Method for biosynthesizing butanone alcohol

Abstract

The invention relates to the technical field of gene engineering, and discloses a method for biosynthesizing butanone alcohol, which is characterized in that 1, 3-butanediol is used as a substrate to produce recombinant escherichia coli of butanone alcohol, and the escherichia coli expresses acetoin reductase, NADH oxidase and hemoglobin through plasmids. According to the method for biologically synthesizing the butanone alcohol, the butanone alcohol is generated by carrying out whole-cell catalysis on the modified escherichia coli from 1, 3-butanediol, so that the green and efficient industrial production of the butanone alcohol is achieved. According to the engineering strain for producing the butanone alcohol by taking 1, 3-butanediol as a substrate, acetoin reductase, NADH oxidase and hemoglobin are subjected to free expression through plasmids, a combination with high butanone alcohol yield and high butanone alcohol conversion rate is screened, the acetoin reductase, the NADH oxidase and the hemoglobin are integrated through a genome, so that protein is stably expressed, and the high yield and stability of the butanone alcohol are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a method for biosynthesizing butanone alcohol. Background Art

[0002] Butanone alcohol, also known as 4-hydroxy-2-butanone, is a small molecule organic compound containing carbonyl and hydroxyl functional groups. It is a colorless to pale yellow liquid with a distinctive odor, a low melting point, and high volatility. As an important chemical intermediate, butanone alcohol has a wide range of applications in various fields. In the pharmaceutical industry, it is used as an intermediate step in the synthesis of various drugs, particularly in the production of vitamin A and other drugs. In flavors and fragrances, it is a key raw material for the synthesis of edible and cosmetic flavors such as raspberry ketone. Butanone alcohol may also be involved in the research of bioactive substances and has the potential to be used in feed additives due to its growth-promoting effects on animals.

[0003] There are various methods for synthesizing butanone alcohol. Common pathways include reducing ketone compounds to produce the corresponding alcohols; generating hydroxyketones through the oxidation of specific alcohols; or hydrolyzing and subsequently converting specific ester compounds. Due to its uniqueness and versatility, butanone alcohol has attracted a large number of manufacturers to enter the market, but chemical synthesis is the primary method. With the development of the pharmaceutical and fine chemical industries, the butanone alcohol market is expected to continue to expand, and technological advances will also promote the development of more efficient and environmentally friendly synthesis processes. Microorganisms, as an environmentally friendly and efficient synthesis method, have attracted widespread attention from various industries, and the synthesis of butanone alcohol through the modification of microorganisms has become a trend. Summary of the Invention

[0004] (1) Technical problems solved

[0005] In response to the deficiencies of the prior art, the present invention provides a method for the biosynthesis of butanone alcohol. By genetic modification, a recombinant Escherichia coli is provided that can produce a high amount of butanone alcohol using 1,3-butanediol as a substrate. The recombinant Escherichia coli contains genetically integrated acetoin reductase, NADH oxidase, and hemoglobin genes that can perform catalytic reactions.

[0006] (2) Technical solution

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solutions: a method for biosynthesizing butanone alcohol, comprising producing recombinant Escherichia coli using 1,3-butanediol as a substrate for producing butanone alcohol, wherein the Escherichia coli expresses acetoin reductase, NADH oxidase, and hemoglobin via a plasmid;

[0008] The acetoin reductase is derived from:

[0009] Serratia plymuthica(SpAR),

[0010] Mangrovibacter plantisponsor (MpAR),

[0011] Tatumel la citrea(TcAR),

[0012] Acinetobacter tandoii (AtAR),

[0013] Proteus mirabi l is(PmAR),

[0014] Ectorhizobium quercum (EqAR);

[0015] The NADH oxidase is derived from Levi lactobaci l lus brevis (LbNOX);

[0016] The hemoglobin was derived from Vitreoscilla (VsHB).

[0017] Preferably, the recombinant Escherichia coli that produces butanone alcohol using 1,3-butanediol as a substrate has acetoin reductase, NADH oxidase and hemoglobin integrated into its genome.

[0018] Preferably, the acetoin reductase, NADH oxidase and hemoglobin are derived from

[0019] Proteus mirabi l is(PmAR),

[0020] Levilactobaci l lus brevis(LbNOX),

[0021] Vitreoscilla (VsHB).

[0022] A method for biosynthesizing butanone alcohol comprises the following steps:

[0023] Step 1: The amino acid sequence of the relevant protein is shown in Table 1. The protein gene is obtained by reverse transcription, codon optimized, and constructed into the corresponding plasmid;

[0024] Step 2: Prepare competent cells of strain S1 by CaCl2 treatment, and heat shock at 42℃ to transfer plasmids

[0025] pET28a-SpAR+LbNOX+VsHB,

[0026] pET28a-MpAR+LbNOX+VsHB,

[0027] pET28a-TcAR+LbNOX+VsHB,

[0028] pET28a-AtAR+LbNOX+VsHB,

[0029] pET28a-PmAR+LbNOX+VsHB,

[0030] pET28a-EqAR+LbNOX+VsHB;

[0031] Transfected with strain S1, cultured overnight at 37°C, single clones were picked, and strains S2, S3, S4, S5, S6, and S7 were obtained (see Table 2). PCR and sequencing were performed for verification, and the verified strains were frozen in glycerol at -80°C.

[0032] Step 3: Frozen strains S1, S2, S3, S4, S5, S6, and S7 were inoculated into 10 ml of LB medium (10.0 g / L peptone, 5.0 g / L yeast extract, 10 g / L sodium chloride, 50 μg / L kanamycin), cultured overnight at 37°C as seed solution, and then transferred to 100 ml of LB medium (containing 50 μg / L kanamycin), cultured at 37°C until OD600 reached 0.6, and induced with 0.6 mM IPTG at 20°C for 16 h.

[0033] Step 4: Collect the cells by centrifugation and dissolve 10 g / L of the cells in 50 mM Tris-HCl buffer (pH 8.0) and 18 g / L 1,3-butanediol. Catalyze at 37°C for 8 hours. Remove the precipitate by centrifugation, extract the supernatant with an equal volume of ethyl acetate, and analyze by gas chromatography to produce S-butanone alcohol.

[0034] (3) Beneficial effects

[0035] Compared with the prior art, the present invention provides a method for biosynthesizing butanone alcohol, which has the following characteristics:

[0036] Beneficial effects:

[0037] This method for the biosynthesis of butanone alcohol uses whole-cell catalysis of 1,3-butanediol in modified Escherichia coli, achieving green and efficient industrial production of butanone alcohol. The engineered butanone alcohol-producing strain, using 1,3-butanediol as a substrate, first expresses acetoin reductase, NADH oxidase, and hemoglobin via plasmids. A combination with high butanone alcohol yield and conversion rate is screened. Finally, genomic integration of acetoin reductase, NADH oxidase, and hemoglobin ensures stable protein expression, achieving high and stable butanone alcohol production. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the synthesis route of S-type butanone alcohol of the present invention;

[0039] Figure 2 Schematic diagram of the plasmid of the present invention;

[0040] Figure 3 This is the electrophoresis diagram of the present invention. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] The present invention provides a technical solution.

[0043] See also Figure 1-3 A method for biosynthesizing butanone alcohol, a recombinant Escherichia coli that produces butanone alcohol using 1,3-butanediol as a substrate, wherein the Escherichia coli expresses acetoin reductase, NADH oxidase and hemoglobin through a plasmid.

[0044] The acetoin reductase is derived from:

[0045] Serratia plymuthica(SpAR),

[0046] Mangrovibacter plantisponsor (MpAR),

[0047] Tatumel la citrea(TcAR),

[0048] Acinetobacter tandoii (AtAR),

[0049] Proteus mirabil is(PmAR),

[0050] Ectorhizobium quercum (EqAR);

[0051] The NADH oxidase is derived from Levi lactobaci l lus brevis (LbNOX);

[0052] The hemoglobin is derived from Vitreoscilla (VsHB);

[0053] The invention relates to a recombinant Escherichia coli that produces butanone alcohol using 1,3-butanediol as a substrate, wherein acetoin reductase, NADH oxidase and hemoglobin are integrated into the Escherichia coli genome.

[0054] The acetoin reductase, NADH oxidase and hemoglobin are respectively derived from

[0055] Proteus mirabi l is(PmAR),

[0056] Levilactobaci l lus brevis(LbNOX),

[0057] Vitreoscilla (VsHB).

[0058] Step 1:

[0059] The amino acid sequences of the related proteins are shown in Table 1. The protein genes were obtained by reverse transcription, codon optimized, and constructed into the corresponding plasmids. The plasmid schematic diagram is shown in Figure 2 shown.

[0060] Table 1 Sequences involved in the study

[0061]

[0062] Table 2 Plasmids and strains used in the study

[0063] strain illustrate source pET28a-SpAR+LbNOX+VsHB Expression plasmid for expression of SpAR in strain S1 The present invention is constructed pET28a-MpAR+LbNOX+VsHB Expression plasmid for expressing MpAR in strain S2 The present invention is constructed pET28a-TcAR+LbNOX+VsHB Expression plasmid for expressing TcAR in strain S2 The present invention is constructed pET28a-AtAR+LbNOX+VsHB Expression plasmid for expressing AtAR in strain S2 The present invention is constructed pET28a-PmAR+LbNOX+VsHB Expression plasmid for expressing PmAR in strain S2 The present invention is constructed pET28a-EqAR+LbNOX+VsHB Expression plasmid for expressing EqAR in strain S2 The present invention is constructed

[0064] strain illustrate source Strain S1(BL21(DE3)) Expression strain This room is preserved strain S2 Strain S1 was transformed with plasmid pET28a-SpAR The present invention is constructed Strain S3 Strain S1 was transformed with plasmid pET28a-MpAR The present invention is constructed Strain S4 Strain S1 was transformed with plasmid pET28a-TcAR The present invention is constructed Strain S5 Strain S1 was transformed with plasmid pET28a-AtAR The present invention is constructed strain S6 Strain S1 was transformed with plasmid pET28a-PmAR The present invention is constructed strain S7 Strain S1 was transformed with plasmid pET28a-EqAR The present invention is constructed

[0065] Step 2:

[0066] The competent cells of strain S1 were prepared by CaCl2 treatment, and the plasmids pET28a-SpAR+LbNOX+VsHB,

[0067] pET28a-MpAR+LbNOX+VsHB,

[0068] pET28a-TcAR+LbNOX+VsHB,

[0069] pET28a-AtAR+LbNOX+VsHB,

[0070] pET28a-PmAR+LbNOX+VsHB,

[0071] pET28a-EqAR+LbNOX+VsHB;

[0072] Transfected with strain S1, cultured at 37°C overnight, picked single clones, and obtained strains S2, S3, S4, S5, S6, and S7 (see Table 2). PCR and sequencing verification were performed, and the verified correct strains were frozen at -80°C using glycerol.

[0073] Step 3:

[0074] Frozen strains S1, S2, S3, S4, S5, S6, and S7 were inoculated into 10 ml LB medium (10.0 g / L peptone, 5.0 g / L yeast extract, 10 g / L sodium chloride, 50 μg / L kanamycin), cultured overnight at 37°C as seed solution, and then transferred into 100 ml LB medium (containing 50 μg / L kanamycin), cultured at 37°C until OD600 reached 0.6, and induced with 0.6 mM IPTG at 20°C for 16 h.

[0075] Step 4:

[0076] The cells were collected by centrifugation, and 10 g / L of the cells were dissolved in 50 mM Tris-HCl buffer (pH 8.0) and 18 g / L 1,3-butanediol, and catalyzed at 37°C for 8 hours. The precipitate was removed by centrifugation, and the supernatant was extracted with an equal volume of ethyl acetate. The generated S-butanone alcohol was detected by gas chromatography, and the results are shown in Table 3 below.

[0077] Table 3 strain S-butanone alcohol (g / L) Strain S1 0.58 strain S2 16.59 Strain S3 15.43 Strain S4 14.20 Strain S5 13.15 strain S6 10.23 strain S7 6.13

[0078] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for biosynthesizing butanone alcohol, characterized in that: A recombinant Escherichia coli that produces butanone alcohol using 1,3-butanediol as a substrate, wherein the Escherichia coli expresses acetoin reductase, NADH oxidase, and hemoglobin via a plasmid; The acetoin reductase is derived from: Serratia plymuthica(SpAR), Mangrovibacter plantisponsor (MpAR), Tatumel la citrea(TcAR), Acinetobacter tandoii (AtAR), Proteus mirabi l is(PmAR), Ectorhizobium quercum (EqAR); The NADH oxidase is derived from Levi lactobaci l lus brevis (LbNOX); The hemoglobin was derived from Vitreoscilla (VsHB).

2. A method for biosynthesizing butanone alcohol according to claim 1, characterized in that: The recombinant Escherichia coli that uses 1,3-butanediol as a substrate to produce butanone alcohol has acetoin reductase, NADH oxidase and hemoglobin integrated into its genome.

3. A method for biosynthesizing butanone alcohol according to claim 2, characterized in that: The acetoin reductase, NADH oxidase and hemoglobin are respectively derived from Proteus mirabi l is(PmAR), Levilactobaci l lus brevis(LbNOX), Vitreoscilla (VsHB).

4. A method for biosynthesizing butanone alcohol according to claim 1, characterized in that: The following steps are included: Step 1: The amino acid sequence of the relevant protein is shown in Table 1. The protein gene is obtained by reverse transcription, codon optimized, and constructed into the corresponding plasmid; Step 2: Prepare competent cells of strain S1 by CaCl2 treatment, and heat shock at 42℃ to transfer plasmids pET28a-SpAR+LbNOX+VsHB, pET28a-MpAR+LbNOX+VsHB, pET28a-TcAR+LbNOX+VsHB, pET28a-AtAR+LbNOX+VsHB, pET28a-PmAR+LbNOX+VsHB, pET28a-EqAR+LbNOX+VsHB; Transfected with strain S1, cultured overnight at 37°C, single clones were picked, and strains S2, S3, S4, S5, S6, and S7 were obtained (see Table 2). PCR and sequencing were performed for verification, and the verified strains were frozen in glycerol at -80°C. Step 3: Frozen strains S1, S2, S3, S4, S5, S6, and S7 were inoculated into 10 ml of LB medium (10.0 g / L peptone, 5.0 g / L yeast extract, 10 g / L sodium chloride, 50 μg / L kanamycin), cultured overnight at 37°C as seed solution, and then transferred to 100 ml of LB medium (containing 50 μg / L kanamycin), cultured at 37°C until OD600 reached 0.6, and induced with 0.6 mM IPTG at 20°C for 16 h. Step 4: Collect the cells by centrifugation and dissolve 10 g / L of the cells in 50 mM Tris-HCl buffer (pH 8.0) and 18 g / L 1,3-butanediol. Catalyze at 37°C for 8 hours. Remove the precipitate by centrifugation, extract the supernatant with an equal volume of ethyl acetate, and analyze by gas chromatography to produce S-butanone alcohol.

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