strains with higher catalytic activity are expressed. FjTAL , HpaBC and aroG Constructed on the low-copy vector pACYC184 to limit its intracellular expression and prevent the rapid transport of highly active enzymes from leading to the accumulation of intermediate metabolites and cytotoxicity; simultaneously, it includes pathway rate-limiting enzymes. Db4CL , ldh and MoRAS Cloning into the medium-to-high copy vector pET28a increases protein abundance and maintains sufficient metabolic flux.

[0077] Conversely, RA06 simultaneously expresses the overexpressed gene in RA02 in the same plasmid, disrupting the dynamic balance of expression levels between the highly active enzyme and the rate-limiting enzyme, resulting in a decrease of approximately 47.5% in rosmarinic acid production compared to RA02.

[0078] (5) The difference between recombinant strains RA02 and RA07 is that RA07 overexpressed the YcjP protein in addition to RA02, and the rosmarinic acid yield of RA07 increased by 39.1 mg / L compared with RA02. This indicates that the YcjP protein further broke through the downstream bottleneck of rosmarinic acid synthesis, and synergistically with the aforementioned optimization, achieved a further increase in yield.

[0079] 3. 7L fermenter verification experiment Take 20 μL of strain RA07 and inoculate it into a 250 mL shake flask containing 50 mL of LB liquid medium containing the corresponding antibiotic. Incubate overnight at 37 ℃ and 150 r / min.

[0080] Subsequently, fed-batch fermentation was carried out in a 7 L fermenter with an initial volume of 3 L of fermentation medium (M9Y liquid medium). During the initial fermentation stage, the inoculum size was 0.3%, the culture temperature was 37 ℃, the stirring speed was 200 r / min, the aeration rate was set to 1 vvm, and the tank pressure was maintained at 0.05 MPa. When the dissolved oxygen (DO) dropped to 30%, the DO was stabilized to 30%–40% by adjusting the stirring speed; simultaneously, 25%–28% ammonia water was automatically added to control the pH of the system at 6.8.

[0081] When the glucose concentration drops below 0.5 g / L during fermentation, fed-batch culture medium is started, and the specific growth rate is controlled; simultaneously, when the OD of the bacterial culture... 600 When the concentration reached 20, IPTG was added to a final concentration of 0.4 mmol / L for induction. After fermentation for 72 h, the organic phase was collected by centrifugation at 12,000 rpm for 10 min. After dilution with ethanol by the corresponding factor, HPLC analysis was performed, and the rosmarinic acid concentration was 6469 mg / L.

[0082] The supplemental culture medium consisted of 40 g / L yeast extract, 400 g / L glucose, and 2.0 g / L magnesium sulfate heptahydrate.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a protein as a 4-coumaric acid coenzyme A ligase, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, This refers to the application of the protein in catalyzing the synthesis of caffeic acid into caffeoyl-CoA, or in the synthesis of rosmarinic acid.

3. A strain that produces rosmarinic acid, characterized in that, The strain was obtained by modifying the host in the following way: modifying the gene encoding aromatic amino acid biotransport regulatory factors in the genome. tyrR、 Phenylalanine synthase encoding gene pheA and the gene encoding pyruvate kinase I pykF Knockout was performed; 4-coumarate-coenzyme A ligase, lactate dehydrogenase, and rosmarinic acid synthase were overexpressed using a first expression vector, and tyrosine ammonia-lyase, 4-hydroxyphenylacetic acid-3-hydroxylase, and DAHP synthase were overexpressed using a second expression vector to construct a heterologous rosmarinic acid synthesis pathway; wherein the copy number of the first expression vector was higher than that of the second expression vector. The amino acid sequence of the 4-coumaric acid coenzyme A ligase is shown in SEQ ID NO.

1.

4. The strain for producing rosmarinic acid as described in claim 3, characterized in that, The replicon of the first expression vector is selected from ColE1 Replicator pMB1 Replicator pUC Replicator or RSF1030 Replicon; the replicon of the second expression vector is selected from: p15A Replicator pSC101 Replicator or F Replicator.

5. The strain for producing rosmarinic acid as described in claim 3, characterized in that, The YcjP protein was also overexpressed in the host; the nucleotide sequence of the gene encoding the YcjP protein is shown in SEQ ID NO.

4.

6. The strain for producing rosmarinic acid as described in claim 3, characterized in that, The host is selected from: Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae.

7. The strain for producing rosmarinic acid as described in claim 3, characterized in that, The 4-coumarate coenzyme A ligase is Db4CL, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2; the lactate dehydrogenase is Ldh, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.8; the rosmarinic acid synthase is MoRAS, and the Genbank encoding gene is FR670523.1; the tyrosine ammonia-lyase is FjTAL, NCBI Reference Sequence: WP_012023194.1; the 4-hydroxyphenylacetic acid-3-hydroxylase is HpaBC, and the nucleotide sequence is shown in SEQ ID NO.3; the encoding gene for the DAHP synthase is aroG. fbr Gene, NCBI Reference Sequence: AXN70009.

1.

8. The use of the strain according to any one of claims 3-7 in the production of rosmarinic acid.

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