Genetically engineered bacteria for producing 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 L-ascorbic acid, and a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium for producing L-ascorbic acid described in the present invention contains a xylose reductase gene (XR), a mannitol dehydrogenase gene (MDH), an S136A mutated L-galactose dehydrogenase gene (LGDH) and a D-arabinose-1,4-lactone oxidase gene (ALO). The present invention obtains an engineered strain for synthesizing L-ascorbic acid, thereby realizing the production of L-ascorbic acid from D-galactose in four steps, greatly shortening the synthetic production pathway of L-ascorbic acid, and improving the yield of L-ascorbic acid.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and relates to a genetic engineering bacterium for producing L-ascorbic acid, and a construction method and application of the genetic engineering bacterium. Background Art

[0002] L-ascorbic acid (LAA), also known as vitamin C, is a hexolactone compound synthesized in plants and most animals. As an important product of plant metabolism, it participates in the antioxidant effect of plants, acts as a coenzyme for certain enzymes, and regulates multiple metabolic pathways and the expression of certain genes.

[0003] Humans and some other primates, such as apes and monkeys, are unable to express and store LAA due to a mutation in the gene encoding L-guluronate-1,4-lactone oxidase (GULO), a key enzyme in the LAA synthesis pathway (L-gulose pathway). They are unable to express and synthesize biologically active GULO protein, and must therefore consume it from daily diets such as fresh fruits and vegetables, otherwise they will develop symptoms of scurvy.

[0004] LAA was originally extracted from plants. In order to reduce production costs, German chemist Reichstein et al. developed the Reichstein method to synthesize LAA. This method includes a five-step chemical catalysis method and a one-step bioconversion method: first, D-glucose is converted into D-sorbitol by high-pressure hydrogenation, and then D-sorbitol is fermented by Gluconobacter oxydans to produce L-sorbose, L-sorbose to diacetone-L-sorbose, to 2-keto-L-gulonic acid (2-KLG), to 2-keto-L-gulonic acid methyl ester, and converted into LAA through four chemical reactions. Compared with the plant extraction method, the Reichstein method significantly reduces the production cost of LAA. However, the disadvantage is that the chemical catalysis steps are complicated, consume a lot of energy, require a large amount of organic solvents, and the reaction conditions are harsh.

[0005] In order to increase the production yield of the LAA precursor 2-KLG, Chinese scientist Yin Guanglin invented the classic two-step fermentation method. First, D-glucose is converted into D-sorbitol by high-pressure hydrogenation, and then converted into L-sorbose by Gluconobacter oxydans. L-sorbose is used as a substrate and converted into the LAA precursor 2-keto-L-gulonic acid (2-KLG) by a mixed bacterial fermentation system consisting of Ketogulonigenium vulgare and Bacillus megaterium. Finally, 2-KLG is converted into LAA through a two-step chemical reaction. This method has become the dominant method for LAA production.

[0006] The two-step fermentation process has obvious disadvantages, such as high material and energy consumption, difficulty in strain selection, complex operation, and poor stability. In order to solve the above problems, researchers turned to constructing recombinant strains through genetic engineering and metabolic engineering to achieve one-step fermentation to produce LAA. Compared with the classic two-step fermentation, one-step fermentation or even one-step single-bacteria fermentation has many advantages, such as: (1) avoiding two sterilizations and thus reducing energy consumption; (2) avoiding two fermentation processes and thus reducing material loss; (3) shortening the fermentation cycle; (4) reducing the difficulty of fermentation regulation; (5) facilitating genetic engineering breeding. At present, most of the research routes for the production of LAA by one-step fermentation are developed on the basis of the classic two-step fermentation method or the new two-step fermentation method, and the relevant strains are transformed through metabolic engineering to achieve one-step fermentation to produce 2-KLG.

[0007] However, whether it is the "classic two-step fermentation method" successfully applied to industrial production or the "new two-step fermentation method" and "one-step fermentation method" proposed later, they cannot completely break away from the chemical synthesis step. From the precursor metabolite 2-KLG to L-ascorbic acid, it also involves two chemical reactions, lactonization and enolization, and still cannot avoid the problems of high energy consumption and pollution of chemical synthesis. Therefore, in recent years, some researchers have proposed to use eukaryotic microbial metabolism such as brewer's yeast and microalgae to synthesize LAA, hoping to completely break away from chemical reactions and achieve complete microbial conversion to produce ascorbic acid.

[0008] It is reported that there are four pathways for the synthesis of LAA in plants, among which the L-galactose pathway is the dominant biosynthetic route, which includes 10 enzymatic steps from D-glucose to the formation of GDP-D-mannose and L-galactose intermediates, and finally the synthesis of LAA. In addition to the main L-galactose pathway, there are three other alternative pathways for the synthesis of LAA: the L-gulose pathway, the D-galacturonic acid pathway, and the inositol pathway. The alternative pathway for LAA includes 10 steps from glucose to the formation of GDP-L-gulose, L-gulonic acid-1,4-lactone intermediates, and finally the synthesis of LAA. The D-galactose pathway includes 11 steps from glucose to GDP-mannose, to cell wall polysaccharides, to the degradation of polysaccharides to D-galactosyl methyl ester, to L-galactose, the formation of L-galactonic acid-1,4-lactone intermediates, and finally the synthesis of LAA. The inositol pathway includes four steps from the formation of inositol to L-gulonic acid-1,4-lactone intermediate and finally the synthesis of LAA.

[0009] Like plants, algae can also synthesize LAA. There were early studies on the development of algae to produce LAA through methods such as mutagenesis, but compared with microbial fermentation, the yield of LAA synthesized by algae was too low and lacked competitiveness.

[0010] Some studies have attempted to introduce the plant L-galactose pathway into yeast. Yeast was long believed to have the ability to synthesize LAA, but it was soon discovered that the product of yeast suspected of LAA was actually iso-vitamin C. Isotretinoin is a 5-carbon analog of LAA, with similar antioxidant properties but without the same physiological function. In view of the similarity between the yeast isotretinoin synthesis pathway and the LAA synthesis pathway in plants, some researchers have used metabolic engineering strategies to transform GDP-mannose, a key intermediate in the plant biosynthesis of LAA, which is naturally produced in yeast and used for cell wall construction. Secondly, the last two steps of the biosynthesis of LAA in plants are similar to the production pathway of isotretinoin in yeast. And it has been demonstrated that yeast strains cultured in the presence of L-galactose can produce LAA. Therefore, by overexpressing genes of the endogenous substrate nonspecific isotretinoin pathway of yeast or exogenously expressing genes of the L-galactose pathway of Arabidopsis, the resulting strains can convert L-galactose, D-glucose and D-galactose into LAA. It is reported that the product titer of L-galactose is about 100 mg / L (DOI: 10.1128 / AEM.70.10.6086-6091.2004), the product titer of D-glucose is 0.1 mg l-1 OD-1 (https: / / doi.org / 10.1371 / journal.pone.0001092), and the product titer of D-galactose is 30 mg l-1 (https: / / doi.org / 10.1186 / 1475-2859-12-59). Since the L-galactose pathway introduced into plants synthesizes LAA, its biosynthetic pathway involves up to 10 steps, which means that the metabolic flux of LAA is low; and this process requires the use of GDP-D-mannose as a precursor for the synthesis of LAA. GDP-D-mannose is used for the construction of yeast cell walls, which will interfere with the natural metabolism of the chassis organism yeast.

[0011] Some researchers have also implanted the plant's D-galacturonic acid pathway into Aspergillus niger, using D-galacturonic acid or citrus peel that can be decomposed into D-galacturonic acid by Aspergillus niger pectinase as fermentation substrates, with a maximum yield of 170 mg / L (https: / / doi.org / 10.1186 / s12934-014-0184-2). However, when this work used D-galacturonic acid as a fermentation substrate to synthesize LAA, D-xylose needed to be added as a co-substrate; and citrus peel needed to be pre-processed before use. The complex composition of citrus peel also increased the difficulty of separating the product, increasing the time and economic cost of the entire LAA production.

[0012] The following patents have been published on the one-step fermentation synthesis of LAA by microorganisms: 1. Authorization announcement number: CN100447249C "Production of ascorbic acid by yeast". The work of this patent is to select L-galactose dehydrogenase, L-galactonic acid-1,4-lactone dehydrogenase, D-arabinose dehydrogenase, D-arabinonic acid-1,4-lactone oxidase, L-gulonic acid-1,4-lactone oxidase and aldonic acid lactonase and recombinant them into yeast of the genus Saccharomyces or Kluyveromyces, and culture the recombinant yeast in a medium containing LAA precursors to produce LAA, and the LAA precursors are selected from L-galactose; D-glucose; L-galactonic acid-1,4-lactone; L-gulonic acid; and L-gulonic acid-1,4-lactone; 2. Authorization announcement number: CN 1058055C "Microbial production of L-ascorbic acid". The organisms in the group consisting of prototheca organisms and chlorella protothecoides species are synthesized and recovered in a fermentation medium at pH 2.5-6.0. This patent mainly solves the problem of extracellular LAA, and at the disclosed pH, there will be no significant degradation of extracellular LAA due to oxidation; 3. Application publication number: CN107164291A "A method for constructing a vitamin C-producing Escherichia coli strain using a plant vitamin C synthesis pathway". Eleven genes from the LAA synthesis pathway in plants are transformed to construct expression elements regulated by a T7 promoter and terminator, and then all the expression elements are constructed into a vector to transform the Escherichia coli strain to achieve one-step synthesis of LAA by Escherichia coli using glucose. Although these patents all achieve one-step fermentation synthesis of LAA by microorganisms, they all use the synthesis pathway of LAA in plants, and their biosynthesis pathway involves multiple steps, and the LAA metabolic flux is low; and the pathway requires the use of GDP-D-mannose as a precursor substance for the synthesis of LAA, and GDP-D-mannose is used for the construction of yeast cell walls, which will interfere with the natural metabolism of the chassis biological yeast. The precursor substances required for the method mentioned in the patent, such as L-galactose, L-galactonic acid-1,4-lactone, etc., are rare sugars or derivatives of rare sugars, which are expensive and difficult to obtain. Summary of the invention

[0013] The primary purpose of the present invention is to provide an engineered bacterium for producing L-ascorbic acid. The engineered bacterium can utilize D-galactose to produce L-ascorbic acid. The synthesis of L-ascorbic acid involves four steps of catalysis and the yield of L-ascorbic acid is high.

[0014] The genetically engineered bacteria for producing L-ascorbic acid of the present invention contain a xylose reductase gene (XR), a mannitol dehydrogenase gene (MDH), an S136A mutated L-galactose dehydrogenase gene (LGDH) and a D-arabinose-1,4-lactone oxidase gene (ALO).

[0015] According to the further characteristics of the genetically engineered bacteria of the present invention, the xylose reductase gene (XR) comes from Hypocrea jecorina, and the protein sequence is shown in SEQ ID NO.1; the mannitol dehydrogenase gene (MDH) comes from Apium graveolens, and the protein sequence is shown in SEQ ID NO.2; the S136A mutated L-galactose dehydrogenase gene (LGDH) comes from Arabidopsis thaliana, and the protein sequence is shown in SEQ ID NO.3; the D-arabinose-1,4-lactone oxidase gene (ALO) comes from Pichia pastoris, and the protein sequence is shown in SEQ ID NO.4.

[0016] According to further features of the genetically engineered bacteria of the present invention, the genetically engineered bacteria contains a vector, and the xylose reductase gene (XR), the mannitol dehydrogenase gene (MDH), the S136A mutated L-galactose dehydrogenase gene (LGDH) and the D-arabinose-1,4-lactone oxidase gene (ALO) are included on the vector.

[0017] 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.

[0018] The invention also provides a method for constructing the genetically engineered bacteria.

[0019] The construction method of the genetically engineered bacteria of the present invention comprises the following steps: using XR gene sequence, MDH gene sequence, LGDH gene sequence and ALO gene sequence as templates respectively, adding respective upstream and downstream primers, and amplifying by conventional PCR to obtain rXR fragment, rMDH fragment, rLGDH fragment and rALO fragment respectively; connecting each amplified fragment between the multiple cloning sites of the vector to obtain XR-MDH-LGDH-ALO recombinant plasmid; after linearizing the recombinant plasmid, integrating it into the Pichia pastoris genome, and screening to obtain a Pichia pastoris recombinant strain that simultaneously expresses XR, MDH, LGDH and ALO.

[0020] According to a further feature of the method for constructing genetically engineered bacteria of the present invention, the method 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, The method comprises the following steps: connecting the fragment to a linearized XR-MDH recombinant plasmid, transforming the ligation product into Escherichia coli, and screening to obtain an XR-MDH-LGDH recombinant plasmid; taking an ALO gene sequence as a template, adding upstream and downstream primers, obtaining an rALO fragment by conventional PCR amplification, connecting the fragment to a linearized XR-MDH-LGDH recombinant plasmid, transforming the ligation product into Escherichia coli, and screening to obtain an XR-MDH-LGDH-ALO recombinant plasmid; transforming the XR-MDH-LGDH-ALO recombinant plasmid into saccharomyces cerevisiae or Escherichia coli, and screening to obtain a saccharomyces cerevisiae recombinant strain or an Escherichia coli recombinant strain simultaneously expressing XR, MDH, LGDH and ALO.

[0021] 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 L-ascorbic acid.

[0022] The present invention also provides a method for producing L-ascorbic acid.

[0023] The method for producing L-ascorbic acid 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 ascorbic acid precursor, and collecting L-ascorbic acid in the fermentation broth.

[0024] According to a further feature of the method for producing L-ascorbic acid of the present invention, the ascorbic acid precursor is selected from one of the following: D-galactose; galactitol; L-galactose; or L-galactonic acid-1,4-lactone.

[0025] The present invention provides the use of the genetically engineered bacteria in preparing a bacterial agent for producing L-galactose or L-galactonic acid-1,4-lactone.

[0026] The genetically engineered bacteria for producing LAA described in the present invention can utilize D-galactose to synthesize L-ascorbic acid through a four-step enzymatic reaction of galactitol, wherein the intermediates are D-galactose, galactitol, and L-galactose L-galactonic acid-1,4-lactone. The biosynthetic pathway involves only four steps, and is a new pathway for synthesizing L-ascorbic acid, named the "galactitol pathway". The biosynthetic process does not divert the natural metabolism of chassis microorganisms, thereby greatly improving the synthesis efficiency of LAA.

[0027] The process of producing L-ascorbic acid by using the 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 the engineered bacteria to make the initial concentration OD 600 =0.05, after culturing at 37°C and 200 r / min for 24 hours, D-galactose is added to an initial concentration of 2 g / L, and then cultured at 37°C to produce L-ascorbic acid. The yield of L-ascorbic acid fermented in the present application can be as high as 300 mg / L or more, which significantly improves the yield of L-ascorbic acid compared with the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The biosynthetic pathway for producing L-ascorbic acid by the engineered bacteria proposed in the present invention is shown.

[0029] Figure 2 The invention detects the production of L-ascorbic acid by the engineered bacteria.

[0030] Figure 3 This is a mass spectrometry identification diagram of L-ascorbic acid produced by the engineered bacteria of the present invention. DETAILED DESCRIPTION

[0031] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0032] like Figure 1As shown, in the present application, the genetically engineered bacteria contain an exogenous xylose reductase gene, and the expressed xylose reductase converts the substrate D-galactose into galactitol; the exogenous mannitol dehydrogenase gene is further integrated, and the expressed mannitol dehydrogenase converts galactitol into L-galactose. The L-galactose dehydrogenase gene with the S136A mutation, the expressed L-galactose dehydrogenase converts L-galactose into L-galactonic acid-1,4-lactone, and the D-arabinose-1,4-lactone oxidase gene is further integrated, and the expressed D-arabinose-1,4-lactone oxidase converts L-galactonic acid-1,4-lactone into L-ascorbic acid. Thus, L-ascorbic acid is synthesized from D-galactose in four steps, which greatly shortens the synthesis steps of L-ascorbic acid and improves the yield of L-ascorbic acid.

[0033] The present invention chemically synthesizes a xylose reductase (XR) gene from Hypocrea jecorina, a mannitol dehydrogenase (MDH) gene from Apium graveolens, an L-galactose dehydrogenase (LGDH) gene with an S136A mutation from Arabidopsis thaliana, and a D-arabinose-1,4-lactone oxidase (ALO) gene from Pichia pastoris. The embodiments of the present invention are described in detail below. This embodiment is implemented on the premise of the technical scheme 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 embodiments.

[0034] Terminology Note:

[0035] The xylose reductase gene is abbreviated as XR; the mannitol dehydrogenase gene is abbreviated as MDH; the S136A mutated L-galactose dehydrogenase gene is abbreviated as LGDH; and the D-arabinose-1,4-lactone oxidase gene is abbreviated as ALO.

[0036] This embodiment includes the following steps:

[0037] Example 1: Full gene synthesis of XR, MDH, LGDH and ALO genes

[0038] The present invention commissioned Shanghai Jierui Bioengineering Co., Ltd. to obtain 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, and the D-arabinose-1,4-lactone oxidase gene (ALO) from Pichia pastoris by means of total gene synthesis.

[0039] Example 2: Construction of recombinant Pichia pastoris GS115 pPIC3.5K XMLA

[0040] (1) Using the vector pGAPZαA as a template, the promoter P was amplified by conventional PCR. GAP -1 fragment. The primer used was P GAP1 -F and P GAP1 -R, as shown in Table 1, the 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.

[0041] (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, as shown in Table 1, the 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 subjected to 1% agarose gel electrophoresis and the corresponding fragment was recovered.

[0042] (3) Using the vector pGAPZαA as a template, the promoter P was amplified by conventional PCR. GAP -3 fragment. The primer used was P GAP2 -F and P GAP3 -R, as shown in Table 1, 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.

[0043] (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, as shown in Table 1, 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.

[0044] (5) Using the vector pGAPZα A 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, as shown in Table 1, 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, and the obtained sequence was subjected to 1% agarose gel electrophoresis to recover the corresponding fragment.

[0045] (6) Using the vector pGAPZα A 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, as shown in Table 1, 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 to recover the corresponding fragment.

[0046] (7) Using the vector pGAPZα A 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, as shown in Table 1, 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 to recover the corresponding fragment.

[0047] (8) The above recovered fragments were used as templates according to the table below, and the upper primer and the lower primer were added respectively to connect the two fragments by overlap extension PCR. The reaction conditions were: 95℃ 2min, 95℃ 20s, 55℃ 20s, 72℃ 10s, a total of 30 cycles; 72℃ 5min. The obtained sequence was subjected to 1% agarose gel electrophoresis and the corresponding fragments were recovered. They were named TP1, TP2 and TP3 respectively.

[0048] Table 1:

[0049] PCR template Primers DNA fragments <![CDATA[T AOX -1、P GAP -2]]> <![CDATA[T AOX1 -F P GAP2 -R]]> TP1 <![CDATA[T AOX -2、P GAP -3]]> <![CDATA[T AOX2 -F P GAP3 -R]]> TP2 <![CDATA[T AOX -3、P GAP -4]]> <![CDATA[T AOX3 -F P GAP4 -R]]> TP3

[0050] (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.

[0051] 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 sequence as homology arm;

[0052] 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 sequence as homology arm;

[0053] 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;

[0054] (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.

[0055] 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 P. pastoris GS115 pPIC3.5K XMLA simultaneously expressing XR, MDH, LGDH and ALO was obtained.

[0056] Table 2 Primer sequences

[0057]

[0058]

[0059] Example 3: Construction of recombinant Saccharomyces cerevisiae INVSc1 pYES2 XMLA rDNA

[0060] (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 pYES2-PXC.

[0061] (2) The phosphoglycerol 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 2F and PGK 2R are shown in Table 2. The terminator CYC1t sequence was amplified using the pESC-URA plasmid. 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 sequences were concatenated into a PGKp-MDH-CYC1t (PMC) expression cassette using the overlap extension PCR method. The PCR conditions are shown in Table 3. Then, the PMC expression cassette and the linearized recombinant vector pYES2-PXC fragment were seamlessly connected using a one-step cloning kit, and the connection product was transformed into E.coli DH5α. The plasmid in the positive clone was extracted to obtain the recombinant vector pYES2-PXMC.

[0062] (3) The phosphoglycerol 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 3F and PGK 3R are shown in Table 2. The terminator CYC1t sequence was amplified using the pESC-URA plasmid. The nucleotide coding sequences of the upstream and downstream primers CYC 3F and CYC 3R are shown in Table 2. The three sequences of PGKp, CYC1t and LGDH were concatenated into a PGKp-LGDH-CYC1t (PLC) expression cassette using the overlap extension PCR method. The PCR conditions are shown in Table 3. Then, the PLC expression cassette and the linearized recombinant vector pYES2-PXMC fragment were seamlessly connected using a one-step cloning kit, and the connection product was transformed into E.coli DH5α. The plasmid in the positive clone was extracted to obtain the recombinant vector pYES2-PXMLC.

[0063] (4) The phosphoglycerol 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 4F and PGK 4R are shown in Table 2. The terminator CYC1t sequence was amplified using the pESC-URA plasmid. 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 sequences were concatenated into a PGKp-ALO-CYC1t (PAC) expression cassette using the overlap extension PCR method. The PCR conditions are shown in Table 3. Then, the PAC expression cassette and the linearized recombinant vector pYES2-PXMLC fragment were seamlessly connected using a one-step cloning kit, and the connection product was transformed into E.coli DH5α. The plasmid in the positive clone was extracted to obtain the recombinant vector pYES2-PXMLAC.

[0064] (5) 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 pYES2-PXMLAC 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 XMLA rDNA. After the pYES2 XMLA 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, and a single colony grew after 3 to 5 days. The single clone was selected for colony PCR verification to obtain the S.cerevisiae INVSc1pYES2 XMLA rDNA engineering bacteria.

[0065] Table 3 Primer sequences

[0066]

[0067]

[0068] Table 4

[0069]

[0070] Example 4 Construction of recombinant Escherichia coli pET XMLA

[0071] (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.

[0072] (2) 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 2F and T7p2R are shown in Table 4; using the MDH sequence in Example 1 as a template, MDH-1F and MDH-1R are respectively the upstream and downstream primers to amplify the MDH-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-2F and T7t-2R are shown in Table 4; using the overlap extension PCR method, the obtained T7p, MDH-1 and T7t three sequences were concatenated into a T7p-MDH-1-T7t (TMT) expression cassette, the PCR conditions are shown in the following table, and then the above-mentioned 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.

[0073] (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 above-mentioned 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.

[0074] (4) 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 4F and T7p4R were shown in Table 4. Using the ALO sequence in Example 1 as a template, ALO-1F and ALO-1R were respectively the upstream and downstream primers to amplify the ALO-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-4F and T7t-4R were shown in Table 4. The obtained T7p, ALO-1 and T7t sequences were concatenated into a T7p-ALO-1-T7t (TAT) expression cassette using the overlap extension PCR method. The PCR conditions were shown in Table 3. 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.

[0075] Table 5 Primer sequences

[0076]

[0077]

[0078] (5) The plasmid pET-XR-MDH-LGDH-ALO was transformed into Escherichia coli BL21 (DE3) to obtain the Escherichia coli genetically engineered strain Escherichia coli pET XMLA for synthesizing LAA.

[0079] The Escherichia coli transformation method of plasmid pET-XR-MDH-LGDH-ALO is as follows: add plasmid pET-XR-MDH-LGDH-ALO to 50 μL Escherichia 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 XMLA.

[0080] Example 5: Production of LAA using the recombinant Pichia pastoris strain P. pastoris GS115 pPIC3.5K XMLA

[0081] A single colony of the recombinant Pichia pastoris strain P. pastoris GS115 pPIC3.5K XMLA was picked up, 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 to an initial concentration of OD600 = 0.05, and after culturing at 30°C and 200 r / min for 24 hours, D-galactose was added to an initial concentration of 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).

[0082] The formula of YPD medium is: yeast extract 10 g / L, peptone 20 g / L, D-glucose 20 g / L.

[0083] Example 6 Production of LAA using S. cerevisiae INVSc1 pYES2 XMLA rDNA engineered bacteria

[0084] A single colony of the recombinant strain of Saccharomyces cerevisiae INVSc1 pYES2 XMLA rDNA was picked up, inoculated into 5 mL of YPD liquid culture medium, and cultured overnight at 30°C and 200 r / min; the overnight culture solution was transferred to 250 mL of YPD liquid culture medium to an initial concentration of OD600 = 0.05, and cultured at 30°C and 200 r / min for 24 hours, then D-galactose was added to an initial concentration of 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).

[0085] The formula of YPD medium is: yeast extract 10 g / L, peptone 20 g / L, D-glucose 20 g / L.

[0086] Example 7 Production of LAA using Escherichia coli pET XMLA

[0087] A single colony of the recombinant Escherichia coli strain Escherichia coli pET XMLA 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 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 D-galactose, take 20mL of bacterial solution in a shake flask at 37°C and 200 rpm for 72 hours, and take samples every 12 hours to detect the LAA content in the supernatant by HPLC. The formula of LB medium is: 10g / L tryptone, 5g / L yeast powder, 10g / L sodium chloride, 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.

[0088] Example 8: HPLC detection of LAA

[0089] The culture system supernatant obtained in Examples 5, 6, and 7 was used for HPLC detection under the following conditions: Waters ACQUITYUPLC BEH C18 column (100 mm*2.1 mm, Particle Sz.1.7 μm), mobile phase acetic acid / water = 1 / 99, flow rate 0.5 mL / min, column temperature 40°C, detection wavelength 243 nm. The retention peak of L-ascorbic acid appeared at 7.0 min. The results are shown in Figure 2 . Test results: LAA production reached more than 300mg / L.

[0090] Example 9: LC-MS / MS Detection of LAA

[0091] 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 is shown in the table below.

[0092] Table 6:

[0093] 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

[0094] Mass spectrometry detection conditions:

[0095] 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.

[0096] The result was L-ascorbic acid, Figure 3 The substance with a molecular weight of 177.03 in the mass spectrum is [C6H8O6] + , which is the mass spectrum peak of L-ascorbic acid.

Claims

1. A genetically engineered bacterium for producing L-ascorbic acid, characterized in that: The genetically engineered bacteria contain a xylose reductase XR gene, a mannitol dehydrogenase MDH gene, an S136A mutated L-galactose dehydrogenase LGDH gene, and a D-arabinose-1,4-lactone oxidase ALO gene; The xylose reductase XR gene is from Hypocrea jecorina, and the protein sequence is shown in SEQ ID NO.1; the mannitol dehydrogenase MDH gene is from Apium graveolens, and the protein sequence is shown in SEQ ID NO.2; the S136A mutated L-galactose dehydrogenase LGDH gene is from Arabidopsis thaliana, and the protein sequence is shown in SEQ ID NO.3; the D-arabinose-1,4-lactone oxidase ALO gene is from Pichia pastoris, and the protein sequence is shown in SEQ ID NO.4; The genetically engineered bacteria contains a vector, and the xylose reductase XR gene, the mannitol dehydrogenase MDH gene, the S136A mutated L-galactose dehydrogenase LGDH gene and the D-arabinose-1,4-lactone oxidase ALO 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: The XR gene sequence, MDH gene sequence, LGDH gene sequence and ALO gene sequence were used as templates, and their respective upstream and downstream primers were added to amplify the rXR fragment, rMDH fragment, rLGDH fragment and rALO fragment respectively through conventional PCR; each amplified fragment was connected to the multiple cloning site of the vector to obtain the XR-MDH-LGDH-ALO recombinant plasmid; after linearization, the recombinant plasmid was integrated into the Pichia pastoris genome, and the Pichia pastoris recombinant strain simultaneously expressing XR, MDH, LGDH and ALO was screened.

3. 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; The XR-MDH-LGDH-ALO recombinant plasmid is transformed into Saccharomyces cerevisiae or Escherichia coli, and a recombinant strain of Saccharomyces cerevisiae or a recombinant strain of Escherichia coli simultaneously expressing XR, MDH, LGDH and ALO is obtained by screening.

4. Use of the genetically engineered bacteria as claimed in claim 1 in preparing a bacterial agent for producing L-ascorbic acid.

5. A method for producing L-ascorbic acid, characterized in that: The genetically engineered bacterium of claim 1 is provided, cultured in a medium containing at least one ascorbic acid precursor, and L-ascorbic acid is collected in the fermentation broth; the ascorbic acid precursor is selected from one of the following: D-galactose; galactitol; L-galactose; or L-galactonic acid-1,4-lactone.

6. Use of the genetically engineered bacteria according to claim 1 in preparing a bacterial agent for producing L-galactose or L-galactonic acid-1,4-lactone.

Images