Genetically engineered bacteria for producing 2-O-α-D-pyranoglucopyranosyl-L-ascorbic acid and its construction method and application

Abstract

The present invention belongs to the field of bioengineering technology, and relates to a genetically engineered bacterium for producing 2-O-α-D-pyranose glucopyranosyl-L-ascorbic acid (AA-2G), and a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium for producing 2-O-α-D-pyranose glucopyranosyl-L-ascorbic acid (AA-2G) described in the present invention contains an α-galactosidase gene (agal), a xylose reductase gene (XR), a mannitol dehydrogenase gene (MDH), an L-galactose dehydrogenase gene (LGDH) with a S136A mutation, a D-arabinose-1,4-lactone oxidase gene (ALO), and a sucrose phosphorylase gene (SPase). The present invention provides a method for producing AA-2G using the recombinant genetically engineered bacterium. The genetically engineered strain realizes the synthesis of AA-2G by whole cells, greatly shortens the synthetic production pathway of AA-2G, and improves the yield of AA-2G.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and relates to a genetically engineered bacterium for producing 2-O-α-D-pyranoglucopyranosyl-L-ascorbic acid (AA-2G), and a construction method and application of the genetically engineered bacterium. Background Art

[0002] L-ascorbic acid (LAA), also known as vitamin C, is an essential nutrient that participates in a variety of physiological activities and plays an important role in maintaining and promoting human health. Since humans cannot synthesize LAA, they must obtain it through daily diet. However, LAA is extremely unstable in aqueous solution and is easily oxidized by oxygen, heat, light, and metal ions. The instability of LAA directly reduces its biological activity and limits its application. Therefore, preparing a new LAA substitute that improves oxidative stability while retaining the biological activity of LAA has always been a research hotspot in academia and industry.

[0003] Various LAA derivatives, such as ascorbic acid (acyl, -phosphate and -sulfate), ascorbyl palmitate, ascorbyl methyl ether and ascorbyl glucoside have been synthesized chemically or biologically. Among LAA derivatives, 2-O-α-D-pyranosyl-L-ascorbic acid (AA-2G) is considered to be superior because it has excellent light resistance, heat resistance, oxidation resistance, and is easily degraded by α-glucosidase to produce LAA and glucose. Therefore, AA-2G has a wide range of uses in the pharmaceutical, food and cosmetic industries.

[0004] At present, the industrial method for producing AA-2G is to use LAA as raw material and synthesize it by enzymatic transglycosylation of glycosyltransferase. According to reports, the glycosyltransferases discovered so far include α-glucosidase (US3763009, 1973), cyclodextrin glycosyltransferase (CGTase) (US5084563, 1992; US5137723, 1992; US5407812, 1995 and US9265781B2, 2016), α-amylase, sucrose phosphorylase and α-isomaltosyl glucosyl sugar forming enzyme (US20060216792, 2006; US8759030, 2014 and CN201510179664, 2015) and dextran sucrase. The current industrial production process of AA-2G involves two independent production units and a mixed system of chemical and biological processes.

[0005] The microbial fermentation synthesis of AA-2G in the published patents mainly focuses on: 1. Adding LAA and glycosyl donors such as maltose, sucrose and cyclodextrin as reaction substrates to the reaction system, and using recombinant glycosyltransferases from different sources as catalysts to synthesize AA-2G, such as the authorization announcement number: CN 108913641 B "A recombinant Escherichia coli and its application"; the authorization announcement number: CN 109988778 B "A sucrose phosphorylase gene and its application". 2. Using site-directed mutagenesis technology to mutate key sites of glycosyltransferases, in order to achieve high conversion rate and high yield of catalytically synthesized AA-2G. For example, the authorization announcement number: CN108018268 B "A cyclodextrin glucosyltransferase mutant for improving AA-2G production"; the authorization announcement number: CN104531629B "A cyclodextrin glucosyltransferase mutant for improving AA-2G conversion rate". 3. Finding new catalytic enzymes, in order to achieve a single catalytic synthesis product with few by-products. For example, US Patents US20060216792 (2006) and US8759030 (2014) reported the use of α-isomaltosyl glucosyl sugar-forming enzymes from Arthrobacter sphaericus as catalysts to prepare AA-2G. Although these patents use different strategies to improve the yield of AA-2G, the time, material and energy consumption problems brought about by the production process are becoming more and more prominent. In order to overcome these problems, it is best to transform the mode of AA-2G production from a mixture of chemical and biological processes to a complete biological process. Summary of the invention

[0006] The primary purpose of the present invention is to provide a genetically engineered bacterium for producing 2-O-α-D-pyranosyl-L-ascorbic acid (AA-2G). The genetically engineered bacterium can synthesize AA-2G in a whole cell using cheap raffinose (also known as raffinose) as a synthetic raw material. The entire synthetic process does not involve a chemical process and is a complete biosynthesis, thereby reforming the synthetic process of AA-2G and solving the problem of high pollution and high energy consumption in the current chemical process for producing AA-2G.

[0007] The genetically engineered bacteria for producing 2-O-α-D-pyranosyl-L-ascorbic acid (AA-2G) of the present invention contain an α-galactosidase gene (agal), a xylose reductase gene (XR), a mannitol dehydrogenase gene (MDH), an S136A mutated L-galactose dehydrogenase gene (LGDH), a D-arabinose-1,4-lactone oxidase gene (ALO) and a sucrose phosphorylase gene (SPase).

[0008] According to the further characteristics of the genetically engineered bacteria of the present invention, the α-galactosidase gene (agal) is from Meiothermus ruber, and the protein sequence is shown in SEQ ID NO.1; the xylose reductase gene (XR) is from Hypocrea jecorina, and the protein sequence is shown in SEQ ID NO.2; the mannitol dehydrogenase gene (MDH) is from Apium graveolens, and the protein sequence is shown in SEQ ID NO.3; the S136A mutated L-galactose dehydrogenase gene (LGDH) is from Arabidopsis thaliana, and the protein sequence is shown in SEQ ID NO.4; the D-arabinose-1,4-lactone oxidase gene (ALO) is from Pichia pastoris, and the protein sequence is shown in SEQ ID NO.5; the sucrose phosphorylase gene (SPase) is from Bifidobacterium longum, and the protein sequence is shown in SEQ ID NO.6.

[0009] According to further features of the genetically engineered bacteria of the present invention, the genetically engineered bacteria contains a vector, and the α-galactosidase gene (agal), xylose reductase gene (XR), mannitol dehydrogenase gene (MDH), S136A mutated L-galactose dehydrogenase gene (LGDH), D-arabinose-1,4-lactone oxidase gene (ALO) and sucrose phosphorylase gene (SPase) are included on the vector.

[0010] According to a further feature of the genetically engineered bacteria of the present invention, the genetically engineered bacteria is selected from: Pichia pastoris, Saccharomyces cerevisiae or Escherichia coli.

[0011] The second object of the present invention is to provide a method for constructing the genetically engineered bacteria.

[0012] Alternatively, the method for constructing the genetically engineered bacteria of the present invention comprises the following steps: using the XR gene sequence, the MDH gene sequence, the LGDH gene sequence and the ALO gene sequence as templates, adding respective upstream and downstream primers, and amplifying by conventional PCR to obtain rXR fragments, rMDH fragments, rLGDH fragments and rALO fragments; connecting each amplified fragment to the multiple cloning sites of the vector to obtain an XR-MDH-LGDH-ALO recombinant plasmid; using the agal gene sequence and the SPase gene sequence as templates, adding respective upstream and downstream primers, and amplifying by conventional PCR to obtain ragal fragments and rSPase fragments; connecting each amplified fragment to the multiple cloning sites of the vector to obtain an agal-SPase recombinant plasmid; after linearizing the two recombinant plasmids, integrating them into the Pichia pastoris genome, and screening to obtain a Pichia pastoris recombinant strain that simultaneously expresses agal, XR, MDH, LGDH, ALO and SPase.

[0013] Alternatively, the construction method of the genetically engineered bacteria of the present invention comprises the following steps: using the XR gene sequence as a template, adding upstream and downstream primers, obtaining the rXR fragment by conventional PCR amplification, connecting to the linearized vector fragment, transforming the connection product into Escherichia coli, and screening to obtain the XR recombinant plasmid; using the MDH gene sequence as a template, adding upstream and downstream primers, obtaining the rMDH fragment by conventional PCR amplification, connecting to the linearized XR recombinant plasmid, transforming the connection product into Escherichia coli, and screening to obtain the XR-MDH recombinant plasmid; using the LGDH gene sequence as a template, adding upstream and downstream primers, obtaining the rLGDH fragment by conventional PCR amplification, connecting to the linearized XR-MDH recombinant plasmid, transforming the connection product into Escherichia coli, and screening to obtain the XR-MDH-LGDH recombinant plasmid; using the ALO gene sequence as a template, adding upstream and downstream primers, obtaining the rALO fragment by conventional PCR amplification, connecting to the linearized XR-MDH-LGDH recombinant plasmid, transforming the connection product into Escherichia coli, and screening to obtain XR-MDH-LGDH-ALO recombinant plasmid; using the SPase gene sequence as a template, adding upstream and downstream primers, obtaining the rSPase fragment through conventional PCR amplification, connecting it to the linearized XR-MDH-LGDH-ALO recombinant plasmid, transforming the ligation product into Escherichia coli, and screening to obtain the XR-MDH-LGDH-ALO-SPase recombinant plasmid; using the agal gene sequence as a template, adding upstream and downstream primers, obtaining the ragal fragment through conventional PCR amplification, connecting it to the linearized XR-MDH-LGDH-ALO-SPase recombinant plasmid, transforming the ligation product into Escherichia coli, and screening to obtain the XR-MDH-LGDH-ALO-SPase-agal recombinant plasmid; transforming the XR-MDH-LGDH-ALO-SPase-agal recombinant plasmid into saccharomyces cerevisiae or Escherichia coli, and screening to obtain the saccharomyces cerevisiae recombinant strain or Escherichia coli recombinant strain simultaneously expressing XR, MDH, LGDH, ALO, SPase and agal.

[0014] The present invention further provides the use of the genetically engineered bacteria, that is, the use of the genetically engineered bacteria of the present invention in preparing a bacterial agent for producing 2-O-α-D-pyranoglucopyranosyl-L-ascorbic acid (AA-2G).

[0015] The present invention also provides a method for producing 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G).

[0016] The method for producing 2-O-α-D-pyranoglucopyranosyl-L-ascorbic acid (AA-2G) of the present invention comprises the following steps: providing the genetically engineered bacteria of the present invention, culturing the bacteria in a culture medium containing at least one AA-2G precursor, and collecting AA-2G in the fermentation broth.

[0017] According to a further feature of the method for producing 2-O-α-D-pyranoglucopyranosyl-L-ascorbic acid (AA-2G) described in the present invention, the AA-2G precursor is selected from one of the following combinations: raffinose; raffinose and L-galactose; raffinose and D-galactose; raffinose and galactitol; raffinose and L-galactosonic acid-1,4-lactone; raffinose and L-ascorbic acid; sucrose and L-galactose; sucrose and D-galactose; sucrose and galactitol; sucrose and L-galactosonic acid-1,4-lactone; sucrose and L-ascorbic acid.

[0018] The present invention provides an engineered bacterium for producing AA-2G, wherein the starting engineered bacterium contains an exogenous α-galactosidase gene, a xylose reductase gene, a mannitol dehydrogenase gene, an S136A mutated L-galactose dehydrogenase gene, a D-arabinose-1,4-lactone oxidase gene and a sucrose phosphorylase gene, thereby obtaining an engineered bacterium for producing AA-2G. The recombinant genetic engineering strain of the present invention realizes the whole-cell synthesis of AA-2G from raffinose through a six-step enzymatic reaction, greatly shortens the synthetic production pathway of AA-2G from sugar, improves the yield of AA-2G, avoids the chemical process of synthesizing AA-2G from sugar, and realizes the full biosynthesis of AA-2G using sugar as a substrate.

[0019] The process of producing AA-2G by using the genetically engineered bacteria of the present invention is simple. It only needs to add glycogen such as glucose and inorganic salts to the fermentation tank at the initial stage, and inoculate a small amount of genetically engineered strains to make the initial concentration OD 600 =0.05, after culturing at 30°C and 200r / min for 24 hours, raffinose is added to an initial concentration of 2g / L, and AA-2G can be fermented and produced at 30°C. In the present application, the yield of AA-2G fermented can reach more than 526mg / L, and AA-2G is directly produced from raffinose, which simplifies the production process, realizes full biosynthesis, improves the yield of AA-2G and reduces production energy consumption. In addition, the fermentation process does not require the addition of expensive raw materials such as coenzymes, saving raw material costs.

[0020] The genetically engineered bacteria provided by the present invention contain exogenous α-galactosidase gene and xylose reductase gene, so the genetically engineered bacteria as a starting strain can synthesize galactitol using raffinose as a substrate.

[0021] The genetically engineered bacteria provided by the present invention contain exogenous α-galactosidase gene, xylose reductase gene and mannitol dehydrogenase gene, so the genetically engineered bacteria as a starting strain can synthesize L-galactose using raffinose as a substrate.

[0022] The genetically engineered bacteria provided by the present invention contain exogenous α-galactosidase gene, xylose reductase gene, mannitol dehydrogenase gene and S136A mutant L-galactose dehydrogenase gene, so the genetically engineered bacteria as a starting strain can synthesize L-galactonic acid-1,4-lactone using raffinose as a substrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure is a flow chart of the biosynthetic pathway for producing AA-2G by the genetically engineered bacteria of the present invention.

[0024] Figure 2 This is a mass spectrometry identification diagram of AA-2G produced by the recombinant genetically engineered bacteria of the present invention. DETAILED DESCRIPTION

[0025] The invention chemically synthesizes an alpha-galactosidase gene (agal) from Meiothermus ruber, a xylose reductase gene (XR) from Hypocrea jecorina, a mannitol dehydrogenase gene (MDH) from Apium graveolens, an L-galactose dehydrogenase gene (LGDH) with S136A mutation from Arabidopsis thaliana, a D-arabinose-1,4-lactone oxidase gene (ALO) from Pichia pastoris, and a sucrose phosphorylase gene (SPase) from Bifidobacterium longum, respectively, connects a promoter and a terminator to the upstream and downstream of the six genes to form a complete expression cassette, integrates the expression cassettes of the six genes into a plasmid through a plasmid method, transforms the plasmid into Escherichia coli, or integrates the six genes into Pichia pastoris or Saccharomyces cerevisiae through homologous recombination. cerevisiae) genome, and obtained a genetically engineered strain that heterologously produces AA-2G.

[0026] like Figure 1As shown, in the present application, the recombinant genetically engineered bacteria contains an exogenous α-galactosidase gene, expresses α-galactosidase, and converts the substrate raffinose (or raffinose) into D-galactose and sucrose; the genetically engineered bacteria contains an exogenous xylose reductase gene, and the expressed xylose reductase converts the substrate D-galactose into galactitol; further, an exogenous mannitol dehydrogenase gene is integrated, and the expressed mannitol dehydrogenase converts galactitol into L-galactose. The S136A mutation L-galactose dehydrogenase gene was further integrated, and the expressed L-galactose dehydrogenase converted L-galactose into L-galactonic acid-1,4-lactone, and the D-arabinose-1,4-lactone oxidase gene was further integrated, and the expressed D-arabinose-1,4-lactone oxidase converted L-galactonic acid-1,4-lactone into L-ascorbic acid. The exogenous sucrose phosphorylase gene integrated by the genetically engineered bacteria, and the expressed sucrose phosphorylase used sucrose as the glycosyl donor, catalyzing the transglycosylation of sucrose (produced by the decomposition of raffinose by α-galactosidase) to L-ascorbic acid, thereby synthesizing AA-2G. Thus, AA-2G was synthesized from raffinose through six-step enzymatic reactions, which greatly shortened the synthesis steps of AA-2G and realized the whole biosynthesis process. The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0027] Terminology Note:

[0028] The α-galactosidase gene is abbreviated as agal; the xylose reductase gene is abbreviated as XR; the mannitol dehydrogenase gene is abbreviated as MDH; the L-galactose dehydrogenase gene with S136A mutation is abbreviated as LGDH; the D-arabinose-1,4-lactone oxidase gene is abbreviated as ALO; and the sucrose phosphorylase gene is abbreviated as SPase.

[0029] Example 1: Full gene synthesis of agal, XR, MDH, LGDH, ALO and SPase genes

[0030] Shanghai Jierui Bioengineering Co., Ltd. was commissioned to use total gene synthesis to obtain the α-galactosidase gene (agal) from Meiothermusruber, the xylose reductase gene (XR) from Hypocrea jecorina, the mannitol dehydrogenase gene (MDH) from Apium graveolens, the S136A mutant L-galactose dehydrogenase gene (LGDH) from Arabidopsis thaliana, the D-arabinose-1,4-lactone oxidase gene (ALO) from Pichia pastoris, and the sucrose phosphorylase gene (SPase) from Bifidobacterium longum.

[0031] Example 2: Construction of recombinant Pichia pastoris GS115 pPIC3.5K XMLASa (1) Using vector pGAPZαA as a template, promoter P was amplified by conventional PCR. GAP -1 fragment. The primer used was P GAP1 -F and P GAP1 -R, used P GAP1 -F contains a Sac I restriction endonuclease cleavage site, P GAP1 -R with XR sequence as homology arm. The reaction conditions were: 95℃2min, 95℃20s, 55℃20s, 72℃10s, 30 cycles in total; 72℃5min, the obtained sequence was subjected to 1% agarose gel electrophoresis to recover the corresponding fragment.

[0032] (2) Using the vector pGAPZαA as a template, the promoter P was amplified by conventional PCR. GAP -2 fragment. The primer used was P GAP2 -F and P GAP2 -R, used P GAP2 -F with T AOX Homology arm, P GAP2 -R with MDH sequence as homology arm. The reaction conditions were: 95℃2min, 95℃20s, 55℃20s, 72℃10s, 30 cycles in total; 72℃5min, the obtained sequence was recovered by 1% agarose gel electrophoresis. (3) Using vector pGAPZαA as template, promoter P was amplified by conventional PCR. GAP -3 fragment. The primer used was P GAP2 -F and P GAP3 -R,P GAP3 -R has LGDH sequence as homology arm. The reaction conditions are: 95℃2min, 95℃20s, 55℃20s, 72℃10s, 30 cycles in total; 72℃5min, the obtained sequence was subjected to 1% agarose gel electrophoresis to recover the corresponding fragment.

[0033] (4) Using the vector pGAPZαA as a template, the promoter P was amplified by conventional PCR. GAP -4 fragment. The primer used was P GAP2 -F and P GAP4 -R,P GAP4 -R with ALO sequence as homology arm. The reaction conditions were: 95℃2min, 95℃20s, 55℃20s, 72℃10s, 30 cycles in total; 72℃5min, the obtained sequence was subjected to 1% agarose gel electrophoresis to recover the corresponding fragment.

[0034] (5) Using the vector pGAPZαA as a template, conventional PCR was used to amplify the promoter T AOX -1 fragment. The primer used was T AOX1 -F and T AOX1 -R,T AOX1 -F with XR sequence as homology arm. AOX1 -R with P GAP2 The sequence was used as the homology arm. The reaction conditions were: 95°C for 2 min, 95°C for 20 s, 55°C for 20 s, 72°C for 10 s, for a total of 30 cycles; 72°C for 5 min. The obtained sequence was subjected to 1% agarose gel electrophoresis to recover the corresponding fragment. (6) Using the vector pGAPZαA as a template, conventional PCR was used to amplify the promoter T AOX -2 fragment. The primer used was T AOX2 -F and T AOX1 -R,T AOX2 -F has MDH sequence as homology arm. The reaction conditions are: 95℃2min, 95℃20s, 55℃20s, 72℃10s, 30 cycles in total; 72℃5min, the obtained sequence was subjected to 1% agarose gel electrophoresis and the corresponding fragment was recovered.

[0035] (7) Using the vector pGAPZαA as a template, conventional PCR was used to amplify the promoter T AOX -3 fragment. The primer used was T AOX3 -F and T AOX1 -R,T AOX3 -F has LGDH sequence as homology arm. The reaction conditions are: 95℃2min, 95℃20s, 55℃20s, 72℃10s, 30 cycles in total; 72℃5min, the obtained sequence was subjected to 1% agarose gel electrophoresis and the corresponding fragment was recovered.

[0036] (8) The above recovered fragments were used as templates according to Table 1 below, and the upper primer and the lower primer were added respectively to connect the two fragments by OVERLAP PCR. The reaction conditions were: 95℃ for 2 min, 95℃ for 20 s, 55℃ for 20 s, 72℃ for 10 s, for a total of 30 cycles; 72℃ for 5 min. The obtained sequence was subjected to 1% agarose gel electrophoresis and the corresponding fragments were recovered. They were named TP1, TP2 and TP3 respectively.

[0037] Table 1:

[0038]

[0039]

[0040] (9) Using the XR gene sequence obtained by chemical synthesis in Example 1 as a template, primers XR-F and XR-R were added to obtain the rXR fragment by conventional PCR amplification. XR-F contained P GAP Sequence as homology arm, XR-R with T AOX -1 sequences served as homology arms.

[0041] The MDH gene sequence obtained by chemical synthesis in Example 1 was used as a template, and primers MDH-F and MDH-R were added to obtain the rMDH fragment by conventional PCR amplification. GAP Sequence as homology arm, MDH-R with T AOX -1 sequences served as homology arms.

[0042] The LGDH gene sequence obtained by chemical synthesis in Example 1 was used as a template, and primers LGDH-F and LGDH-R were added to obtain the rLGDH fragment by conventional PCR amplification. GAP Sequence as homology arm, LGDH-R with T AOX -1 sequences served as homology arms.

[0043] The ALO gene sequence obtained by chemical synthesis in Example 1 was used as a template, and primers ALO-F and ALO-R were added to obtain the rALO fragment by conventional PCR amplification. GAP The sequences were used as homology arms, and ALO-R contained an EcoRI restriction site.

[0044] (10) The eight amplified fragments obtained in steps (1), (8) and (9) were ligated into the multiple cloning sites SacI / EcoRI of the vector pPIC3.5K by In-Fusion recombinase to obtain the pPIC3.5K-XR-MDH-LGDH-ALO plasmid.

[0045] The pPIC3.5K-XR-MDH-LGDH-ALO plasmid verified to be correct was linearized and integrated into the genome of Pichia pastoris GS115 by electroporation. After antibiotic screening, the recombinant Pichia pastoris strain A1 simultaneously expressing XR, MDH, LGDH and ALO was obtained.

[0046] (11) Using the vector pGAPZαA as a template, the promoter P was amplified by conventional PCR. GAP -5 fragment. The primer used was P GAP5 -F and P GAP5 -R, used P GAP5 -F contains EcoRI restriction endonuclease cleavage site, P GAP5 -R with agal sequence as homology arm. The reaction conditions were: 95℃2min, 95℃20s, 55℃20s, 72℃10s, 30 cycles in total; 72℃5min, the obtained sequence was subjected to 1% agarose gel electrophoresis to recover the corresponding fragment.

[0047] (12) Using the vector pGAPZαA as a template, the promoter P was amplified by conventional PCR. GAP -6 fragment. The primer used was P GAP2 -F and P GAP6 -R, used P GAP6 -R with SPase sequence as homology arm. The reaction conditions were: 95℃2min, 95℃20s, 55℃20s, 72℃10s, 30 cycles in total; 72℃5min, the obtained sequence was subjected to 1% agarose gel electrophoresis to recover the corresponding fragment.

[0048] (13) Using the vector pGAPZαA as a template, conventional PCR was used to amplify the promoter T AOX -4 fragment. The primer used was T AOX4 -F and T AOX1 -R,T AOX4 -F has agal sequence as homology arm. The reaction conditions are: 95℃2min, 95℃20s, 55℃20s, 72℃10s, 30 cycles in total; 72℃5min, the obtained sequence was subjected to 1% agarose gel electrophoresis and the corresponding fragment was recovered.

[0049] (14) The recovered P GAP -6 fragment and T AOX -4 fragment as template, adding primers and T AOX4 -F and P GAP6-R, the two fragments were connected together using OVERLAP PCR. The reaction conditions were: 95℃2min, 95℃20s, 55℃20s, 72℃10s, 30 cycles in total; 72℃5min, the obtained sequence was recovered after 1% agarose gel electrophoresis, and named TP4.

[0050] (15) The agal gene sequence obtained by chemical synthesis in Example 1 was used as a template, and primers agal-F and agal-R were added to obtain the ragal fragment by conventional PCR amplification. GAP Sequence as homology arm, agal-R with T AOX -1 sequence as the homology arm; the SPase gene sequence obtained by chemical synthesis in Example 1 was used as a template, and primers SPase-F and SPase-R were added to obtain the rSPase fragment by conventional PCR amplification. SPase-F contained P GAP The sequences were used as homology arms, and SPase-R contained a NotI restriction endonuclease cleavage site.

[0051] Table 2: Primer sequences

[0052]

[0053]

[0054] (16) The four amplified fragments obtained in steps (1), (4) and (5) were connected to the multiple cloning site EcoRI / SalI of the vector pPICZ A by In-Fusion recombinase to obtain the pPICZ A-agal-SPase plasmid.

[0055] The pPICZ A-agal-SPase plasmid verified to be correct was linearized and then integrated into the genome of the Pichia pastoris recombinant strain A1 obtained in Implementation Case 2 by electroporation. After antibiotic screening, the Pichia pastoris recombinant strain P. pastoris GS115 pPIC3.5KXMLASa simultaneously expressing XR, MDH, LGDH, ALO, agal and SPase was obtained.

[0056] Example 3 Construction of recombinant Saccharomyces cerevisiae INVSc1 pYES2 XMLASa rDNA

[0057] (1) The phosphoglycerol kinase promoter (PGKp) sequence was amplified using S.cerevisiae INVSc1 genomic DNA as a template, and the nucleotide coding sequences of the upstream and downstream primers PGK 1F and PGK 1R are shown in Table 2; the terminator CYC1t sequence was amplified using the pESC-URA plasmid, and the nucleotide coding sequences of the upstream and downstream primers CYC 1F and CYC 1R are shown in Table 2; the three sequences of PGKp, XR and CYC1t were concatenated into a PGKp-XR-CYC1t (PXC) expression cassette using the overlap extension PCR method, and the PCR conditions are shown in Table 3, and then the PXC expression cassette and the linearized pYES2 vector fragment were seamlessly connected using a one-step cloning kit (purchased from Novozymes), and the connection product was transformed into E.coli DH5α, and the plasmid in the positive clone was extracted to obtain the recombinant vector 2.

[0058] (2) The phosphoglycerol kinase promoter (PGKp) sequence was amplified using S.cerevisiae INVSc1 genomic DNA as a template, and the nucleotide coding sequences of the upstream and downstream primers PGK 2F and PGK 2R are shown in Table 2; the terminator CYC1t sequence was amplified using the pESC-URA plasmid, and the nucleotide coding sequences of the upstream and downstream primers CYC 2F and CYC 2R are shown in Table 2; the obtained PGKp, CYC1t and MDH three sequences were concatenated into a PGKp-MDH-CYC1t (PMC) expression cassette using the overlap extension PCR method, and the PCR conditions are shown in Table 3, and then the above-mentioned PMC expression cassette and the linearized recombinant vector 2 fragment were seamlessly connected using a one-step cloning kit, and the connection product was transformed into E.coli DH5α, and the plasmid in the positive clone was extracted to obtain the recombinant vector 3.

[0059] (3) Using S.cerevisiae INVSc1 genomic DNA as a template, the phosphoglycerol kinase promoter (PGKp) sequence was amplified, and the upstream and downstream primers PGK 3F and PGK 3R nucleotide coding sequences were shown in Table 2; the terminator CYC1t sequence was amplified using the pESC-URA plasmid, and the upstream and downstream primers CYC 3F and CYC 3R nucleotide coding sequences were shown in Table 2; the obtained PGKp, CYC1t and LGDH three sequences were concatenated into a PGKp-LGDH-CYC1t (PLC) expression cassette using the overlap extension PCR method, and the PCR conditions were shown in Table 3, and then the above-mentioned PLC expression cassette and the linearized recombinant vector 3 fragment were seamlessly connected using a one-step cloning kit, and the connection product was transformed into E.coli DH5α, and the plasmid in the positive clone was extracted to obtain the recombinant vector 4.

[0060] (4) The phosphoglycerol kinase promoter (PGKp) sequence was amplified using S.cerevisiae INVSc1 genomic DNA as a template, and the nucleotide coding sequences of the upstream and downstream primers PGK 4F and PGK 4R are shown in Table 2; the terminator CYC1t sequence was amplified using the pESC-URA plasmid, and the nucleotide coding sequences of the upstream and downstream primers CYC 4F and CYC 4R are shown in Table 2; the obtained PGKp, CYC1t and ALO three-segment sequences were concatenated into a PGKp-ALO-CYC1t (PAC) expression cassette using the overlap extension PCR method, and the PCR conditions are shown in Table 3, and then the above-mentioned PAC expression cassette and the linearized recombinant vector 4 fragment were seamlessly connected using a one-step cloning kit, and the connection product was transformed into E.coli DH5α, and the plasmid in the positive clone was extracted to obtain the recombinant vector 5.

[0061] (5) The phosphoglycerol kinase promoter (PGKp) sequence was amplified using S.cerevisiae INVSc1 genomic DNA as a template, and the nucleotide coding sequences of the upstream and downstream primers PGK 5F and PGK 5R are shown in Table 2; the terminator CYC1t sequence was amplified using the pESC-URA plasmid, and the nucleotide coding sequences of the upstream and downstream primers CYC 5F and CYC 5R are shown in Table 2; the three sequences of PGKp, CYC1t and SPase were concatenated into a PGKp-SPase-CYC1t (PSC) expression cassette using the overlapping extension PCR method, and the PCR conditions are shown in Table 3, and then the PSC expression cassette and the linearized recombinant vector 5 fragment were seamlessly connected using a one-step cloning kit, and the connection product was transformed into E.coli DH5α, and the plasmid in the positive clone was extracted to obtain the recombinant vector 6.

[0062] (6) The phosphoglycerate kinase promoter (PGKp) sequence was amplified using S.cerevisiae INVSc1 genomic DNA as a template. The nucleotide coding sequences of the upstream and downstream primers PGK 6F and PGK 6R are shown in Table 2. The obtained PGKp and agal sequences were concatenated into a PGKp-agal expression cassette using the overlap extension PCR method. The PCR conditions are shown in Table 3. The PGKp-agal expression cassette and the linearized recombinant vector 6 fragment were seamlessly connected using a one-step cloning kit. The connection product was transformed into E.coli DH5α. The plasmid in the positive clone was extracted to obtain the recombinant vector 7.

[0063] (7) Using S.cerevisiae INVSc1 genomic DNA as a template, the core sequence in the rDNA unit was amplified. The upstream and downstream primers rDNA F and rDNA R nucleotide coding sequences are shown in Table 2. After obtaining the PCR product rDNA, the obtained PCR product rDNA and the linearized recombinant vector 7 fragment were seamlessly connected, and the connection product was transformed into E.coli DH5α. The plasmid in the positive clone was extracted to obtain the recombinant vector pYES2 XMLASa rDNA. After the pYES2 XMLASa rDNA vector was linearized with BbuI, the competent S.cerevisiae INVSc1 was transformed by the LiAc / ssDNA method. The transformed system was spread on SCU medium. After 3 to 5 days, a single colony grew out. The single clone was selected for colony PCR verification. The S.cerevisiae INVSc1 pYES2XMLASa rDNA engineering bacteria were obtained.

[0064] Table 3: Primer sequences

[0065]

[0066]

[0067] Table 4

[0068]

[0069] Example 4: Construction of recombinant Escherichia coli pET XMLASa

[0070] (1) Using the pET-28a(+) vector as a template, the terminator T7t sequence was amplified. The nucleotide coding sequences of the upstream and downstream primers T7t-1F and T7t-1R were shown in Table 4. Using the XR sequence in Example 1 as a template, XR-1F and XR-1R were respectively the upstream and downstream primers to amplify the XR-1 sequence. The nucleotide coding sequence was shown in Table 4. The XR-1 and T7t sequences were concatenated into an XR-1-T7t fragment using the overlap extension PCR method. The PCR conditions were shown in Table 3. Then, the XR-1-T7t sequence and the linearized pET-28a(+) vector fragment were seamlessly connected using a one-step cloning kit (purchased from Novozymes). The connection product was transformed into E. coli DH5α. The plasmid in the positive clone was extracted to obtain the recombinant vector pET-XR.

[0071] (2) Using the pET-28a(+) vector as a template, the T7 promoter (T7p) sequence was amplified. The nucleotide coding sequences of the upstream and downstream primers T7p 2F and T7p2R were shown in Table 4. Using the MDH sequence in Example 1 as a template, MDH-1F and MDH-1R were respectively the upstream and downstream primers to amplify the MDH-1 sequence. The nucleotide coding sequence was shown in Table 4. Using the pET-28a(+) vector as a template, the terminator T7t sequence was amplified. The nucleotide coding sequences of the upstream and downstream primers T7t-2F and T7t-2R were shown in Table 4. The three sequences of T7p, MDH-1 and T7t were concatenated into a T7p-MDH-1-T7t (TMT) expression cassette using the overlap extension PCR method. The PCR conditions were shown in the following table. Then, the TMT expression cassette and the linearized recombinant vector pET-XR fragment were seamlessly connected using a one-step cloning kit, and the connection product was transformed into E. coli. In DH5α, the recombinant vector pET-XR-MDH can be obtained by extracting the plasmid from the positive clone.

[0072] (3) Using the pET-28a(+) vector as a template, amplifying the T7 promoter (T7p) sequence, the nucleotide coding sequences of the upstream and downstream primers T7p 3F and T7p3R are shown in Table 4; using the LGDH sequence in Example 1 as a template, LGDH-1F and LGDH-1R are respectively the upstream and downstream primers to amplify the LGDH-1 sequence, the nucleotide coding sequence is shown in Table 4; using the pET-28a(+) vector as a template, amplifying the terminator T7t sequence, the nucleotide coding sequences of the upstream and downstream primers T7t-3F and T7t-3R are shown in Table 4; using the overlap extension PCR method, the obtained T7p, LGDH-1 and T7t three sequences were concatenated into a T7p-LGDH-1-T7t (TLT) expression cassette, the PCR conditions are shown in the following table, and then the TLT expression cassette and the linearized recombinant vector pET-XR-MDH fragment were seamlessly connected using a one-step cloning kit, and the connection product was transformed into E. coli In DH5α, the plasmid from the positive clone can be extracted to obtain the recombinant vector pET-XR-MDH-LGDH.

[0073] (4) Using the pET-28a(+) vector as a template, amplifying the T7 promoter (T7p) sequence, the nucleotide coding sequences of the upstream and downstream primers T7p 4F and T7p4R are shown in Table 4; using the ALO sequence in Example 1 as a template, ALO-1F and ALO-1R are respectively the upstream and downstream primers to amplify the ALO-1 sequence, the nucleotide coding sequence is shown in Table 4; using the pET-28a(+) vector as a template, amplifying the terminator T7t sequence, the nucleotide coding sequences of the upstream and downstream primers T7t-4F and T7t-4R are shown in Table 4; using the overlap extension PCR method, the obtained T7p, ALO-1 and T7t three sequences were concatenated into a T7p-ALO-1-T7t (TAT) expression cassette, the PCR conditions are shown in Table 3, and then the TAT expression cassette and the linearized recombinant vector pET-XR-MDH-LGDH fragment were seamlessly connected using a one-step cloning kit, and the connection product was transformed into E. coli In DH5α, the plasmid from the positive clone can be extracted to obtain the recombinant vector pET-XR-MDH-LGDH-ALO.

[0074] (5) Using the pET-28a(+) vector as a template, amplifying the T7 promoter (T7p) sequence, the nucleotide coding sequences of the upstream and downstream primers T7p 5F and T7p5R are shown in Table 4; using the SPase sequence in Example 1 as a template, SPase-1F and SPase-1R are respectively the upstream and downstream primers to amplify the SPase-1 sequence, the nucleotide coding sequence is shown in Table 4; using the pET-28a(+) vector as a template, amplifying the terminator T7t sequence, the nucleotide coding sequences of the upstream and downstream primers T7t-5F and T7t-5R are shown in Table 4; using the overlap extension PCR method, the obtained T7p, SPase-1 and T7t three sequences were concatenated into a T7p-SPase-1-T7t (TST) expression cassette, the PCR conditions are shown in Table 3, and then the TST expression cassette and the linearized recombinant vector pET-XR-MDH-LGDH-ALO fragment were seamlessly connected using a one-step cloning kit, and the connection product was transformed into E. coli In DH5α, the plasmid from the positive clone can be extracted to obtain the recombinant vector pET-XR-MDH-LGDH-ALO-SPase.

[0075] (6) Using the pET-28a(+) vector as a template, the T7 promoter (T7p) sequence was amplified. The nucleotide coding sequences of the upstream and downstream primers T7p 6F and T7p6R are shown in Table 4. Using the agal sequence in Example 1 as a template, agal-1F and agal-1R were used as the upstream and downstream primers to amplify the agal-1 sequence. The nucleotide coding sequence is shown in Table 4. The obtained T7p, agal-1 and T7t sequences were concatenated into a T7p-agal-1-T7t (TaT) expression cassette using the overlap extension PCR method. The PCR conditions are shown in the following table. Then, the TaT expression cassette and the linearized recombinant vector pET-XR-MDH-GDH-ALO-SPase fragment were seamlessly connected using a one-step cloning kit. The connection product was transformed into E. coli DH5α. The plasmid in the positive clone was extracted to obtain the recombinant plasmid pET-XR-MDH-LGDH-ALO-SPase-agal.

[0076] Table 5 Primer sequences

[0077]

[0078]

[0079] (7) The plasmid pET-XR-MDH-LGDH-ALO-SPase-agal was transformed into Escherichia coli BL21 (DE3) to obtain the Escherichia coli genetically engineered strain Escherichia coli pETXMLASa that synthesizes AA-2G.

[0080] The E. coli transformation method of plasmid pET-XR-MDH-LGDH-ALO-SPase-agal is as follows: add plasmid pET-XR-MDH-LGDH-ALO-SPase-agal to 50 μL E. coli BL21 (DE3) competent cells, ice bath for 30 minutes, and heat shock at 42°C for 90 seconds for transformation. Add 1 ml LB medium, rejuvenate at 37°C for 1 hour, collect bacteria and apply them to kanamycin resistance screening LB plates, and culture at 37°C overnight. After overnight culture, pick the colonies for colony PCR verification, select positive transformants, and perform sequencing verification. The positive transformants with correct sequencing are named strain Escherichia coli pET XMLASa.

[0081] Example 5: Production of AA-2G using the recombinant Pichia pastoris strain P. pastoris GS115 pPIC3.5K XMLASa

[0082] A single colony of the recombinant strain of Pichia pastoris GS115 pPIC3.5K XMLASa was picked and inoculated into 5 mL YPD liquid medium, and cultured overnight at 30°C and 200 r / min; the overnight culture solution was transferred to 250 mL YPD liquid medium, and the initial concentration was OD600 = 0.05. After culturing at 30°C and 200 r / min for 24 hours, raffinose was added to make the initial concentration 2 g / L, and cultured at 30°C for 72 hours (samples were taken after 24 hours, 36 hours, 48 ​​hours and 72 hours, respectively). The formula of the YPD medium is: 10 g / L yeast extract, 20 g / L peptone, and 20 g / L D-glucose.

[0083] Example 6: Production of AA-2G using S. cerevisiae INVSc1 pYES2 XMLASa rDNA engineered bacteria

[0084] A single colony of the recombinant strain of Saccharomyces cerevisiae INVSc1 pYES2 XMLASa rDNA was picked and inoculated into 5 mL YPD liquid medium, and cultured overnight at 30°C and 200 r / min; the overnight culture solution was transferred to 250 mL YPD liquid medium, and the initial concentration was OD600 = 0.05. After culturing at 30°C and 200 r / min for 24 hours, raffinose was added to make the initial concentration 2 g / L, and cultured at 30°C for 72 hours (samples were taken after 24 hours, 36 hours, 48 ​​hours and 72 hours, respectively). The formula of the YPD medium is: 10 g / L yeast extract, 20 g / L peptone, and 20 g / L D-glucose.

[0085] Example 7: Production of AA-2G using Escherichia coli pET XMLASa

[0086] A single colony of the recombinant Escherichia coli pET XMLASa was picked and inoculated into 5 mL LB liquid medium under sterile conditions. The culture was shaken at 37°C and 200 rpm for 8-10 h to obtain a seed solution. The seed solution was inoculated into a 500 mL shake flask containing 100 mL LB medium at 1% (v / v). The culture was shaken at 37°C and 200 rpm for 1.5-2 h. 600When the pH reaches 0.6-0.8, add 100 μL of 0.5M IPTG mother solution, and then shake and culture at 20°C and 200 rpm for 24 hours, and then collect the bacteria at 5000 rpm for 15 minutes. Resuspend the bacteria to 10 OD / mL in M9 medium containing 2g / L raffinose, take 20mL of bacterial solution in a shake flask at 37°C and 200 rpm for 72 hours, and sample every 12 hours to detect the AA-2G content in the supernatant by HPLC. Among them, the formula of LB medium is: tryptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, and the balance is water. The formula of M9 culture medium is: glucose 5g / L, potassium dihydrogen phosphate 3g / L, disodium hydrogen phosphate 6g / L, ammonium chloride 1g, MgSO4·7H2O 246.5mg / L, CaCl2·2H2O 14.7mg / L, FeSO4·7H2O 27.8mg / L, citric acid 2g / L, and the balance is water.

[0087] Example 8: HPLC detection of AA-2G

[0088] The supernatant of the culture system obtained in Examples 5, 6, and 7 was used for HPLC detection under the following conditions: chromatographic column: C18 chromatographic column; 4.6 mm x 250 mm, 5μm (Part NO:059149), mobile phase 0.1M KH2PO4, flow rate 0.8mL / min, column temperature 25℃, detection wavelength 238nm. The retention peak of AA-2G appeared at 8.7min. Detection results: AA-2G yield reached more than 526mg / L.

[0089] Example 9 LC-MS / MS Detection of AA-2G

[0090] Liquid chromatography conditions: chromatographic column: Waters ACQUITY UPLC BEH C18 column (100 mm*2.1 mm, Particle Sz.1.7 μm), column temperature: 40°C, mobile phase: phase A: 0.1% formic acid aqueous solution; phase B: methanol; flow rate: 300 μL / min; mobile phase gradient see Table 6.

[0091] Table 6 HPLC mobile phase

[0092] Time (min) Mobile phase A (%) Mobile phase B (%) 0.00 100 0 3.00 100 0 6.00 10 90 8.00 10 90 8.10 100 0 10.00 100 0

[0093] Mass spectrometry detection conditions:

[0094] Ionization mode: ESI; capillary voltage: 4.5 kV; dryer temperature: 220 °C; nebulizer gas: 1.5 Bar; dryer: 8.0 l / min; scanning mode: first-level full scan.

[0095] The result is AA-2G, attached Figure 2 The substance with a molecular weight of 337.76 in the mass spectrum is [C 12 H 18 O 11 ], which is the mass spectrum peak of AA-2G.

Claims

1. A genetically engineered bacterium for producing 2-O-α-D-pyranosyl-L-ascorbic acid (AA-2G), characterized in that: The genetically engineered bacteria contain an α-galactosidase agal gene, a xylose reductase XR gene, a mannitol dehydrogenase MDH gene, an S136A mutated L-galactose dehydrogenase LGDH gene, a D-arabinose-1,4-lactone oxidase ALO gene, and a sucrose phosphorylase SPase gene; The α-galactosidase agal gene is from Meiothermus ruber, and the protein sequence is shown in SEQ ID NO.1; the xylose reductase XR gene is from Hypocrea jecorina, and the protein sequence is shown in SEQ ID NO.2; the mannitol dehydrogenase MDH gene is from Apium graveolens, and the protein sequence is shown in SEQ ID NO.3; the S136A mutated L-galactose dehydrogenase LGDH gene is from Arabidopsis thaliana, and the protein sequence is shown in SEQ ID NO.4; the D-arabinose-1,4-lactone oxidase ALO gene is from Pichia pastoris, and the protein sequence is shown in SEQ ID NO.5; the sucrose phosphorylase SPase gene is from Bifidobacterium longum, and the protein sequence is shown in SEQ ID NO.6; The genetically engineered bacteria contains a vector, and the α-galactosidase agal gene, xylose reductase XR gene, mannitol dehydrogenase MDH gene, S136A mutated L-galactose dehydrogenase LGDH gene, D-arabinose-1,4-lactone oxidase ALO gene and sucrose phosphorylase SPase gene are included on the vector; the genetically engineered bacteria is selected from: Pichia pastoris, Saccharomyces cerevisiae or Escherichia coli.

2. The method for constructing a genetically engineered bacterium according to claim 1, characterized in that: The following steps are involved: Using the XR gene sequence as a template, adding upstream and downstream primers, amplifying the rXR fragment by conventional PCR, connecting it to the linearized vector fragment, transforming the ligation product into Escherichia coli, and screening to obtain the XR recombinant plasmid; using the MDH gene sequence as a template, adding upstream and downstream primers, amplifying the rMDH fragment by conventional PCR, connecting it to the linearized XR recombinant plasmid, transforming the ligation product into Escherichia coli, and screening to obtain the XR-MDH recombinant plasmid; Using the LGDH gene sequence as a template, adding upstream and downstream primers, amplifying the rLGDH fragment by conventional PCR, connecting it to the linearized XR-MDH recombinant plasmid, transforming the ligation product into Escherichia coli, and screening to obtain the XR-MDH-LGDH recombinant plasmid; Using the ALO gene sequence as a template, adding upstream and downstream primers, obtaining the rALO fragment by conventional PCR amplification, connecting it to the linearized XR-MDH-LGDH recombinant plasmid, transforming the ligation product into Escherichia coli, and screening to obtain the XR-MDH-LGDH-ALO recombinant plasmid; Using the SPase gene sequence as a template, adding upstream and downstream primers, obtaining the rSPase fragment by conventional PCR amplification, connecting it to the linearized XR-MDH-LGDH-ALO recombinant plasmid, transforming the ligation product into Escherichia coli, and screening to obtain the XR-MDH-LGDH-ALO-SPase recombinant plasmid; Using the agal gene sequence as a template, adding upstream and downstream primers, amplifying the ragal fragment by conventional PCR, connecting it to the linearized XR-MDH-LGDH-ALO-SPase recombinant plasmid, transforming the ligation product into Escherichia coli, and screening to obtain the XR-MDH-LGDH-ALO-SPase-agal recombinant plasmid; The XR-MDH-LGDH-ALO-SPase-agal recombinant plasmid is transformed into Saccharomyces cerevisiae or Escherichia coli, and a recombinant Saccharomyces cerevisiae strain or a recombinant Escherichia coli strain simultaneously expressing XR, MDH, LGDH, ALO, SPase and agal is obtained by screening.

3. Use of the genetically engineered bacteria as claimed in claim 1 in preparing a bacterial agent for producing 2-O-α-D-pyranoglucopyranosyl-L-ascorbic acid (AA-2G).

4. A method for producing 2-O-α-D-pyranosyl-L-ascorbic acid (AA-2G), characterized in that: The following steps are involved: The genetically engineered bacteria of claim 1 are provided, cultured in a culture medium containing at least one AA-2G precursor, and AA-2G is collected in a fermentation broth.

5. The method according to claim 4, characterized in that The AA-2G precursor is selected from one of the following combinations: raffinose; raffinose and L-galactose; raffinose and D-galactose; raffinose and galactitol; raffinose and L-galactosonic acid-1,4-lactone; raffinose and L-ascorbic acid; sucrose and L-galactose; sucrose and D-galactose; sucrose and galactitol; sucrose and L-galactosonic acid-1,4-lactone; sucrose and L-ascorbic acid.

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