Methyltransferase mutant, recombinant vector, recombinant bacterium and application of methyltransferase mutant in production of pterostilbene

Abstract

The invention discloses a methyltransferase mutant, a recombinant vector, a recombinant bacterium and application of the methyltransferase mutant in production of pterostilbene, and relates to the technical field of bioengineering. According to the methyltransferase mutant provided by the invention, resveratrol generates pterostilbene under the catalytic action of methyltransferase, and compared with the unmutated methyltransferase, the mutated methyltransferase has the advantages that the affinity of enzyme and a substrate is improved, and the resveratrol is promoted to be converted into pterostilbene. The coding gene of the methyltransferase mutant is transferred into a chassis cell or a chassis strain and is fermented, so that the yield of the pterostilbene can be greatly increased, the efficient synthesis of the plant-derived antioxidant pterostilbene in the microbial chassis cell is realized, and the ever-increasing market demand of the pterostilbene can be met.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and more particularly, to a methyltransferase mutant, a recombinant vector, a recombinant bacterium and their application in the production of pterostilbene. Background Art

[0002] Pterostilbene (C 16 H 16 O3) is a natural styrene compound with strong antioxidant activity and is widely present in plants such as blueberries, grapes, and pine trees. Pterostilbene has broad market prospects in many fields such as health foods, pharmaceuticals, cosmetics, and food additives. As an antioxidant, pterostilbene has high bioavailability and strong stability, making it widely used in the health field. Research shows that pterostilbene can not only effectively scavenge free radicals and reduce oxidative stress, but also has significant biological activities in aspects such as anti-inflammatory, anti-aging, blood sugar reduction, improvement of cardiovascular health, and enhancement of immune function. Pterostilbene is also widely used in high-end anti-aging and whitening skin care products due to its good skin care effect, helping to reduce wrinkles and improve skin elasticity. In addition, pterostilbene also shows potential application prospects in improving the nervous system and protecting eye health. With the in-depth study of its health benefits, the market demand for pterostilbene is increasing continuously.

[0003] Currently, the production methods of pterostilbene mainly include natural extraction and chemical synthesis. Natural extraction is to extract pterostilbene from pterostilbene-source plants (such as blueberries, grapes, and pine bark), but this method has problems such as limited raw material resources, high extraction costs, and low extraction efficiency, and is usually only applicable to the high-end market. The chemical synthesis method uses phloroglucinol as a precursor and synthesizes pterostilbene through selective methylation modification. However, due to the high cost of the substrate phloroglucinol, it is not suitable for large-scale industrial production. Compared with the above methods, the engineering microorganism fermentation method modifies microorganisms through genetic engineering technology to construct an efficient de novo synthesis pathway of pterostilbene, becoming a more promising production method. The advantages of this method are that it can use cheap carbon sources (such as glucose, glycerol, etc.) for fermentation, is not affected by climate and environment, and can carry out large-scale and sustainable production. Secondly, the engineering microorganism fermentation method has a short fermentation cycle and relatively low production costs. In addition, by optimizing the metabolic pathway of microorganisms, the yield of pterostilbene can be significantly increased. At present, although the engineering microorganism fermentation method has gradually received attention in the production of pterostilbene, there is still a lack of microbial strains that can efficiently ferment and produce pterostilbene in the prior art.

[0004] Yarrowia lipolytica has excellent metabolic programmability and industrial production advantages. First, Yarrowia lipolytica can effectively use cheap substrates as carbon sources, has strong tolerance and robustness, and can grow under different environmental conditions. Secondly, Yarrowia lipolytica has significant advantages in heterologous gene expression and can efficiently integrate the biosynthetic pathway of pterostilbene. Thirdly, Yarrowia lipolytica has a good secretion capacity for pterostilbene, which helps to simplify the downstream extraction process and reduce separation costs. In addition, Yarrowia lipolytica is resistant to oxidative stress and has strong antioxidant capacity, which is also consistent with the antioxidant properties of pterostilbene. Finally, Yarrowia lipolytica is a generally recognized as safe (GRAS) yeast certified by the US FDA and is commonly used in food and pharmaceutical industries. Therefore, Yarrowia lipolytica is an ideal microbial platform for the production of pterostilbene.

[0005] At present, there is a problem that the pterostilbene yield and conversion rate cannot be taken into account at the same time. Although the caffeic acid-O-methyltransferase COMT from Arabidopsis thaliana can increase the pterostilbene yield after being transferred into Yarrowia lipolytica compared with the methyltransferase from grape, there is a problem that the conversion rate of pterostilbene is low. Although the conversion rate of pterostilbene is high after the methyltransferase from grape is transferred into Yarrowia lipolytica, the pterostilbene yield is low.

[0006] Based on the above background, the present invention aims to provide a method for engineering Yarrowia lipolytica to synthesize pterostilbene from scratch, so as to meet the growing market demand for pterostilbene.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The object of the present invention is to provide a methyltransferase mutant, a recombinant vector, a recombinant bacterium and the use thereof in producing pterostilbene to solve the above technical problems.

[0009] The present invention is achieved in that:

[0010] In a first aspect, the present invention provides a methyltransferase mutant having at least one of the following mutations compared to a wild-type methyltransferase from grape: Y149H, S225P and N265D, the amino acid sequence of the wild-type methyltransferase being shown in SEQ ID NO:5.

[0011] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned methyltransferase mutant;

[0012] In a preferred embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:4.

[0013] In a third aspect, the present invention provides an expression cassette, which comprises a promoter, the above-mentioned nucleic acid molecule, and a terminator;

[0014] In a preferred embodiment of the application of the present invention, the promoter and the terminator are from Yarrowia lipolytica Po1fk, and Yarrowia lipolytica Po1fk is obtained by knocking out the ku70 gene of Yarrowia lipolytica Po1f;

[0015] In a preferred embodiment of the application of the present invention, the promoter is selected from the promoters P TEFin , P EXP , P TDH1 , P FBA or P GPD of Yarrowia lipolytica; the terminator is selected from the terminators T Xpr2 , T Mig1 , T Pex20 , T Lip1 or T Lip2 of Yarrowia lipolytica, and their coding nucleotide sequences are respectively shown as SEQ ID NO: 6-15.

[0016] In a fourth aspect, the present invention provides a recombinant vector, which comprises the above-mentioned nucleic acid molecule or the above-mentioned expression cassette.

[0017] In a preferred embodiment of the application of the present invention, the recombinant vector further comprises: upstream homologous sequences and downstream homologous sequences of IntA site, IntB site, IntC1 site, IntC3 site, IntD site, IntE site, IntF site, Dga1 site, Lip1 site or Scp2 site from Yarrowia lipolytica Po1fk; the nucleic acid molecule or the expression cassette is inserted between the upstream homologous sequence and the downstream homologous sequence;

[0018] In a preferred embodiment of the application of the present invention, the recombinant vector further comprises at least one of the following expression cassettes: an expression cassette of an enzyme in the tyrosine biosynthetic pathway, an expression cassette of tyrosine ammonia-lyase, an expression cassette of 4-coumaroyl-CoA ligase, and a gene expression cassette of stilbene synthase;

[0019] In a preferred embodiment of the application of the present invention, the enzyme in the tyrosine biosynthetic pathway is selected from at least one of aromatic multifunctional enzyme, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, and chorismate mutase;

[0020] In a preferred embodiment of the application of the present invention, the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase is a mutant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase. Compared with the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of wild-type Yarrowia lipolytica, the mutant 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase has the following mutations: K221L and G139S;

[0021] In a preferred embodiment of the application of the present invention, the copy number of the nucleic acid molecule on the recombinant vector or the above-mentioned expression cassette is 1-5;

[0022] In a preferred embodiment of the application of the present invention, the copy number of the nucleic acid molecule on the recombinant vector or the above-mentioned expression cassette is 3-5;

[0023] In a preferred embodiment of the application of the present invention, the backbone of the recombinant vector is pUrLp or pYLXP';

[0024] In a preferred embodiment of the application of the present invention, the recombinant vector further includes a loxP tag and an orotidine-5'-phosphate decarboxylase encoding gene (Ura3) expression cassette, and the orotidine-5'-phosphate decarboxylase encoding gene expression cassette is located between the loxP tags.

[0025] Fifthly, the present invention provides a recombinant cell or recombinant bacterium, which includes the above-mentioned nucleic acid molecule, the above-mentioned expression cassette or the above-mentioned recombinant vector; the recombinant cell is a non-plant cell;

[0026] In a preferred embodiment of the application of the present invention, the recombinant bacterium is Escherichia coli or yeast;

[0027] In a preferred embodiment of the application of the present invention, the yeast is selected from Candida, Pichia, Saccharomyces cerevisiae or Yarrowia lipolytica;

[0028] In a preferred embodiment of the application of the present invention, the Yarrowia lipolytica is a Yarrowia lipolytica with the ku70 gene knocked out.

[0029] In a preferred embodiment of the application of the present invention, the recombinant cell or recombinant bacterium expresses: the above-mentioned methyltransferase mutant, the enzymes in the tyrosine biosynthetic pathway, tyrosine ammonia-lyase, 4-coumaroyl-CoA ligase and stilbene synthase.

[0030] Sixthly, the present invention provides the application of the methyltransferase mutant, the above-mentioned nucleic acid molecule, the above-mentioned expression cassette, the above-mentioned recombinant vector, the above-mentioned recombinant cell or the above-mentioned recombinant bacterium in the production of pterostilbene.

[0031] Seventhly, the present invention provides a method for producing pterostilbene, which comprises culturing the above-mentioned recombinant cell or recombinant bacterium, and extracting pterostilbene from the culture of the recombinant cell or recombinant bacterium.

[0032] In a preferred embodiment of the application of the present invention, the culture is carried out with shaking at 30°C ± 2°C and 200 - 250 rpm for 3 - 10 days.

[0033] In a preferred embodiment of the application of the present invention, the fermentation medium formula is as follows: glucose 20 - 80 g / L, yeast extract 1 - 10 g / L, peptone 2 - 20 g / L.

[0034] The present invention has the following beneficial effects:

[0035] The present invention provides a methyltransferase mutant. Under the catalytic action of the methyltransferase, resveratrol is converted into pterostilbene. Compared with that before mutation, the mutated methyltransferase improves the affinity between the enzyme and the substrate, and promotes the conversion of resveratrol into pterostilbene. Compared with the recombinant bacterium containing caffeic acid O-methyltransferase COMT derived from Arabidopsis thaliana, the mutated methyltransferase improves the enzyme affinity and the efficiency of resveratrol being catalyzed to produce pterostilbene. Transferring the coding gene of the methyltransferase mutant into a chassis cell or a chassis strain, after fermentation, the yield of pterostilbene can be greatly increased, realizing the efficient synthesis of the plant-derived antioxidant pterostilbene in microbial chassis cells, and meeting the growing market demand for pterostilbene. Brief Description of the Drawings

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0037] Figure 1 It is a de novo synthesis pathway diagram of pterostilbene provided by the present invention;

[0038] Figure 2 It is the construction process of the recombinant plasmid in Example 1 of the present invention;

[0039] Figure 3 It is the map of the site integration plasmid pUrLp-ΔIntA;

[0040] Figure 4 It is the map of the recombinant plasmid pUrLp-ΔIntA-YlARO1-YlARO4-YlARO7 constructed in Example 1 of the present invention;

[0041] Figure 5 It is the map of the recombinant plasmid pUrLp-ΔDga1-FjTAL-(At4CL1~VvSTS) constructed in Example 1 of the present invention;

[0042] Figure 6 The map of the recombinant plasmid pUrLp-ΔIntC3-VvROMT3*3 constructed in Example 1 of the present invention;

[0043] Figure 7 The HPLC map of the recombinant bacterium 3 fermenting to produce pterostilbene provided in Example 2 of the present invention;

[0044] Figure 8 The HPLC maps of the recombinant bacteria fermenting to produce pterostilbene provided in Comparative Examples 1-3, Example 2, and Examples 4-10 of the present invention;

[0045] Figure 9 The vector map of the initial plasmid pYLXP'. Detailed Description of the Invention

[0046] Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0047] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those of ordinary skill in the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J.E. Coligan et al., eds., 1991), each of which is hereby expressly incorporated by reference.

[0048] The term "nucleic acid molecule" refers to a sequence of nucleoside or nucleotide monomers consisting of natural bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid molecules of the present invention can be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA), and can contain natural bases, including adenine, guanine, cytosine, thymine, and uracil. Modified bases can also be included. Examples of such modified bases include nitrogen-containing and deazaanalogues of adenine, guanine, cytosine, thymine, and uracil; and xanthine and hypoxanthine.

[0049] The term "vector" includes any intermediate agent for nucleic acids, which can enable the nucleic acids to be introduced into prokaryotic and / or eukaryotic cells, for example, and integrated into the genome when appropriate. Vectors of this type preferably replicate and / or express in cells. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material, usually circular double-stranded DNA, which can replicate independently of chromosomal DNA. Any plasmid and vector can be used as long as it can replicate and be stable in the host.

[0050] The term "recombinant cell" refers to any cell that can be transformed or transfected with exogenous nucleic acids. The term "recombinant cell" according to the present invention includes prokaryotes (e.g., Escherichia coli) or eukaryotic cells (e.g., mammalian cells, especially human cells, yeast cells and insect cells). Mammalian cells are particularly preferred, such as cells from humans, mice, hamsters, pigs, goats or primates. The cells can be derived from multiple tissue types and include primary cells and cell lines. The nucleic acid can exist in the host cell in single copy form or in two or more copy forms, and in one embodiment, is expressed in the recombinant cell. The recombinant cell can be a competent cell.

[0051] In a first aspect, the present invention provides a methyltransferase mutant, which has at least one of the following mutations compared with the wild-type methyltransferase from grapes: Y149H, S225P and N265D, and the amino acid sequence of the wild-type methyltransferase is shown in SEQ ID NO: 5.

[0052] The inventors found that the methyltransferase from grapes has a better pterostilbene yield after being transferred into Yarrowia lipolytica than the caffeic acid O-methyltransferase COMT from Arabidopsis thaliana.

[0053] Compared with before mutation, the mutated methyltransferase improves the affinity between the enzyme and the substrate, and promotes the conversion of resveratrol to pterostilbene. Transferring the coding gene of the methyltransferase mutant into a chassis cell or a chassis strain, after fermentation, can greatly increase the yield of pterostilbene, realizing the efficient synthesis of the plant-derived antioxidant pterostilbene in Yarrowia lipolytica, and can meet the growing market demand for pterostilbene.

[0054] Simultaneously having three mutations of Y149H, S225P and N265D, it has a higher pterostilbene yield after being transferred into Yarrowia lipolytica than the methyltransferase mutants with single mutations (Y149H, S225P or N265D) or the methyltransferase mutants with double mutations (Y149H and S225P, or Y149H and N265D, or Y149H and N265D). In addition, the methyltransferase mutants with single mutations (Y149H, S225P or N265D) have a higher pterostilbene yield after being transferred into Yarrowia lipolytica than the wild-type methyltransferase.

[0055] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned methyltransferase mutant.

[0056] In the case where the present invention provides the amino acid sequence of the above-mentioned methyltransferase mutant (VvRomT3), those skilled in the art can easily obtain the nucleic acid sequence encoding the above-mentioned methyltransferase mutant according to the degeneracy of codons. For example, the nucleic acid sequence encoding the above-mentioned methyltransferase mutant can be obtained by making corresponding nucleotide mutations on the nucleic acid sequence encoding the wild-type methyltransferase. This is easily achievable for those skilled in the art.

[0057] In a preferred embodiment of the application of the present invention, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 4.

[0058] In a third aspect, the present invention provides an expression cassette, which includes a promoter, the above-mentioned nucleic acid molecule and a terminator;

[0059] In a preferred embodiment of the application of the present invention, the promoter and the terminator are from Yarrowia lipolytica Po1fk, and Yarrowia lipolytica Po1fk is obtained by knocking out the ku70 gene of Yarrowia lipolytica Po1f.

[0060] Yarrowia lipolytica Po1f was purchased from the American Type Culture Collection with the accession number ATCC MYA-2613. Yarrowia lipolytica Po1fΔku70 (MatA, Δku70, leu2-270, ura3-302, xpr2-322, axp1-2), abbreviated as Yarrowia lipolytica Po1fk. Yarrowia lipolytica Po1fk was constructed by knocking out the enzyme-encoding gene ku70 (GenBank accession number: YALI0C08701g) responsible for non-homologous recombination from Yarrowia lipolytica Po1f (published in Kretzschmar A, et al., Current Genetics, 2013, 59(1-2):63-72).

[0061] In a preferred embodiment of the application of the present invention, the promoter is selected from P TEFin , P EXP , P TDH1 , P FBA or P GPD ; the terminator is selected from T Xpr2 , T Mig1 , T Pex20 , T Lip1 or T Lip2 , and their coding nucleotide sequences are shown in SEQ ID NO: 6-15 in sequence.

[0062] In one embodiment, the above expression cassette expressing the methyltransferase mutant is introduced into Yarrowia lipolytica Po1fk in the form of a linearized plasmid, and then integrated into the genome by homologous recombination.

[0063] Fourthly, the present invention provides a recombinant vector, which comprises the above nucleic acid molecule or the above expression cassette.

[0064] In an alternative embodiment, an important feature of the above recombinant vector is that it usually contains an origin of replication, a promoter, a terminator, a marker gene and a translation control element.

[0065] In a preferred embodiment of the application of the present invention, the recombinant vector further comprises: upstream and downstream homologous sequences from the IntA locus, IntB locus, IntC1 locus, IntC3 locus, IntD locus, IntE locus, IntF locus, Dga1 locus, Lip1 locus or Scp2 locus of Yarrowia lipolytica Po1fk; the nucleic acid molecule or expression cassette is inserted between the upstream and downstream homologous sequences.

[0066] Those skilled in the art can insert the above-mentioned expression cassette between the upstream and downstream homologous sequences of any one of the above-mentioned loci in Yarrowia lipolytica according to needs.

[0067] The IntA locus, IntB locus, IntC1 locus, IntC3 locus, IntD locus, IntE locus and IntF locus are non-functional sequences on Yarrowia lipolytica Po1fk. Dga1 is a fatty acid synthesis and metabolism gene. After knocking out Dga1, the accumulation of fatty acids in Yarrowia lipolytica is reduced, preventing competitive inhibition of the synthesis of the target compound. The Lip1 locus or Scp2 locus is a fatty acid degradation and metabolism gene. After knocking out Lip1 or Scp2, the degradation of fatty acids in Yarrowia lipolytica can be reduced.

[0068] In a preferred embodiment of the application of the present invention, the recombinant vector further comprises at least one of the following expression cassettes: an expression cassette of an enzyme in the tyrosine biosynthetic pathway, an expression cassette of tyrosine ammonia-lyase (TAL), an expression cassette of 4-coumaroyl-CoA ligase (4CL1), and a gene expression cassette of stilbene synthase (STS).

[0069] The above-mentioned expression cassette of an enzyme in the tyrosine biosynthetic pathway, the expression cassette of tyrosine ammonia-lyase (TAL), the expression cassette of 4-coumaroyl-CoA ligase (4CL1), and the gene expression cassette of stilbene synthase (STS) are inserted between the upstream and downstream homologous sequences of any one of the above-mentioned loci in Yarrowia lipolytica.

[0070] In a preferred embodiment of the application of the present invention, the enzyme in the tyrosine biosynthetic pathway is selected from at least one of aromatic multifunctional enzyme (YlARO1), 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (YlARO4), and chorismate mutase (YlARO7);

[0071] In the present invention, YlARO1, YlARO4, and YlARO7 are all from Yarrowia lipolytica; TAL is from Flavobacterium johnsoniae, thus abbreviated as FjTAL; 4CL1 is from Arabidopsis thaliana, abbreviated as At4CL1, and STS and RomT3 are both from Vitis vinifera, correspondingly abbreviated as VvSTS and VvRomT3 respectively.

[0072] In the present invention, the Genbank accession numbers of the enzymes YlARO1, YlARO4, and YlARO7 derived from Yarrowia lipolytica are: YALI0C18645g, YALI0B22440g, and YALI0E17479g respectively.

[0073] In one embodiment, three expression cassettes of YlARO1 - YlARO4 - YlARO7 are inserted between the upstream and downstream homologous sequences of the IntA site, and three expression cassettes of FjTAL - (At4CL1 - VvSTS) are inserted between the upstream and downstream homologous sequences of the Dga1 site, where (At4CL1 - VvSTS) refers to an expression cassette that fuses and expresses At4CL1, a rigid linker peptide, and VvSTS. A VvROMT3*3 expression cassette is inserted between the upstream and downstream homologous sequences of the IntC3 site.

[0074] In a preferred embodiment of the application of the present invention, the 3 - deoxy - D - arabino - heptulosonate 7 - phosphate synthase is a mutant 3 - deoxy - D - arabino - heptulosonate 7 - phosphate synthase. Compared with the 3 - deoxy - D - arabino - heptulosonate 7 - phosphate synthase of wild - type Yarrowia lipolytica, the mutant 3 - deoxy - D - arabino - heptulosonate 7 - phosphate synthase has the following mutation: K221L. The chorismate mutase is a mutant chorismate mutase. Compared with the chorismate mutase of wild - type Yarrowia lipolytica, the mutant chorismate mutase has the following mutation: G139S.

[0075] By introducing specific site mutations, the feedback inhibition of the tyrosine biosynthesis pathway was lifted. The above mutations effectively alleviated the feedback inhibition of L - tyrosine in the tyrosine biosynthesis pathway on 3 - deoxy - D - arabino - heptulosonate 7 - phosphate synthase (YlARO4) and chorismate mutase. The mutant 3 - deoxy - D - arabino - heptulosonate 7 - phosphate synthase and chorismate mutase have higher substrate specificity and improved enzyme activity.

[0076] The present invention constructs a recombinant Yarrowia lipolytica strain for highly efficient fermentation production of pterostilbene using glucose as a substrate by relieving the feedback inhibition of the tyrosine biosynthesis pathway, fusing the expression of 4-coumarate ligase and stilbene synthase, and mutating trans-resveratrol dimethyltransferase (i.e., methyltransferase). Experiments have proven that this recombinant strain can significantly increase the final yield of pterostilbene when fermented in a 24-deep well plate for 96 hours, achieving the highly efficient synthesis of the plant-derived antioxidant pterostilbene in Yarrowia lipolytica.

[0077] In a preferred embodiment of the application of the present invention, the copy number of the nucleic acid molecule on the recombinant vector or the above-mentioned expression cassette is 1-5;

[0078] In a preferred embodiment of the application of the present invention, the copy number of the nucleic acid molecule on the recombinant vector or the above-mentioned expression cassette is 3-5; when the copy number of the above-mentioned expression cassette on the recombinant vector is 3-5, a higher pterostilbene yield can be obtained.

[0079] In a preferred embodiment of the application of the present invention, the backbone of the recombinant vector is pUrLp or pYLXP'.

[0080] In a preferred embodiment of the application of the present invention, the recombinant vector further includes a loxP tag and an orotidine-5'-phosphate decarboxylase encoding gene (Ura3) expression cassette, and the orotidine-5'-phosphate decarboxylase encoding gene expression cassette is located between the loxP tags.

[0081] Preferably, there is a rigid linker peptide encoding gene between the expression cassette of 4-coumaroyl-CoA ligase and the expression cassette of stilbene synthase;

[0082] Preferably, the rigid linker peptide is EAAAK; through the rigid linker peptide, the activities of 4-coumaroyl-CoA ligase and stilbene synthase are enhanced, the distance between the substrate and the enzyme is shortened, and the loss of intermediates is reduced.

[0083] Preferably, the nucleotide sequence of the encoding gene of tyrosine ammonia-lyase is as shown in SEQ ID No: 1, the nucleotide sequence of the encoding gene of 4-coumaroyl-CoA ligase is as shown in SEQ ID No: 2, and the nucleotide sequence of the encoding gene of stilbene synthase is as shown in SEQ ID No: 3.

[0084] In a fifth aspect, the present invention provides a recombinant cell or recombinant bacterium, which includes the above-mentioned nucleic acid molecule, the above-mentioned expression cassette or the above-mentioned recombinant vector; the recombinant cell is a non-plant cell;

[0085] In a preferred embodiment of the application of the present invention, the recombinant bacterium is Escherichia coli or yeast;

[0086] In a preferred embodiment of the application of the present invention, the yeast is selected from Candida, Pichia pastoris, Saccharomyces cerevisiae or Yarrowia lipolytica;

[0087] In a preferred embodiment of the application of the present invention, the Yarrowia lipolytica is the Yarrowia lipolytica with the ku70 gene knocked out.

[0088] In a preferred embodiment of the application of the present invention, the recombinant cell or recombinant bacterium expresses: the above-mentioned methyltransferase mutant, the enzymes in the tyrosine biosynthetic pathway, tyrosine ammonia-lyase, 4-coumaroyl-CoA ligase and stilbene synthase.

[0089] In a sixth aspect, the present invention provides the application of the methyltransferase mutant, the above-mentioned nucleic acid molecule, the above-mentioned expression cassette, the above-mentioned recombinant vector, the above-mentioned recombinant cell or the above-mentioned recombinant bacterium in the production of pterostilbene.

[0090] In one embodiment, the above-mentioned nucleic acid molecule is introduced into the target cell;

[0091] The above-mentioned recombinant vector is introduced into the target cell, and the vector contains the coding gene encoding the methyltransferase mutant or contains the expression cassette.

[0092] In one embodiment, first, a recombinant vector expressing the methyltransferase mutant, a vector expressing the enzymes in the tyrosine biosynthetic pathway, a vector expressing tyrosine ammonia-lyase, a vector expressing 4-coumaroyl-CoA ligase and a vector expressing stilbene synthase are constructed respectively. Preferably, 4-coumaroyl-CoA ligase and stilbene synthase are fused and expressed, and a rigid linker peptide is arranged between 4-coumaroyl-CoA ligase and stilbene synthase. Then the above-mentioned vectors are introduced into Yarrowia lipolytica by genetic engineering methods respectively.

[0093] In a seventh aspect, the present invention provides a method for producing pterostilbene, culturing the above-mentioned recombinant cell or recombinant bacterium, and extracting pterostilbene from the culture of the recombinant cell or recombinant bacterium.

[0094] The morphology of the recombinant bacterium is, for example, the culture of Yarrowia lipolytica, for example, the bacterial agent. The culture protection is not limited to being obtained by solid fermentation or liquid fermentation.

[0095] The culture of Yarrowia lipolytica includes but is not limited to at least one of the concentrate, paste, dry matter, liquid matter, and diluent of Yarrowia lipolytica. The dry matter includes but is not limited to spray-dried matter, freeze-dried matter, vacuum-dried matter, drum-dried matter, etc.

[0096] In a preferred embodiment of the application of the present invention, it is cultured with shaking at 30 °C and 200-250 rpm for 3-10 days.

[0097] In a preferred embodiment of the application of the present invention, the fermentation medium formula is: glucose 20 - 80 g / L, yeast extract 1 - 10 g / L, peptone 2 - 20 g / L.

[0098] For example, pterostilbene is extracted by cryogenic grinding treatment with a methanol solution containing grinding particles, centrifuging to obtain the supernatant, and separating through high performance liquid chromatography.

[0099] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0100] The features and performance of the present invention will be further described in detail below in conjunction with the embodiments.

[0101] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0102] Yarrowia lipolytica Po1f was purchased from the American Type Culture Collection, with the accession number ATCC MYA - 2613. Yarrowia lipolytica Po1fΔku70 (MatA, Δku70, leu2 - 270, ura3 - 302, xpr2 - 322, axp1 - 2), abbreviated as Yarrowia lipolytica Po1fk. Yarrowia lipolytica Po1fk was constructed by knocking out the enzyme - encoding gene ku70 (GenBank accession number: YALI0C08701g) responsible for non - homologous recombination from Yarrowia lipolytica Po1f (published in Kretzschmar A, et al., Current Genetics, 2013, 59(1 - 2):63 - 72).

[0103] Example 1

[0104] This example provides a method for constructing a recombinant vector.

[0105] (I) Preparation of the target gene

[0106] According to the nucleotide sequences of the encoding genes of FjTAL, At4CL1, VvSTS, and VvRomT3 (UniProt ID: A5FKY3, Q42524, P28343, B6VJS4) provided on UniProt, after codon optimization, GenScript Biotech Corporation was commissioned to synthesize the optimized encoding genes of FjTAL, At4CL1, VvSTS, and VvRomT3 (SEQ ID No: 1, 2, 3, 4).

[0107] Using the genomic DNA of Yarrowia lipolytica Po1fk as a template, YlARO1, YlARO4, and YlARO7 were amplified.

[0108] Using the genomic DNA of Yarrowia lipolytica Po1fk as a template, the promoters P TEFin , P EXP , P TDH1 , P FBA , P GPD sequences and the terminators T Xpr2 , T Mig1 , T Pex20 , T Lip1 , T Lip2 sequences were amplified. The nucleotide sequences of P TEFin , P EXP , P TDH1 , P FBA , P GPD , T Xpr2 , T Mig1 , T Pex20 , T Lip1 , T Lip2 are shown in SEQ ID NO: 6 - 15 in sequence.

[0109] (II) Construction of Recombinant Plasmids

[0110] The recombinant plasmids and their uses are shown in Table 1; the primers used for constructing the recombinant plasmids are shown in Table 2.

[0111] 1. Construction of Recombinant Plasmid pYLXP'-YlARO1

[0112] The recombinant plasmid pYLXP'-YlARO1 uses the initial plasmid pYLXP' as a backbone, and the YlARO1 gene is inserted between the promoter P TEFin and the terminator T Xpr2 to assemble the YlARO1 gene expression cassette (P TEFin -YlARO1-T Xpr2 ).

[0113] The initial plasmid pYLXP' contains the promoter P endogenous to Yarrowia lipolytica TEFin and the terminator T Xpr2 , and single or multiple fragments can be inserted by double digestion with restriction enzymes SnaBI and KpnI followed by Gibson assembly technology; the initial plasmid pYL31 contains the promoter P FBA and the terminator T Pex20 , and single or multiple fragments can be inserted by single digestion with restriction enzyme BamHI followed by Gibson assembly technology; the initial plasmid pYL24 contains the promoter P TDH1 and the terminator T Mig1 , and single or multiple fragments can be inserted by double digestion with restriction enzymes XbaI and KpnI followed by Gibson assembly technology.

[0114] Using XP-YlARO1-F and XP-YlARO1-R as primers and the genomic DNA of Yarrowia lipolytica Po1fk as a template, the YlARO1 gene was amplified.

[0115] The above PCR amplification system is as follows:

[0116]

[0117] Among them, 2×Phanta Max Master Mix (Dye Plus) was purchased from Nanjing Novizan Biotech Co., Ltd.

[0118] The procedure of the above PCR is as follows: denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C (extension time = target fragment length / 1 kb, unit: min), and repeat 34 cycles.

[0119] Use Gel Extraction Kit agarose gel recovery kit (purchased from Yeasen Biotech Co., Ltd.) to purify and recover each fragment.

[0120] After double digestion of the initial plasmid pYLXP' with restriction enzymes SnaBI and KpnI from TaKaRa, the linearized initial plasmid pYLXP' was recovered by agarose gel electrophoresis gel recovery.

[0121] The vector map of the initial plasmid pYLXP' is referred to Figure 9As shown, the linearized initial plasmid pYLXP' and the YlARO1 gene prepared in this example were subjected to Gibson assembly using the Seamless Cloning Kit of Shanghai Beyotime Biotechnology Co., Ltd., and the YlARO1 gene was inserted into the promoter P of the plasmid pYLXP' TEFin and the terminator T Xpr2 therebetween, thereby assembling the YlARO1 gene expression cassette (P TEFin -YlARO1-T Xpr2 ). The reaction system is shown in the following table. After incubating the reaction system at 37 °C for 30 min, a circular recombinant vector was obtained.

[0122] The system for Gibson assembly is as follows in the table:

[0123]

[0124]

[0125] Among them, the usage amounts of the linearized vector (x) and the inserted fragment (y) can be calculated by the following formula: The optimal usage amount of each fragment or linearized vector = [0.02 × the number of base pairs of the fragment or linearized vector] ng.

[0126] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, screened by an ampicillin-resistant plate, and verified by colony PCR and sequencing to obtain the recombinant plasmid pYLXP'-YlARO1.

[0127] Similarly, recombinant plasmids pYL31-YlARO4, pYL24-YlARO7, pYL24-FjTAL, pYLXP'-(At4CL1~VvSTS), pYLXP'-VvROM3, pYL31-VvROM3, and pYL24-VvROM3 can be constructed by the above method.

[0128] 2. Construction of the recombinant plasmid pUrLp-ΔIntA-YlARO1-YlARO4-YlARO7

[0129] The recombinant plasmid pUrLp-ΔIntA-YlARO1-YlARO4-YlARO7 uses pUrLp as the backbone, inserts a 1 kb homologous arm IntA-up upstream of the start codon at the IntA site and a 1 kb homologous arm IntA-dn downstream of the stop codon in Yarrowia lipolytica Po1fk, and also inserts the YlARO1 gene expression cassette (P TEFin -YlARO1-T Xpr2 ) and the YlARO4 gene expression cassette (P FBA-YlARO4-T Pex20 )、YlARO7 gene expression cassette (P TDH1 -YlARO7-T Mig1 ), two loxP tags and orotidine-5'-phosphate decarboxylase encoding gene expression cassette (loxP-P TEFin -URA3-T Xpr2 -loxP) are also between the upstream and downstream homologous arms of the IntA locus.

[0130] The initial plasmid pUrLp contains two loxP tags and orotidine-5'-phosphate decarboxylase encoding gene expression cassette (loxP-P TEFin -URA3-T Xpr2 -loxP). Using the Gibson assembly technology mentioned in Title 1 of this example, the 1 kb homologous arm IntA-up upstream of the start codon and the 1 kb homologous arm IntA-dn downstream of the stop codon of the IntA locus can be inserted successively on the initial plasmid pUrLp to obtain the site integration plasmid pUrLp-ΔIntA( Figure 3 ).

[0131] The site integration plasmid pUrLp-ΔIntA was digested with the restriction enzymes NheI / SalI, and the linearized site integration plasmid pUrLp-ΔIntA was recovered. Then, the plasmid carrying the target gene expression cassette constructed based on pYLXP', pYL31, and pYL24 was digested with the restriction enzymes AvrII / SalI, and the linearized target gene expression cassette was recovered. Since NheI and AvrII are isocaudamers, the linearized site integration plasmid pUrLp-ΔIntA and the linearized target gene expression cassette can be ligated into a complete recombinant plasmid by the action of T4 DNA ligase. By ligating multiple times, a recombinant plasmid that can express multiple target genes simultaneously can be constructed. The specific principle is shown in Figure 2 .

[0132] After digesting the plasmid pUrLp-ΔIntA with the restriction enzymes NheI and SalI from TaKaRa, the linearized pUrLp-ΔIntA plasmid was recovered by agarose gel electrophoresis gel extraction.

[0133] After digesting the plasmid pYLXP'-YlARO1 with the restriction enzymes AvrII and SalI from TaKaRa, the linearized YlARO1 gene expression cassette (P TEFin -YlARO1-T Xpr2 ) was recovered by agarose gel electrophoresis gel extraction.

[0134] The linearized pUrLp-ΔIntA plasmid and the YlARO1 gene expression cassette (PTEFin -YlARO1-T Xpr2 ) The ligation was achieved using the T4 DNA Ligase from Shanghai Beyotime Biotechnology Co., Ltd. to obtain a circular recombinant vector.

[0135] The T4 DNA Ligase reaction system is as follows:

[0136] Component Volume T4 DNA Ligase Mix 2 μL Linearized vector (pUrLp-ΔIntA) x ng <![CDATA[Insert fragment (P TEFin -YlARO1-T Xpr2 )]]> y ng Distilled water Make up the volume to 10 μl

[0137] Among them, the usage amounts of the linearized vector (x) and the inserted fragment (y) can be calculated by the following formula: The optimal usage amount of each fragment or linearized vector = [0.02 × the number of base pairs of the fragment or linearized vector] ng.

[0138] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, screened by an ampicillin-resistant plate, and verified by restriction digestion and sequencing to obtain the positive recombinant plasmid pUrLp-ΔIntA-YlARO1.

[0139] After digesting the plasmid pUrLp-ΔIntA-YlARO1 with the restriction endonucleases NheI and SalI from TaKaRa, the linearized pUrLp-ΔIntA-YlARO1 plasmid was recovered by agarose gel electrophoresis and gel extraction.

[0140] After digesting the plasmid pYL31-YlARO4 with the restriction endonucleases AvrII and SalI from TaKaRa, the linearized YlARO4 gene expression cassette (P FBA -YlARO4-T Pex20 ) was recovered.

[0141] The linearized pUrLp-ΔIntA-YlARO1 plasmid and the YlARO4 gene expression cassette (P FBA -YlARO4-T Pex20 ) were ligated using the T4 DNA Ligase from Shanghai Beyotime Biotechnology Co., Ltd. to obtain a circular recombinant vector.

[0142] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, screened by an ampicillin-resistant plate, and verified by restriction digestion and sequencing to obtain the positive recombinant plasmid pUrLp-ΔIntA-YlARO1-YlARO4.

[0143] After digesting the plasmid pUrLp-ΔIntA-YlARO1-YlARO4 with the restriction endonucleases NheI and SalI from TaKaRa, the linearized pUrLp-ΔIntA-YlARO1-YlARO4 plasmid was recovered by agarose gel electrophoresis and gel extraction.

[0144] After digesting plasmid pYL24 - YlARO7 with the restriction endonucleases AvrII and SalI from TaKaRa, the linearized YlARO7 gene expression cassette (P TDH1 -YlARO7-T Mig1 ) was recovered by agarose gel electrophoresis.

[0145] The linearized plasmid pUrLp - ΔIntA - YlARO1 - YlARO4 and the YlARO7 gene expression cassette (P TDH1 -YlARO7-T Mig1 ) were ligated using T4 DNA Ligase from Shanghai Beyotime Biotechnology Co., Ltd. to obtain a circular recombinant vector.

[0146] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, screened by ampicillin - resistant plates, and verified by restriction digestion and sequencing to obtain the positive recombinant plasmid pUrLp - ΔIntA - YlARO1 - YlARO4 - YlARO7( Figure 4 ).

[0147] After digesting plasmid pUrLp - ΔIntA - YlARO1 - YlARO4 - YlARO7 with the restriction endonuclease AvrII from TaKaRa, the linearized plasmid pUrLp - ΔIntA - YlARO1 - YlARO4 - YlARO7 was recovered by agarose gel electrophoresis. This linearized plasmid can be used for the construction of subsequent recombinant Yarrowia lipolytica strains.

[0148] Similarly, the linearized recombinant plasmids pUrLp - ΔDga1 - FjTAL - (At4CL1~VvSTS)( Figure 5 ), pUrLp - ΔIntC3 - VvROMT3*3( Figure 6 ) can be constructed by the above method. The copy number of VvROMT3 in pUrLp - ΔIntC3 - VvROMT3*3 is 3.

[0149] Table 1 Recombinant plasmids and their uses

[0150]

[0151] Table 2 Primer sequences

[0152]

[0153]

[0154] Example 2

[0155] Construct a recombinant Yarrowia lipolytica strain for producing pterostilbene, Figure 1 which is the de novo synthesis roadmap of pterostilbene.

[0156] (I) Construction of recombinant strain 1

[0157] The linearized plasmid pUrLp-ΔIntA-YlARO1-YlARO4-YlARO7 containing the gene expression cassettes of YlARO1, YlARO4, and YlARO7 was introduced into Yarrowia lipolytica Po1fk. The gene expression cassettes of YlARO1, YlARO4, and YlARO7 were integrated into the genomic IntA locus by homologous recombination to obtain positive clones. Then, through the Cre-loxP technology, one loxP tag and the orotidine-5'-phosphate decarboxylase-encoding gene expression cassette (P TEFin -URA3-T Xpr2 ) were lost, and recombinant strain 1 was obtained.

[0158] The specific method is as follows:

[0159] 1. Yarrowia lipolytica Po1fk was cultured overnight in YPD liquid medium (containing 2% peptone, 1% yeast extract, and 2% glucose) to prepare competent cells.

[0160] 2. Take 1 mL of the overnight cultured bacterial solution, centrifuge it at low speed, discard the supernatant, and thoroughly mix the cell pellet, 95 μL of 50% PEG4000, and 5 μL of 2 M lithium acetate solution to form competent cells.

[0161] 3. After thoroughly mixing 5 μL of the recovered linearized plasmid pUrLp-ΔIntA-YlARO1-YlARO4-YlARO7 and 5 μL of boiled and denatured salmon sperm DNA (ssDNA), add them to the competent cells prepared in the previous step; incubate in a 30°C water bath for 45 minutes, with vortex shaking for 15 seconds every 15 minutes during this period; then perform a heat shock at 39°C for 10 minutes.

[0162] 4. Spread the heat-shocked competent cells on the corresponding screening plates. Use the screening medium CSM-Ura to screen for positive clones and perform colony PCR identification.

[0163] 5. Inoculate the positive clones with correct PCR identification into YPD liquid medium and culture them overnight to prepare competent cells. Introduce the circular plasmid pYLXP'-Cre into the positive clones. The Cre enzyme expressed in plasmid form can recognize two loxP tags with the same sequence on the genome, causing homologous recombination between the two loxP tags, thereby losing one loxP tag and the orotidine-5'-phosphate decarboxylase-encoding gene expression cassette (P TEFin-URA3-T Xpr2 ), thereby restoring the Ura nutritional selection marker. Subsequently, the competent cells were spread on the selection medium CSM-Leu and cultured at 30 °C for 2 days.

[0164] 6. Streak the single colonies from the CSM-Leu plate on the YPD plate containing 5-fluoroorotic acid and the CSM-Ura plate simultaneously, and observe the growth of the cells. 5-Fluoroorotic acid itself is non-toxic to yeast cells, but the functional Ura3 gene can encode and express orotidine-5'-monophosphate decarboxylase (OMP decarboxylase), which converts 5-fluoroorotic acid into a toxic form (such as 5-fluorouracil), thereby preventing yeast cells from growing. Therefore, after the loss of the Ura3 gene in step 5 above, the cells without the Ura3 gene can survive. Select the single colonies that can grow on the YPD plate containing 5-fluoroorotic acid but fail to grow on the CSM-Ura plate, and verify them by colony PCR.

[0165] 7. Inoculate the colonies successfully verified by PCR into the YPD liquid medium. After overnight culture, the circular plasmid pYLXP'-Cre can be lost, thereby restoring the Leu nutritional selection marker. The strain that simultaneously restores the Ura nutritional selection marker and the Leu nutritional selection marker is named recombinant strain 1.

[0166] Among them, the selection medium CSM-Ura contains: glucose 20 g / L, YNB (yeast nitrogen base without amino acids) 6.7 g / L, CSM-Ura (complete supplement mixture excluding uracil) 0.67 g / L, agar powder 23 g / L. The selection medium CSM-Leu contains: glucose 20 g / L, YNB (yeast nitrogen base without amino acids) 6.7 g / L, CSM-Leu (complete supplement mixture excluding leucine) 0.67 g / L, agar powder 23 g / L. The YPD plate containing 5-fluoroorotic acid (containing 5-fluoroorotic acid 1 g / L, peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L and agar powder 23 g / L).

[0167] (II) Construction of recombinant strain 2

[0168] The linearized plasmid pUrLp-ΔDga1-FjTAL-(At4CL1~VvSTS) containing the FjTAL, (At4CL1~VvSTS) gene expression cassette was introduced into recombinant strain 1. The FjTAL, (At4CL1~VvSTS) gene expression cassette was integrated into the genomic Dga1 locus by homologous recombination to obtain positive clones, and then one loxP tag and the orotidine-5'-phosphate decarboxylase encoding gene expression cassette (P TEFin -URA3-T Xpr2 ) were lost by the Cre-loxP technology to obtain recombinant strain 2.

[0169] (3) Construction of Recombinant Strain 3

[0170] The linearized plasmid pUrLp-ΔIntC3-VvROMT3*3 containing three VvROMT3 (triple mutant (Y149H, S225P, N265D)) gene expression cassettes was introduced into recombinant strain 2. The three VvROMT3 gene expression cassettes were integrated into the genomic IntC3 locus by homologous recombination to obtain positive clones. Then, one loxP tag and the orotidine-5'-phosphate decarboxylase encoding gene expression cassette (P TEFin -URA3-T Xpr2 ) were lost by Cre-loxP technology to obtain recombinant strain 3.

[0171] Example 3

[0172] Application of Recombinant Yarrowia lipolytica Strains in the Production of Pterostilbene

[0173] (1) Cultivation of Recombinant Strains

[0174] Recombinant strain 3 in Example 2 was used to produce pterostilbene. The specific method was as follows: Recombinant strain 3 was activated and cultured in YPD liquid medium (containing 2% peptone, 1% yeast extract, 2% glucose) at 30 °C and 250 rpm for 16 h to obtain a seed solution. The seed solution was inoculated into 3.5 mL of fermentation medium to make the initial OD600 0.2, and shaken at 30 °C and 250 rpm for 4 days. The fermentation medium formula was: glucose 20 - 80 g / L, yeast extract 1 - 10 g / L, peptone 2 - 20 g / L.

[0175] (2) Qualitative and Quantitative Analysis of Pterostilbene

[0176] 1. Extraction of Pterostilbene

[0177] To achieve complete extraction of pterostilbene, 100 μL of fermentation broth was resuspended in 700 μL of methanol solution containing grinding particles. Grinding treatment was carried out at -20 °C using a cryogenic grinder for a total of 20 cycles (each cycle for 40 seconds), and then centrifuged at 12,000 rpm for 3 minutes. After centrifugation, the supernatant was taken for high performance liquid chromatography (HPLC) analysis.

[0178] 2. HPLC Analysis of Pterostilbene

[0179] An Agilent 1260 Infinity II high performance liquid chromatography system equipped with a variable wavelength detector (VWD) was used, and the chromatographic column was a C18 column (4.6 mm × 100 mm). The mobile phase consisted of phase A (aqueous solution containing 0.1% formic acid) and phase B (methanol), and the gradient elution program was as follows: the initial proportion of phase B was 15%, linearly increased to 90% within 15 minutes, then maintained for 3 minutes, increased to 100% at 20 minutes, then maintained for 2 minutes, and returned to the initial proportion of 15% at 27 minutes. The flow rate was set at 0.8 mL / min, the column temperature was 40 °C, the detection wavelength was 305 nm, and the injection volume was 10 μL. A standard product provided by Shanghai Macklin Biochemical Co., Ltd. was used for qualitative and quantitative analysis of pterostilbene, and the retention time of pterostilbene was 14.7 min.

[0180] (III) Beneficial effects of recombinant bacterium 3

[0181] After 4 days of fermentation in a 24-deep well plate, the final optical density OD600 of recombinant bacterium 3 reached 38.8, the final yield of resveratrol reached 140.4 mg / L, the final yield of pinosylvin monomethyl ether reached 1.0 mg / L, and the final yield of pterostilbene reached 198.9 mg / L ( Figure 7 ), that is, 198.9 mg of pterostilbene was produced per liter of fermentation broth, realizing the efficient synthesis of the plant-derived antioxidant pterostilbene in Yarrowia lipolytica.

[0182] Example 4

[0183] Compared with Example 2, the difference was only that based on recombinant bacterium 2 of Example 2, the double mutant VvRomT2 (Y149H, S225P) was introduced into Yarrowia lipolytica recombinant bacterium 2.

[0184] Other construction steps were the same as those in Example 2. Pterostilbene was fermented and extracted according to the method of Example 3, and the yield of pterostilbene was detected.

[0185] Example 5

[0186] Compared with Example 2, the difference was only that based on recombinant bacterium 2 of Example 2, the double mutant VvRomT2 (Y149H, N265D) was introduced into Yarrowia lipolytica recombinant bacterium 2.

[0187] Other construction steps were the same as those in Example 2. Pterostilbene was fermented and extracted according to the method of Example 3, and the yield of pterostilbene was detected.

[0188] Example 6

[0189] Compared with Example 2, the difference was only that based on recombinant bacterium 2 of Example 2, the double mutant VvRomT2 (S225P, N265D) was introduced into Yarrowia lipolytica recombinant bacterium 2.

[0190] Other construction steps are the same as those in Example 2. Ferment and extract pterostilbene according to the method in Example 3, and detect the yield of pterostilbene.

[0191] Example 7

[0192] Compared with Example 2, the only difference is that based on the recombinant strain 2 in Example 2, the triple mutant VvRomT3 (Y149H, S225P, N265D) is introduced into the Yarrowia lipolytica recombinant strain 2, and the copy number of VvRomT3 in the expression cassette is 1, while the copy number of VvRomT3 in the expression cassette of Example 2 is 3.

[0193] Other construction steps are the same as those in Example 2. Ferment and extract pterostilbene according to the method in Example 3, and detect the yield of pterostilbene.

[0194] Example 8

[0195] Compared with Example 2, the only difference is that based on the recombinant strain 2 in Example 2, the single mutant VvRomT1 (Y149H) is introduced into the Yarrowia lipolytica recombinant strain 2. Other construction steps are the same as those in Example 2. Ferment and extract pterostilbene according to the method in Example 3, and detect the yield of pterostilbene.

[0196] Example 9

[0197] Compared with Example 2, the only difference is that based on the recombinant strain 2 in Example 2, the single mutant VvRomT1 (S225P) is introduced into the Yarrowia lipolytica recombinant strain 2. Other construction steps are the same as those in Example 2. Ferment and extract pterostilbene according to the method in Example 3, and detect the yield of pterostilbene.

[0198] Example 10

[0199] Compared with Example 2, the only difference is that based on the recombinant strain 2 in Example 2, the single mutant VvRomT1 (N265D) is introduced into the Yarrowia lipolytica recombinant strain 2. Other construction steps are the same as those in Example 2. Ferment and extract pterostilbene according to the method in Example 3, and detect the yield of pterostilbene.

[0200] Comparative Example 1

[0201] Compared with Example 2, the only difference is that the wild-type VvRomT from grapes is introduced into Yarrowia lipolytica. Other construction steps are the same as those in Example 2. Ferment and extract pterostilbene according to the method in Example 3, and detect the yield of pterostilbene ( Figure 8 ). The results are as follows:

[0202] Table 3 Pterostilbene yields of Comparative Example 1 and Example 2

[0203]

[0204] Comparative Example 2

[0205] Compared with Example 2, the difference is only that caffeic acid O-methyltransferase COMT derived from Arabidopsis thaliana is introduced into Yarrowia lipolytica, and other construction steps are the same as those in Example 2. Fermentation and extraction of pterostilbene are carried out according to the method of Example 3, and the yield of pterostilbene is detected ( Figure 8 ). The results are as follows:

[0206] Table 4 Pterostilbene yields of Comparative Example 2 and Example 2

[0207]

[0208] Experimental Example 1

[0209] The pterostilbene yields after fermentation of the recombinant bacteria after single mutation, double mutation of VvRomT2, or triple mutation of VvRomT3 were detected respectively ( Figure 8 ). The results are as follows:

[0210] Table 5 Pterostilbene yields of Comparative Examples 1-2 and Examples

[0211]

[0212] The above results show that the recombinant bacterium 3 constructed in Example 2 can express: the mutated 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (YlARO4), chorismate mutase, and the triple-mutated VvRomT3 (Y149H, S225P, N265D). The recombinant bacterium 3 has the highest pterostilbene yield after fermentation, reaching 198 mg / L. The pterostilbene yield obtained by fermenting the recombinant bacterium containing the triple-mutated methyltransferase (Example 7) is significantly higher than that of the recombinant bacterium containing the single- and double-mutated methyltransferases. The pterostilbene yield obtained by fermenting the recombinant bacterium containing the single-mutated methyltransferase is significantly higher than that of the recombinant bacterium containing the wild-type methyltransferase. Although the pterostilbene yield obtained by fermenting the recombinant bacterium containing the single-mutated methyltransferase is lower than that of the recombinant bacterium containing Arabidopsis caffeic acid O-methyltransferase COMT (Comparative Example 2) after fermentation, Comparative Example 2 has problems such as more other products, high pinosylvin yield, and low pterostilbene conversion rate, while the pinosylvin yield of the recombinant bacterium with a single mutation site is extremely low and the pterostilbene conversion rate is high.

[0213] Therefore, compared with before the mutation, the mutated methyltransferase improves the affinity between the enzyme and the substrate and promotes the conversion of resveratrol to pterostilbene. Compared with the recombinant bacterium containing Arabidopsis-derived caffeic acid O-methyltransferase COMT, the mutated methyltransferase improves the affinity between the enzyme and the substrate and increases the efficiency of resveratrol being catalyzed to form pterostilbene.

[0214] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A methyltransferase mutant, characterized in that Compared with the wild-type methyltransferase from grape, the wild-type methyltransferase has at least one of the following mutations: Y149H, S225P and N265D. The amino acid sequence of the wild-type methyltransferase is shown in SEQ ID NO:

5.

2. A nucleic acid molecule, characterized in that It encodes the methyltransferase mutant according to claim 1; Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

4.

3. An expression cassette, characterized in that It comprises a promoter, the nucleic acid molecule according to claim 2 and a terminator; Preferably, the promoter and the terminator are from Yarrowia lipolytica Polifk, and the Yarrowialipolytica Polifk is constructed by knocking out the ku70 gene of Yarrowia lipolytica Polif; Preferably, the promoter is selected from the promoter P of Yarrowia lipolytica TEFin , P EXP , P TDH1 , P FBA or P GPD ; The terminator is selected from the terminator T of Yarrowia lipolytica Xpr2 , T Mig1 , T Pex20 , T Lip1 or T Lip2 The encoding nucleotide sequences are shown in SEQ ID NOs: 6-15.

4. A recombinant vector, characterized in that: It comprises the nucleic acid molecule of claim 2 or the expression cassette of claim 3; Preferably, the recombinant vector further comprises: an upstream homologous sequence and a downstream homologous sequence from the IntA site, IntB site, IntC1 site, IntC3 site, IntD site, IntE site, IntF site, Dga1 site, Lip1 site or Scp2 site of Yarrowia lipolytica Po1fk; the nucleic acid molecule or the expression cassette is inserted between the upstream homologous sequence and the downstream homologous sequence; Preferably, the recombinant vector further comprises at least one of the following expression cassettes: an expression cassette for an enzyme of the tyrosine biosynthetic pathway, an expression cassette for tyrosine ammonia lyase, an expression cassette for 4-coumaroyl-CoA ligase, and an expression cassette for stilbene synthase; Preferably, the enzyme of the tyrosine biosynthesis pathway is selected from at least one of aromatic multifunctional enzyme, 3-deoxy-7-phosphoheptulate synthase and chorismate mutase; Preferably, the 3-deoxy-7-phosphoheptoneate synthase is a mutant 3-deoxy-7-phosphoheptoneate synthase, and compared with the 3-deoxy-7-phosphoheptoneate synthase of the wild-type Yarrowia lipolytica, the mutant 3-deoxy-7-phosphoheptoneate synthase has the following mutation: K221L; the chorismate mutase is a mutant chorismate mutase, and compared with the chorismate mutase of the wild-type Yarrowia lipolytica, the mutant chorismate mutase has the following mutation: G139S; Preferably, the copy number of the nucleic acid molecule or the expression cassette according to claim 3 on the recombinant vector is 1-5; Preferably, the copy number of the nucleic acid molecule or the expression cassette according to claim 3 on the recombinant vector is 3-5; Preferably, the backbone of the recombinant vector is pUrLp or pYLXP'; Preferably, the recombinant vector further comprises a loxP tag and an orotidine-5'-phosphate decarboxylase encoding gene (Ura3) expression cassette, and the orotidine-5'-phosphate decarboxylase encoding gene expression cassette is located between the loxP tags; Preferably, a rigidly connected peptide encoding gene is provided between the expression cassette of the 4-coumaroyl-CoA ligase and the expression cassette of the stilbene synthase; Preferably, the rigid linker peptide is EAAAK; Preferably, the nucleotide sequence of the gene encoding the tyrosine ammonia lyase is shown as SEQ ID No: 1, the nucleotide sequence of the gene encoding the 4-coumaroyl-CoA ligase is shown as SEQ ID No: 2, and the nucleotide sequence of the gene encoding the stilbene synthase is shown as SEQ ID No:

3.

5. A recombinant cell or recombinant bacterium, characterized in that: The recombinant cell or recombinant bacterium comprises the nucleic acid molecule according to claim 2, the expression cassette according to claim 3 or the recombinant vector according to claim 4; the recombinant cell is a non-plant cell; Preferably, the recombinant bacteria is Escherichia coli or yeast; Preferably, the yeast is selected from Candida, Pichia pastoris, Saccharomyces cerevisiae or Yarrowia lipolytica; Preferably, the Yarrowia lipolytica is a Yarrowia lipolytica in which the ku70 gene is knocked out.

6. The recombinant cell or recombinant bacterium according to claim 5, characterized in that: The recombinant cell or recombinant bacterium expresses: the methyltransferase mutant according to claim 1, enzymes of the tyrosine biosynthesis pathway, tyrosine ammonia lyase, 4-coumaroyl-CoA ligase and stilbene synthase.

7. Use of the methyltransferase mutant according to claim 1, the nucleic acid molecule according to claim 2, the expression cassette according to claim 3, the recombinant vector according to claim 4, or the recombinant cell or recombinant bacteria according to any one of claims 5-6 in the production of pterostilbene.

8. A method for producing pterostilbene, characterized in that: Cultivating the recombinant cell or recombinant bacteria according to any one of claims 5 to 6, and extracting pterostilbene from the culture of the recombinant cell or recombinant bacteria.

9. The method for producing pterostilbene according to claim 8, characterized in that: Culture at 30°C ± 2°C, 200-250 rpm with shaking for 3-10 days.

10. The method for producing pterostilbene according to claim 8, characterized in that: The formula of the fermentation medium is: glucose 20-80 g / L, yeast extract 1-10 g / L, and peptone 2-20 g / L.

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