Recombinant expression vector for synthesizing outer membrane protein of cyanidin-3-O-glucoside, recombinant microorganism and preparation method of recombinant microorganism

Abstract

The invention relates to a recombinant expression vector for synthesizing outer membrane protein of cyanidin-3-O-glucoside, a recombinant microorganism and a preparation method of the recombinant microorganism. The recombinant expression vector contains a gene sequence for coding the outer membrane protein. The outer membrane protein is used for constructing a recombinant expression vector and a recombinant microorganism, efflux transport of cyanidin-3-O-glucoside is promoted, and the yield of cyanidin-3-O-glucoside is increased.

Description

Technical Field

[0001] The invention relates to application of outer membrane protein derived from Escherichia coli in enhancing biosynthesis of cyanidin-3-O-glucoside, belonging to the technical field of biosynthesis. Background Art

[0002] Cyanidin 3-O-glucoside (C3G) is a polyphenolic flavonoid compound that is widely present in nature. It is a product of plant secondary metabolism and a water-soluble natural pigment. C3G has multiple physiological functions such as anti-oxidation, anti-inflammatory, and cognitive regulation. It has broad application prospects in the fields of pharmaceutical industry, food processing, cosmetics manufacturing, solar cells, etc.

[0003] At present, the production methods of C3G mainly include traditional extraction method and biosynthesis method. The traditional extraction method uses organic solvents to extract C3G from plant raw materials, but this method is easily restricted by land supply and climatic conditions. The biosynthesis method uses microorganisms or animal and plant cells for production. The reaction conditions are mild and controllable, and it is a potential supplement to the traditional extraction method. Among them, the use of recombinant Escherichia coli and catechins as precursors can achieve high-yield synthesis of C3G, but its output is far lower than the demand for industrial production, and the optimization effect at the levels of genes, enzymes, metabolism, fermentation, etc. is limited (see, Non-Patent Literature 1).

[0004] Transporter engineering has been widely used to solve problems such as insufficient substrate uptake, low product secretion efficiency, and leakage of metabolic intermediates in microbial synthesis, so as to improve the tolerance of microorganisms to toxic compounds and increase the total product yield. For example, some compounds that can be used as advanced biofuels are toxic to host cells. Transporters are used to transfer intracellular products to the extracellular space, reduce the toxicity of the products to host cells, and relieve their inhibitory effects on product synthesis, which is beneficial to obtain high-concentration products (see Non-Patent Document 2).

[0005] Escherichia coli outer membrane proteins (OMPs) are a class of periplasmic space proteins embedded in the outer membrane and periplasmic space of cells and anchored on the outer membrane. Many outer membrane proteins have a β-barrel structure, which is composed of multiple parallel β-pleated sheets to form a central pore. This structure enables outer membrane proteins to allow specific molecules to pass through while maintaining the integrity of the outer membrane. Therefore, outer membrane proteins have important physiological functions, such as supporting the outer membrane skeleton of cells, controlling the entry and exit of small molecules and secretory proteins, and participating in antibiotic resistance.

[0006] Compared with the transport proteins on the inner membrane, the outer membrane proteins have higher specificity and selectivity, and can distinguish more substrates and transport them specifically. In the process of antibiotic resistance, some outer membrane proteins can form complexes with periplasmic space proteins and inner membrane proteins to form active efflux pumps to pump drugs or toxic small molecules out of the cell, which is called efflux.

[0007] Research on increasing anthocyanin production by transporter engineering has made some progress (see Non-Patent Document 3). In the related research on the synthesis of C3G by E. coli, it has gradually become clear that overexpression of inner membrane transporter can effectively increase C3G production (see Non-Patent Document 4), but exploration of outer membrane proteins is rare. Therefore, exploring new outer membrane proteins that can promote C3G efflux in the E. coli system is of great significance to further increase the biosynthetic yield of C3G.

[0008] Prior art literature

[0009] Non-patent literature 1: Yan Y, Chemler J, Huang L, et al. Metabolic engineering of anthocyanin biosynthesis in Escherichia coli [J]. Applied and Environmental Microbiology, 2005, 71 (7): 3617-3623.

[0010] Non-patent literature 2: Zhao Y, Wu B-H, Liu ZN, et al. Combinatorial optimization of resveratrol production in engineered E. coli [J]. Journal of Agricultural and Food Chemistry, 2018, 66(51): 13444-13453.

[0011] Non-patent document 3: Cress BF, Leitz QD, Kim DC, et al. CRISPRi-mediatedmetabolic engineering of E.coli for O-methylated anthocyanin production[J]. Microbial Cell Factories, 2017, 16(1):10.

[0012] Non-patent document 4: Lim CG, Wong L, BhanN, et al. 2015. Development ofarecombinantEscherichia coli strain for overproduction of the plant pigmentanthocyanin[J]. Applied and Environmental Microbiology, 2015, 81: 6276-6284. Summary of the invention

[0013] Problem that the invention aims to solve

[0014] When using recombinant strains to synthesize C3G, it is extremely challenging to find a suitable outer membrane protein in the host strain that can efflux and transport heterologous compounds. By exploring the efflux transport mechanism of C3G in E. coli, it is expected to solve the problem of C3G accumulation in cells and increase its production.

[0015] Solutions for solving problems

[0016] The inventors have found through research that the use of outer membrane protein OmpL to prepare recombinant expression vectors and recombinant microorganisms can promote the efflux transport of C3G, solve the problem of intracellular accumulation of C3G, and increase its yield. Specifically, the present invention provides a recombinant expression vector of an outer membrane protein for synthesizing cyanidin-3-O-glucoside, which contains a gene sequence for encoding the outer membrane protein.

[0017] In the recombinant expression vector of the present invention, the outer membrane protein is selected from any one of the following (i) to (ii):

[0018] (i) a polypeptide OmpL having an amino acid sequence as shown in SEQ ID NO: 1;

[0019] (ii) A polypeptide mutant comprising the amino acid sequence shown in SEQ ID NO: 1, wherein the polypeptide mutant has one or more amino acids substituted, repeated, deleted or added at one or more positions of the sequence shown in SEQ ID NO: 3, and has the function of the polypeptide OmpL.

[0020] In the recombinant expression vector of the present invention, the recombinant expression vector may contain a gene sequence encoding the outer membrane protein as shown in any one of the following (iii) to (v):

[0021] (iii) a polynucleotide encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1;

[0022] (iv) a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 2;

[0023] (v) a polynucleotide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 2, and encoding a polypeptide having the function of polypeptide OmpL.

[0024] The recombinant expression vector of the present invention may also contain an expression vector skeleton for overexpressing the outer membrane protein gene sequence in a host cell.

[0025] The present invention also provides a recombinant microorganism for synthesizing cyanidin-3-O-glucoside. The recombinant microorganism contains a host cell and the recombinant expression vector is transformed in the host cell.

[0026] In the recombinant microorganism of the present invention, the recombinant microorganism may further comprise a recombinant plasmid transformed into the host cell.

[0027] In the recombinant microorganism of the present invention, the recombinant plasmid may contain the gene sequence of enzymes related to the cyanidin-3-O-glucoside metabolic pathway and can be expressed in the host cell.

[0028] In the recombinant microorganism of the present invention, the enzyme related to the cyanidin-3-O-glucose metabolic pathway may be anthocyanidin synthase ANS and / or 3-O-glucosyltransferase 3GT.

[0029] In the recombinant microorganism of the present invention, the host cell may be derived from a microorganism of the genus Escherichia, Brevibacterium, Corynebacterium, Bacillus, Saccharomyces, or Lactococcus.

[0030] In the recombinant microorganism of the present invention, the host cell may be derived from at least one of the group consisting of Escherichia coli, Lactococcus lactis, Corynebacterium glutamicum and Saccharomyces cerevisiae.

[0031] The present invention also provides a method for preparing a recombinant microorganism for synthesizing cyanidin-3-O-glucoside, the method comprising the following steps:

[0032] 1) replicating the gene sequence encoding the outer membrane protein;

[0033] 2) constructing a recombinant expression vector comprising the gene sequence described in step 1);

[0034] 3) The recombinant expression vector obtained in step 2) and the recombinant plasmid encoding the key enzymes in the cyanidin-3-O-glucoside metabolic pathway are transformed into host cells to obtain a recombinant microorganism for synthesizing the cyanidin-3-O-glucoside.

[0035] The present invention also provides a method for producing cyanidin-3-O-glucoside, which comprises using bacterial cells of the above-mentioned recombinant microorganism to overexpress the above-mentioned outer membrane protein to promote the synthesis of the precursor substance added to the culture system into the cyanidin-3-O-glucoside and then transport it to the outside of the bacterial cells.

[0036] In the method for producing cyanidin-3-O-glucoside of the present invention, the precursor substance may be (+)-catechin.

[0037] The present invention also provides an application of an outer membrane protein in the production of anthocyanins. The outer membrane protein may be the outer membrane protein mentioned above, and the anthocyanin is a polyphenolic flavonoid compound containing 2-phenylbenzopyranyl cation. Preferably, the anthocyanin is cyanidin-3-O-glucoside.

[0038] Effects of the Invention

[0039] The present invention is based on the new discovery that the outer membrane protein OmpL is related to the regulation of C3G transport. By constructing a recombinant expression vector and overexpressing the ompL gene, the extracellular transport efficiency of C3G synthesized by the host cell is effectively improved. Fermentation experiments have shown that compared with the control strain without overexpression of ompL, the recombinant strain with overexpression of ompL significantly enhances the production capacity of C3G.

[0040] The present invention constructs a recombinant strain by utilizing the gene of the outer membrane protein OmpL from Escherichia coli, and the genes of the key enzymes PhANS (from petunia) and At3GT (from Arabidopsis) in the C3G metabolic pathway. The strain exhibits stable C3G growth performance and high synthesis efficiency under the condition of adding (+)-catechin, and can achieve the purpose of high C3G production.

[0041] The present invention overexpresses the outer membrane protein ompL gene in host cells to efficiently transport C3G and other polyphenolic flavonoid compounds with similar structures synthesized by (+)-catechin in host cells to the outside of the cells. This method provides a new direction for increasing the production of C3G and similar compounds by utilizing transport regulation mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The flowchart is a process for catalyzing the synthesis of C3G using precursor (+)-catechin as substrate. C3G can be transported to the extracellular space via OmpL located on the outer membrane.

[0043] Figure 2 This is the enzyme digestion verification diagram of the recombinant expression vector pACYCDuet-1-ompL in the screened sample single colonies (#1, #2), where M is Marker.

[0044] Figure 3 This is the plasmid map of the recombinant expression vector pACYCDuet-1-ompL of the outer membrane protein OmpL from Escherichia coli.

[0045] Figure 4 Comparison of the extracellular C3G content in the fermentation samples of the control strain without overexpression of the ompL gene and the recombinant strain with overexpression after feeding (+)-catechin.

[0046] Figure 5 The figure shows the growth comparison of the control strain without overexpression of ompL gene and the recombinant strain with overexpression under non-induction conditions (left figure) and induction conditions (right figure).

[0047] Figure 6 Comparison of the extracellular C3G content in the fermentation samples after feeding (+)-catechin to the control strain, the recombinant strain overexpressing the multidrug resistance transporter gene mdtH, and the recombinant strain overexpressing the combination of the ompL gene and the multidrug resistance transporter gene mdtH.

[0048] Figure 7 Comparison of the growth of the control strain, the recombinant strain overexpressing the multidrug resistance transporter gene mdtH, and the recombinant strain overexpressing the ompL gene and the multidrug resistance transporter gene mdtH under non-induction conditions (left figure) and induction conditions (right figure).

[0049] Figure 8 This is a comparison of the content of biosynthesized C3G when other outer membrane proteins are expressed in combination with the multidrug resistance transporter MdtH in Comparative Example 2. DETAILED DESCRIPTION

[0050] definition

[0051] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and are amino acid polymers of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0052] As used in the present invention, the term "coding gene" refers to a DNA molecule that can guide protein synthesis through certain rules. The process by which a coding gene guides protein synthesis generally includes a transcription process using double-stranded DNA as a template and a translation process using mRNA as a template. A coding gene contains a CDS sequence (Coding Sequence) that can guide the production of mRNA encoding a protein. In some embodiments, the protein coding gene of the present invention is a gene encoding an anthocyanin efflux protein. In some embodiments, the protein coding gene of the present invention is a gene encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or a mutant that retains its anthocyanin efflux protein function.

[0053] As used herein, the term "mutant" refers to a polynucleotide or polypeptide that contains an alteration (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions relative to a "wild-type" or "compared" polynucleotide or polypeptide. Wherein, substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to the removal of a nucleotide or amino acid occupying a position. Insertion refers to the addition of a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position.

[0054] As used in the present invention, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a separate fragment or a component of a larger nucleotide sequence structure, which is derived from a nucleotide sequence separated at least once in quantity or concentration, and can be identified, manipulated, and restored by standard molecular biology methods (e.g., using a cloning vector) and its component nucleotide sequences. When a nucleotide sequence is represented by a DNA sequence (bases are A, T, G, C), this also includes an RNA sequence (bases are A, U, G, C), in which "U" replaces "T". In other words, "polynucleotide" refers to a nucleotide polymer removed from other nucleotides (separate fragments or entire fragments), or can be a component or ingredient of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

[0055] As used herein, the terms "sequence identity" and "percent identity" refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. Two or more sequences or subsequences have a "sequence identity" or "percent identity" of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotides when compared and aligned with maximum correspondence using a sequence comparison algorithm or measured by visual inspection. In certain embodiments, the sequences are substantially identical over the entire length of either or both of the compared biopolymers (e.g., polynucleotides).

[0056] As used herein, the term "transformation" has a meaning generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The method of transformation includes any method of introducing nucleic acid into a cell, including but not limited to electroporation, calcium phosphate precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method, etc.

[0057] Unless otherwise defined in the present invention or clearly indicated by the background, all technical and scientific terms in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0058] Synthesis of Cyanidin-3-O-Glucoside

[0059] By introducing heterologous plant synthesis pathway genes into microbial cells, the synthesis of C3G in microorganisms can be achieved, and the microbial strains suitable for synthesizing C3G of the present invention are not limited. Preferably, the strain is derived from microorganisms of the genus Escherichia, Brevibacterium, Corynebacterium, Bacillus, Saccharomyces, and Lactococcus. More preferably, the strain is derived from Escherichia coli, Corynebacterium glutamicum, Lactococcus lactis, and Saccharomyces cerevisiae. Most preferably, the strain is derived from Escherichia coli.

[0060] Escherichia coli has been used as a host cell to synthesize anthocyanins for a long time. For example, in Escherichia coli, heterologous plant gene metabolic pathways of flavanone 3β-hydroxylase (F3H) from apple, dihydroflavonol 4-reductase (DFR) from anthurium, anthocyanidin synthase (ANS) from apple, and UDP-glucose flavonoid-3-O-glucosyltransferase (3GT) from petunia were constructed, and C3G can be synthesized using naringenin or eriodictyol as substrates, but pelargonidin 3-O-glucoside (P3G) is also synthesized as a byproduct.

[0061] For the synthesis of C3G by E. coli, the enzymes encoded by homologous genes from different plants have different kinetic and thermodynamic activities when catalyzing the reaction. The study found that compared with heterologous ANS from snapdragon, gerbera, and apple, ANS from petunia had the highest yield of C3G in E. coli. Subsequently, AT3GT from Arabidopsis was fused to the N-terminus of ANS from petunia, and it was found that the expression of the fusion enzyme further increased the total yield of anthocyanins. In addition, the yield of anthocyanins synthesized by E. coli can be further improved by optimizing the combination of strategies such as carbon metabolism regulation, culture medium optimization, gene expression optimization, mixed culture, screening of ANS and 3GT from different plant sources, and enhancement of UDP-glucose supply. In particular, when (+)-catechin is used as a substrate in E. coli, a higher anthocyanin yield can be obtained.

[0062] Lactococcus lactis is a generally recognized as safe food-grade microorganism (GRAS) because it is not pathogenic to humans and animals. Using flavan-3-ols as substrates, engineered Lactococcus lactis can quickly convert flavonoids in green tea into C3G, Delphinidin3-O-glucoside (D3G), and methylated orange and yellow pyranocyanidins. Although the Gram-positive bacteria Lactococcus lactis can stably produce anthocyanidins at lower pH values ​​and higher flavonoid concentrations, the total amount of pigment obtained is much lower than the C3G content in engineered Escherichia coli.

[0063] Corynebacterium glutamicum is a gram-positive bacteria, by regulating the expression of anthocyanidin synthase gene ANS and 3-O-glucosyltransferase gene 3GT, regulating the supply of cofactor UDP-glucose, optimizing fermentation conditions and other strategies, in Corynebacterium glutamicum, catechins can be used as substrate to synthesize C3G. However, because C3G is unstable under neutral conditions, and Corynebacterium glutamicum shows high sensitivity to low pH, a series of physiological damages can occur in cells during fermentation, thereby affecting cell metabolism, therefore Corynebacterium glutamicum is utilized as host bacteria to synthesize C3G and is restricted. Escherichia coli does not have this problem, by reducing the pH value (for example, pH is 5.0) of the culture medium used during the synthesis of C3G, the instability of C3G under neutral conditions can be reduced.

[0064] Saccharomyces cerevisiae has also been used to synthesize C3G. Since the total synthesis pathway of most anthocyanins requires the action of multiple plant CYP enzymes, and these enzymes are usually difficult to express in bacterial host cells, Saccharomyces cerevisiae is considered to be a good host strain for heterologous synthesis of anthocyanins. Some studies have used dihydroflavonols as substrates to obtain C3G by expressing plant-derived anthocyanin transporter MdGSTF6 and anthocyanin synthesis pathway enzymes FaDFR, PhANS0, Dc3GT, and knocking out the endogenous anthocyanin degradation enzyme gene EXG1 in yeast. Other studies have increased the production of anthocyanins in eukaryotic cells by expressing a de novo synthesis pathway in Saccharomyces cerevisiae to obtain anthocyanins from glucose, but it is still lower than the C3G content obtained by engineering Escherichia coli.

[0065] Therefore, among the microorganisms that synthesize C3G, the highest yield is obtained using E. coli as the chassis strain. However, the synthesized C3G accumulates excessively in E. coli cells, which is not conducive to the improvement of C3G production.

[0066] Outer membrane protein

[0067] The outer membrane protein of the present invention is a protein located on the outer membrane of a cell and capable of transporting intracellular C3G to the outside of the cell.

[0068] The inventors screened typical outer membrane protein genes in different taxonomic families based on the E. coli BL21 (DE3) synthesizing C3G, introduced them into the E. coli chassis strain synthesizing C3G, and screened the outer membrane proteins that can promote the efflux of C3G using fermentation tests.

[0069] The study found that when catechins are used as substrates, the extracellular C3G production of E. coli can be increased by overexpression of endogenous outer membrane protein genes of E. coli, such as ompN, ompL, slp, cirA, nlpE, mltA and slyB. Preferably, the efflux effect of C3G can be significantly improved by overexpression of the ompL gene. For the specific process, see Figure 1 .

[0070] Anthocyanin synthases from different sources have different effects on the use of outer membrane proteins to synthesize C3G organisms. Anthocyanin synthases used for Escherichia coli to synthesize C3G include ANS derived from snapdragon, gerbera, apple, petunia (Petuniahybrida), red clover (Trifoliumpratense), etc., among which ANS derived from petunia is preferably more conducive to the biosynthesis of C3G. More preferably, the synthase obtained by fusion of At3GT from Arabidopsis thaliana and ANS from petunia can further increase the yield of C3G. Most preferably, while overexpressing the outer membrane protein gene, the fusion enzyme of At3GT from Arabidopsis thaliana and ANS from petunia is used to synthesize C3G, which can more significantly increase the extracellular emission of C3G.

[0071] In some embodiments, the outer membrane protein of the present invention uses catechin as a substrate. Because it has a high substrate specificity for catechin, it can achieve efficient efflux of C3G and increase the yield of C3G.

[0072] In some embodiments, the outer membrane protein of the present invention is the polypeptide OmpL.

[0073] In some embodiments, the outer membrane protein of the present invention is a polypeptide having the same function of promoting the extracellular release of C3G as the polypeptide OmpL.

[0074] In some embodiments, the outer membrane protein of the present invention is a polypeptide OmpL having an amino acid sequence as shown in SEQ ID NO:1.

[0075] In some embodiments, the outer membrane protein of the present invention is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 and having the same function of promoting the extracellular release of C3G.

[0076] In some embodiments, the present invention provides a polypeptide OmpL mutant, which is a mutant having one or more amino acids substituted, repeated, deleted or added at one or more positions of the sequence shown in SEQ ID NO: 1, and maintaining the function of the OmpL protein. Preferably, the mutant has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity (including all ranges and percentages between these values) with the OmpL protein. The OmpL mutant can be used as a transporter of C3G to improve the ability of microorganisms to expel C3G.

[0077] Recombinant expression vector

[0078] As a DNA construct, the vector contains a DNA sequence for connection, which is operably connected to a suitable sequence to express the target gene in a suitable host. Any vector can be used in the present invention, including plasmids, viruses, phages and transposons. The vector of the present invention includes but is not limited to chromosomes, non-chromosomes and synthetic DNA sequences, for example, bacterial plasmids, phage DNA, yeast plasmids and vectors derived from a combination of plasmids and phage DNA, from DNA of viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40 and pseudorabies. Preferably, the vector of the present invention is a high expression level vector pACYCDuet-1 (Addgene catalog number: 71147), which contains a P15A replicon, a chloramphenicol resistance gene and a strong T7 promoter, and can achieve efficient expression.

[0079] A recombinant expression vector is a DNA structure for expressing a polynucleotide encoding a desired polypeptide, for example, and may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers, etc., ii) a structural or coding sequence that is transcribed into mRNA and translated into protein, and iii) a transcriptional subunit of appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors can be constructed in any suitable manner.

[0080] In some embodiments, the recombinant expression vector of the present invention contains a gene sequence for encoding the outer membrane protein of the present invention. Preferably, the outer membrane protein is a polypeptide OmpL. More preferably, the outer membrane protein is a polypeptide mutant comprising the amino acid sequence shown in SEQ ID NO: 1. Most preferably, the outer membrane protein is a polypeptide having the amino acid sequence shown in SEQ ID NO: 1.

[0081] In some embodiments, the recombinant expression vector of the present invention contains a polynucleotide encoding the polypeptide OmpL.

[0082] The polynucleotide encoding the polypeptide OmpL of the present invention is further operably connected to a regulatory sequence and used to construct a nucleic acid construct, a recombinant expression vector, a recombinant host cell, etc. that expresses the OmpL protein. The polynucleotide, nucleic acid construct, recombinant expression vector, recombinant host cell, etc. can promote the efficient discharge of C3G to the extracellular space by expressing the OmpL protein that has the function of transporting C3G, thereby increasing the substrate conversion rate and C3G yield.

[0083] In some embodiments, the recombinant expression vector of the present invention contains a polynucleotide encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO:1.

[0084] In some embodiments, the recombinant expression vector of the present invention contains a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO:2.

[0085] In some embodiments, the recombinant expression vector of the present invention contains a nucleotide sequence encoding a polypeptide having the function of an OmpL protein and having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity (including all ranges and percentages between these values) with the sequence shown in SEQ ID NO: 2. Preferably, the sequence identity is at least 97%, more preferably, the sequence identity is at least 98%, and most preferably, the sequence identity is at least 99%.

[0086] In some embodiments, the recombinant expression vector of the present invention further contains expression elements for overexpressing the outer membrane protein gene sequence in host cells, including a T7 promoter, a T7 terminator, a ribosome binding site, and a lac operator.

[0087] Host cells

[0088] The host cell of the present invention is any cell type that is easily transformed, transfected, transduced, etc. with a nucleic acid construct or expression vector comprising a polynucleotide. Recombinant host cells include host cells that are different from the parent cell after the introduction of a polynucleotide, nucleic acid construct or recombinant expression vector encoding a C3G transporter. Recombinant host cells are specifically achieved by transformation.

[0089] Preferably, the host cell of the present invention is derived from a microorganism of the genus Escherichia, Brevibacterium, Corynebacterium, Bacillus, Saccharomyces cerevisiae, or Lactococcus. More preferably, the host cell is selected from at least one of the group consisting of bacterial cells of Escherichia coli, Corynebacterium glutamicum, Lactococcus lactis, or Saccharomyces cerevisiae. Most preferably, the host cell is an Escherichia coli cell.

[0090] In some embodiments, the host cells of the present invention transport C3G and other polyphenolic flavonoids with similar structures synthesized by (+)-catechin in the host cells to the extracellular space by overexpressing the outer membrane protein OmpL.

[0091] Recombinant microorganisms

[0092] The recombinant microorganism of the present invention is obtained by recombining or transforming the microorganism, and the recombinant or transforming method includes but is not limited to increasing the copy number of protein coding genes, modifying the sequence of protein coding genes, etc. The recombinant gene includes a protein coding sequence operably connected to an expression control sequence, including but not limited to a gene with a modified protein coding sequence. The recombinant gene is stored in the genome of the microorganism, the plasmid in the microorganism, or the bacteriophage in the microorganism.

[0093] Preferably, the recombinant microorganism of the present invention is a recombinant microorganism obtained by genetically engineering one or more of the following microorganisms: microorganisms of the genus Escherichia, Brevibacterium, Corynebacterium, Bacillus, Saccharomyces, and Lactococcus. More preferably, the microorganism is derived from Escherichia coli, Corynebacterium glutamicum, Lactococcus lactis, and Saccharomyces cerevisiae. Most preferably, the microorganism is derived from Escherichia coli.

[0094] In some embodiments, the recombinant microorganism of the present invention contains a host cell, and a recombinant expression vector transformed into the host cell.

[0095] In some embodiments, the recombinant microorganism of the present invention further comprises a recombinant plasmid transformed into a host cell.

[0096] In some embodiments, the recombinant microorganism of the present invention carries the gene of anthocyanidin synthase ANS and / or 3-O-glucosyltransferase 3GT. The recombinant microorganism exhibits stable growth performance and high synthesis efficiency under the condition of adding (+)-catechin, thereby achieving the goal of high production of C3G.

[0097] Method for preparing recombinant microorganism

[0098] The present invention also relates to a method for preparing a recombinant microorganism for efficiently expressing key enzymes in the C3G metabolic pathway.

[0099] The method for preparing the recombinant microorganism of the present invention comprises the following steps:

[0100] 1) Copy the gene sequence encoding the outer membrane protein.

[0101] The outer membrane protein is an outer membrane protein that promotes the efflux of C3G.

[0102] Preferably, the outer membrane protein is an outer membrane protein that has high substrate specificity for catechins and can achieve efficient efflux of C3G. More preferably, the outer membrane protein is the above-mentioned polypeptide OmpL, a polypeptide having the same function of promoting the extracellular emission of C3G as the polypeptide OmpL, a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, or a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 1 and having the same function of promoting the extracellular emission of C3G. Most preferably, the outer membrane protein is a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1.

[0103] In some embodiments, the coding sequence of the gene of the outer membrane protein is PCR amplified. With catechin as a substrate, from the perspective of improving the extracellular discharge effect of C3G and obtaining a higher yield of C3G, it is preferred to perform PCR amplification on the endogenous outer membrane protein gene of Escherichia coli, for example, outer membrane protein genes such as ompN, ompL, slp, cirA, nlpE, mltA and slyB. More preferably, the coding sequence of the gene of the outer membrane protein OmpL derived from Escherichia coli (i.e., ompL) is PCR amplified to obtain a more excellent extracellular discharge effect of C3G.

[0104] The PCR amplification of the present invention can be performed using conventional methods in the art.

[0105] Preferably, the coding sequence of the target gene ompL is amplified using Escherichia coli BL21 (DE3) genomic DNA as a template.

[0106] 2) Constructing a recombinant expression vector comprising the gene sequence described in step 1).

[0107] The recombinant expression vector of the present invention can be constructed using conventional methods in the art.

[0108] In order to clone the target gene into the expression vector, the vector is usually treated with restriction endonucleases. Then, the products after enzyme cleavage are detected by electrophoresis and the products are recovered and purified. The amplified product is connected to the enzyme-cleaved vector, thereby cloning the coding sequence of the outer membrane protein gene into the expression vector.

[0109] In some embodiments, the ligation product is transferred into competent cells, single colonies are obtained through resistance screening, colony PCR detection and enzyme digestion verification are performed on the target gene to obtain a successfully constructed recombinant expression vector for overexpression of the target gene.

[0110] 3) The recombinant expression vector obtained in step 2) and the recombinant plasmid encoding the key enzymes of the C3G metabolic pathway are transformed into host cells to obtain a recombinant microorganism for synthesizing C3G.

[0111] In this step, the recombinant microorganism that synthesizes C3G can use conventional methods in the art.

[0112] In some embodiments, solid culture medium is first used to screen resistant colonies of host cells, and host cells with better growth status are obtained by liquid culture. Then, the host cells are washed and incubated, and recombinant plasmids and recombinant expression vectors are added thereto, mixed, and incubated on ice. After a short heat shock, pre-cooled culture medium is added, and the transformed cells are incubated to recover the cells and replicate the plasmids. Resistance screening is performed to obtain transformed colonies with antibiotic resistance genes, and a recombinant microorganism that overexpresses the target gene for producing C3G is successfully constructed.

[0113] Preferably, the plasmid used in the above method for constructing a recombinant microorganism is the recombinant plasmid pETM6-At3GT-m-PhANS (see Non-Patent Document 3).

[0114] The present invention constructs a recombinant microorganism for efficiently expressing key enzymes in the C3G metabolic pathway through the above method.

[0115] Production method of C3G

[0116] The present invention utilizes the bacterial cells of the recombinant microorganism to overexpress the outer membrane protein, thereby promoting the precursor substance added to the culture system to synthesize C3G and then transport it to the outside of the bacterial cell, thereby increasing the extracellular production of C3G. Preferably, the precursor substance is (+)-catechin. The outer membrane protein is more likely to bind to the substrate (+)-catechin, promote the transport of the substrate into the cell, that is, promote the absorption of the substrate, thereby increasing the synthesis of C3G.

[0117] Overexpression of outer membrane protein can be achieved by adding an inducer. The inducer used in the present invention is not limited, preferably, the inducer is IPTG.

[0118] Overexpression of outer membrane proteins usually inhibits the growth of host cells and promotes the efflux of C3G. However, different outer membrane proteins have different effects on the efflux of C3G. Overexpression of some outer membrane proteins can lead to leakage of intracellular substances; overexpression of some outer membrane proteins can occupy more membrane area and affect the normal metabolism of cells; overexpression of some outer membrane proteins can change the structure of the outer membrane proteins themselves, hindering the recognition and binding of C3G with other transport proteins. Preferably, when the precursor substance is (+)-catechin, the outer membrane protein gene is ompL, ompN or slp, and overexpression of these genes can significantly improve the efflux of C3G. More preferably, the outer membrane protein gene is ompL, and overexpression of ompL makes the efflux effect of C3G more excellent.

[0119] In some embodiments, the present invention also uses a multidrug resistance (MDR) transporter in combination with an outer membrane protein to improve the transport effect of C3G. Preferably, an endogenous multidrug resistance transporter of Escherichia coli is used in combination with the outer membrane protein. More preferably, when catechin is used as a substrate, an endogenous multidrug resistance transporter gene of Escherichia coli, such as mdtH, yhiHJ or sugE, is used in combination with the outer membrane protein gene to further promote C3G efflux.

[0120] The present invention also provides a method for improving C3G production, wherein the method utilizes the above recombinant microorganism and solves the problem of C3G sensitivity to pH value by a two-step culture method, thereby increasing C3G production.

[0121] The improved method comprises the following steps:

[0122] 1) The recombinant microorganism is cultured in a liquid culture medium to induce the expression of key enzymes in the C3G synthesis pathway and overexpress the outer membrane protein OmpL, thereby providing a culture environment suitable for improving the efficiency of C3G synthesis.

[0123] 2) The culture medium is replaced with a modified M9 culture medium with a suitable pH value to provide an acidic environment suitable for C3G synthesis and improve the synthesis efficiency of C3G. Preferably, the pH value of the modified M9 culture medium is 4.5 to 5.5. More preferably, the pH value is 5.0.

[0124] Since C3G is relatively stable under acidic conditions and easily decomposed under neutral or alkaline conditions, the above two-step culture method can solve the problem of C3G's sensitivity to pH value, allowing C3G to be produced efficiently and stably.

[0125] The outer membrane protein of the invention can be used to produce anthocyanin and improve the yield of anthocyanin.

[0126] Anthocyanins are polyphenolic flavonoid compounds containing 2-phenylbenzopyran cations. Different anthocyanins are combined with different sugar groups to form different anthocyanins. Common anthocyanins include cyanidin, pelargonidin, delphinidin, and peony pigment, and the commonly used sugar groups include glucose, rhamnose, galactose, etc. Cyanidin-3-O-glucoside (C3G) is one of the most widely studied anthocyanins. C3G has strong antioxidant and anti-inflammatory functions, can inhibit oxidative stress and neuroinflammatory reactions, prevent neurotoxicity caused by lipopolysaccharide, H2O2, ethanol, kainic acid, acrolein, glutamate and scopolamine, and protect the central nervous system from damage. Studies have shown that C3G can also maintain the metabolism of glucose, fatty acids and lipids by regulating different genes and signaling pathways, protecting healthy individuals from obesity-related problems. In addition, clinical studies have also shown that C3G has multiple pharmacological activities such as anti-cancer, regulating intestinal flora, promoting platelet activation and aggregation, and protecting vision, and has great commercial application value in the field of medicine and health. Other polyphenolic flavonoid compounds with similar structures besides C3G also have the above-mentioned functions of C3G.

[0127] The present invention also provides a method for producing anthocyanins, which comprises the above-mentioned process of preparing the recombinant microorganism and the process of preparing C3G using the recombinant microorganism.

[0128] The method of the present invention utilizes the above-mentioned recombinant microorganism to overexpress outer membrane protein, and efficiently transports C3G and other polyphenolic flavonoid compounds with similar structures synthesized by (+)-catechin in host cells to the outside of cells, thereby increasing the production of C3G and anthocyanins with similar structures.

[0129] The present invention is further explained below in conjunction with examples. It should be noted that these examples are only used to describe the present invention and the present invention is not limited thereto.

[0130] Example

[0131] <Experimental Materials>

[0132] Vector: pACYCDuet-1 (Addgene catalog number: 71147).

[0133] Strain: E. coli BL21(DE3) (Invitrogen, Non-Patent Document 3).

[0134] Cells: E. coli DH5α cells (Novagen, Non-Patent Document 3).

[0135] Plasmid: pETM6-At3GT-m-PhANS (Non-Patent Document 3).

[0136] Primers, DNA Marker, and isopropyl-β-D-thiogalactoside (IPTG) were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0137] Prime STAR DNA polymerase and restriction endonucleases were purchased from Takara Biotechnology (Dalian) Co., Ltd.

[0138] One-step PCR Cloning kit and one-step directional cloning kit were purchased from Suzhou Nearshore Protein Technology Co., Ltd.

[0139] T4 DNA ligase was purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0140] Bacterial genomic DNA extraction kit, plasmid extraction kit, ordinary agarose gel DNA recovery kit, nucleic acid dye, DNA Loading Buffer, 2× Taq DNA polymerase were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0141] <Reagents>

[0142] Vitamin B1 hydrochloride (Thiamine HCl) was purchased from Shanghai Blue Season Biotechnology Co., Ltd.

[0143] Glucose, magnesium chloride hexahydrate (MgCl2·6H2O), and sodium chloride (NaCl) were purchased from Tianjin Komiou Chemical Reagent Co., Ltd.

[0144] Calcium chloride (CaCl2), magnesium sulfate (MgSO4·7H2O), and ammonium chloride (NH4Cl) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0145] Agar powder was purchased from Beijing Biotoda Technology Co., Ltd.

[0146] Agarose was purchased from Beijing Zhijie Fangyuan Technology Co., Ltd.

[0147] Ampicillin, chloramphenicol, kanamycin, and (+)-catechin were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0148] Dimethylformamide (DMF), methanol, anhydrous ethanol (analytical grade), concentrated hydrochloric acid, and glycerol (analytical grade) were purchased from Tianjin Tianli Chemical Reagent Co., Ltd.

[0149] Cyanidin 3-O-glucoside (C3G) was purchased from Beijing Merida Technology Co., Ltd.

[0150] Acetonitrile, methanol and formic acid (all chromatographically pure) were purchased from MacLean Biochemical Technology Co., Ltd.

[0151] <Instruments and Equipment>

[0152] High-pressure steam sterilizer SX-500 was purchased from Tianmei (China) Scientific Instrument Co., Ltd.

[0153] The multifunctional microplate reader Infinite MNANO was purchased from Swiss Tecan (Shanghai) Trading Co., Ltd.

[0154] PCR instrument T20 was purchased from Hangzhou Langji Scientific Instrument Co., Ltd.

[0155] Electrophoresis apparatus JY600C, horizontal electrophoresis tank JY-SPCT, and UV gel imaging analysis system JY04S-3C were purchased from Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd.

[0156] The gene introduction instrument SCIENTZ-2C was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.

[0157] Ultra-micro-volume UV-visible spectrophotometer B-500 was purchased from Shanghai Yuanxi Instrument Co., Ltd.

[0158] Liquid chromatography (HPLC) LC2000 was purchased from Shanghai Tianmei Scientific Instrument Co., Ltd.

[0159] Blue light transilluminator BRC-1 was purchased from Lepte Scientific Instrument (Beijing) Co., Ltd.

[0160] It should be noted that the conventional plasmid and vector construction, protein expression method and cell transformation methods involved in the present invention can also refer to the molecular biology and genetic methods known in the art, for example, can also refer to the corresponding methods recorded in public publications such as "Current Protocols in Molecular Biology (published by Wiley)" and "Molecular Cloning: A Laboratory Manual (published by Cold Spring Harbor Laboratory)".

[0161] <Preparation of culture medium>

[0162] LB liquid medium: Weigh 20.0 g of LB broth powder, dissolve it thoroughly in 1 L of deionized water, sterilize it at 121 °C for 20 min, and store it at room temperature. When preparing solid medium, 2% (m / v) agar powder can be added additionally to the above formula.

[0163] SOC medium: 10 mM NaCl, 2% (m / v) tryptone, 2.5 mM KCl, 0.5% (m / v) yeast extract, dissolve in deionized water, autoclave (121 °C, 20 min), after the solution returns to room temperature, add 2 mL of 40% sterile glucose solution, sterile MgCl2·6H2O with a final concentration of 10 mM, mix well and dispense into sterile centrifuge tubes, store at 4 °C.

[0164] Modified M9 liquid medium: Weigh 1.5 g of KH2PO4, 6.4 g of Na2HPO4, 0.25 g of NaCl, 0.5 g of NH4Cl, 2.0 g of glucose into 450 mL of deionized water, after dissolution, slowly add 1 mL of sterile MgSO4 (1 M), 0.5 mL of CaCl2 (0.1 M), 0.2 mL of vitamin B1 (5 mg / mL) in sequence, adjust the pH to 5.0 with HCl and then make up the volume to 500 mL, sterilize by filtration through a 0.22 μm filter membrane, store in the dark at 4 °C.

[0165] <Determination of the content of C3G by HPLC>

[0166] Use a high performance liquid chromatograph to quantitatively analyze the fermentation supernatant, and determine the yield of extracellular C3G in Escherichia coli and the residual amount of the substrate (+)-catechin.

[0167] Detection conditions:

[0168] Use a Unitary-C18 chromatographic column (250 mm × 4.6 mm, 5 μm), ultraviolet detector, detect (+)-catechin at a wavelength of 280 nm, and detect C3G with a diode array detector at a wavelength of 520 nm.

[0169] The injection volume for detection is 10 μL, the flow rate is 1.0 mL / min, and the mobile phase is acetonitrile + 0.1% formic acid (mobile phase A) and water + 0.1% formic acid (mobile phase B).

[0170] The elution program is 10%-40% A (0-10 min), 40%-60% A (10-15 min), 60%-90% A (15-20 min), 90%-10% A (20-25 min), 10% A (25-30 min).

[0171] The standard curve is set with 5 points, the correlation coefficient R 2The content of C3G in the sample was quantified by the peak area integration at 520 nm relative to the standard curve. The residual amount of (+)-catechin in the sample was quantified by the peak area integration at 280 nm relative to the standard curve.

[0172] <Growth curve test>

[0173] The turbidity method is used to indicate the growth concentration of bacteria, that is, the absorbance value OD of the cell culture at different time points at a wavelength of 600nm is detected by an enzyme marker 600 .

[0174] Different recombinant E. coli were inoculated into LB liquid medium containing corresponding antibiotics (80 mg / L ampicillin, 25 mg / L chloramphenicol) and cultured overnight at 37°C and 200 rpm for 12-16 hours. The next day, the overnight cultured bacterial solution was transferred to 50 mL fresh LB medium containing corresponding antibiotics at a ratio of 2% (v / v), and the initial turbidity of different cell culture solutions was adjusted to be consistent. After the transfer, the culture was continued at 37°C and 200 rpm until the OD 600 When the OD value was about 0.6, IPTG was added to induce the gene expression of the outer membrane protein OmpL at a final concentration of 0.5 mM. After adding the inducer, the culture was continued at 30°C and 200 rpm, and samples were taken at regular intervals to measure the OD value of the samples. 600 The growth curve of the recombinant strain was plotted. The recombinant strain without IPTG induction was cultured at 37°C and 200 rpm, and samples were taken regularly after transfer to measure the OD of the samples. 600 And draw the strain growth curve.

[0175] Example 1 Construction of recombinant expression vector of outer membrane protein OmpL

[0176] First, the outer membrane protein ompL gene coding sequence was cloned.

[0177] Through systematic identification of the outer membrane protein database and transcriptome temporal analysis, an outer membrane protein OmpL involved in transporting C3G was found in Escherichia coli. The coding sequence of the gene of the outer membrane protein OmpL from Escherichia coli (i.e., ompL) was amplified by PCR, and the reaction system of PCR amplification is shown in Table 1 below:

[0178] Table 1

[0179]

[0180] The reaction program of PCR amplification was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 90 s, 30 cycles; and maximum extension at 72°C for 5 min.

[0181] To obtain an appropriate amount of DNA template, Escherichia coli BL21 (DE3) bacterial solution was used and a genomic DNA extraction kit was used for extraction. The genomic DNA was extracted according to the instructions. Then, the extracted DNA was verified by agarose gel electrophoresis, and then the concentration and quality of the DNA were detected using an ultra-micro UV spectrophotometer.

[0182] The forward primer and reverse primer are designed as specific overlapping complementary primers, and the specific sequences are as follows (the underlined bases are homologous to the two ends of the expression vector backbone):

[0183] ompL-F:5′- taagaaggagatatacatatg atgaaaaatattaatgcaataa-3′ (SEQ ID NO: 3)

[0184] ompL-R:5′- ggtttctttaccagactcgag tcagaagaaatacttcgtccc-3′ (SEQ ID NO: 4)

[0185] In the above PCR amplification reaction procedure, the coding sequence of the target gene ompL (the start codon is ATG and the stop codon is TGA) is amplified using PrimeSTAR high-fidelity polymerase with Escherichia coli BL21 (DE3) genomic DNA as a template. The coding sequence can be transcribed and translated to obtain the transport protein OmpL with the amino acid sequence shown in SEQ ID NO: 1 as follows:

[0186] MKNINAIILLSSLTSASVFAGAYVENREAYNLASDQGEVMLRVGYNFDMGAGIMLTNTYTFQREDEL

[0187] KHGYNEIEGWYPLFKPTDKLTIQPGGLINDKSIGSGGAVYLDVNYKFVPWFNLTVRNRYNHNNYSS

[0188] TDLSGELDNDTYEIGTYWNFKITDKFSYTFEPHYFIRVNDFNSSNGKDHHWEITNTFRYRINEHWLPYFELRWLDRNVEPYHREQNQIRIGTKYFF(SEQ ID NO: 1)

[0189] After the reaction procedure was completed, the quality of the amplified product was evaluated by 1.2% agarose gel electrophoresis, and then the product was recovered and purified using an agarose gel recovery kit.

[0190] The vector pACYCDuet-1 (Addgene catalog number: 71147) is used as an expression vector, which contains a P15A replicon, a chloramphenicol resistance gene, and a strong T7 promoter, which can achieve efficient expression. In order to clone the target gene, that is, the coding sequence of the outer membrane protein OmpL (SEQ ID NO: 2) into the pACYCDuet-1 expression vector, the pACYCDuet-1 vector is first treated with double enzyme digestion of restriction endonucleases NdeI and XhoI (react at 37°C for 1.5 hours). Then, the size of the enzyme-digested product is detected by 1% agarose gel electrophoresis, and the product is subsequently recovered and purified using an agarose gel recovery kit. Next, a professional recombinase is used. Plus connect the amplified product with the pACYCDuet-1 vector after enzyme digestion. This process is carried out at 50°C and the reaction time is 30 minutes. Through these steps, the coding sequence of the outer membrane protein ompL gene is cloned into the pACYCDuet-1 expression vector.

[0191] atgaaaaatattaatgcaataattttattatcttctctgacctctgcctcagtattcgctggcgcatacgtcgaaaacagggaggcatataatctcgcctccgatcag

[0192] ggcgaggttatgctacgtgtgggttataacttcgatatgggcgcgggtattatgttaaccaatacctacacctttcagcgagaagatgaactaaaacatggatat

[0193] aacgaaatcgaaggttggtatccgttatttaaaccaaccgataaattaaccatccagcccggtggcttaattaatgataagagtatcggttcgggtggtgcagtgt

[0194] atctggacgtcaactataaatttgtaccatggtttaatctgacagtacgaaatcgctataaccataataactatagttcaacagatttgagcggggaactggataat

[0195] aatgacacctatgaaattggtacctactggaattttaaaatcaccgataagttctcctatacatttgagccacattattttatacgcgtaaatgactttaatagcagca

[0196] atggaaaagatcatcactgggaaatcactaacaccttccgttatcgcattaatgaacactggctgccttatttcgaattacgctggttagatcgtaatgtcgaaccgtaccaccgcgagcaaaaccagattcgtatcgggacgaagtatttcttctga (SEQ ID NO: 2)

[0197] The ligation product was transformed into competent E. coli DH5α cells, and these cells were spread on a screening plate containing chloramphenicol and cultured overnight in a 37°C constant temperature incubator until a single colony appeared. Afterwards, a single colony was picked and the ompL gene on the recombinant expression vector was detected by colony PCR using ompL-F primer (SEQ ID NO: 3) and T7-Term primer (SEQ ID NO: 5), and enzyme digestion was performed to verify the results. Figure 2 shown.

[0198] The above T7-Term primer is complementary to the T7 terminator on the vector backbone, and the specific sequence is as follows:

[0199] T7-Term:5′- gctagttattgctcagcggt -3' (SEQ ID NO: 5)

[0200] Depend on Figure 2 It can be seen that after colony PCR amplification and enzyme digestion, the two single colonies showed consistent results in electrophoresis, with two bands appearing. The smaller band was located between 1000 bp and 2000 bp, which was consistent with the chain length of ompL, indicating that the recombinant expression vector overexpressing ompL was successfully constructed.

[0201] The positive clones that were verified to be correct were sequenced and named pACYCDuet-1-ompL, i.e., the recombinant expression vector of the outer membrane protein OmpL, and stored in a refrigerator at -20°C for future use. Its structure is as follows Figure 3 shown.

[0202] Example 2 Construction of recombinant microorganisms overexpressing the ompL gene for C3G synthesis

[0203] During the construction process, the recombinant plasmid pETM6-At3GT-m-PhANS and the recombinant expression vector containing the outer membrane protein ompL gene coding sequence (i.e., pACYCDuet-1-ompL) were co-transformed into Escherichia coli BL21 (DE3) by chemical transformation. The recombinant microorganism constructed in this way can successfully achieve overexpression of the ompL gene and can efficiently express key enzymes in the C3G metabolic pathway.

[0204] The specific steps of the construction method are as follows:

[0205] Step 1: Inoculate the E. coli BL21 (DE3) glycerol bacteria to be transformed into LB liquid culture medium containing 80 mg / L ampicillin and 25 mg / L chloramphenicol, and culture overnight at 37°C and 200 rpm to increase the number of colonies and cell viability.

[0206] Step 2: Take an appropriate amount of cells from the overnight bacterial solution and inoculate them into fresh LB liquid medium. Adjust the culture temperature to 37°C and control the culture speed to 200 rpm until the OD of the cells reaches 600 Reach about 0.6 to ensure that cells are in optimal growth state.

[0207] Step 3: Take the cell culture medium, centrifuge it, remove the supernatant, and then wash the cells with pre-cooled 0.1M CaCl2 solution to remove the residual culture medium and other impurities.

[0208] Step 4: Resuspend the cells in pre-chilled 0.1 M CaCl2 solution and incubate on ice for 5 minutes to improve the transfection efficiency and stability of the cells to the plasmid.

[0209] Step 5: Add 100 ng of each pETM6-At3GT-m-PhANS recombinant plasmid and pACYCDuet-1-ompL recombinant expression vector to 20 μL of cell suspension and mix gently to allow the plasmid to fully contact the cells. Then incubate on ice for 15 minutes to promote transformation and stability of the plasmid.

[0210] Step 6: Briefly heat shock at 42°C for 30 seconds, then immediately add pre-cooled SOC medium and incubate the transformed cells at 37°C for 40 minutes to promote cell recovery and plasmid replication.

[0211] Step 7: Spread the transformed cells evenly on a screening plate containing 80 mg / L ampicillin and 25 mg / L chloramphenicol, and culture at 37°C to allow the growth and formation of transformed colonies carrying antibiotic resistance genes.

[0212] Through the above steps, a recombinant strain overexpressing the ompL gene for efficient production of C3G was constructed.

[0213] Example 3 Application of outer membrane protein OmpL in C3G biosynthesis

[0214] (A) Comparison of C3G biosynthesis in the control strain without overexpression of the ompL gene and the recombinant strain with overexpression

[0215] Since C3G is more stable under acidic conditions and easily decomposed under neutral and alkaline conditions, C3G is produced by the aforementioned two-step culture method. Among them, the culture medium is changed to a modified M9 culture medium with a pH of 5.0 to obtain an acidic environment suitable for C3G synthesis. This method can solve the problem of C3G's sensitivity to pH value and improve the synthesis efficiency of C3G.

[0216] In order to verify the effect of overexpressed outer membrane protein OmpL in C3G biosynthesis, the control strain (Control group) without overexpression of ompL gene and the recombinant strain (OmpL group) with overexpression were cultured separately. The control strain contained pETM6-At3GT-m-PhANS and empty vector pACYCDuet-1, and the recombinant strain contained pETM6-At3GT-m-PhANS and pACYCDuet-1-ompL.

[0217] After the induction culture was completed, (+)-catechin dissolved in dimethylformamide and ethanol was added for precursor feeding, and the ratio of the precursor feeding solution was dimethylformamide:ethanol=8:2, v / v. Subsequently, at different time points after the addition of (+)-catechin, fermentation samples of the control strain and the recombinant strain, i.e., fermentation broth containing bacterial cells, were collected. Then, the C3G content in the fermentation sample was determined by HPLC.

[0218] The fermentation samples of the control strain and the recombinant strain were analyzed by HPLC to compare the synthesis of C3G at different time points to verify the effect of overexpression of the ompL gene on C3G biosynthesis.

[0219] The following are the specific experimental steps:

[0220] (1) The strains obtained above were inoculated into different test tubes containing 3 mL of LB liquid medium and cultured overnight at 37°C and 200 rpm.

[0221] (2) Take the overnight bacterial culture and transfer it to 50 mL of fresh LB liquid medium at a ratio of 1:50 (v / v), and culture it at 37°C and 200 rpm until the OD 600 A value of 0.6 is used to ensure that cells are in the appropriate growth phase.

[0222] (3) In OD 600After the value reaches the desired range, add the inducer IPTG with a final concentration of 0.5 mM to induce the expression of the target gene. Continue to culture at 30°C and 200 rpm for 3 hours to promote the expression of the target gene and the increase of protein production.

[0223] (4) The induced bacterial solution was transferred to a 50 mL sterile centrifuge tube and centrifuged at 4°C and 4000 rpm for 10 min to collect the cells.

[0224] (5) Resuspend the cells in 10 mL of fresh modified M9 medium, and add the substrate catechin (final concentration 0.5 g / L) and auxiliary factors (final concentration 0.1 mM 2-oxoglutaric acid, 2.5 mM sodium ascorbate) to provide precursors for C3G synthesis. Continue to culture at 30°C and 200 rpm for 20 hours to promote the accumulation of the target product.

[0225] (6) Fermentation samples were collected at different time points after the addition of (+)-catechin. The fermentation sample was mixed evenly with acidified methanol (1%, i.e., 1 mL of analytical grade concentrated hydrochloric acid was added to 100 mL of methanol) at a ratio of 1:1 (volume ratio). Subsequently, the mixture was centrifuged at 12,000 rpm for 10 min at room temperature, and the supernatant was taken, i.e., the extracellular C3G detection sample; 400 μL of 80% acidified anhydrous ethanol (1% HCl) was added to the precipitate, and the mixture was placed in an ultrasonic cleaner, ultrasonicated at 40°C for 15 min, and centrifuged at 12,000 rpm for 10 min, and the supernatant was taken, i.e., the intracellular C3G detection sample.

[0226] The above experimental results are as follows Figure 4 shown.

[0227] Depend on Figure 4 It can be seen that after 6 hours of fermentation, the maximum synthesis of C3G by the recombinant strain (OmpL group) was increased by 95% compared with the control strain (Control group). The experimental results prove that overexpression of the ompL gene can promote the excretion of C3G to the extracellular space and significantly increase the synthesis of C3G.

[0228] (II) Comparison of cell growth between the control strain without overexpression of ompL gene and the recombinant strain with overexpression

[0229] First, the obtained strains were inoculated into conical flasks containing 50 mL of LB liquid medium and cultured at 37°C and 200 rpm for 8 hours. 600 The cell growth status was evaluated by PCR using the PCR products, i.e., the cell growth status under non-inducing conditions.

[0230] The obtained strains were inoculated into conical flasks containing 50 mL LB liquid medium and cultured at 37 °C and 200 rpm until OD600 The final concentration of IPTG was 0.5 mM, and the cells were cultured at 30 °C and 200 rpm. The OD of the bacterial solution was monitored. 600 The values ​​were used to assess cell growth conditions, i.e., cell growth conditions under induction conditions.

[0231] like Figure 5 As shown in the figure, under non-induction conditions, the growth curves of the control strain (Control group) and the recombinant strain (OmpL group) basically overlapped, with no obvious differences. Under induction conditions, after adding the inducer IPTG to induce OmpL expression for a period of time, the turbidity value of the bacterial solution of the recombinant strain (OmpL group) was lower than that of the control strain (Control group), indicating that the overexpression of the outer membrane protein OmpL inhibited the growth of host cells.

[0232] By overexpressing ompL in the recombinant strain and performing quantitative analysis, it can be clearly seen that the extracellular production of C3G increased significantly. This discovery reveals the key role of ompL in the transport regulation of C3G, and effectively promotes the synthesis efficiency of C3G by increasing the transport rate of C3G.

[0233] (III) Comparison of C3G biosynthesis in the control strain without overexpression of the ompL gene and the recombinant strain with overexpression of the ompL gene when used simultaneously with other transporters

[0234] In order to verify that the outer membrane protein OmpL can be used in combination with other transport proteins to increase the biosynthetic yield of C3G, the multidrug resistance transporter MdtH (encoded by the mdtH gene) was selected, which was found in previous studies and can effectively enhance the efflux transport of C3G when used alone.

[0235] First, according to the method of Example 1, Escherichia coli BL21 (DE3) was used as the starting strain and pACYCDuet-1 plasmid was used as the vector to construct a recombinant expression vector for overexpressing the multidrug resistance transporter MdtH, named pACYCDuet-1-mdtH. The sequences of the forward primer mdtH-F and the reverse primer mdtH-R used to amplify mdtH using the Escherichia coli genome as a template are as follows:

[0236] mdtH-F:5'-aaggaggttatacatatgtcccgcgtgtcgc-3' (SEQ ID NO: 6)

[0237] mdtH-R:5'-agctgcgggtaccttattagtgatggtgatg-3' (SEQ ID NO: 7)

[0238] Secondly, using Escherichia coli BL21 (DE3) as the starting strain and pACYCDuet-1-mdtH plasmid as the vector, the vector was digested with restriction endonuclease Nco I, and the target fragment ompL was connected to the vector pACYCDuet-1-MdtH by homologous recombination. After transformation, enzyme digestion verification and sequencing verification were performed to construct a recombinant expression vector that overexpresses the outer membrane protein OmpL and the multidrug resistance transporter protein MdtH, named pACYCDuet-1-mdtH-ompL.

[0239] The forward primer and reverse primer used to amplify the target fragment ompL using the E. coli genome as a template are ompL-F-NcoI (SEQ ID NO: 8) and ompL-R-EcoRI (SEQ ID NO: 9), respectively.

[0240] ompL-F-NcoI:5'-ttaataaggagatataccatgaaaaatattaatgca ataattttattatc-3' (SEQ ID NO: 8)

[0241] ompL-R-EcoRI:5'-caggcgcgccgagctcgaattctcagaagaaatacttcgtcccgatacg-3' (SEQ ID NO: 9)

[0242] Then, according to the method of Example 2, pACYCDuet-1-mdtH and pETM6-At3GT-m-PhANS were co-transformed into Escherichia coli BL21 (DE3) competent cells to obtain a recombinant strain (MdtH group) that overexpressed the Escherichia coli multidrug resistance transporter MdtH. pACYCDuet-1-mdtH-ompL and pETM6-At3GT-m-PhANS were co-transformed into Escherichia coli BL21 (DE3) competent cells to obtain a recombinant strain (MdtH-OmpL group) that combined overexpressed the Escherichia coli multidrug resistance transporter MdtH and the outer membrane protein OmpL. The control strain (Control group) was obtained by co-transforming pETM6-At3GT-m-PhANS and the pACYCDuet-1 empty vector that did not carry the outer membrane protein gene and the multidrug resistance transporter gene into Escherichia coli BL21 (DE3) competent cells.

[0243] Next, according to the "two-step method" experimental steps (1) to (6) in Example 3 (I), (+)-catechin was used as a substrate for the biosynthesis of C3G, and the extracellular C3G production of the MdtH group, MdtH-OmpL group, and Control group was determined by HPLC. The experimental results are as follows: Figure 6shown.

[0244] Depend on Figure 6 It can be seen that after 6 hours of fermentation, the maximum synthesis of C3G in the recombinant strain (MdtH-OmpL group) was 2.13 times higher than that in the control strain (Control group) and 0.83 times higher than that in the recombinant strain (MdtH group). The experimental results show that the combined overexpression of the ompL gene and mdtH can further promote the excretion of C3G to the extracellular space, thereby significantly increasing the synthesis of C3G.

[0245] (IV) Comparison of cell growth between the control strain without overexpression of the ompL gene and the recombinant strain with overexpression when used with other transporters

[0246] First, the recombinant strain (MdtH-OmpL group), recombinant strain (MdtH group) and control strain (Control group) were inoculated into conical flasks containing 50 mL LB liquid medium and cultured at 37 °C and 200 rpm for 8 h. The OD value of the bacterial solution was monitored. 600 The values ​​were used to evaluate cell growth conditions, i.e., cell growth conditions under non-inducing conditions.

[0247] The recombinant strain (MdtH-OmpL group), recombinant strain (MdtH group) and control strain (Control group) were inoculated into conical flasks containing 50 mL LB liquid medium and cultured at 37 °C and 200 rpm until OD 600 The final concentration of IPTG was 0.5 mM, and the cells were cultured at 30 °C and 200 rpm. The OD of the bacterial solution was monitored. 600 The values ​​were used to evaluate the cell growth status, i.e., the cell growth status under induction conditions.

[0248] like Figure 7 As shown in the figure, under non-induction conditions, the growth curves of the recombinant strain (MdtH-OmpL group), the recombinant strain (MdtH group) and the control strain (Control group) basically overlapped, and no significant differences were found. Under induction conditions, after the induction agent IPTG was added to induce expression for a period of time, the turbidity values ​​of the bacterial solution of the recombinant strain (MdtH group) and the recombinant strain (MdtH-OmpL group) were lower than those of the control strain (Control group), and the turbidity value of the bacterial solution of the recombinant strain (MdtH-OmpL group) was lower than that of the recombinant strain (MdtH group), indicating that the overexpression of the multidrug resistance transporter MdtH slightly inhibited the growth of host cells, while the combined overexpression of the outer membrane protein OmpL and the multidrug resistance transporter MdtH inhibited the growth of host cells to a higher degree.

[0249] By combining the overexpression of mdtH and ompL in the recombinant strain and performing quantitative analysis, it can be clearly seen that the extracellular production of C3G increased significantly after combined overexpression. This discovery reveals the key role of ompL in the transport regulation of C3G, and effectively promotes the synthesis of C3G by increasing the transport rate of C3G.

[0250] Comparative Example 1 Effects of other outer membrane proteins on C3G biosynthesis

[0251] We selected TolC, a conserved outer membrane channel protein widely present in Gram-negative bacteria, and compared it with OmpL to compare the extracellular C3G production of Escherichia coli after gene overexpression. The specific operation is as follows:

[0252] First, according to the method of Example 1, Escherichia coli BL21 (DE3) was used as the starting strain and pACYCDuet-1 plasmid was used as the vector to construct a recombinant expression vector for overexpressing the outer membrane channel protein TolC, named pACYCDuet-1-tolC. The sequences of the forward primer tolC-F and the reverse primer tolC-R used for amplifying tolC using the Escherichia coli genome as a template are as follows:

[0253] tolC-F:5'-taataaggagatataccatgaagaaattgctcccca-3' (SEQ ID NO: 10)

[0254] tolC-R:5'-caggcgcgccgagctcgaattctcagttacggaaagggttatg-3' (SEQ ID NO: 11)

[0255] Then, according to the method of Example 2, pACYCDuet-1-tolC and pETM6-At3GT-m-PhANS were co-transformed into E. coli BL21 (DE3) competent cells to obtain a C3G synthetic strain that overexpressed the E. coli outer membrane protein gene. The control strain was the same as that in Example 3, which was obtained by co-transforming the C3G synthetic pathway plasmid pETM6-At3GT-m-PhANS and the pACYCDuet-1 empty vector without the outer membrane protein gene into E. coli BL21 (DE3) competent cells.

[0256] Next, according to the "two-step method" experimental steps (1) to (6) in Example 3 (I), (+)-catechin was used as a substrate for the biosynthesis of C3G. The extracellular C3G production of the strain overexpressing tolC and ompL and the control strain was determined by HPLC. The experimental results are shown in Table 2

[0257] Table 2

[0258]

[0259] (Note: The formula for calculating the multiple increase of extracellular C3G production of the overexpression strain compared with the control strain is:

[0260] (Extracellular C3G production of overexpression strain - extracellular C3G production of control strain) / extracellular C3G production of control strain. )

[0261] As can be seen from Table 2, after overexpression of the ompL gene, the unit OD 600 The extracellular C3G production of the overexpressed strain was 0.91 times higher than that of the control strain, while the extracellular C3G production of the strain after overexpressing the tolC gene was only 0.17 times higher than that of the control strain. It was proved that the ompL gene had a more significant C3G extracellular emission effect than the tolC gene, which was beneficial to improve the biosynthesis of C3G. The reason for this may be that OmpL promoted the efflux of C3G on the one hand, and on the other hand, it had the function of binding the substrate (+)-catechin, which could promote the transport of the substrate into the cell, that is, promote the absorption of the substrate, thereby improving the synthesis of C3G.

[0262] The tolC gene overexpression strain has limited effect on promoting C3G efflux. The reason may be that the overexpression of tolC leads to leakage of intracellular substances, which affects the absorption of substrates by cells on the one hand and the normal metabolism of bacteria on the other hand. Although the efflux of C3G is enhanced, the overall synthesis of C3G may decrease, so the increase in the production of products discharged to the extracellular space is small. In addition, in the process of C3G efflux, TolC may not be a key outer membrane protein. Its overexpression occupies cell resources and has little significance for increasing the extracellular production of C3G.

[0263] Comparative Example 2 Effects of combined overexpression of other outer membrane proteins and the multidrug resistance transporter MdtH on C3G biosynthesis

[0264] The other six outer membrane protein encoding genes of E. coli, ompN, slp, cirA, nlpE, mltA and slyB, were selected to compare the extracellular C3G production of E. coli after overexpression in combination with the gene mdtH. The specific operation is as follows:

[0265] First, using E. coli BL21 (DE3) as the starting strain and pACYCDuet-1-mdtH plasmid as the vector, a recombinant expression vector for combined overexpression of the multidrug resistance transporter MdtH and the outer membrane protein gene X was constructed, named pACYCDuet-1-mdtH-X, where X represents ompN, slp, cirA, nlpE, mltA, and slyB. The sequences of the forward primer XF and reverse primer XR used to amplify these genes using the E. coli genome as a template are as follows:

[0266] OmpN-F:5'-gatataccatgggcaaaagcaaagtactggcacttttaa-3' (SEQ ID NO: 12)

[0267] OmpN-R:5'-gagctcgaattcttagaactgataaaccagacctaaagc-3' (SEQ ID NO: 13)

[0268] Slp-F:5'-gatataccatgggcaacatgacaaaaggtgcactcatcc-3' (SEQ ID NO: 14)

[0269] Slp-R:5'-gagctcgaattcttatttgaccagctcaggtgttacc-3' (SEQ ID NO: 15)

[0270] CirA-F:5'-gatataccatgggctttaggttgaaccctttcgtacggg-3' (SEQ ID NO: 16)

[0271] CirA-R:5'-gagctcgaattctcagaagcgataatccactgcc-3' (SEQ ID NO: 17)

[0272] NlpE-F:5'-gatataccatggtgaaaaaagcgatagtgacag-3' (SEQ ID NO: 18)

[0273] NlpE-R:5'-gagctcgaattcttactgccccaaactactgcaatcc-3' (SEQ ID NO: 19)

[0274] MltA-F:5'-gatataccatgggcaaaggacgttgggtaaagtaccttctttatg-3' (SEQ ID NO: 20)

[0275] MltA-R:5'-gagctcgaattctcagccgctaaagacgttacc-3' (SEQ ID NO: 21)

[0276] SlyB-F:5'-gatataccatgggcattaaacgcgtattggttgtttcaatgg-3' (SEQ ID NO: 22)

[0277] SlyB-R:5'-gagctcgaattcttagcgcggagaaacggtc-3' (SEQ ID NO: 23)

[0278] Then, according to the method of Example 2, pACYCDuet-1-mdtH-X and pETM6-At3GT-m-PhANS were co-transformed into Escherichia coli BL21 (DE3) competent cells, respectively, to obtain a C3G synthetic strain overexpressing the co-expression module of Escherichia coli multidrug resistance transporter MdtH and outer membrane protein X. Among them, there are two control strains, one of which is obtained by co-transforming the C3G synthetic pathway plasmid pETM6-At3GT-m-PhANS and the pACYCDuet-1 empty vector without the multidrug resistance transporter MdtH and outer membrane protein expression module into Escherichia coli BL21 (DE3) competent cells; the other is obtained by co-transforming the C3G synthetic pathway plasmid pETM6-At3GT-m-PhANS and the pACYCDuet-1-mdtH plasmid that only overexpresses the Escherichia coli multidrug resistance transporter MdtH into Escherichia coli BL21 (DE3) competent cells.

[0279] Next, according to the "two-step method" experimental steps (1) to (6) in Example 3 (I), (+)-catechin was used as a substrate for the biosynthesis of C3G, and the extracellular C3G production of the strain overexpressing ompL, ompN, slp, cirA, nlpE, mltA and slyB and the control strain was determined by HPLC. The experimental results are as follows: Figure 8 shown.

[0280] Depend on Figure 8 It can be seen that among the seven selected outer membrane proteins, after overexpression of the ompL gene, the unit OD 600 The extracellular C3G production of the overexpression strain was 73% higher than that of the control strain pACYCDuet-1-mdth. However, after overexpressing ompN, slp, and cirA, the extracellular C3G production of the strain did not increase significantly compared with the control strain. After overexpressing nlpE, mltA, and slyB genes, the extracellular C3G production of the strain decreased compared with the control strain.

[0281] Therefore, overexpression of the ompL gene significantly increased the extracellular C3G production of E. coli compared to overexpression of the ompN, slp, cirA, nlpE, mltA and slyB genes, proving that the ompL gene has a more significant C3G extracellular emission effect than other genes, which is beneficial to improving the biosynthesis of C3G. The reason for this may be that OmpL can form pores on the outer membrane to help bacteria absorb and discharge nutrients and metabolites.

[0282] The efflux effect of C3G in the strain overexpressing the ompN, slp, cirA, nlpE, mltA and slyB genes was poorer than that of ompL. The possible reasons are: (1) the overexpression of outer membrane proteins occupied a large membrane area, affecting the normal metabolism of cells; (2) the change of the outer membrane protein structure hindered the recognition and binding of the product C3G with other transport proteins.

[0283] Comparative Example 3 Effects of overexpression of ompL on the biosynthesis of anthocyanin synthase C3G from different sources

[0284] The key enzyme XANS of C3G metabolic pathway from different sources (X = Tp, Ph; Tp from Trifolium pragmatense; Ph from Petunia hybrida) was selected and combined with At3GT (from Arabidopsis thaliana) to construct the recombinant plasmid pETM6-At3GT-m-XANS.

[0285] First, according to the method of Example 2, pACYCDuet-1-ompL and pETM6-At3GT-m-XANS were co-transformed into E. coli BL21 (DE3) competent cells to obtain a C3G synthetic strain that overexpressed the E. coli outer membrane protein gene. Among them, the control strain was obtained by co-transforming the C3G synthetic pathway plasmid pETM6-At3GT-m-XANS and the pACYCDuet-1 empty vector without the outer membrane protein gene into E. coli BL21 (DE3) competent cells.

[0286] Then, according to the "two-step method" experimental steps (1) to (6) in Example 3 (I), the biosynthesis of C3G was carried out using (+)-catechin as a substrate, and the extracellular C3G production of the C3G synthesis strain overexpressing the Escherichia coli outer membrane protein gene and the control strain under different sources of ANS was determined by HPLC. The experimental results are shown in Table 3.

[0287] Table 3

[0288]

[0289] (Note: The formula for calculating the multiple increase of extracellular C3G production of the overexpression strain compared with the control strain is:

[0290] (Extracellular C3G production of overexpression strain - extracellular C3G production of control strain) / extracellular C3G production of control strain. )

[0291] As can be seen from Table 3, overexpression of the ompL gene has a very significant effect on the extracellular emission of C3G in C3G biosynthesis strains of ANS from different sources, which is beneficial to improving the biosynthesis of C3G.

[0292] However, the overexpression of the ompL gene had different effects on the extracellular emission of C3G in C3G biosynthetic strains of ANS from different sources. After overexpressing the ompL gene, the extracellular C3G production of the C3G biosynthetic strain of PhANS was 0.91 times higher than that of the control strain, while the extracellular C3G production of the C3G biosynthetic strain of TpANS was 0.57 times higher than that of the control strain. The reason for this may be that compared with PhANS, the C3G biosynthetic strain of TpANS already had a higher C3G production when ompL was not overexpressed, so the effect of overexpressing ompL on the extracellular production of C3G was relatively low, but it still significantly increased the production of extracellular C3G.

[0293] Industrial Applicability

[0294] The present invention discovered that the outer membrane protein OmpL is related to the transport regulation of C3G. By constructing a recombinant expression vector and overexpressing the ompL gene, the extracellular transport efficiency of C3G synthesized by the host cell is effectively improved, thereby significantly enhancing the production capacity of C3G. The present invention provides a new way to improve the biosynthesis of polyphenolic flavonoid compounds, and has good industrial value and commercial prospects.

Claims

1. A recombinant expression vector for synthesizing outer membrane protein of cyanidin-3-O-glucoside, characterized in that: The recombinant expression vector contains a gene sequence for encoding the outer membrane protein, and the outer membrane protein is selected from any one of the following (i) to (ii): (i) a polypeptide OmpL having an amino acid sequence as shown in SEQ ID NO: 1; (ii) A polypeptide mutant comprising the amino acid sequence shown in SEQ ID NO: 1, wherein one or more amino acids are substituted, repeated, deleted or added at one or more positions of the sequence shown in SEQ ID NO: 3, and has the function of polypeptide OmpL.

2. The recombinant expression vector according to claim 1, wherein The recombinant expression vector contains a gene sequence encoding the outer membrane protein as shown in any one of the following (iii) to (v): (iii) a polynucleotide encoding a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1; (iv) a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 2; (v) a polynucleotide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 2, and encoding a polypeptide having the function of polypeptide OmpL.

3. A recombinant microorganism for synthesizing cyanidin-3-O-glucoside, characterized in that: The recombinant microorganism contains a host cell, and the recombinant expression vector according to claim 1 or 2 transformed in the host cell.

4. The recombinant microorganism according to claim 3, wherein The recombinant microorganism further comprises a recombinant plasmid transformed into the host cell. Preferably, the recombinant plasmid contains the gene sequence of enzymes related to the cyanidin-3-O-glucoside metabolic pathway and can be expressed in the host cell.

5. The recombinant microorganism according to claim 4, wherein The enzymes related to the cyanidin-3-O-glucoside metabolic pathway are anthocyanidin synthase ANS and / or 3-O-glucoside transferase 3GT.

6. The recombinant microorganism according to any one of claims 3 to 5, wherein The host cell is derived from microorganisms of the genus Escherichia, Brevibacterium, Corynebacterium, Bacillus, Saccharomyces, or Lactococcus.

7. A method for preparing a recombinant microorganism according to any one of claims 3 to 6, characterized in that: The method comprises the following steps: 1) replicating the gene sequence encoding the outer membrane protein; 2) constructing a recombinant expression vector comprising the gene sequence described in step 1); 3) The recombinant expression vector obtained in step 2) and the recombinant plasmid encoding the key enzymes in the cyanidin-3-O-glucoside metabolic pathway are transformed into host cells to obtain a recombinant microorganism for synthesizing the cyanidin-3-O-glucoside.

8. A method for producing cyanidin-3-O-glucoside, characterized in that: The method comprises using bacterial cells of the recombinant microorganism according to any one of claims 3 to 6 to overexpress the outer membrane protein, so as to promote the synthesis of the precursor substance added to the culture system into the cyanidin-3-O-glucoside and then transport it to the outside of the bacterial cell.

9. The method according to claim 8, wherein: The precursor substance is (+)-catechin.

10. Use of an outer membrane protein in producing anthocyanins, characterized in that: The outer membrane protein is the outer membrane protein described in claim 1, and the anthocyanin is a polyphenolic flavonoid compound containing 2-phenylbenzopyranyl cation. Preferably, the anthocyanin is cyanidin-3-O-glucoside.

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