Saccharomyces cerevisiae engineering bacterium for efficient heterologous synthesis of zealexin A1 and construction method of saccharomyces cerevisiae engineering bacterium

Abstract

The invention discloses a saccharomyces cerevisiae engineering bacterium for efficient heterologous synthesis of zealexin A1 and a construction method of the saccharomyces cerevisiae engineering bacterium. The construction method comprises the following steps: introducing a co-expression vector with a combined and modified (S)-beta-macrocarpal synthase encoding gene t14BdML2 and an optimized cytochrome P450 oxidase encoding gene ZmCYP71Z18 expression cassette into saccharomyces cerevisiae; the saccharomyces cerevisiae engineering bacteria for efficient heterologous synthesis of zealexin A1 are obtained. The yield of the zealexin A1 obtained through fermentation of the engineering bacteria is 119.31 mg / L, and an efficient and sustainable method is provided for production of the zealexin A1.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to an engineering yeast of Saccharomyces cerevisiae for efficiently heterologously synthesizing zealexin A1 and a construction method thereof. Background Art

[0002] Zealexin A1 is a sesquiterpene phytoalexin, which not only has antifungal and insecticidal activities, but also has the effect of enhancing plant drought resistance. It can widely inhibit the growth of fungal pathogens such as Rhizopus microsporus, Aspergillus flavus, Fusarium graminearum and Fusarium verticillium. Its many biological activities make it have broad application prospects in the fields of agriculture and food preservation. Due to the low content of zealexin A1 in plants, complex extraction process, high extraction cost and high consumption of natural resources, the feasibility of extracting zealexin A1 from natural resources is low. In 2016, Mao et al. first analyzed the biosynthetic pathway of zealexin A1 after in vitro verification and in vivo verification in Escherichia coli. First, (S)-β-macroene synthase ZmTPS6 or ZmTPS11 catalyzes the sesquiterpene precursor farnesyl pyrophosphate (FPP) to form (S)-β-macroene, and then cytochrome P450 oxidase ZmCYP71Z18 catalyzes the oxidation of C15 in (S)-β-macroene to form zealexin A1. In 2020, Ding et al. further discovered that ZmTPS6, ZmTPS11, ZmTPS12 and ZmTPS13 all have the function of catalyzing FPP to produce (S)-β-macroene. The three cytochrome P450 oxidases ZmCYP71Z16, ZmCYP71Z18 and ZmCYP71Z19 all have the function of catalyzing (S)-β-macroene to produce zealexin A1. With the development of synthetic biology and metabolic engineering, heterologous synthesis of Saccharomyces cerevisiae provides an economical, efficient and sustainable method for producing terpenoids. So far, there are no reports on the efficient heterologous synthesis of zealexin A1 in Saccharomyces cerevisiae. Summary of the invention

[0003] The first object of the present invention is to provide a method for constructing an engineered strain of Saccharomyces cerevisiae that can efficiently heterologously synthesize zealexin A1.

[0004] The second object of the present invention is to provide an engineered strain of Saccharomyces cerevisiae that can efficiently heterologously synthesize zealexin A1.

[0005] The third object of the present invention is to provide an application of an engineered strain of Saccharomyces cerevisiae in producing zealexin A1.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A method for constructing an engineered strain of Saccharomyces cerevisiae for efficient heterologous synthesis of zealexin A1 comprises the following steps:

[0008] A co-expression vector having a combined modified (S)-β-macrocarpene synthase encoding gene t14BdML2 and an optimized cytochrome P450 oxidase encoding gene ZmCYP71Z18 expression cassette is introduced into Saccharomyces cerevisiae to obtain a recombinant bacterium capable of efficiently heterologously synthesizing zealexin A1;

[0009] The (S)-β-macrocarpene synthase encoding gene t14BdML2 of the combined transformation is BdTPS11 L398M / I440V / T443S The first 14 amino acids of the amino acid sequence were truncated at the N-terminus and fused to the endogenous yeast farnesyl pyrophosphate synthase ERG20 via a linker peptide GGGS;

[0010] The promoter of the expression cassette is P GAL1 or P GAL10 The promoter and terminator are T CYC1 or T ADH1 However, it should be understood that the present invention is not limited to the promoter and terminator, and may also be any other suitable promoter and terminator.

[0011] The vector is a pESC-URA vector plasmid. However, it should be understood that the present invention is not limited to this vector plasmid, and may also be any other suitable vector plasmid.

[0012] The nucleotide sequence of the combined modified (S)-β-macrocarpene synthase encoding gene t14BdML2 is shown in SEQ ID No. 1;

[0013] The nucleotide sequence of the optimized cytochrome P450 oxidase encoding gene ZmCYP71Z18 is shown in SEQ ID No. 2;

[0014] The above method constructs an engineered yeast strain for efficiently heterologously synthesizing zealexin A1.

[0015] The use of the above-mentioned engineered yeast Saccharomyces cerevisiae in the production of zealexin A1 comprises the following steps:

[0016] The recombinant bacteria are cultured in a fermentation medium to obtain a fermentation product; the fermentation product is extracted using an organic solvent, and an organic phase is collected.

[0017] The fermentation medium comprises 6.7 g / L of amino-free yeast nitrogen source YNB, 20 g / L of glucose, and 2 g / L of uracil-deficient mixed powder.

[0018] The organic solvent is ethyl acetate.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] Experiments have shown that co-expression of the (S)-β-macrocarpene synthase t14BdML2 and the cytochrome P450 oxidase ZmCYP71Z18 of the present invention can increase the yield of zealexin A1; the cerevisiae engineering strain for efficient heterologous synthesis of zealexin A1 constructed by the present invention lays a foundation for the industrial production of zealexin A1. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a plasmid schematic diagram of the zealexin A1 Saccharomyces cerevisiae expression vector;

[0022] Figure 2 GC-MS detection graph of zealexin A1 Saccharomyces cerevisiae expression strain; A is the GC graph of the fermentation product, B is the GC graph of the methyl-esterified zealexin A1 standard, C is the MS graph of zealexin A1 methyl ester in the fermentation product, and D is the MS graph of the methyl-esterified zealexin A1 standard. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0024] The experiments in the following examples without specifying specific conditions were generally carried out under conventional conditions.

[0025] Example 1 Preparation of target gene

[0026] Gene BdTPS11 used in the present invention L398M / I440V / T443S The original sequence was derived from Brachypodium distachyon, in which position 398 was mutated from leucine to methionine, position 440 was mutated from isoleucine to valine, and position 443 was mutated from threonine to serine. The gene ZmCYP71Z18 was derived from corn and synthesized by the company after codon optimization. The endogenous farnesyl pyrophosphate synthase gene ERG20 of Saccharomyces cerevisiae was amplified using Saccharomyces cerevisiae genomic DNA as a template.

[0027] Example 2 Construction of Zealexin A1 Saccharomyces cerevisiae expression vector

[0028] BdTPS11 L398M / I440V / T443SAs a template, primers t14BdML2-F and Bd-R were used to amplify the first gene fragment required for the combined transformation, with the bases corresponding to the 14 amino acids at the N-terminus removed, the bases corresponding to the start codon, and the bases corresponding to the connecting peptide GGGS; primers ERG20-F and primers EL2-R were used to amplify the second gene fragment required for the combined transformation. Overlap extension PCR was used to fuse the two fragments to obtain the combined transformation gene fragment. The BM seamless cloning kit of Bomed Bio was used for homologous recombination reaction to assemble the combined transformation gene fragment into the P of pESC-URA. GAL1 Promoter and T CYC1 The codon-optimized ZmCYP71Z18 was assembled in the P GAL10 Promoter and T ADH1 The co-expression vector of the combined modified (S)-β-macrocarpon synthase t14BdML2 and cytochrome P450 oxidase ZmCYP71Z18 was constructed between the terminators. The recombinant product was transformed into the competent E. coli DH5α, then spread on the LB solid medium containing ampicillin, and cultured overnight in a 37°C incubator. After the transformants grew out, they were verified by colony PCR, and the plasmids in the strains with the correct length of the target band were extracted and sequenced by the company to ensure the correctness of the plasmids, and finally the zealexin A1 expression vector pt14BdML2-ZmCYP was obtained.

[0029] Example 3 Construction of Zealexin A1 Saccharomyces cerevisiae expression strain

[0030] The plasmid verified by sequencing was transformed into the Saccharomyces cerevisiae strain by lithium acetate transformation method, and was spread on SD-URA (uracil-deficient synthesis) solid medium and cultured in a 30°C incubator for 2-3 days. After the transformants grew out, they were verified by colony PCR to obtain the recombinant Saccharomyces cerevisiae strain t14BdML2-ZmCYP.

[0031] Example 4 Recombinant strain fermentation and product detection

[0032] A single colony of the recombinant Saccharomyces cerevisiae strain t14BdML2-ZmCYP was selected and inoculated into 10 mL SD-URA liquid medium and cultured in a shaker at 30°C for 18 h. An appropriate amount of the culture solution was transferred into 50 mL SD-URA liquid medium to adjust the OD of the medium to 600 The fermentation medium was incubated at 30°C in a shaker for 96 h. At 30 h, a galactose solution with a final concentration of 10 g / L was added to the fermentation medium. At 36 h and 60 h, 500 μL of anhydrous ethanol was added to the fermentation medium as a supplementary carbon source.

[0033] After the fermentation is completed, add organic solvent and grinding beads to the fermentation broth, use an oscillator to oscillate at high speed for 30 minutes, centrifuge at 6000rpm for 5 minutes, take 1mL of the upper organic phase into a brown sample bottle, and quickly blow dry under mild nitrogen. Subsequently, add equal volumes of methanol and trimethylsilyldiazomethane (TMSD) to carry out derivatization reaction. After the reaction is completed, filter the reaction solution through a membrane to remove impurities and detect it by GC-MS. The identification of Zealexin A1 is based on the zealexin A1 standard provided by Professor Arata Yajima's research group and the mass spectrometry data in the literature. The quantification of zealexin A1 is based on the α-linolenic acid standard. The test results are as follows Figure 1 As shown, the production of zealexin A1 was 119.31 mg / L.

[0034] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for constructing an engineered strain of Saccharomyces cerevisiae for efficient heterologous synthesis of zealexin A1, comprising the following steps: A co-expression vector having a combined modified (S)-β-macrocarpene synthase encoding gene t14BdML2 and an optimized cytochrome P450 oxidase encoding gene ZmCYP71Z18 expression cassette is introduced into Saccharomyces cerevisiae to obtain a recombinant bacterium capable of efficiently heterologously synthesizing zealexinA1; The (S)-β-macrocarpene synthase encoding gene t14BdML2 of the combined transformation is BdTPS11 L398M / I440V / T443S The first 14 amino acids of the amino acid sequence were truncated at the N-terminus and fused to the endogenous yeast farnesyl pyrophosphate synthase ERG20 via a linker peptide GGGS; The nucleotide sequence of the combined modified (S)-β-macrocarpene synthase encoding gene t14BdML2 is shown in SEQ ID No. 1; The nucleotide sequence of the optimized cytochrome P450 oxidase encoding gene ZmCYP71Z18 is shown in SEQ ID No.

2.

2. The method for constructing an engineered strain of Saccharomyces cerevisiae for efficient heterologous synthesis of zealexin A1 according to claim 1, characterized in that: The promoter of the expression cassette is P GAL1 or P GAL10 The promoter and terminator are T CYC1 or T ADH1 Terminator.

3. The method for constructing an engineered strain of Saccharomyces cerevisiae for efficient heterologous synthesis of zealexin A1 according to claim 1, characterized in that: The vector is a pESC-URA vector plasmid.

4. An engineered strain of Saccharomyces cerevisiae for efficiently heterologously synthesizing zealexin A1 constructed by the method of claim 1.

5. Use of the engineered yeast Saccharomyces cerevisiae according to claim 4 in producing zealexin A1.

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