Recombinant escherichia coli for producing Shinorine and preparation method thereof

Abstract

The invention discloses recombinant escherichia coli for producing Shinorine and a preparation method of the recombinant escherichia coli. The invention belongs to the field of synthetic biology, and particularly relates to recombinant escherichia coli for producing Shinorine and a preparation method of the recombinant escherichia coli. The recombinant strain provided by the invention can be used for synthesizing the mycosporine-like amino acid Shinorine by using glucose as a raw material, the capability of producing the Shinorine is obviously improved, and the recombinant strain can be used for large-scale preparation of the mycosporine-like amino acid Shinorine. Compared with the traditional method for extracting from algae, the microbial fermentation method disclosed by the invention has the advantages of short production period, no restriction by climate and environment, high yield and the like.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biology, and in particular relates to a recombinant Escherichia coli for producing Shinorine and a preparation method thereof. Background Art

[0002] Ultraviolet (UV) radiation is ubiquitous in our daily lives. Long-term exposure to UV radiation can have profound health consequences. UV radiation can be categorized by wavelength into UV-A (approximately 315nm to 400nm), UV-B (approximately 280nm to 315nm), and UV-C (approximately 100nm to 280nm). Most UV-A radiation reportedly penetrates the ozone layer and cloud layers and is deeply absorbed by the skin, causing carcinogenic effects and skin aging. A smaller amount of UV-B radiation reaches the Earth's surface and is shallowly absorbed by the skin, making it the primary cause of sunburn. UV-C radiation generally cannot penetrate the atmosphere or the ozone layer. At the molecular level, UV radiation can oxidize proteins and lipids, rendering them inoperable, and directly damage DNA through the formation of cyclobutane pyrimidine dimers and 6-4 photoproducts. Furthermore, UV radiation indirectly leads to the formation of reactive oxygen species, further impairing cellular function.

[0003] In response to the damage caused by ultraviolet radiation, the World Health Organization recommends using sunscreens with a protection factor greater than or equal to 15 to protect the skin. The mechanism of action of the active ingredients of chemical sunscreens is usually based on the conjugation effect formed by the benzene ring and the adjacent carbonyl group in its structure; this structure can absorb high-energy ultraviolet rays and cause the electrons to enter an excited state. When the electrons return to the ground state, the energy is released in a form that is safer for the human body. The ultraviolet absorbing ingredients of chemical sunscreens include avobenzone, octocrylene, octylmethoxycinnamate, para-aminobenzoic acid, and benzoic acid. -While chemical sunscreens effectively absorb UV rays, they also raise health concerns. Side effects of chemical sunscreens include burning, dermatitis, acne, and allergic reactions. Excessive use of some chemical sunscreen ingredients has been shown to lead to absorption and accumulation in the body. Furthermore, chemical sunscreens can have negative ecological impacts. For example, high concentrations of chemical sunscreen ingredients such as octocrylene and 3-(4-methylbenzylidene) camphor have been found in some marine organisms. This accumulation not only affects the development and reproduction of marine organisms but also poses food safety concerns through biomagnification in the food chain. Compared to chemical sunscreens, physical sunscreens (such as titanium dioxide and zinc oxide) are relatively safe and provide UV protection through scattering and reflection. However, physical sunscreens are not popular with consumers because they leave a white residue on the skin. In recent years, consumers have shown a preference for natural sunscreens, which are safer and more aesthetically pleasing.

[0004] Mycosporine-like amino acids (MAAs) are a class of natural products that can absorb ultraviolet radiation. As a strategy to protect against ultraviolet radiation, these natural products are widely found in various marine, freshwater, and terrestrial organisms, such as corals, red algae, microalgae, cyanobacteria, fungi, etc. Shinorine, as a MAA, can absorb ultraviolet radiation and prevent it from damaging skin cells. At the same time, due to the presence of its polar groups, it has good water solubility and stability, making it suitable for use in various types of sunscreen products. However, despite its huge potential, its large-scale development is still subject to various limitations. At present, domestic research on Shinorine is still in its initial stage, with the following main problems: (1) Raw material cost, traditional extraction requires large-scale cultivation and harvesting of algae; (2) Separation and purification are difficult, requiring complex product separation processes and expensive production equipment; (3) The chiral center of Shinorine increases the difficulty of large-scale chemical synthesis; (4) In terms of microbial synthesis, there has been a small amount of work on microbial synthesis of Shinorine through metabolic engineering, with a Shinorine yield of 2077 mg / L (CN112292452B). There is no metabolic engineering strategy for synthesizing shinorine using glucose as the sole raw material. Therefore, improving the efficiency of shinorine biosynthesis is the only way to develop a production model with broad application prospects. Summary of the Invention

[0005] The main problem to be solved by the present invention is how to achieve a dynamic balance of metabolic flow in Escherichia coli cells so that they can efficiently synthesize Shinorine using glucose as a raw material.

[0006] In order to solve the above problems, the present invention provides a method for constructing recombinant bacteria.

[0007] The method for constructing a recombinant bacterium provided by the present invention comprises the following transformation of the recipient bacterium:

[0008] M1), knocking out the transaldolase B (talB) gene in the recipient bacteria;

[0009] M2), knocking out the transaldolase A (talA) gene in the recipient bacteria;

[0010] M3), knock out the wild-type promoter that drives the expression of 6-phosphate glucose dehydrogenase (zwf) gene in the recipient bacteria and replace it with promoter P 119 ;

[0011] M4), knocking out the glucose-6-phosphate isomerase (pgi) gene in the recipient bacteria;

[0012] M5), knock out the wild-type promoter that drives the expression of the transketolase A (tktA) gene in the recipient bacteria and replace it with promoter P CPA1 ;

[0013] M6), knock out the wild-type promoter that drives the expression of the transketolase B (tktB) gene in the recipient bacteria and replace it with promoter P CPA1 ;

[0014] M7) Introduce the mycosporine-like amino acid shinorine synthesis gene cluster (ScMAAs) genes into the recipient bacteria.

[0015] M7) The mycosporine-like amino acid shinorine synthesis gene cluster (ScMAAs) may be derived from Scytonema cf. Crispum.

[0016] The recipient bacteria may be Escherichia coli.

[0017] Furthermore, the method further comprises transforming the recombinant bacteria as follows:

[0018] M8), knocking out the L-methionine transcription factor (metJ) gene in the recipient bacteria;

[0019] M9), knock out the wild-type promoter that drives the expression of the L-methionine adenosyltransferase (metK) gene in the recipient bacteria and replace it with promoter P 119 ;

[0020] M10), knocking out the pyruvate formate lyase (pflB) gene in the recipient strain and replacing it with S-adenosylhomocysteine ​​nucleosidase (mtn) and S-ribosylhomocysteine ​​lyase (luxS) genes;

[0021] The S-adenosylhomocysteine ​​nucleosidase (mtn) and S-ribosylhomocysteine ​​lyase (luxS) described in M10) are derived from Escherichia coli BW25113 and are synthesized by P CPA1 The promoter initiates expression.

[0022] Furthermore, the method further comprises transforming the recombinant bacteria as follows:

[0023] M11), the wild-type promoter that drives the expression of the phosphogluconate dehydratase (edd) gene in the recipient bacteria was knocked out and replaced with the promoter P J23105 ;

[0024] M12), knocking out the pyruvate oxidase (poxB) gene in the recipient strain and replacing it with phosphoglycerate dehydrogenase (serA), phospho-L-serine aminotransferase (serC), and phospho-L-serine phosphorylase (serB) genes;

[0025] The phosphoglycerate dehydrogenase (serA), phospho-L-serine aminotransferase (serC) and phospho-L-serine phosphorylase (serB) described in M12) are derived from Escherichia coli BW25113 and are prepared by P J23105 Promoter to start expression; M13), knock out the L-serine deaminase III (tdcG) gene in the recipient bacteria;

[0026] M14), knocking out the L-serine deaminase I (sdaA) gene in the recipient strain;

[0027] M15) and knocked out the L-serine deaminase II (sdaB) gene in the recipient bacteria.

[0028] Furthermore, the method further comprises transforming the recombinant bacteria as follows:

[0029] M16), knocking out the L-serine hydroxymethyltransferase (glyA) gene in the recipient bacteria and replacing it with the L-serine hydroxymethyltransferase (glyA) gene and methionine synthase (metH) gene.

[0030] The L-serine hydroxymethyltransferase (glyA) gene and methionine synthase (metH) gene described in M16) are derived from Escherichia coli BW25113 and are connected by a linker to form a fusion protein encoding gene. CPA1 The promoter initiates expression.

[0031] Herein, 1) the Genbank number of the 6-phosphate glucose dehydrogenase is AKT72612.1, the Genbank number of its encoding gene is Gene ID: 946370, and the update date is 2024.07.06;

[0032] 2) The Genbank number of the transaldolase A is NP_416959.1, the Genbank number of the gene encoding it is Gene ID: 947006, and the update date is 2024.07.06;

[0033] 3) The Genbank number of the transaldolase B is NP_414549.1, the Genbank number of its encoding gene is GeneID:944748, and the update date is 2024.07.06;

[0034] 4) The Genbank number of the transketolase A is YP_026188.1, the Genbank number of its encoding gene is GeneID:947420, and the update date is 2024.07.06;

[0035] 5) The Genbank number of the transketolase B is NP_416960.1, the Genbank number of its encoding gene is GeneID:945865, and the update date is 2024.07.06;

[0036] 6) The Genbank number of the glucose-6-phosphate isomerase is NP_418449.1, the Genbank number of its encoding gene is Gene ID: 948535, and the update date is 2024.07.06;

[0037] 7) The Genbank number of the L-methionine adenosyltransferase is NP_417417.1, the Genbank number of its encoding gene is Gene ID: 945389, and the update date is 2024.07.06;

[0038] 8) The Genbank number of the Shinorine synthesis gene cluster is KX021866, and the update date is 2024.07.06;

[0039] 9) The Genbank number of the L-methionine synthesis transcription factor is NP_418373, the Genbank number of its encoding gene is Gene ID: 948435, and the update date is 2024.07.06;

[0040] 10) The Genbank number of the pyruvate formate lyase is NP_415423.1, the Genbank number of the gene encoding it is Gene ID: 945514, and the update date is 2024.07.06;

[0041] 11) The Genbank number of the S-adenosylhomocysteine ​​nucleosidase is NP_414701.1, the Genbank number of its encoding gene is Gene ID: 948542, and the update date is 2024.07.06;

[0042] 12) The Genbank number of the S-ribosylhomocysteine ​​lyase is NP_417172.1, the Genbank number of its encoding gene is Gene ID: 947168, and the update date is 2024.07.06;

[0043] 13) The Genbank number of the phosphogluconate dehydratase is NP_416365.1, the Genbank number of its encoding gene is Gene ID: 946362, and the update date is 2024.07.06;

[0044] 14) The Genbank number of the pyruvate oxidase is CAD6018048.1, the Genbank number of its encoding gene is Gene ID: 946132, and the update date is 2024.07.06;

[0045] 15) The Genbank number of the phosphoglycerate dehydrogenase is NP_417388.1, the Genbank number of its encoding gene is Gene ID: 945258, and the update date is 2024.07.06;

[0046] 16) The Genbank number of the phospho-L-serine phosphorylase is NP_418805.1, the Genbank number of its encoding gene is Gene ID: 948913, and the update date is 2024.07.06;

[0047] 17) The Genbank number of the phospho-L-serine aminotransferase is NP_415427.1, the Genbank number of its encoding gene is Gene ID: 945527, and the update date is 2024.07.06;

[0048] 18) The Genbank number of the L-serine deaminase III is YP_026204.1, the Genbank number of its encoding gene is Gene ID: 2847724, and the update date is 2024.07.06;

[0049] 19) The Genbank number of the L-serine deaminase I is NP_416328.1, the Genbank number of its encoding gene is Gene ID: 946331, and the update date is 2024.07.06;

[0050] 20) The Genbank number of the L-serine deaminase II is NP_417277.1, the Genbank number of its encoding gene is Gene ID: 947262, and the update date is 2024.07.06;

[0051] 21) The Genbank number of the L-serine hydroxymethyltransferase is NP_417046.1, the Genbank number of its encoding gene is Gene ID: 947022, and the update date is 2024.07.06;

[0052] 22) The Genbank number of the methionine synthase is NP_418443.1, the Genbank number of its encoding gene is Gene ID: 948522, and the update date is 2024.07.06;

[0053] The P CPA1 A promoter is any of the following DNA molecules:

[0054] 1) A DNA molecule having a nucleotide sequence of one chain as SEQ ID No: 1 in the sequence listing,

[0055] 2) a DNA molecule that is more than 80% identical to the DNA molecule of 1) and has promoter function;

[0056] The P 119 A promoter is any of the following DNA molecules:

[0057] 1) A DNA molecule having a nucleotide sequence of one chain as SEQ ID No: 2 in the sequence listing,

[0058] 2) a DNA molecule that is more than 80% identical to the DNA molecule of 1) and has promoter function;

[0059] The P J23105 A promoter is any of the following DNA molecules:

[0060] 1) A DNA molecule having a nucleotide sequence of one chain as SEQ ID No: 3 in the sequence listing,

[0061] 2) a DNA molecule that is more than 80% identical to the DNA molecule of 1) and has promoter function;

[0062] The linker is any of the following DNA molecules:

[0063] 1) A DNA molecule having a nucleotide sequence of one chain as SEQ ID No: 4 in the sequence listing;

[0064] The present invention also provides a recombinant bacterium constructed by the method described above.

[0065] The present invention also provides a bacterial agent, which contains the above-mentioned recombinant Escherichia coli or a culture containing the above-mentioned recombinant Escherichia coli.

[0066] The term "culture" refers to any liquid or solid product (i.e., fermentation product) containing a microbial population after artificial inoculation and cultivation. This refers to a product obtained by growing and / or amplifying microorganisms. It can be a biologically pure culture of the microorganism or contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process.

[0067] The present invention also provides the application of the above method in any of the following:

[0068] W1) Application in the production of mycosporin-like amino acid Shinorine;

[0069] W2) Application in the preparation of products producing mycosporin-like amino acid Shinorine.

[0070] The present invention also provides the use of the recombinant Escherichia coli described above in any of the following:

[0071] W1) Application in the production of mycosporin-like amino acid Shinorine;

[0072] W2) Application in the preparation of products producing mycosporin-like amino acid Shinorine.

[0073] The present invention also provides a method for preparing mycosporine-like amino acids, comprising using glucose, L-methionine, glycine, L-serine and / or glucose, glycine, L-serine and / or glucose, glycine and / or glucose as substrates, inoculating the recombinant bacteria described above into a culture medium containing the substrates, and whole-cell catalysis to obtain the mycosporine-like amino acid Shinorine.

[0074] The growth medium of the recombinant bacteria can be as follows: 5g / L yeast powder, 5g / L glycerol, 25mM Na2HPO4, 25mMKH2PO4, 50mM NH4Cl, 5mM Na2SO4, 2mM MgSO4, 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μM ZnSO4, 2μM CoCl2, 2μM NiCl2, 2μM Na2Mo4, 2μM Na2SeO3 and 2μM H3BO3.

[0075] The culture medium containing the substrate may be as follows:

[0076] 1) Composition and final concentrations of medium B: 48 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 187 mM NH4Cl, 100 g / L glucose, 25 g / L glycine, 25 g / L L-serine, 10 g / L L-methionine.

[0077] 2) Composition and final concentration of C medium: 48 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 187 mM NH4Cl, 100 g / L glucose, 25 g / L glycine, 25 g / L L-serine.

[0078] 3) Composition and final concentration of D medium: 48 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 187 mM NH4Cl, 100 g / L glucose, 25 g / L glycine.

[0079] 4) Composition and final concentration of E medium: 48 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 187 mM NH4Cl, 100 g / L glucose.

[0080] The present invention transforms the recipient bacteria including 1), 2), 3) and 4):

[0081] 1) Knock out the transaldolase A and transaldolase B genes in biological cells, and enhance the glucose-6-phosphate dehydrogenase, transketolase A (transketolase 1), and transketolase B (transketolase 2) genes in biological cells.

[0082] 2) Introducing or enhancing the Shinorine synthesis gene cluster into biological cells.

[0083] 3) Knock out the gene for the DNA-binding transcriptional repressor MetJ in the biological cells, enhance the gene for the L-methionine adenosyltransferase in the biological cells, and replace the gene encoding pyruvate formate-lyase with genes encoding S-adenosylhomocysteine ​​nucleosidase and S-ribosylhomocysteine ​​lyase.

[0084] 4) Knockout the genes encoding L-serine deaminase I, L-serine deaminase II, and L-serine deaminase III in the biological cells, enhance the gene encoding phosphogluconate dehydratase in the biological cells, and replace the gene encoding pyruvate oxidase with genes encoding phosphoglycerate dehydrogenase, phosphoserinephosphatase, and phosphoserine aminotransferase.

[0085] 5) Strengthen the genes of L-serine hydroxymethyltransferase and methionine synthase in biological cells.

[0086] The above method 1) strengthens the glucose uptake pathway, blocks the glycolysis reaction, strengthens the pentose phosphate pathway, and blocks the pathway of sedoheptulose-7-phosphate, thereby improving substrate utilization efficiency; method 2) provides the microorganism with the ability to synthesize Shinorine from glucose, glycine, L-serine, and L-methionine; method 3) provides or enhances the microorganism's ability to synthesize Shinorine from glucose, glycine, and L-serine; method 4) provides or enhances the microorganism's ability to synthesize Shinorine from glucose and glycine; method 5) provides or enhances the microorganism's ability to synthesize Shinorine from glucose.

[0087] The beneficial technical effects achieved by the present invention are as follows:

[0088] The recombinant bacteria constructed in this invention can synthesize the mycosporin-like amino acid shinorine using glucose as a raw material, significantly improving its production capacity and enabling large-scale production of shinorine. Compared with traditional algae extraction methods, the microbial fermentation method of this invention offers advantages such as a shorter production cycle, unrestricted climate, and higher yields. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 Comparison of the ability of recombinant strains MS6-1 and MS6-0 to produce shinorine.

[0090] Figure 2 Comparison of the ability of recombinant bacteria MS9-1, MS9-0 and MS6-1 to produce shinorine.

[0091] Figure 3 Comparison of the ability of recombinant bacteria MS14-1, MS14-0 and MS9-1 to produce shinorine.

[0092] Figure 4 Comparison of the ability of recombinant bacteria MS15-1, MS15-0 and MS14-1 to produce shinorine. DETAILED DESCRIPTION

[0093] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0094] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0095] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0096] Escherichia coli BW25113 (CGSC#:7636), plasmid pKD46 (CGSC#:7739), and pCP20 (CGSC#:7629) in the following examples were purchased from the Escherichia coli Genetic Collection at Yale University (CGSC), USA, at https: / / ecgrc.net.

[0097] Escherichia coli MC02 (CGMCC No. 34378) can be obtained from CGMCC at: https: / / cgmcc.net.

[0098] The primers used in the present invention are shown in Table 1. The strain information used is shown in Table 2.

[0099] Table 1. Primers used in this patent

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] Note: Please underline the homology arms or homology sequences in the primers, or distinguish between uppercase and lowercase letters.

[0106] Table 2. Strains used in the present invention

[0107]

[0108]

[0109] Example 1. Construction of recombinant Escherichia coli MS6-1 and production of Shinorine

[0110] First, we generated a basic strain, MS10-1, for the production of mycosporin-like amino acids, based on Escherichia coli MC02. It can synthesize shinorine from glucose, glycine, L-serine, and L-methionine. This strain primarily blocks the sedoheptulose-7-phosphate consumption pathway, enhances the expression of several key enzymes in the pentose phosphate pathway, and blocks the conversion of glucose-6-phosphate to fructose-6-phosphate. The strain was constructed as follows; the primers used are listed in Table 1.

[0111] 1. Knockout of the transaldolase B (talB) gene

[0112] First, the transaldolase talB gene of Escherichia coli MC02 was knocked out to obtain the recombinant strain MS1. The specific steps are as follows:

[0113] (1-a) Preparation of the targeting fragment talB up-kan-talB down

[0114] PCR amplification was performed using talB-1 / talB-2 as primers and the frt-kan-frt (SEQ ID No: 5) screening marker fragment as a template to obtain the targeting fragment talB up-kan-talB down.

[0115] (1-b) Preparation of host bacteria containing pKD46 plasmid

[0116] Plasmid pKD46 was transformed into E. coli MC02 by the calcium chloride transformation method. After overnight culture at 30°C on LB plates containing ampicillin, clones were selected to obtain E. coli MC02-pKD46 containing plasmid pKD46. A single clone of MC02-pKD46 was inoculated into LB medium containing 100 μg / mL and 2 g / L L-arabinose and cultured to the logarithmic phase (OD 600nm =0.6-0.8), and then washed with pre-chilled 10% glycerol to prepare MC02-pKD46 competent cells. After induction with L-arabinose, MC02-pKD46 expresses the three recombinant proteins Gam, Beta, and Exo of λ phage, thereby enhancing its homologous recombination ability.

[0117] (1-c) Homologous recombination

[0118] The targeting fragment talB up-kan-talB down prepared in (1-a) was transformed into the competent cells of MC02-pKD46 prepared in (1-b) by electroporation. The electroporated cells were plated on LB solid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C. A single colony was then selected and identified using primers talB-3 and kan-R; the positive band was approximately 1400 bp in size. The selected positive colony was designated as recombinant strain MS1-kan.

[0119] (1-d) Elimination of resistance

[0120] Plasmid pcP20 was transformed into M1-kan prepared in (1-c) using the calcium chloride method. The transformed cells were plated onto LB solid medium containing 100 μg / mL ampicillin and cultured overnight at 30°C. A single colony was then picked and inoculated onto LB medium containing 100 μg / mL ampicillin and 2 g / L L-arabinose and cultured at 30°C. The bacterial suspension was then streaked onto LB solid medium without antibiotics and onto LB solid medium containing 50 μg / mL kanamycin, respectively, and cultured overnight at 42°C. After confirming that kanamycin resistance had been eliminated, a single colony was picked from the LB solid medium without antibiotics and inoculated onto LB solid medium containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, or without antibiotics, respectively, and cultured overnight at 30°C. Positive clones were identified as those that grew in the medium without antibiotics but not in the medium containing ampicillin and kanamycin. PCR amplification was performed using primers talB-3 and talB-2. The positive clone was about 400 bp. The positive clone obtained by screening was named recombinant strain MS1.

[0121] The recombinant bacterium MS1 is a strain in which the talB gene is knocked out. The Genbank number of the transaldolase B is NP_414549.1, and the Genbank number of the gene encoding it is Gene ID: 944748 (2023.7.6).

[0122] 2. Knockout of the transaldolase A (hereafter referred to as talA) gene

[0123] The recombinant strain MS1 ​​obtained in step (1) was used as the starting strain, and the transaldolase gene talA was knocked out to obtain the recombinant strain MS2. The specific steps are as follows:

[0124] (2-a) Preparation of the targeting fragment talA up-kan-talA down:

[0125] PCR amplification was performed using talA-1 / talA-2 as primers and the frt-kan-frt (SEQ ID No: 5) screening marker fragment as a template to obtain the targeting fragment talA up-kan-talA down.

[0126] (2-b) Follow the steps of (1) (steps 1-b to 1-d), except that the primers talB-1 / talB-2 / talB-3 are replaced with talA-1 / talA-2 / talA-3. The targeting fragment talA up-kan-talA down is obtained, and the transformed strain is replaced with MS1 instead of MC02.

[0127] In step (2-c), PCR amplification and identification were performed using talA-3 / Kan-R as primers, and the amplified target band of about 1400 bp was considered positive;

[0128] In step (2-d), PCR amplification and identification were performed using talA-3 / talA-2 as primers. Amplification of a target band of about 400 bp was considered positive, and the positive clone obtained by screening was named recombinant bacteria MS2.

[0129] The Genbank number of the transaldolase A is NP_416959.1, the Genbank number of its encoding gene is GeneID:947006, and the update date is 2024.07.06;

[0130] 3. Enhanced expression of the 6-phosphate glucose dehydrogenase (zwf) gene by promoter replacement

[0131] The recombinant strain MS2 obtained in step (2) was used as the starting strain. Starting from the recombinant strain MS2, the promoter of the 6-phosphate glucose dehydrogenase zwf gene in the strain (GenBank: CP032667.1, positions 1936315-1936449, updated on 08-OCT-2018) was replaced with the constitutive promoter P of Escherichia coli. 119 (SEQ ID No: 2), and recombinant Escherichia coli MS3 was obtained by the following steps:

[0132] (3-a) Targeting fragment zwf up-kan-P 119 -ZWF down preparation

[0133] The DNA fragments synthesized by GeneScript (GenScript) are as follows: from 5' to 3', they contain the 70 bp homology arm fragment upstream of the zwf gene (denoted as zwf up, see Table 1), the frt-kan-frt screening marker fragment (SEQ ID No: 5), the P 119 The promoter fragment (SEQ ID No: 2) and the first 70 bp fragment of the zwf gene (denoted as sequence zwf down, see Table 1) were amplified by PCR using zwf-1 / zwf-2 as primers and the gene-synthesized DNA fragment as a template to obtain the targeting fragment zwf up-kan-P 119 -zwf down.

[0134] (3-b) The subsequent steps were similar to those in (1) (steps 1-b to 1-d), except that the primers used in step 1-c were changed to zwf-3 / Kan-R to identify a positive clone of approximately 1400 bp. The primers used in step 1-d were changed to zwf-3 / zwf-2 to identify a positive clone of approximately 400 bp. The positive clone obtained by screening was named recombinant strain MS3.

[0135] The NCBI Reference Sequence number of the 6-phosphate glucose dehydrogenase (zwf) is AKT72612.1, the Genbank number of its encoding gene is Gene ID: 946370, and the update date is 2024.07.06;

[0136] 4. Knockout of the 6-phosphoglucose isomerase (pgi) gene

[0137] First, starting from the recombinant strain MS3, its 6-phosphoglucose isomerase gene pgi was knocked out to obtain the recombinant strain MS4. The specific steps are as follows:

[0138] (4-a) Preparation of the targeting fragment pGI-up-kan-pGI-down:

[0139] PCR amplification was performed using pgi-1 / pgi-2 as primers and the frt-kan-frt (SEQ ID No: 5) screening marker fragment as a template to obtain the targeting fragment pgi up-kan-pgi down.

[0140] (4-b) Follow the steps of (1) (steps 1-b to 1-d), except that the primers talB-1 / talB-2 / talB-3 are replaced with pgi-1 / pgi-2 / pgi-3. The targeting fragment pgi up-kan-pgi down is obtained, and the transformed strain is replaced with MS3 instead of MC02.

[0141] In step (4-c), PCR amplification and identification were performed using pgi-3 / Kan-R as primers, and the amplified target band of about 1400 bp was considered positive;

[0142] In step (4-d), PCR amplification and identification were performed using pgi-3 / pgi-2 as primers. Amplification of a target band of about 400 bp was considered positive, and the positive clone obtained by screening was named recombinant bacteria MS4.

[0143] The Genbank number of the glucose-6-phosphate isomerase (hereinafter referred to as pgi) is NP_418449.1, the Genbank number of its encoding gene is Gene ID: 948535, and the update date is 2024.07.06;

[0144] 5. Enhanced expression of the transketolase A (tktA) gene by promoter replacement

[0145] Referring to the steps in step 3, on the basis of the recombinant strain MS4, the promoter of the transketolase A (tktA) gene (GenBank: NC_000913.3, positions 3083616-3083767, updated on 09-MAR-2022) was replaced with the Escherichia coli constitutive promoter P CPA1 , and obtain recombinant E. coli MS5. The specific steps are as follows:

[0146] (5-a) Targeting fragment tktA up-kan-P CPA1 Preparation of -tktA down:

[0147] The DNA fragments synthesized by GeneScript (GenScript) are as follows: from 5' to 3', they contain the 70 bp homology arm fragment upstream of the tktA gene (denoted as tktA up, see Table 1), the frt-kan-frt selection marker fragment (SEQ ID No: 5), the P CPA1 The promoter fragment (SEQ ID No: 1) and the first 70 bp fragment of the tktA gene (denoted as sequence tktA down, see Table 1) were amplified by PCR using tktA-1 / tktA-2 as primers and the gene-synthesized DNA fragment as a template to obtain the targeting fragment tktA up-kan-P CPA1 -tktAdown.

[0148] (5-b) The subsequent steps were similar to those in Example 1 (3) (steps 3-b to 3-d), except that the primers zwf-1 / zwf-2 / zwf-3 were replaced with tktA-1 / tktA-2 / tktA-3. The target fragment tktA up-kan-P was obtained. CPA1 -tktA down, the transformed strain was replaced by MS2 to MS4.

[0149] In step (5-c), PCR amplification and identification were performed using tktA-3 / Kan-R as primers, and the amplified target band of about 1400 bp was considered positive;

[0150] In step (5-d), PCR amplification and identification were performed using tktA-3 / tktA-2 as primers. A target band of about 400 bp was amplified as positive, and the positive clone obtained by screening was named recombinant bacteria MS5.

[0151] The Genbank number of the transketolase A (hereinafter referred to as tktA) is YP_026188.1, the Genbank number of its encoding gene is Gene ID: 947420, and the update date is 2024.07.06;

[0152] 6. Enhanced expression of the transketolase B (tktB) gene by promoter replacement

[0153] Referring to the steps in step 3, on the basis of the recombinant strain MS5, the promoter of the transketolase A (tktB) gene (GenBank: NC_000913.3 (2579617-2579635) 09-MAR-2022) was replaced with the Escherichia coli constitutive promoter P CPA1 , and obtain recombinant E. coli MS6. The specific steps are as follows:

[0154] (6-a) Targeting fragment tktB up-kan-P CPA1 Preparation of -tktB down:

[0155] The DNA fragments synthesized (GenScript) were as follows: from 5' to 3', they contained the 70 bp homology arm fragment upstream of the tktB gene (denoted as tktB up, see Table 1), the frt-kan-frt screening marker fragment (SEQ ID No: 5), the P CPA1 The promoter fragment (SEQ ID No: 1) and the first 70 bp fragment of the tktB gene (denoted as sequence tktB down, see Table 1) were amplified by PCR using tktB-1 / tktB-2 as primers and the gene-synthesized DNA fragment as a template to obtain the targeting fragment tktB up-kan-P CPA1 -tktBdown.

[0156] (6-b) The subsequent steps were similar to those in Example 1 (3) (steps 3-b to 3-d), except that the primers zwf-1 / zwf-2 / zwf-3 were replaced with tktB-1 / tktB-2 / tktB-3. CPA1 -tktB down, the transformed strain was replaced by MS2 to MS5.

[0157] In step (6-c), PCR amplification and identification were performed using tktB-3 / Kan-R as primers, and the amplified target band of about 1400 bp was considered positive;

[0158] In step (6-d), PCR amplification and identification were performed using tktB-3 / tktB-2 as primers. A target band of about 400 bp was amplified as positive, and the positive clone obtained by screening was named recombinant bacteria MS6.

[0159] The Genbank number of the transketolase B (hereinafter referred to as tktB) is NP_416960.1, the Genbank number of its encoding gene is Gene ID: 945865, and the update date is 2024.07.06;

[0160] 7. Construction of expression vector for mycosporin-like amino acid synthesis gene from Scytonema cf. Crispum.

[0161] The mycosporine-like amino acid shinorine synthesis gene cluster (nucleotide sequence as SEQ ID No: 7) derived from Scytonemacf. Crispum and codon-optimized in Escherichia coli was artificially synthesized. The expression of the gene cluster takes shinorine as the main product.

[0162] The fragment ScMAAs was amplified by PCR from the above-synthesized gene cluster using primers ScMAAs-01F and ScMAAs-01R, and the target fragment ScMAAs was recovered by agarose gel electrophoresis.

[0163] Using plasmid pTrc99a (Beijing Zhuangmeng International Biogene Technology Co., Ltd., catalog number ZK1610) as a template, primers pTrc99a-01F and pTrc99a-01R were used for PCR amplification and recovery to obtain the large vector fragment pTrc. Using the Gibson assembly method (Gibson DG, Young L, et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. methods. 2009; 6(5): 343-345), the pTrc fragment and ScMAAs were Gibson ligated and transformed into Escherichia coli DH5α competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd., catalog number TSC-C01). After recovery, the cells were evenly plated onto the corresponding resistance LB plates and cultured overnight at 37°C. Clones were selected and verified for successful assembly by PCR amplification using primers RV-M and pTrcHis-R, and sequencing was performed. Finally, positive clones were obtained through screening and the plasmid was extracted and named pTrc99a-ScMAAs.

[0164] The structure of the recombinant vector pTrc99a-ScMAAs is described as follows: a DNA fragment with the sequence of SEQ ID No: 7 is inserted between 5'-agcggataacaatttcacacaggaaacaga-3' and 5'-gagtcgacctgcaggcatgcaagcttggct-3' of the starting vector pTrc99a, while keeping the other sequences of the starting vector pTrc99a unchanged.

[0165] 8. Construction of production strain MS6-1

[0166] The plasmid pTrc99a-ScMAAs constructed in step 7 of Example 1 was transferred to the recombinant bacterium MS6 obtained in step (6) of Example 1 by the calcium chloride transformation method. After overnight culture at 37°C on an LB plate containing 100 μg / mL ampicillin, clones were selected to obtain a production strain containing the plasmid pTrc99a-ScMAAs, which was named recombinant bacterium MS16-1.

[0167] 9. Construction of strain MS6-0

[0168] The plasmid pTrc99a was transferred into the recombinant bacterium MS6 obtained in step (12) of Example 1 by the calcium chloride transformation method. After overnight culture at 37°C on an LB plate containing 100 μg / mL ampicillin, clones were selected to obtain a recombinant bacterium containing the plasmid pTrc99a but not the Shinorine synthesis gene. This strain was used as a control and was named recombinant bacterium MS6-0.

[0169] 2. Preparation of Shinorine Using Glucose, Glycine, L-Serine, and L-Methionine as Substrates

[0170] 1. Preparation of culture medium:

[0171] The composition and final concentrations of medium A are: 5 g / L yeast powder, 5 g / L glycerol, 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5 mM Na2SO4, 2 mM MgSO4, 50 μM FeCl3, 20 μM CaCl2, 10 μM MnCl2, 10 μM ZnSO4, 2 μM CoCl2, 2 μM NiCl2, 2 μM Na2Mo4, 2 μM Na2SeO3 and 2 μM H3BO3.

[0172] The composition and final concentrations of medium B are: 48 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 187 mM NH4Cl, 100 g / L glucose, 25 g / L glycine, 25 g / L L-serine, and 10 g / L L-methionine.

[0173] 2. Preparation of Shinorine

[0174] The experiment was repeated three times, and the specific steps for each experimental repetition were as follows:

[0175] 1) Culture of bacteria and induction of related enzymes

[0176] The strain MS6-1 obtained in step 1 was cultured overnight and inoculated into 20 ml of medium A (medium A containing ampicillin at a final concentration of 50 mg / L) at a 1% inoculum size. The culture was then incubated at 30°C and 220 rpm for 3-4 h until the OD 600nm After the pH value was 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and the culture was continued at 30°C and 220 rpm for 16 hours. The bacteria were collected by centrifugation at 10,000 g for 10 minutes to obtain MS6-1 bacteria.

[0177] According to the above method, MS6-0 was cultured and collected using A medium (A medium containing ampicillin at a final concentration of 50 mg / L) to obtain MS6-0 bacteria.

[0178] 2) Whole-cell catalytic production of Shinorine

[0179] The 30 mg (i.e. 1×10 11 cfu) dry weight of MS6-1 and MS6-0 bacteria were resuspended in shake flasks containing 20 mL of B medium and cultured at 37°C for 24 h.

[0180] 3) Product collection and testing

[0181] Centrifuge 0.5 ml of the whole-cell catalytic sample obtained in step 2) at 4000 g and 4°C for 20 min. Collect the extracellular product using the following steps:

[0182] 350 μL of the supernatant obtained after centrifugation was vacuum dried, and then 700 μL of methanol was added for reconstitution. The mixture was then centrifuged at 10,000 g for 10 min. 600 μL of the supernatant was vacuum dried, and then 300 μL of deionized water was added for reconstitution. The reconstituted solution was centrifuged at 18,000 g for 2 min. The supernatant was filtered through a 0.22 μm filter to obtain the filtrate, which was the sample to be tested.

[0183] The shinorine content in the samples was determined using high-performance liquid chromatography (HPLC). HPLC analysis was performed using an Agilent Eclipse XDB-C18 column (5 μm, 4.6 × 250 mm) with a mobile phase consisting of methanol and water (methanol:water volume ratio of 84:16), a column temperature of 30°C, a flow rate of 1 mL / min, and a UV detector.

[0184] The results showed that the yield of Shinorine of recombinant strain MS6-0 was 0, and the average yield of Shinorine of engineered strain MS6-1 was 25.82±0.73 g / L. Figure 1Therefore, the engineered strain MS6-1 can synthesize Shinorine from glucose, L-methionine, glycine, and L-serine.

[0185] Example 2: Construction of recombinant Escherichia coli MS9-1 and production of Shinorine

[0186] 1. Starting with Escherichia coli MS6, a strain MS9-1 was generated for the production of mycosporine-like amino acids. It can synthesize mycosporine-like amino acids shinorine from glucose, glycine, and L-serine. This strain primarily enhances the L-methionine cycle. The primers used for construction of this strain are shown in Table 1. The construction method is as follows:

[0187] 1. Enhancement of L-methionine adenosyltransferase (metK) gene expression by promoter replacement

[0188] Referring to step (3) in Example 1, on the basis of the recombinant strain MS6, the promoter of the L-methionine adenosyltransferase (metK) gene (GenBank: NC_000913.3, positions 3086686-3086705, updated on 09-MAR-2022) was replaced with the Escherichia coli constitutive promoter P 119 , and recombinant E. coli MS7 was obtained. The difference is that the gene synthesis fragments are as follows: from 5' to 3', they are composed of metk up, frt-kan-frt screening marker fragment, P 119 Promoter fragment (SEQ ID No: 2), metk down, primer zwf-1 / zwf-2 / zwf-3 combination was replaced with metk-1 / metk-2 / metk-3 in sequence, and host strain MS2 was replaced with MS6.

[0189] The sequence of the upstream homology arm of metk is metk up, see Table 1; the sequence of the downstream homology arm of metk is metk down, see Table 1;

[0190] PCR amplification was performed using metk-3 / Kan-R as primers, and the amplified target band of about 1400 bp was considered positive;

[0191] PCR amplification and identification were performed using metk-2 / metk-3 as primers. The amplified target band of about 400 bp was considered positive, and the positive clone obtained by screening was named recombinant bacteria MS7.

[0192] The Genbank number of the L-methionine adenosyltransferase (hereinafter referred to as metK) is NP_417417.1, the Genbank number of its encoding gene is Gene ID: 945389, and the update date is 2024.7.6.

[0193] 2. Knockout of the L-methionine synthesis transcription factor (hereafter referred to as metJ) gene

[0194] Referring to step (1) in Example 1, on the basis of MS7, the metJ gene, a transcription factor for L-methionine synthesis, was knocked out to obtain the recombinant bacterium MS8.

[0195] The differences are that the host strain MC02 was replaced with MS7, and the primer combination talB-1 / talB-2 / talB-3 was replaced with metJ-1 / metJ-2 / metJ-3. The resulting targeting fragment was metj up-kan-metj down; the relevant primer sequences are shown in Table 1.

[0196] PCR amplification was performed using metJ-2 / Kan-R as primers, and the amplified target band of about 1400 bp was considered positive;

[0197] PCR amplification and identification were performed using metJ-2 / metJ-3 as primers, and the amplified target band of about 400 bp was considered positive.

[0198] The positive clone obtained by screening was named recombinant bacteria MS8, the Genbank number of the L-methionine synthesis transcription factor is NP_418373, the Genbank number of its encoding gene is Gene ID: 948435, and the update date is: 2024.7.6.

[0199] 3. The gene encoding pyruvate formate lyase (hereinafter referred to as pflB) was replaced with genes encoding S-adenosylhomocysteine ​​nucleosidase (hereinafter referred to as mtn) and S-ribosylhomocysteine ​​lyase (hereinafter referred to as luxS).

[0200] Using strain MS8 as the starting strain, the poxB gene was replaced by the pflB gene, and the constitutive promoter P CPA1 To express the mtn and luxs genes, the recombinant strain MS9 was obtained. The specific steps are as follows:

[0201] (3-a) Targeting fragment pflB up-kan-P CPA1 -mtn-luxS-pflB down build:

[0202] The DNA fragments synthesized (GenScript) were as follows: from 5' to 3', they contained the 70 bp homology arm fragment upstream of the pflB gene (denoted as pflB up, see Table 1), the frt-kan-frt selection marker fragment (SEQ ID No: 5), the P CPA1The promoter fragment (SEQ ID No: 2), mtn gene, luxs gene, TrrnB terminator fragment (SEQ ID No: 6), and the 70 bp downstream fragment of the pflB gene (denoted as sequence poxB down, see Table 1) were amplified by PCR using pflB-1 / pflB-2 as primers and the gene-synthesized DNA fragment as a template to obtain the targeting fragment pflB up-kan-P CPA1 -mtn-luxS-pflB down. The above targeting fragment can be used to replace the pflB gene with the mtn and luxs genes, and use the promoter P CPA1 To express mtn and luxs genes.

[0203] (3-b) The subsequent steps are as described in (1) of Example 1 (steps 1-b to 1-d), except that the primers talB-1 / talB-2 / talB-3 are replaced with pflB-1 / pflB-2 / pflB-3.

[0204] PCR amplification was performed using pflB-3 / Kan-R as primers, and the amplified target band of about 1400 bp was considered positive;

[0205] PCR amplification and identification were performed using pflB-2 / pflB-3 as primers, and the amplified target band of about 1500 bp was considered positive.

[0206] The positive clone obtained by screening was named recombinant bacteria MS9, the Genbank number of the pyruvate formate lyase (hereinafter referred to as pflB) is NP_415423.1, the Genbank number of its encoding gene is Gene ID: 945514, and the update date is: 2024.7.6.

[0207] The Genbank number of the S-adenosylhomocysteine ​​nucleosidase (hereinafter referred to as mtn) is NP_414701.1, the Genbank number of its encoding gene is Gene ID: 948542, and the update date is 2024.7.6.

[0208] The Genbank number of the S-ribosylhomocysteine ​​lyase (hereinafter referred to as luxS) is NP_417172.1, the Genbank number of its encoding gene is Gene ID: 947168, and the update date is 2024.7.6.

[0209] 4. Construction of production strain MS9-1

[0210] Starting from the recombinant bacterium MS9, the plasmid pTrc99a-ScMAAs containing the shinorine synthesis gene cluster was transferred. The specific steps were as described in 8 of Example 1, and it was named MS9-1.

[0211] (5) Construction of strain MS9-0

[0212] Starting from the recombinant bacteria MS9, the plasmid pTrc99a was transferred, and the specific steps were referred to 9 in Example 1, and it was named MS9-0.

[0213] 2. Preparation of Shinorine Using Glucose, Glycine, and L-Serine as Substrates

[0214] 1. Preparation of culture medium:

[0215] The composition and final concentration of medium A are the same as those of medium A in Example 1.

[0216] The composition and final concentrations of C medium are: 48 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 187 mM NH4Cl, 100 g / L glucose, 25 g / L glycine, and 25 g / L L-serine.

[0217] 2. Preparation of Shinorine

[0218] The experiment was repeated three times, and the specific steps for each experimental repetition were as follows:

[0219] 1. Culture of bacteria and induction of related enzymes:

[0220] The strain MS9-1 obtained in step 1 was cultured overnight and inoculated into 20 ml of medium A (medium A containing ampicillin at a final concentration of 50 mg / L) at a 1% inoculum size. The culture was then cultured at 30°C and 220 rpm for 3-4 h until the OD 600nm After the pH value was 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and the culture was continued at 30°C and 220 rpm for 16 hours. The bacteria were collected by centrifugation at 10,000 g for 10 minutes to obtain MS9-1 bacteria.

[0221] According to the above method, MS9-0 was cultured and collected using A medium (A medium containing ampicillin at a final concentration of 50 mg / L) to obtain MS9-0 bacterial cells.

[0222] 2. Whole-cell catalytic production of shinorine

[0223] The 30 mg (i.e. 1×10 11 cfu) dry weight of MS9-1 and MS9-0 bacteria were resuspended in shake flasks containing 20 mL of culture medium and cultured at 37°C for 24 h.

[0224] 3. Product collection and testing

[0225] 0.5 ml of the whole cell catalysis sample obtained in 2) was centrifuged at 4000 g and 4° C. for 20 min. The extracellular and intracellular products were collected and detected in the same manner as in Example 1.

[0226] The results showed that the average Shinorine production of engineered strain MS9-0 was 0, the average Shinorine production of engineered strain MS6-1 was 15.45±1.96 g / L, and the average Shinorine production of engineered strain MS9-1 was 28.86±3.57 g / L, as follows: Figure 2 Therefore, the engineered strain MS9-1 can efficiently synthesize Shinorine from glucose, glycine, and L-serine without the need for additional L-methionine.

[0227] Example 3: Construction of recombinant Escherichia coli MS14-1 and production of Shinorine

[0228] First, a strain, MS14-1, was generated from Escherichia coli MS9 for the production of mycosporine-like amino acids. It can synthesize the mycosporine-like amino acid shinorine from glucose and glycine. This strain primarily enhances the L-serine biosynthesis pathway. The primers used for the construction of this strain are shown in Table 1. The construction method is as follows:

[0229] 1. Enhanced expression of the phosphogluconate dehydratase (edd) gene by promoter replacement

[0230] Referring to step 3 in Example 1, on the basis of the recombinant strain MS9, the promoter of the phosphogluconate dehydratase (edd) gene (GenBank: NC_000913.3, positions 1934605-1934826, updated on 09-MAR-2022) was replaced with the Escherichia coli constitutive promoter P J23105 , and recombinant E. coli MS10 was obtained. The difference is that the gene synthesis fragments are as follows: from 5' to 3', they are composed of the following: edd up, frt-kan-frt screening marker fragment, P J23105 Promoter fragment (SEQ ID No: 3), edd down, primer combination zwf-1 / zwf-2 / zwf-3 was replaced with edd-1 / edd-2 / edd-3 in sequence, and host strain MS2 was replaced with MS9.

[0231] The sequence of the upstream homology arm of edd is edd up, see Table 1; the sequence of the downstream homology arm of edd is edd down, see Table 1;

[0232] PCR amplification was performed using edd-3 / Kan-R as primers, and the amplified target band of about 1400 bp was considered positive;

[0233] PCR amplification and identification were performed using edd-2 / edd-3 as primers, and the amplified target band of about 400 bp was considered positive.

[0234] The positive clone obtained by screening was named recombinant bacteria MS10, the Genbank number of the phosphogluconate dehydratase (hereinafter referred to as edd) is NP_416365.1, the Genbank number of its encoding gene is Gene ID: 946362, and the update date is: 2024.7.6.

[0235] 2. Replace the gene encoding pyruvate oxidase (hereinafter referred to as poxB) with genes encoding phosphoglycerate dehydrogenase (hereinafter referred to as serA), phospho-L-serine phosphorylase (serB), and phospho-L-serine aminotransferase (serC)

[0236] Using strain MS10 as the starting strain, the poxB gene was replaced with serA, serB, and serC genes, and the constitutive promoter P was used. J23105 To express serA, serB, and serC genes, the recombinant strain MS11 was obtained. The specific steps are as follows:

[0237] (2-a) Targeting fragment poxB up-kan-P J23105- Preparation of serABC-poxB down:

[0238] The DNA fragments synthesized (GenScript) were as follows: from 5' to 3', they contained the 70 bp homology arm fragment upstream of the poxB gene (denoted as poxB up, see Table 1), the frt-kan-frt selection marker fragment (SEQ ID No: 5), the P J23105 The promoter fragment (SEQ ID No: 3), serA gene, serB gene, serC gene, TrrrnB gene (SEQ ID No: 6), and the 70 bp downstream fragment of the poxB gene (denoted as sequence poxB down, see Table 1) were amplified by PCR using lpxM-1 / lpxM-2 as primers and the gene-synthesized DNA fragment as a template to obtain the targeting fragment poxB up-kan-P J23105- serABC-poxB down.

[0239] (2-b) The subsequent steps were similar to those in Example 1 (steps 1-b to 1-d), except that the primers talB-1 / talB-2 / talB-3 were replaced with poxB-1 / poxB-2 / poxB-3. J23105-serABC-poxB down, the transformed strain was replaced by MC02 and MS10.

[0240] PCR amplification was performed using poxB-3 / Kan-R as primers, and the amplified target band of about 1400 bp was considered positive;

[0241] PCR amplification was performed using poxB-2 / poxB-3 as primers, and the amplified target band of about 3500 bp was considered positive.

[0242] The positive clone obtained by screening was named recombinant bacteria MS11, the Genbank number of the phosphoglycerate dehydrogenase was NP_417388.1, the Genbank number of its encoding gene was Gene ID: 945258, and the update date was: 2024.7.6.

[0243] The Genbank number of the phospho-L-serine phosphorylase (hereinafter referred to as serB) is NP_418805.1, the Genbank number of its encoding gene is Gene ID: 948913, and the update date is 2024.7.6.

[0244] The Genbank number of the phospho-L-serine aminotransferase (hereinafter referred to as serC) is NP_415427.1, the Genbank number of its encoding gene is Gene ID: 945527, and the update date is: 2024.7.6.

[0245] 3. Knockout of the L-serine deaminase I (hereinafter referred to as sdaA) gene

[0246] Referring to step 1 in Example 1, on the basis of MS11, the L-serine deaminase IsdaA gene was knocked out to obtain the recombinant strain MS12.

[0247] The differences are that the host strain MC02 was replaced with MS11, and the primer combination talB-1 / talB-2 / talB-3 was replaced with sdaA-1 / sdaA-2 / sdaA-3. The resulting targeting fragment was sdaA up-kan-sdaA down; the relevant primer sequences are shown in Table 1.

[0248] PCR amplification was performed using sdaA-2 / Kan-R as primers, and the amplified target band of about 1400 bp was considered positive;

[0249] PCR amplification was performed using sdaA-2 / sdaA-3 as primers, and the amplified target band of about 400 bp was considered positive.

[0250] The positive clone obtained by screening was named recombinant bacteria MS12, the Genbank number of the L-serine deaminase I (hereinafter referred to as sdaA) is NP_416328.1, the Genbank number of its encoding gene is Gene ID: 946331, and the update date is: 2024.7.6.

[0251] 4. Knockout of the L-serine deaminase II (sdaB) gene

[0252] Referring to step 1 in Example 1, on the basis of MS12, the L-serine deaminase IsdaA gene was knocked out to obtain the recombinant strain MS13.

[0253] The differences are that the host strain MC02 was replaced with MS12, and the primer combination talB-1 / talB-2 / talB-3 was replaced with sdaB-1 / sdaB-2 / sdaB-3. The resulting targeting fragment was sdaB up-kan-sdaB down; the relevant primer sequences are shown in Table 1.

[0254] PCR amplification was performed using sdaB-2 / Kan-R as primers, and the amplified target band of about 1400 bp was considered positive;

[0255] PCR amplification was performed using sdaB-2 / sdaB-3 as primers, and the amplified target band of about 400 bp was considered positive.

[0256] The positive clone obtained by screening was named recombinant bacteria MS13, the Genbank number of the L-serine deaminase II (hereinafter referred to as sdaB) is NP_417277.1, the Genbank number of its encoding gene is Gene ID: 947262, and the update date is: 2024.7.6.

[0257] 5. Knockout of the L-serine deaminase III (hereinafter referred to as tdcG) gene

[0258] Referring to step 1 in Example 1, on the basis of MS13, the L-serine deaminase IsdaA gene was knocked out to obtain the recombinant strain MS14.

[0259] The differences are that the host strain MC02 was replaced with MS13, and the primer combination talB-1 / talB-2 / talB-3 was replaced with tdcG-1 / tdcG-2 / tdcG-3. The resulting targeting fragment was tdcG up-kan-tdcG down; the relevant primer sequences are shown in Table 1.

[0260] PCR amplification was performed using tdcG-2 / Kan-R as primers, and the amplified target band of about 1400 bp was considered positive;

[0261] PCR amplification and identification were performed using tdcG-2 / tdcG-3 as primers, and the amplified target band of about 400 bp was considered positive.

[0262] The positive clone obtained by screening was named recombinant bacteria MS14, the Genbank number of the L-serine deaminase III (hereinafter referred to as tdcG) is YP_026204.1, the Genbank number of its encoding gene is Gene ID: 2847724, and the update date is: 2024.7.6.

[0263] 6. Construction of production strain MS14-1

[0264] Starting from the recombinant strain MS14, the plasmid pTrc99a-ScMAAs containing the shinorine synthesis gene cluster was transferred. The specific steps were as described in 8 of Example 1, and it was named MS14-1.

[0265] 7. Construction of strain MS14-0

[0266] Starting from the recombinant bacteria MS14, the plasmid pTrc99a was transferred, and the specific steps were referred to 9 in Example 1, and it was named MS14-0.

[0267] 2. Preparation of Shinorine Using Glucose and Glycine as Substrates

[0268] 1. Preparation of culture medium:

[0269] The composition and final concentration of medium A are the same as those of medium A in Example 1.

[0270] The composition and final concentration of D medium are: 48 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 187 mM NH4Cl, 100 g / L glucose, 25 g / L glycine.

[0271] 2. Preparation of Shinorine

[0272] The experiment was repeated three times, and the specific steps for each experimental repetition were as follows:

[0273] 1. Culture of bacteria and induction of related enzymes:

[0274] The strain MS14-1 obtained in step 1 was cultured overnight and inoculated into 20 ml of medium A (medium A containing ampicillin at a final concentration of 50 mg / L) at a 1% inoculum size. The culture was then incubated at 30°C and 220 rpm for 3-4 h until the OD 600nmAfter the pH value was 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and the culture was continued at 30°C and 220 rpm for 16 hours. The bacteria were collected by centrifugation at 10,000 g for 10 minutes to obtain MS14-1 bacteria.

[0275] According to the above method, MS14-0 was cultured and collected using A medium (A medium containing ampicillin at a final concentration of 50 mg / L) to obtain MS14-0 bacterial cells.

[0276] 2. Whole-cell catalytic production of shinorine

[0277] The 30 mg (i.e. 1×10 11 cfu) dry weight of MS14-1 and MS14-0 bacteria were resuspended in a shake flask containing 20 mL of C medium and cultured at 37°C for 24 h.

[0278] 3. Product collection and testing

[0279] 0.5 ml of the whole-cell catalytic sample obtained in step 2 was centrifuged at 4000 g and 4° C. for 20 min. The extracellular and intracellular products were collected and detected in the same manner as in Example 1.

[0280] The results showed that the average Shinorine production of the engineered strain MS14-0 was 0, and the average Shinorine production of MS9-1 was 5.76±0.77 g / L, which was much lower than the average Shinorine production of the engineered strain MS14-1, which was 30.94±2.52 g / L. Figure 3 Therefore, the engineered strain MS14-1 can efficiently synthesize Shinorine from glucose and glycine without the need for additional L-methionine and L-serine.

[0281] Example 4: Construction of recombinant Escherichia coli MS15-1 and production of Shinorine

[0282] 1. This example uses Escherichia coli MS14 as a starting point to generate a basic strain, MS15-1, for the production of mycosporine-like amino acids. This strain can synthesize the mycosporine-like amino acid shinorine from glucose. This strain primarily enhances the glycine biosynthesis pathway. The primers used to construct this strain are shown in Table 1. The construction method is as follows:

[0283] 1. Replace the gene encoding L-serine hydroxymethyltransferase (hereinafter referred to as glyA) with a fusion protein gene of glyA and methionine synthetase (hereinafter referred to as metH) (hereinafter referred to as glyA-metH)

[0284] Using strain MS14 as the starting strain, the glyA gene was replaced with the glyA-metH gene, and the constitutive promoter P was used to express the gene. CPA1 To express the glyA-metH gene, the recombinant strain MS15 was obtained. The specific steps were as follows: step 2 in Example 3. The difference was that the gene synthesis fragments were as follows: from 5' to 3', they were composed of the following: glyA up, frt-kan-frt screening marker fragment, P CPA1 The promoter fragment (SEQ ID No: 2), metH gene (excluding the TAA stop codon), linker fragment (SEQ ID No: 4), glyA gene, glyA down, primer poxB-1 / poxB-2 / poxB-3 combination were replaced with glyA-1 / glyA-2 / glyA-3 in sequence, and the host strain MS10 was replaced with MS14.

[0285] The sequence of the glyA upstream homology arm is glyA up, see Table 1; the sequence of the glyA downstream homology arm is glyA down, see Table 1;

[0286] PCR amplification was performed using glyA-3 / Kan-R as primers, and the amplified target band of about 1400 bp was considered positive;

[0287] PCR amplification was performed using glyA-2 / glyA-3 as primers, and the amplified target band of about 5000 bp was considered positive.

[0288] The positive clone obtained by screening was named recombinant bacteria MS15, the Genbank number of the L-serine hydroxymethyltransferase is NP_417046.1, the Genbank number of the gene encoding it is Gene ID: 947022, updated on July 6, 2024; the Genbank number of the methionine synthase is NP_418443.1, the Genbank number of the gene encoding it is Gene ID: 948522, updated on July 6, 2024.

[0289] 2. Construction of production strain MS15-1

[0290] Starting from the recombinant strain MS15, the plasmid pTrc99a-ScMAAs containing the shinorine synthesis gene cluster was transferred. The specific steps were as described in 8 of Example 1, and it was named MS15-1.

[0291] 3. Construction of strain MS15-0

[0292] Starting from the recombinant bacteria MS15, the plasmid pTrc99a was transferred, and the specific steps were referred to 9 in Example 1, and it was named MS15-0.

[0293] 2. Preparation of Shinorine Using Glucose as Substrate

[0294] 1. Preparation of culture medium

[0295] The composition and final concentration of medium A are the same as those of medium A in Example 1.

[0296] The composition and final concentration of E medium: 48 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 187 mM NH4Cl, 100 g / L glucose.

[0297] 2. Preparation of Shinorine

[0298] The experiment was repeated three times, and the specific steps for each experimental repetition were as follows:

[0299] 1) Culture of bacteria and induction of related enzymes:

[0300] The strain MS15-1 obtained in step 1 was cultured overnight and inoculated into 20 ml of medium A (medium A containing ampicillin at a final concentration of 50 mg / L) at a 1% inoculum size. The culture was then incubated at 30°C and 220 rpm for 3-4 h until the OD 600nm After the pH value was 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and the culture was continued at 30°C and 220 rpm for 16 hours. The bacteria were collected by centrifugation at 10,000 g for 10 minutes to obtain MS15-1 bacteria.

[0301] According to the above method, MS15-0 was cultured and collected using A medium (A medium containing ampicillin at a final concentration of 50 mg / L) to obtain MS15-0 bacteria.

[0302] 2) Whole-cell catalytic production of shinorine

[0303] The 30 mg (i.e. 1×10 11 cfu) dry weight of MS15-1 and MS15-0 bacteria were resuspended in shake flasks containing 20 mL of E medium and cultured at 37°C for 24 h.

[0304] 3) Product collection and testing

[0305] 0.5 ml of the whole cell catalysis sample obtained in 2) was centrifuged at 4000 g and 4° C. for 20 min. The extracellular and intracellular products were collected and detected in the same manner as in Example 1.

[0306] The results showed that the average Shinorine production of the engineered strain MS15-0 was 0, and the average Shinorine production of MS14-1 was 0.88±0.09 g / L, which were much lower than the average Shinorine production of the engineered strain MS15-1, 33.88±1.68 g / L. Figure 4 As shown. Therefore, using the engineered strain MS15-1, there is no need to add L-methionine, L-serine and glycine, and shinorine can be efficiently synthesized from glucose. The present invention is described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention and without conducting unnecessary experiments, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art.

Claims

1. A method for constructing a recombinant bacterium, characterized in that: The method comprises transforming the recipient bacteria as follows: M1), knocking out the transaldolase B (talB) gene in the recipient bacteria; M2), knocking out the transaldolase A (talA) gene in the recipient bacteria; M3), knock out the wild-type promoter that drives the expression of 6-phosphate glucose dehydrogenase (zwf) gene in the recipient bacteria and replace it with promoter P 119 ; M4), knocking out the glucose-6-phosphate isomerase (pgi) gene in the recipient bacteria; M5), knock out the wild-type promoter that drives the expression of the transketolase A (tktA) gene in the recipient bacteria and replace it with promoter P CPA1 ; M6), knock out the wild-type promoter that drives the expression of the transketolase B (tktB) gene in the recipient bacteria and replace it with promoter P CPA1 ; M7) Introduce the mycosporine-like amino acid shinorine synthesis gene cluster (ScMAAs) genes into the recipient bacteria.

2. The method according to claim 1, characterized in that The method further comprises transforming the recombinant bacteria as follows: M8), knocking out the L-methionine transcription factor (metJ) gene in the recipient bacteria; M9), knock out the wild-type promoter that drives the expression of the L-methionine adenosyltransferase (metK) gene in the recipient bacteria and replace it with promoter P 119 ; M10), knocking out the pyruvate formate lyase (pflB) gene in the recipient strain and replacing it with S-adenosylhomocysteine ​​nucleosidase (mtn) and S-ribosylhomocysteine ​​lyase (luxS) genes; wherein the S-adenosylhomocysteine ​​nucleosidase (mtn) and S-ribosylhomocysteine ​​lyase (luxS) described in M10) are synthesized by P CPA1 The promoter initiates expression.

3. The method according to claim 1 or 2, characterized in that: The method further comprises transforming the recombinant bacteria as follows: M11), the wild-type promoter that drives the expression of the phosphogluconate dehydratase (edd) gene in the recipient bacteria was knocked out and replaced with the promoter P J23105 ; M12), knocking out the pyruvate oxidase (poxB) gene in the recipient strain and replacing it with phosphoglycerate dehydrogenase (serA), phospho-L-serine aminotransferase (serC), and phospho-L-serine phosphorylase (serB) genes; M12) described phosphoglycerate dehydrogenase (serA), phospho-L-serine aminotransferase (serC) and phospho-L-serine phosphorylase (serB) are produced by P J23105 The promoter initiates expression; M13), knocking out the L-serine deaminase III (tdcG) gene in the recipient strain; M14), knocking out the L-serine deaminase I (sdaA) gene in the recipient strain; M15) and knocked out the L-serine deaminase II (sdaB) gene in the recipient bacteria.

4. The method according to any one of claims 1 to 3, characterized in that: The method further comprises transforming the recombinant bacteria as follows: M16), knocking out the L-serine hydroxymethyltransferase (glyA) gene in the recipient bacteria and replacing it with the L-serine hydroxymethyltransferase (glyA) gene and the methionine synthase (metH) gene; Wherein the L-serine hydroxymethyltransferase (glyA) gene and methionine synthetase (metH) gene described in M16) are connected by a linker to form a fusion expression protein gene, and the fusion protein is expressed by P CPA1 The promoter initiates expression.

5. The method according to any one of claims 1 to 4, characterized in that: 1) The Genbank number of the 6-phosphate glucose dehydrogenase is AKT72612.1, the Genbank number of its encoding gene is Gene ID: 946370, and the update date is 2024.07.06; 2) The Genbank number of the transaldolase A is NP_416959.1, the Genbank number of the gene encoding it is Gene ID: 947006, and the update date is 2024.07.06; 3) The Genbank number of the transaldolase B is NP_414549.1, the Genbank number of its encoding gene is GeneID:944748, and the update date is 2024.07.06; 4) The Genbank number of the transketolase A is YP_026188.1, the Genbank number of its encoding gene is GeneID:947420, and the update date is 2024.07.06; 5) The Genbank number of the transketolase B is NP_416960.1, the Genbank number of its encoding gene is GeneID:945865, and the update date is 2024.07.06; 6) The Genbank number of the glucose-6-phosphate isomerase is NP_418449.1, the Genbank number of its encoding gene is Gene ID: 948535, and the update date is 2024.07.06; 7) The Genbank number of the L-methionine adenosyltransferase is NP_417417.1, the Genbank number of its encoding gene is Gene ID: 945389, and the update date is 2024.07.06; 8) The Genbank number of the Shinorine synthesis gene cluster is KX021866, and the update date is 2024.07.06; 9) The Genbank number of the L-methionine synthesis transcription factor is NP_418373, the Genbank number of its encoding gene is Gene ID: 948435, and the update date is 2024.07.06; 10) The Genbank number of the pyruvate formate lyase is NP_415423.1, the Genbank number of the gene encoding it is Gene ID: 945514, and the update date is 2024.07.06; 11) The Genbank number of the S-adenosylhomocysteine ​​nucleosidase is NP_414701.1, the Genbank number of its encoding gene is Gene ID: 948542, and the update date is 2024.07.06; 12) The Genbank number of the S-ribosylhomocysteine ​​lyase is NP_417172.1, the Genbank number of its encoding gene is Gene ID: 947168, and the update date is 2024.07.06; 13) The Genbank number of the phosphogluconate dehydratase is NP_416365.1, the Genbank number of its encoding gene is Gene ID: 946362, and the update date is 2024.07.06; 14) The Genbank number of the pyruvate oxidase is CAD6018048.1, the Genbank number of its encoding gene is GeneID:946132, and the update date is 2024.07.06; 15) The Genbank number of the phosphoglycerate dehydrogenase is NP_417388.1, the Genbank number of its encoding gene is Gene ID: 945258, and the update date is 2024.07.06; 16) The Genbank number of the phospho-L-serine phosphorylase is NP_418805.1, the Genbank number of its encoding gene is Gene ID: 948913, and the update date is 2024.07.06; 17) The Genbank number of the phospho-L-serine aminotransferase is NP_415427.1, the Genbank number of its encoding gene is Gene ID: 945527, and the update date is 2024.07.06; 18) The Genbank number of the L-serine deaminase III is YP_026204.1, the Genbank number of its encoding gene is Gene ID: 2847724, and the update date is 2024.07.06; 19) The Genbank number of the L-serine deaminase I is NP_416328.1, the Genbank number of its encoding gene is Gene ID: 946331, and the update date is 2024.07.06; 20) The Genbank number of the L-serine deaminase II is NP_417277.1, the Genbank number of its encoding gene is Gene ID: 947262, and the update date is 2024.07.06; 21) The Genbank number of the L-serine hydroxymethyltransferase is NP_417046.1, the Genbank number of its encoding gene is Gene ID: 947022, and the update date is 2024.07.06; 22) The Genbank number of the methionine synthase is NP_418443.1, the Genbank number of its encoding gene is GeneID:948522, and the update date is 2024.07.06; The P CPA1 A promoter is any of the following DNA molecules: 1) A DNA molecule having a nucleotide sequence of one chain as SEQ ID No: 1 in the sequence listing, 2) a DNA molecule that is more than 80% identical to the DNA molecule of 1) and has promoter function; The P 119 A promoter is any of the following DNA molecules: 1) A DNA molecule having a nucleotide sequence of one chain as SEQ ID No: 2 in the sequence listing, 2) a DNA molecule that is more than 80% identical to the DNA molecule of 1) and has promoter function; The P J23105 A promoter is any of the following DNA molecules: 1) A DNA molecule having a nucleotide sequence of one chain as SEQ ID No: 3 in the sequence listing, 2) a DNA molecule that is more than 80% identical to the DNA molecule of 1) and has promoter function; The linker is a DNA molecule whose nucleotide sequence in one chain is SEQ ID No: 4 in the sequence list.

6. The recombinant bacteria constructed according to any one of claims 1 to 5.

7. Use of the method according to any one of claims 1 to 5 in W1) or W2): W1) Application in the production of mycosporin-like amino acid Shinorine; W2) Application in the preparation of products producing mycosporin-like amino acid Shinorine.

8. Use of the recombinant Escherichia coli according to any one of claim 6 in W1) or W2): W1) Application in the production of mycosporin-like amino acid Shinorine; W2) Application in the preparation of products producing mycosporin-like amino acid Shinorine.

9. A method for preparing a mycosporine-like amino acid, comprising inoculating the recombinant bacterium according to claim 6 into a culture medium containing glucose, L-methionine, glycine, L-serine and / or glucose, glycine and / or glucose as substrates, and catalyzing the production of the mycosporine-like amino acid Shinorine by whole-cell catalysis.

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