Construction method and application of yeast engineering bacteria for producing 1-aminocyclopropane-1-carboxylic acid

Abstract

The invention belongs to the technical field of biological engineering, and particularly relates to a construction method and application of yeast engineering bacteria for producing 1-aminocyclopropane-1-carboxylic acid (ACC). Pyruvate carboxylase gene RoPYC, yeast SAM enzyme gene Sam2 and ACS synthase gene GmACS are overexpressed in saccharomyces cerevisiae, so that the 1-aminocyclopropane-1-carboxylic acid (ACC) is obtained; a path for biosynthesizing 1-aminocyclopropane-1-carboxylic acid (ACC) by taking glucose as a substrate is designed, a saccharomyces cerevisiae engineering bacterium for producing ACC is constructed, the ACC can be produced by large-scale fermentation, and the highest yield can reach 50.56 g / L. The method has the advantages of low environmental influence, easiness in large-scale production expansion, high production efficiency and the like, has a good economic prospect, and lays a foundation for artificial efficient biosynthesis of ACC.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and relates to the construction of engineering bacteria, in particular to the construction of a yeast engineering bacterium producing 1-aminocyclopropane-1-carboxylic acid. Background Art

[0002] 1-Aminocyclopropanecarboxylic acid (ACC) is a natural non-protein amino acid with unique biological activities. In plants, ACC is an important precursor for ethylene synthesis and has a significant regulatory effect on the growth and development process of plants. In addition, ACC has excellent physiological regulatory effects on animals such as silkworms and mice, and is a new type of dual plant and animal growth regulator. ACC also shows broad application prospects in the fields of medicine, chemical industry, and agriculture, such as being used as a precursor for the anti-epileptic drug valproic acid, a raw material for the synthesis of targeted tumor drugs, and an anti-pollution agent for antibacterial and antifungal medical devices.

[0003] Currently, ACC is mainly prepared by chemical synthesis. In recent years, the synthesis research of 1-aminocyclopropane-1-carboxylic acid has received extensive attention and has become a relatively active research field in organic chemistry. Organic synthesis workers have gradually developed some synthetic routes with advantages such as easily available raw materials, mild reaction conditions, and simple operations, providing a sufficient material basis for research in the fields of medicine, life science, biology, and agricultural science. However, the chemical synthesis of ACC still has disadvantages such as low reaction yield, complex steps, many by-products, and a large amount of solid waste, which seriously hinders the large-scale production and application of ACC.

[0004] Biosynthesis methods have shown many advantages in the field of chemical synthesis and have gradually become an important part of modern chemical research and industrial applications. First of all, biosynthesis methods have high selectivity and specificity. Biocatalysts, especially enzymes, can catalyze specific substrates under mild conditions, significantly reducing the generation of by-products. This high selectivity not only improves the purity of the target product but also reduces the costs of subsequent separation and purification. Secondly, biosynthesis methods generally have a lower environmental impact. Compared with traditional chemical synthesis methods, renewable raw materials are usually used in the biosynthesis process, the reaction conditions are mild, and the products do not contain harmful chemicals. This makes biosynthesis more in line with the concept of sustainable development within the framework of green chemistry. Patent 202110358488.5 discloses a lycoris aurea 1-aminocyclopropane-1-carboxylic acid synthase, its encoding gene, and applications. The 1-aminocyclopropane-1-carboxylic acid synthase of the lycoris aurea plant of the genus Lycoris has good enzyme activity and can catalyze the synthesis of 1-aminocyclopropane-1-carboxylic acid from S-adenosylmethionine, and the yield of 1-aminocyclopropane-1-carboxylic acid can reach up to 986 mg / L at most.

[0005] Therefore, using the biosynthesis method to prepare ACC has the advantages of low environmental impact, easy scale-up production, and high production efficiency, has good economic prospects, and has become one of the important choices for the development of ACC synthesis technology. Summary of the Invention

[0006] To solve the above problems, the present invention proposes a method for constructing a yeast engineering bacterium producing 1-aminocyclopropane-1-carboxylic acid and its applications.

[0007] The technical solution of the present invention is realized as follows:

[0008] The method for constructing the engineered yeast strain of the present invention is as follows: Using yeast as the chassis bacterium, relying on the yeast glucose metabolism pathway, by combinatorially constructing constitutive strong promoter and inducible promoter gene expression cassettes, introducing the pyruvate carboxylase gene RoPYC from Rhizopus oryzae, the enhanced yeast SAM enzyme gene Sam2, and the plant-derived 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene GmACS, and expressing them in yeast, changing the glucose metabolic flux, so that the engineered bacterium uses glucose as the initial substrate, overexpresses the pyruvate carboxylase gene RoPYC in the tricarboxylic acid cycle through the combinatorial promoter to enhance the supply of succinyl coenzyme A, increases the high serine to homocysteine pathway, and improves the production of L-methionine; then induces the expression of the enhanced yeast SAM enzyme gene Sam2 by ethanol to produce SAM enzyme, and catalyzes the synthesis of S-adenosylmethionine from L-methionine by SAM enzyme; finally, induces the expression of the plant-derived ACC synthase (ACS) enzyme gene GmACS by ethanol to produce ACS enzyme, and catalyzes the synthesis of 1-aminocyclopropane-1-carboxylic acid from S-adenosylmethionine, and finally obtains an engineered yeast strain with high-efficiency synthesis of 1-aminocyclopropane-1-carboxylic acid.

[0009] Based on this, the present application proposes a method for constructing an engineered yeast strain producing 1-aminocyclopropane-1-carboxylic acid (ACC), and the steps are as follows:

[0010] (1) Construction of the ACC1 gene cassette recombinant vector:

[0011] Using the yeast strain genome as a template, performing PCR amplification with primers LEU(Dn)+pADH1-F and LEU2-R to obtain the downstream homologous arm fragment;

[0012] Using the plasmid pHDE-Cas9 as a template, performing PCR amplification with primers RoPYC+LEU(up)-F and Kan MX+HindⅢ-R to obtain the RoPYC fragment;

[0013] Using the plasmid UAS TEF1+CIT1+CLB2 as a template, performing PCR amplification with primers UAS+PTDH3-R and UAS+KANMX-F to obtain the UAS fragment;

[0014] Using the obtained downstream homologous arm fragment, RoPYC fragment, and UAS fragment as templates, performing fusion PCR amplification with primers LEU2-up-F and UAS+PTDH3-R to obtain the ACC1 gene cassette;

[0015] (2) Construction of the ACC2 gene cassette recombinant vector:

[0016] Using the yeast strain genome as a template, the promoter TDH3 fragment was obtained by PCR amplification with primers pTDH3+UAS-F and pTDH3+sPn50-R;

[0017] Using the artificially synthesized Sam2 as a template, the Sam2 fragment was obtained by PCR amplification with primers sPn50+EGFP-R and sPn50+pTDH3-F;

[0018] Using the plasmid pT4-CMV-GFP as a template, the EGFP fragment was obtained by PCR amplification with primers EGFP+Sam2-F and EGFP+tPFK1-R;

[0019] Using the obtained TDH3 fragment, Sam2 fragment and EGFP fragment as templates, the ACC2 gene cassette was obtained by fusion PCR amplification with primers pTDH3+UAS-F and EGFP+tPFK1-R;

[0020] (3) Construction of the ACC3 gene cassette recombinant vector:

[0021] Using the yeast strain genome as a template, the upstream homologous arm fragment was obtained by PCR amplification with primers LEU2-up-F and LEU2(up)+Kan MX-R;

[0022] Using the yeast strain genome as a template, the tPFK1 fragment was obtained by PCR amplification with primers tPFK1+EGFP-F and PNUGT31+tPFK1+SmaI-R;

[0023] Using the artificially synthesized GmACS as a template, the GmACS fragment was obtained by PCR amplification with primers PNUGT31+tPFK1-F and pADH1+LEU2(Dn)-R;

[0024] Using the obtained upstream homologous arm fragment, tPFK1 fragment and GmACS fragment as templates, the ACC3 gene cassette was obtained by fusion PCR amplification with primers tPFK1+EGFP-F and LEU2-R;

[0025] (4) The ACC1 gene cassette recombinant vector in step (1), the ACC2 gene cassette recombinant vector in step (2) and the ACC3 gene cassette recombinant vector in step (3) were linearized to obtain the ACC1 linearized fragment, the ACC2 linearized fragment and the ACC3 linearized fragment respectively;

[0026] (5) The ACC1 linearized fragment, the ACC2 linearized fragment and the ACC3 linearized fragment in step (4) were co-transformed into yeast cells, and the obtained positive transformants were the yeast engineering bacteria producing ACC.

[0027] Preferably, the above yeast strain is Saccharomyces cerevisiae W303a.

[0028] Preferably, the nucleotide sequence of RoPYC in the above step (1) is as shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3; the forward primer for fusion PCR amplification is LEU2-up-F, and its nucleotide sequence is as shown in SEQ ID No.10, and the reverse primer is UAS+PTDH3-R, and its nucleotide sequence is as shown in SEQ ID No.11.

[0029] Preferably, the nucleotide sequence of the artificially synthesized Sam2 in the above step (2) is as shown in SEQ ID No.4 or SEQ ID No.5 or SEQ ID No.6; the forward primer for fusion PCR amplification is pTDH3+UAS-F, and its nucleotide sequence is as shown in SEQ ID No.12, and the reverse primer is EGFP+tPFK1-R, and its nucleotide sequence is as shown in SEQ ID No.13.

[0030] Preferably, the nucleotide sequence of the artificially synthesized GmACS in the above step (3) is as shown in SEQ ID No.7 or SEQ ID No.8 or SEQ ID No.9; the forward primer for fusion PCR amplification is tPFK1+EGFP-F, and its nucleotide sequence is as shown in SEQ ID No.14, and the reverse primer is LEU2-R, and its nucleotide sequence is as shown in SEQ ID No.15.

[0031] Preferably, the above intermediate vector is pEASY.

[0032] Preferably, the co-transformation method in the above step (5) is electrotransformation or lithium acetate transformation; the yeast cells are Saccharomyces cerevisiae W303a.

[0033] Furthermore, the reaction system of the above fusion PCR is 50 μL: 1 μL of template, 2 μL each of 10 mM upstream primer and 10 mM downstream primer, 25 μL of enzyme mixture, and made up to 50 μL with deionized water; the PCR reaction program is: 94 °C, 5 min; 94 °C, 30 s, 56 °C, 1.5 min, 72 °C, 1 min, for 35 cycles; 72 °C, 7 min.

[0034] In the second aspect, an engineered bacterium constructed by the method for constructing the above yeast engineered bacterium producing 1-aminocyclopropane-1-carboxylic acid (ACC).

[0035] In the third aspect, the application of the above method for constructing a yeast engineered bacterium producing 1-aminocyclopropane-1-carboxylic acid (ACC) or the above engineered bacterium in the fermentation production of ACC.

[0036] Preferably, in the above fermentation production, the engineered bacterial seed solution is inoculated into the fermentation medium at a ratio of 2-8% and fermented at 25-30°C for 24-48 h to achieve the synthesis of 1-aminocyclopropane-1-carboxylic acid.

[0037] The present invention has the following beneficial effects:

[0038] By overexpressing the pyruvate carboxylase gene RoPYC, yeast SAM enzyme gene Sam2, and ACS synthase gene GmACS in Saccharomyces cerevisiae, the present invention designs a biosynthetic pathway for 1-aminocyclopropane-1-carboxylic acid (ACC) using glucose as a substrate, constructs an engineered Saccharomyces cerevisiae strain producing ACC, and can ferment and produce ACC on a large scale, with the highest yield reaching 50.56 g / L. The present invention has the advantages of low environmental impact, easy scale-up production, high production efficiency, etc., and has good economic prospects, laying a foundation for the artificial and efficient biosynthesis of ACC. Description of the Drawings

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0040] Figure 1 It is the gel electrophoresis diagram of the ACC1 linearized fragment, ACC2 linearized fragment, and ACC3 linearized fragment.

[0041] Figure 2 It is the ion exchange chromatography separation and purification of 1-aminocyclopropane-1-carboxylic acid (ACC) in the fermentation broth of Application Example 5.

[0042] Figure 3 It is the HR-MS detection diagram of ACC in the fermentation broth of Application Example 5. Detailed Embodiments

[0043] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0044] Unless otherwise specified, the test methods used in the following experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0045] The method for constructing the engineered yeast strain of the present invention is as follows: Using yeast as the chassis bacterium, relying on the yeast glucose metabolism pathway, by combinatorially constructing constitutive strong promoter and inducible promoter gene expression cassettes, introducing the pyruvate carboxylase gene RoPYC from Rhizopus oryzae, the enhanced yeast SAM enzyme gene Sam2, and the plant-derived 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene GmACS, enabling their expression in yeast, changing the glucose metabolic flux, so that the engineered bacterium uses glucose as the initial substrate, overexpressing the pyruvate carboxylase gene RoPYC in the tricarboxylic acid cycle through the combinatorial promoter to enhance the supply of succinyl coenzyme A, increasing the high serine to homocysteine pathway, and increasing the production of L-methionine; then inducing the expression of the enhanced yeast SAM enzyme gene Sam2 by ethanol to produce SAM enzyme, and catalyzing the synthesis of S-adenosylmethionine from L-methionine by the SAM enzyme; finally, inducing the expression of the plant-derived ACC synthase (ACS) enzyme gene GmACS by ethanol to produce ACS enzyme, and catalyzing the synthesis of 1-aminocyclopropane-1-carboxylic acid from S-adenosylmethionine, ultimately obtaining an engineered yeast strain with high-efficiency synthesis of 1-aminocyclopropane-1-carboxylic acid.

[0046] The target genes used in this experiment are the pyruvate carboxylase gene RoPYC, the mutant enhanced yeast SAM enzyme gene Sam2, and the mutant ACS (ACC synthase) enzyme gene GmACS. The above target genes are all obtained by artificial synthesis after codon optimization of Saccharomyces cerevisiae; among them, the nucleic acid sequence of RoPYC is SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3; the nucleic acid sequence of the enhanced yeast SAM enzyme gene Sam2 is SEQ ID No.4 or SEQ ID No.5 or SEQ ID No.6; the nucleic acid sequence of the enhanced ACS (ACC synthase) enzyme gene GmACS is shown as SEQ ID No.7 or SEQ ID No.8 or SEQ IDNo.9.

[0047] The method for constructing the engineered yeast strain proposed by the present invention is applicable to all Saccharomyces cerevisiae strains. The original Saccharomyces cerevisiae strain used in this application is yeast W303a, purchased from Shanghai Beinuo Biotechnology Co., Ltd.

[0048] Example 1

[0049] The method for constructing the gene expression cassette for constructing the ACC-synthesizing engineered Saccharomyces cerevisiae strain is as follows:

[0050] (1) Construction of the ACC1 gene cassette recombinant vector:

[0051] Using the genome of yeast strain W303a as a template, PCR amplification was performed with primers LEU(Dn)+pADH1-F and LEU2-R to obtain the downstream fragment of the homologous arm;

[0052] Using the plasmid pHDE-Cas9 as a template, PCR amplification was performed with primers RoPYC+LEU(up)-F and Kan MX+HindⅢ-R to obtain the RoPYC fragment;

[0053] Using the plasmid UAS TEF1+CIT1+CLB2 as a template, PCR amplification was performed with primers UAS+PTDH3-R and UAS+KANMX-F to obtain the UAS fragment;

[0054] Using the obtained downstream fragment of the homologous arm, RoPYC fragment, and UAS fragment as templates, fusion PCR amplification was performed with primers LEU2-up-F (SEQ ID No.10) and UAS+PTDH3-R (SEQ ID No.11) to obtain the ACC1 gene cassette;

[0055] The ACC1 gene cassette was ligated to the pEASY vector through the pEASY-Blunt Cloning Kit to construct the ACC1 gene cassette recombinant vector.

[0056] (2) Construction of the ACC2 gene cassette recombinant vector:

[0057] The yeast genomic DNA extraction kit was used to obtain the genome of yeast strain W303a. Using this as a template, primers pTDH3+UAS-F and pTDH3+sPn50-R were used for amplification to obtain the promoter TDH3 fragment;

[0058] Using the synthesized gene sequence Sam2 as a template, primers sPn50+EGFP-R and sPn50+pTDH3-F were used for amplification to obtain the Sam2 fragment;

[0059] Using the plasmid pT4-CMV-GFP as a template, primers EGFP+Sam2-F and EGFP+tPFK1-R were used for amplification to obtain the EGFP fragment;

[0060] Using the obtained TDH3 fragment, Sam2 fragment, and EGFP fragment as templates, fusion PCR amplification was performed with primers pTDH3+UAS-F (SEQ ID No.12) and EGFP+tPFK1-R (SEQ ID No.13) to obtain the ACC2 gene cassette;

[0061] The ACC2 gene cassette was ligated to the pEASY vector through the pEASY-Blunt Cloning Kit to construct the ACC2 gene cassette recombinant vector.

[0062] (3) Construction of the ACC3 gene cassette recombinant vector

[0063] Using the genome of yeast strain W303a as a template, PCR amplification was carried out with primers LEU2-up-F and LEU2(up)+Kan MX-R to obtain the upstream homologous arm fragment;

[0064] Using the genome of yeast strain W303a as a template, PCR amplification was carried out with primers tPFK1+EGFP-F and primer PNUGT31+tPFK1+SmaI-R to obtain the tPFK1 fragment;

[0065] Using GmACS as a template, PCR amplification was carried out with primers PNUGT31+tPFK1-F and pADH1+LEU2(Dn)-R to obtain the GmACS fragment;

[0066] Using the obtained tPFK1, GmACS, and upstream homologous arm fragment as templates, primers tPFK1+EGFP-F (SEQ ID No.14) and LEU2-R (SEQ ID No.15) were used for fusion PCR amplification to obtain the ACC3 gene cassette;

[0067] The ACC3 gene cassette was ligated to the pEASY vector through the pEASY-Blunt Cloning Kit to construct the ACC3 gene cassette recombinant vector.

[0068] The above PCR was carried out using the Q5 High-Fidelity DNA Polymerases cloning kit, and the PCR reaction system was 50 μL for each: the amount of each template was 25 ng - 100 ng. 2 μL of 10 mM upstream primer and 2 μL of 10 mM downstream primer, 25 μL of enzyme mixture, and deionized water was added to make up 50 μL; the enzyme mixture was the corresponding reagent in the Q5 High-Fidelity DNA Polymerases cloning kit; the PCR reaction program was: 94 °C, 5 min; 94 °C, 30 s, 56 °C, 1.5 min, 72 °C, 1 min, 35 cycles; 72 °C, 7 min.

[0069] The sequences of the above primers related to gene cassette construction are shown in Table 1.

[0070] Table 1 Primer sequences related to gene cassette construction

[0071]

[0072] Example 2

[0073] A method for constructing an engineered Saccharomyces cerevisiae strain for synthesizing ACC:

[0074] 1. Linearization of the vector

[0075] Using the obtained recombinant vector of the ACC1 gene cassette as a template, the ACC-1 linearized fragment was obtained by PCR amplification with primers LEU2-up-F and UAS+pTDH3-R.

[0076] Using the obtained recombinant vector of the ACC2 gene cassette as a template, the ACC-2 linearized fragment was obtained by PCR amplification with primers pTDH3+UAS-F and EGFP+tPFK1-R.

[0077] Using the obtained recombinant vector of the ACC3 gene cassette as a template, the ACC-3 linearized fragment was obtained by PCR amplification with primers tPFK1+EGFP-F and LEU2-R.

[0078] The above PCR cloning was performed using the Q5 High-Fidelity DNA Polymerases cloning kit to obtain the linearized fragment. The PCR reaction system was all 50 μL: consisting of 1 μL of template, 2 μL each of 10 mM upstream primer and 10 mM downstream primer, 25 μL of enzyme mixture, and deionized water was added to make up 50 μL; the enzyme mixture was the corresponding reagent in the Q5 High-Fidelity DNA Polymerases cloning kit; the PCR reaction program was: 94 °C for 5 min; 94 °C for 30 s, 56 °C for 1.5 min, 72 °C for 1 min, 35 cycles; 72 °C for 7 min. After PCR, gel electrophoresis was carried out, and after confirming successful amplification, the target band was recovered.

[0079] The results were as Figure 1 shown. The gel electrophoresis results of the ACC1 linearized fragment, ACC2 linearized fragment, and ACC3 linearized fragment (1 is ACC1, 2 is ACC2, 3 is ACC3) indicated that the corresponding gene expression cassettes had been successfully constructed.

[0080] 2. Yeast transformation

[0081] The linearized fragments of the recombinant vectors of the above-obtained ACC1 gene cassette, ACC2 gene cassette, and ACC3 gene cassette were transferred into Saccharomyces cerevisiae W303a, and the transformation methods could use electroporation and lithium acetate transformation. The transformed bacterial solution was spread on the YPD1010 solid plate and cultured at 30 °C until transformants grew out. Positive transformants were picked for fermentation detection to obtain the Saccharomyces cerevisiae engineering bacteria producing 1-aminocyclopropane-1-carboxylic acid.

[0082] The formula of the YPD1010 fermentation medium was 20 g of glucose, 20 g of peptone, 10 g of yeast extract powder, 0.2 g of adenine, 1000 g of water, and 20 g of agar.

[0083] 3. Shake flask fermentation

[0084] Pick the monoclonal strain activated on the plate and prepare the fermentation seed liquid in the corresponding liquid screening medium (30 °C, 250 r / min, 16 h); centrifuge to collect the thalli, transfer them to a 100 mL Erlenmeyer flask containing 10 mL of YPD1012 liquid medium, adjust the OD to 0.1, and culture with shaking at 30 °C and 250 r / min for 4 d.

[0085] The formula of YPD1012 medium is 20 g of glucose, 20 g of peptone, 10 g of yeast extract powder, 0.2 g of adenine, and 1000 g of water.

[0086] Application Example 1

[0087] Application of the engineered Saccharomyces cerevisiae in the fermentation production of 1-aminocyclopropane-1-carboxylic acid, the steps are as follows:

[0088] (1) Seed culture: The culture temperature is 30 °C, maintain the culture pH at 6.0 ± 0.2 by automatically adding 20% ammonia water solution, and maintain the dissolved oxygen value of the medium at 85% by adjusting the stirring speed or ventilation volume.

[0089] The seed medium used in seed culture is: 60 g / L of glucose, 10 g / L of yeast extract, 10 g / L of peptone, 2 g / L of ammonium sulfate, 20 g / L of potassium dihydrogen phosphate, 10 g / L of methionine, 0.2 g / L of adenine, and 1000 g of water.

[0090] (2) Fermentation culture in a 5 L fermenter: Prepare 3 L of medium according to the following ratio, sterilize, and inoculate. The inoculation amount is 2%, the culture temperature is 30 °C, maintain the culture pH at 6.0 ± 0.2 by automatically adding 20% ammonia water solution, maintain the dissolved oxygen value of the medium during fermentation at 35% by adjusting the stirring speed or ventilation volume, add 30% glucose solution to control the glucose concentration in the tank at 30 g / L, and the fermentation cycle is 24 h.

[0091] The fermentation medium used in fermentation culture is: 80 g / L of glucose, 10 g / L of yeast extract, 15 g / L of peptone, 5 g / L of ammonium sulfate, 20 g / L of potassium dihydrogen phosphate, and 1000 g of water.

[0092] Application Example 2

[0093] Application of the engineered Saccharomyces cerevisiae in the fermentation production of 1-aminocyclopropane-1-carboxylic acid, the steps are as follows:

[0094] (1) Seed culture: The culture temperature is 30 °C, maintain the culture pH at 6.0 ± 0.2 by automatically adding 20% ammonia water solution, and maintain the dissolved oxygen value of the medium at 85% by adjusting the stirring speed or ventilation volume.

[0095] The seed medium used in seed culture is as follows: 80 g / L of glucose, 4 g / L of yeast extract, 20 g / L of peptone, 2 g / L of ammonium sulfate, 38 g / L of potassium dihydrogen phosphate, 30 g / L of methionine, 0.2 g / L of adenine, and 1000 g of water.

[0096] (2) Fermentation culture in a 5 L fermenter: Prepare 3 L of medium according to the following ratio, sterilize it, and inoculate. The inoculation amount is 3%, the culture temperature is 30 °C, maintain the culture pH at 6.0 ± 0.2 by automatically adding 20% ammonia water solution, maintain the dissolved oxygen value of the medium fermentation at 35% by adjusting the stirring speed or ventilation volume, control the glucose concentration in the tank at 40 g / L by adding 30% glucose solution, and the fermentation period is 30 h.

[0097] The fermentation medium used in fermentation culture is as follows: 70 g / L of glucose, 6 g / L of yeast extract, 10 g / L of peptone, 5 g / L of ammonium sulfate, 20 g / L of potassium dihydrogen phosphate, and 1000 g of water.

[0098] Application Example 3

[0099] Application of the engineered Saccharomyces cerevisiae in the fermentation production of 1-aminocyclopropane-1-carboxylic acid, the steps are as follows:

[0100] (1) Seed culture: The culture temperature is 30 °C, maintain the culture pH at 6.0 ± 0.2 by automatically adding 20% ammonia water solution, and maintain the dissolved oxygen value of the medium at 85% by adjusting the stirring speed or ventilation volume.

[0101] The seed medium used in seed culture is as follows: 80 g / L of glucose, 4 g / L of yeast extract, 20 g / L of peptone, 2 g / L of ammonium sulfate, 38 g / L of potassium dihydrogen phosphate, 30 g / L of methionine, 0.2 g / L of adenine, and 1000 g of water.

[0102] (2) Fermentation culture in a 5 L fermenter: Prepare 3 L of medium according to the following ratio, sterilize it, and inoculate. The inoculation amount is 5%, the culture temperature is 30 °C, maintain the culture pH at 6.0 ± 0.2 by automatically adding 20% ammonia water solution, maintain the dissolved oxygen value of the medium fermentation at 35% by adjusting the stirring speed or ventilation volume, control the glucose concentration in the tank at 50 g / L by adding 30% glucose solution, and the fermentation period is 36 h.

[0103] The fermentation medium used in fermentation culture is as follows: 90 g / L of glucose, 10 g / L of yeast extract, 20 g / L of peptone, 2 g / L of ammonium sulfate, 38 g / L of potassium dihydrogen phosphate, and 1000 g of water.

[0104] Application Example 4

[0105] Application of the engineered Saccharomyces cerevisiae in the fermentation production of 1-aminocyclopropane-1-carboxylic acid, the steps are as follows:

[0106] (1) Seed culture: The culture temperature was 30°C, the culture pH was maintained at 6.0±0.2 by automatically adding 20% ammonia solution, and the dissolved oxygen value of the culture medium was maintained at 85% by adjusting the stirring speed or ventilation volume.

[0107] The seed culture medium used in the seed culture was: 90 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, 2 g / L ammonium sulfate, 30 g / L potassium dihydrogen phosphate, 30 g / L methionine, 0.5 g / L adenine, and 1000 g water.

[0108] (2) Fermentation in a 5-L fermenter: Prepare 3 L of culture medium according to the following proportions, sterilize, and inoculate. The inoculum size is 8%, the culture temperature is 30°C, the culture pH is maintained at 6.0 ± 0.2 by automatically adding 20% ammonia solution, the dissolved oxygen value of the culture medium is maintained at 35% by adjusting the stirring speed or ventilation volume, and the glucose concentration in the tank is controlled at 80 g / L by adding 30% glucose solution. The fermentation period is 48 h.

[0109] The fermentation medium used in the fermentation culture is: 90 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, 2 g / L ammonium sulfate, 38 g / L potassium dihydrogen phosphate, and 1000 g water.

[0110] Application Example 5

[0111] The application of engineered yeast Saccharomyces cerevisiae in the fermentation production of 1-aminocyclopropane-1-carboxylic acid comprises the following steps:

[0112] (1) Seed culture: The culture temperature was 30°C, the culture pH was maintained at 6.0±0.2 by automatically adding 20% ammonia solution, and the dissolved oxygen value of the culture medium was maintained at 85% by adjusting the stirring speed or ventilation volume.

[0113] The seed culture medium used in the seed culture was: 90 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, 2 g / L ammonium sulfate, 30 g / L potassium dihydrogen phosphate, 30 g / L methionine, 0.5 g / L adenine, and 1000 g water.

[0114] (2) Fermentation in a 5-L fermenter: Prepare 3 L of culture medium according to the following proportions, sterilize, and inoculate. The inoculum size is 8%, the culture temperature is 30°C, the culture pH is maintained at 6.0 ± 0.2 by automatically adding 20% ammonia solution, the dissolved oxygen content of the culture medium is maintained at 35% by adjusting the stirring speed or ventilation rate, and the glucose concentration in the tank is controlled at 90 g / L by adding 30% glucose solution. The fermentation period is 48 h.

[0115] The fermentation medium used in fermentation culture is as follows: 90 g / L of glucose, 10 g / L of yeast extract, 20 g / L of peptone, 2 g / L of ammonium sulfate, 38 g / L of potassium dihydrogen phosphate, and 1000 g of water.

[0116] Application Example 6

[0117] The application of the engineered Saccharomyces cerevisiae in the fermentation production of 1-aminocyclopropane-1-carboxylic acid is as follows:

[0118] (1) Seed culture: The culture temperature is 30 °C, the culture pH is maintained at 6.0 ± 0.2 by automatically adding 20% ammonia water solution, and the dissolved oxygen value of the culture medium is maintained at 85% by adjusting the stirring speed or ventilation volume.

[0119] The seed medium used in seed culture is as follows: 90 g / L of glucose, 10 g / L of yeast extract, 20 g / L of peptone, 2 g / L of ammonium sulfate, 30 g / L of potassium dihydrogen phosphate, 30 g / L of methionine, 0.5 g / L of adenine, and 1000 g of water.

[0120] (2) Fermentation culture in a 5 L fermenter: Prepare 3 L of medium according to the following ratio, sterilize it, and inoculate. The inoculation amount is 8%, the culture temperature is 30 °C, the culture pH is maintained at 6.0 ± 0.2 by automatically adding 20% ammonia water solution, the dissolved oxygen value of the fermentation of the culture medium is maintained at 35% by adjusting the stirring speed or ventilation volume, add 30% glucose solution to control the glucose concentration in the tank at 100 g / L, and the fermentation cycle is 48 h.

[0121] The fermentation medium used in fermentation culture is as follows: 90 g / L of glucose, 20 g / L of yeast extract, 30 g / L of peptone, 6 g / L of ammonium sulfate, 45 g / L of potassium dihydrogen phosphate, and 1000 g of water.

[0122] Implementation Effect Example

[0123] 1. Isolation and purification of ACC in the fermentation broth of Application Example 5

[0124] Ion exchange chromatography separation and purification scheme and parameters for 1-aminocyclopropane-1-carboxylic acid (ACC) in the fermentation broth of Application 5:

[0125] Use an AminoPac PA-10 ion exchange column, elution system: gradient elution with 0.25 mol / L NaOH, 1.00 mol / L NaAc, and water;

[0126] Operating parameters: flow rate 0.25 mL / min, column temperature 30 °C, injection volume 25 μL.

[0127] The results are as Figure 2As shown, a single-component sample was obtained from the fermentation broth, which was the sample of the separation and purification of ACC, and was verified as ACC by high-resolution mass spectrometry detection.

[0128] 2. HR-MS Detection of ACC in the Fermentation Broth of Application Example 5

[0129] After the purified sample solution passed through a 0.22 μm organic membrane, 1-aminocyclopropane-1-carboxylic acid was analyzed by high-performance liquid chromatography. The detection conditions were as follows: The Agilent 6545A QTOF mass spectrometer was controlled by the control software (LC / MS Data Acquisition, Version B.08.00) based on the Auto MS / MS mode to collect primary and secondary mass spectrometry data. The MS collection rate was 6 spectrum / s, and the MS2 collection rate was 12 spectrum / s. The secondary collision energy of the sample was detected at 10 v and 40 v respectively, and 12 ions in the primary spectrum were selected for secondary scanning. The primary mass scanning range was m / z (50 - 1300), and the secondary mass scanning range was m / z (20 - 1300). The positive mode was used for collection respectively, and the data storage format was centroid; the ESI ion source parameters were set as follows: ion source drying gas temperature (Gas Temp): 320 °C, nitrogen flow rate (Gas Flow): 8 L / min, sheath gas flow rate (Sheath Gas Flow): 12 L / min, sheath gas temperature (Sheath Gas Temp): 350 °C; capillary voltage (VCap): 4000 V (positive ion mode), 3500 V (negative ion mode).

[0130] The results are as Figure 3 shown. The molecular weight of ACC in the fermentation broth was 102.0927, and the molecule was C4H8NO2, which was consistent with the molecular weight and molecular formula of ACC reported in the literature.

[0131] 3. Analysis of the Fermentation Yield of 1-Aminocyclopropane-1-Carboxylic Acid

[0132] The detection method for the yield of 1-aminocyclopropane-1-carboxylic acid was as follows: Take 2 mL of the cultured fermentation broth, centrifuge at 12000×g for 3 min, wash with sterile water, add an appropriate amount of glass beads (0.5 mm) and 1 mL of extraction solution (water: n-butanol: trifluoroacetic acid = 1:10:2), oscillate and break for 5 min, ultrasonicate in ice water for 30 min, centrifuge at 10000×g for 5 min, and take the supernatant (extract twice and combine the supernatants); after the supernatant passed through a 0.22 μm organic membrane, the yield of 1-aminocyclopropane-1-carboxylic acid was analyzed by high-performance liquid chromatography. The detection conditions were: DAD detector, Waters C18 chromatographic column (250 mm × 4.6 mm, 5 μm), mobile phase: methanol: water: trifluoroacetic acid (50:50:0.8, v / v), flow rate 0.1 mL / min, column temperature 30 °C, detection wavelength 276 nm; three biological replicate tests were performed for each sample to be tested; the standard of 1-aminocyclopropane-1-carboxylic acid was used for quantitative analysis.

[0133] Table 2 ACC content in the fermentation of engineered Saccharomyces cerevisiae

[0134]

[0135] The fermentation broth of the above Application Examples 1-6 was taken to detect the ACC content. The results are shown in Table 2. It can be seen from Table 2 that the engineered Saccharomyces cerevisiae constructed by the present invention can ferment and produce ACC, and the highest yield is 50.56 g / L.

[0136] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a yeast engineered strain producing 1-aminocyclopropane-1-carboxylic acid, characterized in that, The steps are as follows: (1)Construction of the ACC1 gene cassette recombinant vector: Using the yeast strain genome as a template, PCR amplification was performed with primers LEU (Dn) + pADH1-F and LEU2-R to obtain the downstream fragment of the homologous arm; Using plasmid pHDE-Cas9 as a template, primers RoPYC +LEU(up)-F and Kan MX+Hind Ⅲ-R were used for PCR amplification to obtain RoPYC fragment; Using plasmid UAS TEF1+CIT1+CLB2 as a template, PCR amplification was carried out using primers UAS+PTDH3-R and UAS+KANMX-F to obtain the UAS fragment; Using the obtained downstream fragment of the homologous arm, RoPYC fragment and the UAS fragment as templates, the ACC1 gene cassette was obtained by fusion PCR amplification with primers LEU2-up-F and UAS+PTDH3-R; (2)Construction of the ACC2 gene cassette recombinant vector: Using the yeast strain genome as a template, PCR amplification was performed with primers pTDH3 + UAS-F and pTDH3 + sPn50-R to obtain the promoter TDH3 fragment; Using the artificially synthesized Sam2 as a template, PCR amplification was carried out using primers sPn50+EGFP-R and sPn50+pTDH3-F to obtain Sam2 fragment; Using plasmid pT4-CMV-GFP as a template, PCR amplification was performed with primers EGFP+ Sam2 -F and EGFP+tPFK1-R to obtain the EGFP fragment; Using the obtained TDH3 fragment, Sam2 fragment and the EGFP fragment as templates, the primers pTDH3+UAS-F and EGFP+tPFK1-R were used for fusion PCR amplification to obtain the ACC2 gene cassette; (3)Construction of the ACC3 gene cassette recombinant vector: Using the yeast strain genome as a template, PCR amplification was performed with primers Leu2-up-F and LEU2 (up) + Kan MX-R to obtain the upstream fragment of the homologous arm; Using the yeast strain genome as a template, PCR amplification was performed with primers tPFK1 + EGFP-F and PNUGT31 + tPFK1 + SmaI-R to obtain the tPFK1 fragment; Using the artificially synthesized GmACS as a template, PCR amplification was carried out using primers PNUGT31 + tPFK1-F and pADH1 + LEU2(Dn)-R to obtain GmACS fragment; The obtained upstream fragment of the homologous arm, tPFK1 fragment and GmACS fragment were used as templates, and primers tPFK1+EGFP-F and LEU2-R were used for fusion PCR amplification to obtain the ACC3 gene cassette; (4)The ACC1 gene cassette recombinant vector in step (1), the ACC2 gene cassette recombinant vector in step (2), and the ACC3 gene cassette recombinant vector in step (3) were linearized to obtain the ACC1 linearized fragment, the ACC2 linearized fragment, and the ACC3 linearized fragment, respectively; (5)The ACC1 linearized fragment, the ACC2 linearized fragment, and the ACC3 linearized fragment in step (4) were co-transformed into yeast cells, and the obtained positive transformants were the yeast engineering bacteria producing ACC.

2. The construction method of the engineered yeast strain for producing 1-aminocyclopropane-1-carboxylic acid according to claim 1, characterized in that: In the step (1) RoPYC The nucleotide sequence is as shown in SEQ ID No.1 or SEQ ID No.2 or SEQ ID No.3; the forward primer for fusion PCR amplification is LEU2-up-F, and the nucleotide sequence is as shown in SEQ ID No.10; the reverse primer is UAS+PTDH3-R, and the nucleotide sequence is as shown in SEQ ID No.

11.

3. The method for constructing a yeast engineering bacterium for producing 1-aminocyclopropane-1-carboxylic acid according to claim 2, wherein: The artificially synthesized Sam2 nucleotide sequence in step (2) is shown in SEQ ID No.4 or SEQ ID No.5 or SEQ ID No.6; The forward primer for fusion PCR amplification is pTDH3 + UAS-F, and its nucleotide sequence is as shown in SEQ ID No. 12; the reverse primer is EGFP + tPFK1-R, and its nucleotide sequence is as shown in SEQ ID No.

13.

4. The method for constructing a yeast engineering bacterium for producing 1-aminocyclopropane-1-carboxylic acid according to claim 3, characterized in that: The artificially synthesized GmACS nucleotide sequence in step (3) is as shown in SEQ ID No.7 or SEQ ID No.8 or SEQ ID No.9; the forward primer for fusion PCR amplification is tPFK1+EGFP-F, and the nucleotide sequence is as shown in SEQ ID No.14; the reverse primer is LEU2-R, and the nucleotide sequence is as shown in SEQ ID No.

15.

5. The method for constructing a yeast engineering strain for producing 1-aminocyclopropane-1-carboxylic acid according to claim 4, wherein: The intermediate vector is the pEASY vector.

6. The method for constructing a yeast engineering bacterium for producing 1-aminocyclopropane-1-carboxylic acid according to claim 5, characterized in that: The co-transformation method in step (5) is electrotransformation or lithium acetate transformation; the yeast cells are Saccharomyces cerevisiae W303a.

7. The method for constructing a yeast engineering bacterium for producing 1-aminocyclopropane-1-carboxylic acid according to claim 6, characterized in that: The yeast strain is Saccharomyces cerevisiae W303a; the reaction system of fusion PCR is 50 μL: 1 μL of template, 2 μL each of 10 mM upstream primer and 10 mM downstream primer, 25 μL of enzyme mixture, and deionized water was added to make up 50 μL; the PCR reaction program is: 94°C for 5 min; 94°C for 30 s, 56°C for 1.5 min, 72°C for 1 min, 35 cycles; 72°C for 7 min.

8. An engineered bacterium constructed by the method for constructing a yeast engineering bacterium producing 1-aminocyclopropane-1-carboxylic acid according to any one of claims 1-7.

9. Use of the engineered bacterium according to claim 8 in the fermentative production of 1-aminocyclopropane-1-carboxylic acid.

10. The application according to claim 9, wherein The steps are as follows: Inoculate the engineered bacterium seed liquid into the fermentation medium at a ratio of 2-8% and ferment and culture at 25-30°C for 24-48 h to achieve the synthesis of 1-aminocyclopropane-1-carboxylic acid.

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