Saccharomyces cerevisiae engineering bacterium for producing cinnamyl alcohol as well as construction method and application thereof

Abstract

The invention belongs to the field of microbial genetic engineering, and particularly relates to a saccharomyces cerevisiae engineering bacterium for producing cinnamyl alcohol as well as a construction method and application of the saccharomyces cerevisiae engineering bacterium. The recombinant saccharomyces cerevisiae comprises a phenylalanine ammonia lyase gene Arval derived from anoectochilus roxburghii, a phosphopantetheine ethylamine phosphotransferase gene Bssfp derived from bacillus subtilis, and a reductase gene Nicar derived from nocardia aralgarica, or a reductase gene Nicar of nocardia otitis media, or a carboxylic acid reductase gene Mpar of mycobacterium phlei. According to the invention, the total synthesis of cinnamyl alcohol in the saccharomyces cerevisiae is realized for the first time, and the cinnamyl alcohol produced by using the recombinant saccharomyces cerevisiae can reach 30.30 mg / L.

Description

Technical Field

[0001] The invention belongs to the field of microbial genetic engineering, and particularly relates to an engineering yeast for producing cinnamyl alcohol, a construction method and an application thereof. Background Art

[0002] Cinnamyl alcohol is also known as cinnamyl alcohol, 3-phenyl-2-propene-1-ol, β-phenylpropenol, etc. Its molecular formula is C9H 10 O, relative molecular weight 134.18. Cinnamyl alcohol is an aromatic alcohol with a mild, long-lasting and comfortable aroma similar to hyacinth and ointment, and also has a certain sweetness. Cinnamyl alcohol has many important functions and uses: first, cinnamyl alcohol can be used to prepare flavors, can also be used as a fixative, and can also be used to synthesize spices such as cinnamyl acetate and cinnamyl methyl acrylate; second, cinnamyl alcohol has good anti-inflammatory and antibacterial activity; third, cinnamyl alcohol is an important pharmaceutical intermediate, which is used to prepare fungal infection drugs naftifine, anticancer substances toremifene and calcium antagonist cilnidipine. At present, the sales of cinnamyl alcohol products in the global market have surged and are in short supply.

[0003] The existing technology mainly prepares cinnamyl alcohol by chemical synthesis, but the chemical synthesis method has many by-products, is difficult to purify, and the extraction waste has a large environmental pollution, which is not the best method for preparing cinnamyl alcohol. For this reason, technicians also try to extract cinnamyl alcohol from storax oil, Peruvian sesame oil and cinnamon oil by saponification, but the content of cinnamyl alcohol in natural raw materials is low, and there are problems such as low extraction yield and great extraction difficulty. Therefore, it is necessary to find a method for preparing cinnamyl alcohol with high extraction efficiency and environmental friendliness.

[0004] In recent years, biosynthesis technology has been widely used in the production and preparation of various types of compounds due to its advantages such as mild reaction conditions, environmental friendliness, high specificity, fast preparation speed, high yield, and sustainable production. Biosynthesis technology is mainly the process of specific engineered bacteria using substrates through a series of biosynthetic pathways to produce compounds, but there are currently no reports on engineered bacteria for the efficient preparation of cinnamyl alcohol.

[0005] The present invention aims to solve at least one of the above problems. Summary of the invention

[0006] The present invention aims to use genetic engineering technology to provide a method for constructing a plasmid expression engineering bacteria method, using a ura3 gene knockout strain KMLY1-8 of wild-type saccharomyces cerevisiae KMLY1-2 as a chassis cell, cloning a phenylalanine ammonia lyase gene Arpal from Anoectochilus roxburghii, a phosphopantetheinyl phosphotransferase gene Bssfp from Bacillus subtilis, and a carboxylic acid reductase gene Nicar from Nocardia iowensis, or a carboxylic acid reductase gene Nocar from Nocardia otitidiscaviarum, or a carboxylic acid reductase gene Mpcar from Mycolicibacterium phlei into a plasmid pY26TEF-GPD, and then constructing a plasmid expression engineering bacteria method, and the engineering bacteria obtained by the method can efficiently produce cinnamyl alcohol. The present invention has great application potential in the field of cinnamyl alcohol biosynthesis.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The first invention of the present invention provides an engineered yeast of cinnamyl alcohol, wherein the engineered bacteria is a recombinant yeast of cinnamyl alcohol, and the recombinant yeast of cinnamyl alcohol comprises a phenylalanine ammonia lyase gene Arpal from Anoectochilus roxburghii, a phosphopantetheinyl phosphotransferase gene Bssfp from Bacillus subtilis, and a reductase gene Nicar from Nocardia iocardia, a reductase gene Nocar from Nocardia otitidis, or a carboxylic acid reductase gene Mpcar from Mycobacterium phlei.

[0009] The second aspect of the present invention provides a method for constructing an engineered yeast strain for producing cinnamyl alcohol, wherein the ura3 gene knockout strain KMLY1-8 of wild-type Saccharomyces cerevisiae KMLY1-2 is used as a chassis strain, and the phenylalanine ammonia lyase gene Arpal from Anoectochilus roxburghii, the phosphopantetheinyl phosphotransferase gene Bssfp from Bacillus subtilis, and the reductase gene Nicar from Nocardia iodica, or the reductase gene Nocar from Nocardia otitidis, or the carboxylic acid reductase gene Mpcar from Mycobacterium phlei are integrated into the genome of the chassis strain, and the construction method comprises the following specific steps:

[0010] Step (1), knocking out the ura3 gene in the wild-type Saccharomyces cerevisiae KMLY1-2 to obtain a chassis strain named Saccharomyces cerevisiae KMLY1-8;

[0011] Step (2), preparing Saccharomyces cerevisiae KMLY1-8 into competent cells;

[0012] Step (3), constructing Arpal-Bssfp double gene expression plasmid;

[0013] Step (4), using the Arpal-Bssfp double gene expression plasmid obtained in step (3) as a template to construct the Arpal-Bssfp-Nicar three-gene expression plasmid, in the transformation system, using the PEG-lithium acetate chemical transformation method to transform the Arpal-Bssfp-Nicar three-gene expression plasmid into the KMLY1-8 competent cells obtained in step (2), picking a single colony, and using primers PY-vF (PAL) and +PY-vR (PAL), primers PY-Vf and PY-Vr, and primers MCS1-vF1 and MCS1-vR1 to perform colony PCR verification, and verifying that the correct strain is the cinnamyl alcohol-producing Saccharomyces cerevisiae engineered strain, named KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-NiCAR;

[0014] Or, the Arpal-Bssfp double gene expression plasmid obtained in step (3) is used as a template to construct the Arpal-Bssfp-Nocar triple gene expression plasmid, and in the transformation system, the Arpal-Bssfp-Nocar triple gene expression plasmid is transformed into the KMLY1-8 competent cells obtained in step (2) using the PEG-lithium acetate chemical transformation method, and a single colony is picked, and the primers PY-vF (PAL) and +PY-vR (PAL), primers PY-Vf and PY-Vr, and primers MCS1-vF1 and MCS1-vR1 are used to perform colony PCR verification, and the correct strain is the cinnamyl alcohol-producing Saccharomyces cerevisiae engineered strain, which is named KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-NoCAR;

[0015] Alternatively, the Arpal-Bssfp double gene expression plasmid obtained in step (3) is used as a template to construct the Arpal-Bssfp-Mpcar triple gene expression plasmid. In the transformation system, the Arpal-Bssfp-Mpcar triple gene expression plasmid is transformed into the KMLY1-8 competent cells obtained in step (2) using the PEG-lithium acetate chemical transformation method. A single colony is picked and colony PCR verification is performed using primers PY-vF (PAL) and +PY-vR (PAL), primers PY-Vf and PY-Vr, and primers MCS1-vF1 and MCS1-vR1, respectively. The correct strain is the engineered yeast of Saccharomyces cerevisiae producing cinnamyl alcohol, which is named KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-MpCAR.

[0016] The above-mentioned brewer's yeast KMLY1-2 was isolated and obtained by the inventor in the early stage and deposited in the China Center for Type Culture Collection with the deposit number CCTCC M 2018457.

[0017] Furthermore, the method for knocking out the ura3 gene in the wild-type Saccharomyces cerevisiae KMLY1-2 in the step (1) is: constructing a recombinant plasmid p3052-ura3-sgRNA, and in the transformation system, using the PEG-lithium acetate chemical transformation method to transform the plasmid pCfB2312 expressing the Cas9 protein, the recombinant plasmid p3052-ura3-sgRNA and the donor DNA into the KMLY1-2 strain competent cells, and using primers Ura3-vF1 and Ura3-vR1 to perform colony PCR verification on a single colony, and the successfully verified strain is the Saccharomyces cerevisiae KMLY1-2 ura3 gene knockout strain, Saccharomyces cerevisiae KMLY1-8.

[0018] Furthermore, the construction method of the Arpal-Bssfp dual gene expression plasmid in the step (3) is as follows: codon optimization is performed on the phenylalanine ammonia lyase gene Arpal from golden thread orchid, and then the promoter pADH1 (as shown in SEQ ID No.31) and the terminator tPDC1 (as shown in SEQ ID No.32) are synthesized at its 5′ and 3′ ends respectively, and the synthesized DNA is used as a template (as shown in SEQ ID No.33), and primers PAL-F1① and PAL-R1① are used to perform PCR amplification to obtain the gene Arpal expression cassette; codon optimization is performed on the phosphopantetheinyl phosphotransferase gene Bssfp from Bacillus subtilis, and the synthesized DNA is used as a template (as shown in SEQ ID No.34), primers BsSFP-F1 and BsSFP-R1 were used to perform PCR amplification to obtain the coding region of gene Bssfp; plasmid pY26TFE-GPD was used as a template, primers PY26-F1① and PY26-R1① were used to perform PCR amplification to obtain a linearized expression vector; genes Arpal, Bssfp and the linearized vector were seamlessly cloned, and plasmids from positive transformants were extracted to obtain Arpal-Bssfp double gene expression plasmid, named

[0019] pY26TEF-GPD-ArPAL-BsSFP.

[0020] Furthermore, the construction method of the Arpal-Bssfp-Nicar three-gene expression plasmid in the step (4) is as follows: codon optimization is performed on the carboxylate reductase gene Nicar of Nocardia iodica, and the synthesized DNA is used as a template (as shown in SEQID No.35), and primers NiCAR-F1 and NiCAR-R1 are used to perform PCR amplification to obtain the coding region of the gene Nicar; the Arpal-Bssfp double-gene expression plasmid prepared in step (3) is used as a template, and primers MCS1-F1 and MCS1-R1 are used to perform PCR amplification to obtain a linearized expression vector; the gene Nicar and the linearized vector are seamlessly cloned, and the plasmid in the positive transformant is extracted to obtain the Arpal-Bssfp-Nicar three-gene expression plasmid;

[0021] The method for constructing the Arpal-Bssfp-Nocar triple gene expression plasmid is as follows: codon optimization is performed on the carboxylate reductase gene Nocar of Nocardia otitis media, and the synthesized DNA is used as a template (as shown in SEQ ID No.36) and primers NoCAR-F1 and NoCAR-R1 are used to perform PCR amplification to obtain the gene Nocar expression frame; the Arpal-Bssfp double gene expression plasmid prepared in step (3) is used as a template and primers MCS1-F1 and MCS1-R1 are used to perform PCR amplification to obtain a linearized expression vector; the gene Nocar and the linearized vector are seamlessly cloned, and the plasmid in the positive transformant is extracted to obtain the Arpal-Bssfp-Nocar triple gene expression plasmid;

[0022] The construction method of the Arpal-Bssfp-Mpcar three-gene expression plasmid is as follows: codon optimization is performed on the carboxylate reductase gene Mpcar of Mycobacterium phlei, and the synthesized DNA is used as a template (as shown in SEQ ID No. 37), and primers MpCAR-F1 and MpCAR-R1 are used to perform PCR amplification to obtain the gene Mpcar coding region; the Arpal-Bssfp double-gene expression plasmid prepared in step (3) is used as a template, and primers MCS1-F1 and MCS1-R1 are used to perform PCR amplification to obtain a linearized expression vector; the gene Mpcar and the linearized vector are seamlessly cloned, and the plasmid in the positive transformant is extracted to obtain the Arpal-Bssfp-Mpcar three-gene expression plasmid.

[0023] Furthermore, the construction method of the recombinant plasmid p3052-ura3-sgRNA is as follows: find the ura3 gene coding frame from the whole genome of Saccharomyces cerevisiae KMLY1-2, design sgRNA online, synthesize 50bp DNA single strands Ura3-20bp-F1, Ura3-20bp-R1, anneal the two 50bp DNA single strands to form a 50bp DNA double strand, use primers 3052F and 3052R to linearize the plasmid pCfB3052, and then seamlessly clone and connect it with the 50bp DNA double strand, and extract the plasmid in the positive transformant to obtain the recombinant plasmid p3052-ura3-sgRNA.

[0024] Furthermore, the PCR reaction procedure for obtaining the Arpal expression cassette by PCR amplification in step (3) is as follows: pre-denaturation at 95°C for 4 min, denaturation at 95°C for 15 s, annealing at 50.9°C for 15 s, extension at 72°C for 3 min, and after 30 cycles, extension at 72°C for 5 min, and then purification of the PCR product using a universal DNA purification recovery kit to obtain the Arpal expression cassette; the PCR reaction procedure for obtaining the Bssfp expression cassette by PCR amplification is as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 50.9°C for 15 s, extension at 72°C for 3 min, and extension at 72°C for 5 min. Anneal at ℃ for 15s, extend at 72℃ for 50s. After 30 cycles, extend at 72℃ for 5min, and purify the PCR product using a universal DNA purification and recovery kit to obtain the gene Bssfp expression frame; PCR amplification to obtain the linearized expression vector. The PCR reaction procedure is: pre-denaturation at 95℃ for 3min, denaturation at 95℃ for 15s, annealing at 50.9℃ for 15s, and extension at 72℃ for 7min. After 30 cycles, extend at 72℃ for 5min, and purify the PCR product using a universal DNA purification and recovery kit to obtain the linearized expression vector.

[0025] Furthermore, in the construction of the Arpal-Bssfp-Nicar three-gene expression plasmid in step (4), the reaction procedure for PCR amplification to obtain the Nicar gene is: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 49°C for 15 seconds, extension at 72°C for 50 seconds, and after 30 cycles, extension at 72°C for 5 minutes, and then purification of the PCR product using a universal DNA purification and recovery kit to obtain the Nicar coding region;

[0026] In the construction of the Arpal-Bssfp-Nocar three-gene expression plasmid, the reaction procedure for PCR amplification to obtain the gene Nocar was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 49°C annealing for 15 s, 72°C extension for 50 s, 30 cycles, 72°C extension for 5 min, and then a universal DNA purification and recovery kit was used to purify the PCR product to obtain the coding region of the gene Nocar;

[0027] In the construction of the Arpal-Bssfp-Nocar three-gene expression plasmid, the PCR amplification was performed to obtain the PCR reaction program of the gene Mpcar: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 49°C annealing for 15 s, 72°C extension for 50 s, and after 30 cycles, 72°C extension for 5 min, and then the PCR product was purified using a universal DNA purification and recovery kit to obtain the gene Mpcar coding region;

[0028] The reaction procedure for PCR amplification to obtain a linearized expression vector in the construction of the Arpal-Bssfp-Nocar three-gene expression plasmid is as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 51.2°C for 15 s, extension at 72°C for 9 min, and after 30 cycles, extension at 72°C for 5 min, purifying the PCR product using a universal DNA purification recovery kit to obtain a linearized expression vector.

[0029] The third aspect of the present invention also provides use of the engineered yeast Saccharomyces cerevisiae described in the first aspect for producing cinnamyl alcohol.

[0030] The fourth aspect of the present invention also provides a method for producing cinnamyl alcohol, comprising fermenting and culturing the engineered yeast Saccharomyces cerevisiae described in the first aspect to obtain cinnamyl alcohol from the culture.

[0031] Furthermore, the culture process is to carry out shake flask fermentation culture in SC-URA liquid culture medium for 24 hours.

[0032] The biosynthetic pathway of cinnamyl alcohol of the present invention is as follows: phenylalanine (Phe) generates cinnamic acid under the action of phenylalanine ammonia lyase (PAL), cinnamic acid forms cinnamaldehyde under the catalysis of carboxylic acid reductase (CAR), and finally cinnamaldehyde is reduced by cinnamyl alcohol dehydrogenase (CAD) under the action of reduced coenzyme II to produce cinnamyl alcohol. Carboxylic acid reductase (CAR) requires activation by phosphopantetheine transferase (SFP).

[0033] The present invention has the following beneficial effects:

[0034] (1) The present invention constructed three engineered strains of Saccharomyces cerevisiae producing cinnamyl alcohol, namely KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-NiCAR, KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-NoCAR, and KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-MpCAR. Among them, KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-MpCAR had the highest yield of 30.30 mg / L.

[0035] (2) The present invention realizes the total synthesis of cinnamyl alcohol in Saccharomyces cerevisiae for the first time. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a comparison chart of the cinnamyl alcohol production of the recombinant strains KMLY1-8 / pY26TEF-GPD-BsSFP-ArPAL-NiCAR, KMLY1-8 / pY26TEF-GPD-BsSFP-ArPAL-NoCAR, KMLY1-8 / pY26TEF-GPD-BsSFP-ArPAL-MpCAR and the control strain KMLY1-8 / pY26TEF-GPD constructed in a specific embodiment of the present invention. DETAILED DESCRIPTION

[0037] The present invention is further described below by way of examples, but is not limited to these examples.

[0038] The experimental methods without specific conditions in the examples are usually carried out under conventional conditions and conditions described in the manual, or under conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all commercially available unless otherwise specified.

[0039] The SC-URA culture medium used in the specific embodiment of the present invention is formulated as follows: 1.34 g YNB (Yeast nitrogen base, without amino acids, without ammonium sulfate), 0.5 g ammonium sulfate, add distilled water to make up the volume to 78 mL, adjust the pH to 5.8, make up the volume to 79 mL, and sterilize at 121° C. for 20 min; when the temperature drops to 55° C., add 10 mL 10× dropout solution, 10 mL 20% separately sterilized glucose solution, 1 mL 1 μmol / L filter-sterilized Fe(NH4)2(SO4)2 solution, and 0.2 mL 0.02% filter-sterilized D-Biotin; the formula of 10× dropout solution is: 0.02 g L-Histidine HCl, 0.1 g L-Leucine, 0.02 g L-Methionine, 0.03 g L-Tyrosine, 0.05 g L-Glutamine and 0.02 g L-Arginine were diluted to 100 mL with distilled water and sterilized at 121°C for 20 min.

[0040] The high performance liquid chromatography (HPLC) detection conditions were as follows: chromatographic column: Agilent Eclipse SB-C18 (4.6×150 μm, 5 μm); mobile phase: acetonitrile-0.1% formic acid water (30:70); flow rate: 0.8 mL / min; column temperature: 30°C; injection volume: 20 μL; detection wavelength: 254 nm.

[0041] Saccharomyces cerevisiae competent cells were prepared as follows:

[0042] (1) Take the -80°C Saccharomyces cerevisiae KMLY1-2 or KMLY1-8 strain, inoculate it into yeast extract peptone dextrose liquid medium (YPD) at a 1% (v / v) inoculum, and culture it overnight at 30°C and 150 rpm in a shaking incubator;

[0043] (2) Take 10 μL of bacterial solution and add it to 990 μL of sterile water. 600 Measure the bacterial count, OD 600 When the value is 0.1, the bacterial concentration is about 1×10 6 CFU / mL;

[0044] (3) After inoculation in 50 mL of YPD medium, the culture was incubated at 30°C and 150 rpm for about 4 h. The bacterial concentration was about 2.5 × 10 8 CFU / mL;

[0045] (4) Centrifuge the bacterial solution at 3000 g for 5 min to collect the cells, and then resuspend the precipitate in 25 mL of sterile water; centrifuge at 3000 g for 5 min, discard the supernatant, and resuspend the cells in 1 mL of sterile water;

[0046] (5) Transfer the bacterial suspension to a 1.5 mL centrifuge tube and centrifuge at 13,000 g for 30 seconds, then discard the supernatant;

[0047] (6) Resuspend the cells in 1 mL of sterile water, pipette 100 μL into a 1.5 mL centrifuge tube, centrifuge at 13,000 g for 30 s, discard the supernatant, and prepare Saccharomyces cerevisiae competent cells. Place the cells on ice for later use.

[0048] The chemical transformation method of Saccharomyces cerevisiae PEG-lithium acetate is as follows:

[0049] (1) Prepare the conversion system according to the formula in Table 1;

[0050] (2) Mix all 360 μL of the transformation system with the competent cells, and shake the mixture vigorously to mix it thoroughly with the bacterial solution;

[0051] (3) Place the mixed system in a water bath at 42°C for 40 min;

[0052] (4) Centrifuge at 12000g for 30 seconds, discard the supernatant, and resuspend the cells in 1 mL of YPD medium;

[0053] (5) Incubate the bacterial suspension at 30°C for 2-3 h. After centrifugation, spread the bacteria on SC-URA solid plates and culture at 30°C for 48 h. After transformants grow, perform colony PCR verification.

[0054] Table 1 Conversion system formula

[0055]

[0056]

[0057]

[0058] Table 2 Primer sequence list

[0059] Note: The lowercase letters of the primer sequences in the table are the homologous sequences of the plasmid in seamless cloning.

[0060] In the specific implementation manner of the present invention, the primer sequences used are shown in Table 2 above.

[0061] In addition, in the specific embodiment of the present invention, Saccharomyces cerevisiae KMLY1-2 was isolated and obtained by the inventor in the early stage and deposited in the China Center for Type Culture Collection with the deposit number of CCTCC M 2018457. The expression plasmid pY26TEF-GPD was purchased from Wuhan Miaoling Biotechnology Co., Ltd. The gene editing plasmids pCfB3052 and pCfB2312 were purchased from Addgene. The seamless cloning kit (CloneExpress Ultra One Step Cloning Kit) was purchased from Novozymes, and the operation method was performed according to the kit instructions. The results in the following examples are the average values ​​of three replicates unless otherwise specified.

[0062] Example 1: Construction of KMLY1-8 strain and KMLY1-8 / pY26TEF-GPD strain

[0063] The coding frame of the ura3 gene was found from the whole genome of Saccharomyces cerevisiae KMLY1-2, and sgRNA was designed online. The sequence included 15 bp sequence homologous to linearized pCfB3052, 20 bp gRNA, and 15 bp sequence homologous to linearized pCfB3052. The following two designed 50 bp DNA single strands were sent to Shanghai Biotech Co., Ltd. for synthesis:

[0064] Ura3-20bp-F1: 5′-gaaagataaatgatcGGTGTGGGTTTAGATGACAAgttttagagctagaa-3′ (as shown in SEQ ID No. 38);

[0065] Ura3-20bp-R1: 5′-ttctagctctaaaacTTGTCATCTAAACCCACACCgatcatttatctttc-3′ (as shown in SEQ ID No. 39).

[0066] The lowercase letters in the sequence are homologous to the linearized pCfB3052, and the uppercase letters are the 20 bp gRNA sequence. Two 50 bp DNA single strands were annealed to form a 50 bp DNA double strand. The plasmid pCfB3052 was linearized using primers 3052F and 3052R, and then seamlessly cloned and connected with the 50 bp DNA double strand. The recombinant plasmid p3052-ura3-sgRNA was extracted from the positive transformants.

[0067] Using the genomic DNA of Saccharomyces cerevisiae KMLY1-2 as a template, the upstream homology arm of the ura3 gene was amplified using primers Ura3-uF1 and Ura3-uR1, and the downstream homology arm of the ura3 gene was amplified using Ura3-dF1 and Ura3-dR1. The upstream homology arm of the ura3 gene was subjected to agarose gel electrophoresis, and the upstream and downstream homology arm fragments were purified and recovered. Using the purified upstream and downstream homology arms as templates, and using Ura3-uF1 and Ura3-dR1 as primers, the donor DNA fragment was obtained by overlapping PCR, and then gel excision, purification and recovery were performed.

[0068] The transformation system was configured according to Table 1, and the plasmid pCfB2312 expressing Cas9 protein, the recombinant plasmid p3052-ura3-sgRNA and donor DNA were co-transformed into KMLY1-2 competent cells using the PEG-lithium acetate chemical transformation method. The primers Ura3-vF1 and Ura3-vR1 were used to perform colony PCR verification on single colonies, and the successfully verified strain was the Saccharomyces cerevisiae KMLY1-2 ura3 gene knockout strain, namely KMLY1-8.

[0069] The transformation system was configured according to Table 1, and the plasmid pY26TEF-GPD was transformed into KMLY1-8 competent cells using the PEG-lithium acetate chemical transformation method. Single colonies were picked and colony PCR was performed using primers PY-vF (PAL) and PY-vR (PAL). The correct strain was the control strain KMLY1-8 / pY26TEF-GPD.

[0070] Example 2: Construction of Arpal-Bssfp dual-gene engineering bacteria

[0071] The phenylalanine ammonia lyase gene Arpal from Anoectochilus roxburghii was selected for codon optimization, and then the promoter pADH1 (SEQ ID No.31) and the terminator tPDC1 (SEQ ID No.32) were added to its 5' and 3' ends respectively, and chemically synthesized by Nanjing GenScript Biotechnology Co., Ltd. The above chemically synthesized DNA was used as a template (SEQ ID No.33), and the primers PAL-F1① and PAL-R1① were used for PCR amplification to obtain the gene Arpal expression cassette. The PCR reaction program was: 95℃ pre-denaturation for 4min, 95℃ denaturation for 15s, 50.9℃ annealing for 15s, 72℃ extension for 3min, and 72℃ extension for 5min after 30 cycles. The PCR product was purified using the universal DNA purification and recovery kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain the gene Arpal expression cassette.

[0072] The phosphopantetheine phosphotransferase gene Bssfp from Bacillus subtilis was selected for codon optimization, and the synthesized DNA was used as a template (as shown in SEQ ID No. 34) and chemically synthesized by Nanjing GenScript Biotechnology Co., Ltd. The primers BsSFP-F1 and BsSFP-R1 were used for PCR amplification to obtain the coding region of the gene Bssfp. The PCR reaction program was: 95°C pre-denaturation for 3min, 95°C denaturation for 15s, 49°C annealing for 15s, 72°C extension for 50s, and after 30 cycles, 72°C extension for 5min. The PCR product was purified using the universal DNA purification and recovery kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain the coding region of the gene Bssfp.

[0073] Plasmid pY26TFE-GPD was used as a template and primers PY26-F1① and PY26-R1① were used to amplify the linearized expression vector. The PCR reaction procedure was: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 50.9℃ annealing for 15s, 72℃ extension for 7min, and after 30 cycles, 72℃ extension for 5min. The PCR product was purified using the universal DNA purification and recovery kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain the linearized expression vector.

[0074] The Arpal, Bssfp genes and the linearized vector were seamlessly cloned, and the plasmids in the positive transformants were extracted and sent to Shanghai Biotech Co., Ltd. for sequencing verification, and the Arpal-Bssfp dual gene expression plasmid pY26TEF-GPD-ArPAL-BsSFP was obtained. The transformation system was configured according to Table 1, and the plasmid was transformed into KMLY1-8 competent cells using the PEG-lithium acetate chemical transformation method. Single colonies were picked and verified by colony PCR using primers PY-vF (PAL) and +PY-vR (PAL), as well as PY-Vf and PY-Vr. The correct strain was the Arpal and Bssfp gene overexpression strain KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP.

[0075] Example 3: Construction of Arpal-Bssfp-Nicar three-gene engineering bacteria

[0076] The carboxylic acid reductase gene Nicar from Nocardia iowensis was selected for codon optimization, and the synthesized DNA was used as a template (as shown in SEQ ID No.35) and chemically synthesized by Nanjing GenScript Biotechnology Co., Ltd. The Nicar coding region was obtained by PCR amplification using primers NiCAR-F1 and NiCAR-R1. The PCR reaction procedure was: 95°C pre-denaturation for 3min, 95°C denaturation for 15s, 49°C annealing for 15s, 72°C extension for 50s, and 72°C extension for 5min after 30 cycles. The PCR product was purified using the universal DNA purification and recovery kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain the Nicar coding region.

[0077] Plasmid pY26TEF-GPD-ArPAL-BsSFP was used as a template and primers MCS1-F1 and MCS1-R1 were used for PCR amplification to obtain a linear expression vector. The PCR reaction program was: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 51.2°C annealing for 15 s, 72°C extension for 9 min, and after 30 cycles, 72°C extension for 5 min. The PCR product was purified using the universal DNA purification and recovery kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain a linear expression vector.

[0078] The gene Nicar and the linearized vector were seamlessly cloned in the same manner as in Example 2 to obtain the three-gene expression plasmid pY26TEF-GPD-ArPAL-BsSFP-NiCAR. The transformation system was configured according to the formula in Table 1, and the plasmid was transformed into KMLY1-8 competent cells using the PEG-lithium acetate chemical transformation method. A single colony was picked and colony PCR verification was performed using primers MCS1-vF1 and MCS1-vR1. The correct strain was the three-gene overexpression strain KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-NiCAR.

[0079] Example 4: Construction of Arpal-Bssfp-Nocar three-gene engineering bacteria

[0080] The carboxylic acid reductase gene Nocar from Nocardia otitidiscaviarum was selected for codon optimization, and the synthesized DNA was used as a template (as shown in SEQ ID No.36) and chemically synthesized by Nanjing GenScript Biotechnology Co., Ltd. The coding region of the gene Nocar was obtained by PCR amplification using primers NoCAR-F1 and NoCAR-R1. The PCR reaction procedure was: 95°C pre-denaturation for 3min, 95°C denaturation for 15s, 49°C annealing for 15s, 72°C extension for 50s, and after 30 cycles, 72°C extension for 5min. The PCR product was purified using the universal DNA purification and recovery kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain the coding region of the gene Nocar.

[0081] Using plasmid pY26TEF-GPD-ArPAL-BsSFP as a template, primers MCS1-F1 and MCS1-R1 were used for PCR amplification to obtain a linearized expression vector, and the specific method was the same as in Example 3. Gene Nocar and the linearized vector were seamlessly cloned, and the method was consistent with that in Example 2 to obtain the three-gene expression plasmid pY26TEF-GPD-ArPAL-BsSFP-NiCAR. According to the method in Example 3, pY26TEF-GPD-ArPAL-BsSFP-NiCAR was transformed into KMLY1-8, and colony PCR verification was performed using primers MCS1-vF1 and MCS1-vR1. The correct strain was the three-gene overexpression strain KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-NoCAR.

[0082] Example 5: Construction of Arpal-Bssfp-Mpcar three-gene engineering bacteria

[0083] The carboxylic acid reductase gene Mpcar from Mycolicibacterium phlei was selected for codon optimization, and the synthesized DNA was used as a template (as shown in SEQ ID No. 37) and chemically synthesized by Nanjing GenScript Biotechnology Co., Ltd. The coding region of the gene Mpcar was obtained by PCR amplification using primers MpCAR-F1 and MpCAR-R1. The PCR reaction procedure was: 95°C pre-denaturation for 3min, 95°C denaturation for 15s, 49°C annealing for 15s, 72°C extension for 50s, and 72°C extension for 5min after 30 cycles. The PCR product was purified using the Tiangen Universal DNA Purification and Recovery Kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to obtain the coding region of the gene Mpcar.

[0084] Using plasmid pY26TEF-GPD-ArPAL-BsSFP as a template, primers MCS1-F1 and MCS1-R1 were used for PCR amplification to obtain a linearized expression vector, and the specific method was the same as in Example 3. The gene Mpcar and the linearized vector were seamlessly cloned, and the method was consistent with that in Example 2 to obtain the three-gene expression plasmid pY26TEF-GPD-ArPAL-BsSFP-MpCAR. According to the method in Example 3, pY26TEF-GPD-ArPAL-BsSFP-MpCAR was transformed into KMLY1-8, and primers MCS1-vF1 and MCS1-vR1 were used for colony PCR verification, and the correct strain was verified to be the three-gene overexpression strain KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-MpCAR.

[0085] Example 6: Determination of cinnamyl alcohol production by engineered strains of Saccharomyces cerevisiae

[0086] The recombinant strains KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-NiCAR, KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-NoCAR, KMLY1-8

[0087] / pY26TEF-GPD-ArPAL-BsSFP-MpCAR and the control strain KMLY1-8 / pY26TEF-GPD were inoculated into 5 mL SC-URA liquid medium at a 2% inoculum, and activated and cultured at 30°C and 200 rpm for 24 h. Then, the activated bacteria were inoculated into 50 mL SC-URA liquid medium at a 2% inoculum, and cultured at 30°C and 200 rpm for 24 h. The OD of the bacterial suspension was 600 The value was 1.8 to 2.0; the bacteria were collected by centrifugation at 6000 rpm for 5 min, the supernatant was discarded, and then 5 mL of sterile water was added to the centrifuge tube for resuspending to obtain a recombinant bacterial suspension; the bacterial suspension was transferred to 200 mL of SC-URA liquid culture medium and cultured at 30°C and 200 rpm for 18-24 h to make the OD of the bacterial suspension 600 The value is 1.8-2.0; centrifuge at 6000rpm for 5min to collect the cells, discard the supernatant, resuspend the cells in 10mL SC-URA medium containing 1g / L phenylalanine, transfer to a test tube, and culture at 30℃ and 200rpm for 24h; collect the culture solution, add grinding beads and grind it in a low-temperature high-speed grinder, centrifuge at 12000rpm for 1min, and take the supernatant for HPLC analysis after the centrifugation. The yield of cinnamyl alcohol is as follows Figure 1As shown, no cinnamyl alcohol production was detected in the control strain, KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-NiCAR produced 23.45 mg / L of cinnamyl alcohol, KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-NoCAR produced 7.99 mg / L of cinnamyl alcohol, and KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-MpCAR produced 30.30 mg / L of cinnamyl alcohol, which was the highest, indicating that KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-MpCAR was the optimal strain for producing cinnamyl alcohol.

[0088] It should be understood that after reading the above content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

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Claims

1. An engineered strain of Saccharomyces cerevisiae producing cinnamyl alcohol, characterized in that: The engineered bacteria is a recombinant Saccharomyces cerevisiae, which includes the phenylalanine ammonia lyase gene Arpal from Anoectochilus roxburghii, the phosphopantetheinyl phosphotransferase gene Bssfp from Bacillus subtilis, and the reductase gene Nicar from Nocardia iocardia, the reductase gene Nocar from Nocardia otitidis, or the carboxylic acid reductase gene Mpcar from Mycobacterium phlei.

2. A method for constructing an engineered strain of Saccharomyces cerevisiae that produces cinnamyl alcohol, characterized in that: The ura3 gene knockout strain KMLY1-8 of wild-type Saccharomyces cerevisiae KMLY1-2 is used as a chassis strain, and the phenylalanine ammonia lyase gene Arpal from Anoectochilus roxburghii, the phosphopantetheinyl phosphotransferase gene Bssfp from Bacillus subtilis, and the reductase gene Nicar from Nocardia iocardiae, or the reductase gene Nocar from Nocardia otitidis, or the carboxylic acid reductase gene Mpcar from Mycobacterium phlei are integrated into the genome of the chassis strain, and the construction method comprises the following specific steps: Step (1), knocking out the ura3 gene in the wild-type Saccharomyces cerevisiae KMLY1-2 to obtain a chassis strain named Saccharomyces cerevisiae KMLY1-8; Step (2), preparing Saccharomyces cerevisiae KMLY1-8 into competent cells; Step (3), constructing Arpal-Bssfp double gene expression plasmid; Step (4), using the Arpal-Bssfp double gene expression plasmid obtained in step (3) as a template to construct the Arpal-Bssfp-Nicar three-gene expression plasmid, in the transformation system, using the PEG-lithium acetate chemical transformation method to transform the Arpal-Bssfp-Nicar three-gene expression plasmid into the KMLY1-8 competent cells obtained in step (2), picking a single colony, and using primers PY-vF (PAL) and +PY-vR (PAL), primers PY-Vf and PY-Vr, and primers MCS1-vF1 and MCS1-vR1 to perform colony PCR verification, and verifying that the correct strain is the cinnamyl alcohol-producing Saccharomyces cerevisiae engineered strain, named KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-NiCAR; Or, the Arpal-Bssfp double gene expression plasmid obtained in step (3) is used as a template to construct the Arpal-Bssfp-Nocar triple gene expression plasmid, and in the transformation system, the Arpal-Bssfp-Nocar triple gene expression plasmid is transformed into the KMLY1-8 competent cells obtained in step (2) using the PEG-lithium acetate chemical transformation method, and a single colony is picked, and the primers PY-vF (PAL) and +PY-vR (PAL), primers PY-Vf and PY-Vr, and primers MCS1-vF1 and MCS1-vR1 are used to perform colony PCR verification, and the correct strain is the cinnamyl alcohol-producing Saccharomyces cerevisiae engineered strain, which is named KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-NoCAR; Alternatively, the Arpal-Bssfp double gene expression plasmid obtained in step (3) is used as a template to construct the Arpal-Bssfp-Mpcar triple gene expression plasmid. In the transformation system, the Arpal-Bssfp-Mpcar triple gene expression plasmid is transformed into the KMLY1-8 competent cells obtained in step (2) using the PEG-lithium acetate chemical transformation method. A single colony is picked and colony PCR verification is performed using primers PY-vF (PAL) and +PY-vR (PAL), primers PY-Vf and PY-Vr, and primers MCS1-vF1 and MCS1-vR1, respectively. The correct strain is the engineered yeast of Saccharomyces cerevisiae producing cinnamyl alcohol, which is named KMLY1-8 / pY26TEF-GPD-ArPAL-BsSFP-MpCAR.

3. The construction method according to claim 2, characterized in that: The method for knocking out the ura3 gene in the wild-type Saccharomyces cerevisiae KMLY1-2 in the step (1) is as follows: constructing a recombinant plasmid p3052-ura3-sgRNA, using a PEG-lithium acetate chemical transformation method to transform the plasmid pCfB2312 expressing the Cas9 protein, the recombinant plasmid p3052-ura3-sgRNA and the donor DNA into the KMLY1-2 strain competent cells in the transformation system, using primers Ura3-vF1 and Ura3-vR1 to perform colony PCR verification on a single colony, and the successfully verified strain is the Saccharomyces cerevisiae KMLY1-2 ura3 gene knockout strain, Saccharomyces cerevisiae KMLY1-8.

4. The construction method according to claim 2, characterized in that: The construction method of the Arpal-Bssfp dual gene expression plasmid in the step (3) is as follows: codon optimization is performed on the phenylalanine ammonia lyase gene Arpal from golden thread orchid, and then the promoter pADH1 and the terminator tPDC1 are synthesized at the 5′ and 3′ ends thereof, respectively, and the synthesized DNA is used as a template to perform PCR amplification using primers PAL-F1① and PAL-R1① to obtain the gene Arpal expression cassette; The phosphopantetheine phosphotransferase gene Bssfp from Bacillus subtilis was codon optimized, and the synthesized DNA was used as a template to amplify the coding region of the gene Bssfp using primers BsSFP-F1 and BsSFP-R1; Using plasmid pY26TFE-GPD as a template, primers PY26-F1① and PY26-R1① were used for PCR amplification to obtain a linearized expression vector; The Arpal, Bssfp genes and the linearized vector were seamlessly cloned, and the plasmids in the positive transformants were extracted to obtain the Arpal-Bssfp double gene expression plasmid, which was named pY26TEF-GPD-ArPAL-BsSFP.

5. The construction method according to claim 2, characterized in that: In the step (4) The construction method of the Arpal-Bssfp-Nicar three-gene expression plasmid is as follows: codon optimization is performed on the carboxylic acid reductase gene Nicar of Nocardia iodica, and the synthesized DNA is used as a template, and primers NoCAR-F1 and NoCAR-R1 are used to perform PCR amplification to obtain the coding region of the gene Nicar; the Arpal-Bssfp double-gene expression plasmid prepared in step (3) is used as a template, and primers MCS1-F1 and MCS1-R1 are used to perform PCR amplification to obtain a linearized expression vector; the gene Nicar and the linearized vector are seamlessly cloned, and the plasmid in the positive transformant is extracted to obtain the Arpal-Bssfp-Nicar three-gene expression plasmid; The construction method of the Arpal-Bssfp-Nocar three-gene expression plasmid is as follows: codon optimization of the carboxylic acid reductase gene Nocar of Nocardia otitidis caviae is performed, and the synthesized DNA is used as a template to perform PCR amplification using primers NiCAR-F1 and NiCAR-R1 to obtain the coding region of the gene Nocar; Using the Arpal-Bssfp double gene expression plasmid prepared in step (3) as a template, using primers MCS1-F1 and MCS1-R1 to perform PCR amplification to obtain a linearized expression vector; seamlessly cloning the gene Nocar and the linearized vector, and extracting the plasmid from the positive transformant to obtain the Arpal-Bssfp-Nocar triple gene expression plasmid; The construction method of the Arpal-Bssfp-Mpcar three-gene expression plasmid is as follows: codon optimization is performed on the carboxylate reductase gene Mpcar of Mycobacterium phlei, and the synthesized DNA is used as a template, and primers MpCAR-F1 and MpCAR-R1 are used to perform PCR amplification to obtain the gene Mpcar coding region; the Arpal-Bssfp double-gene expression plasmid prepared in step (3) is used as a template, and primers MCS1-F1 and MCS1-R1 are used to perform PCR amplification to obtain a linearized expression vector; the gene Mpcar and the linearized vector are seamlessly cloned, and the plasmid in the positive transformant is extracted to obtain the Arpal-Bssfp-Mpca three-gene expression plasmid.

6. The construction method according to claim 2, characterized in that: The construction method of the recombinant plasmid p3052-ura3-sgRNA is as follows: the ura3 gene coding frame is found from the whole genome of Saccharomyces cerevisiae KMLY1-2, sgRNA is designed online, 50bp DNA single strands Ura3-20bp-F1 and Ura3-20bp-R1 are synthesized, two 50bp DNA single strands are annealed to form a 50bp DNA double strand, plasmid pCfB3052 is linearized using primers 3052F and 3052R, and then it is seamlessly cloned and connected with the 50bp DNA double strand, and the plasmid in the positive transformant is extracted to obtain the recombinant plasmid p3052-ura3-sgRNA.

7. The construction method according to claim 4, characterized in that: The PCR reaction procedure for PCR amplification to obtain the Arpal gene expression cassette in step (3) is as follows: pre-denaturation at 95°C for 4 minutes, denaturation at 95°C for 15 seconds, annealing at 50.9°C for 15 seconds, extension at 72°C for 3 minutes, and extension at 72°C for 5 minutes after 30 cycles, and then purification of the PCR product using a universal DNA purification recovery kit to obtain the Arpal gene expression cassette; The PCR reaction program for PCR amplification to obtain the gene Bssfp expression cassette is as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 49°C annealing for 15 s, 72°C extension for 50 s, 30 cycles, 72°C extension for 5 min, and then purification of the PCR product using a universal DNA purification recovery kit to obtain the gene Bssfp expression cassette; The linearized expression vector was obtained by PCR amplification. The PCR reaction procedure was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 50.9°C for 15 s, and extension at 72°C for 7 min. After 30 cycles, the PCR product was purified using a universal DNA purification recovery kit to obtain the linearized expression vector.

8. The construction method according to claim 5, characterized in that: Step (4) In the construction of the Arpal-Bssfp-Nicar three-gene expression plasmid, the reaction procedure for PCR amplification to obtain the Nicar gene was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 49°C annealing for 15 s, 72°C extension for 50 s, and after 30 cycles, 72°C extension for 5 min, and then the PCR product was purified using a universal DNA purification and recovery kit to obtain the Nicar expression cassette; In the construction of the Arpal-Bssfp-Nocar three-gene expression plasmid, the reaction procedure for PCR amplification to obtain the gene Nocar was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 49°C annealing for 15 s, 72°C extension for 50 s, 30 cycles, 72°C extension for 5 min, and then a universal DNA purification and recovery kit was used to purify the PCR product to obtain the gene Nocar expression cassette; In the construction of the Arpal-Bssfp-Nocar three-gene expression plasmid, the PCR amplification was performed to obtain the PCR reaction program of the gene Mpcar: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 49°C annealing for 15 s, 72°C extension for 50 s, and after 30 cycles, 72°C extension for 5 min, and then the PCR product was purified using a universal DNA purification and recovery kit to obtain the gene Mpcar expression cassette; The reaction procedure for PCR amplification to obtain a linearized expression vector in the construction of the Arpal-Bssfp-Nocar three-gene expression plasmid is as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 51.2°C for 15 s, extension at 72°C for 9 min, and after 30 cycles, extension at 72°C for 5 min, purifying the PCR product using a universal DNA purification recovery kit to obtain a linearized expression vector.

9. Use of the engineered yeast Saccharomyces cerevisiae according to claim 1 in producing cinnamyl alcohol.

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