CN112322514B

CN112322514B - Method for enhancing tolerance and yield of glycerol-producing candida 2-phenethyl alcohol

Info

Publication number: CN112322514B
Application number: CN202011251954.1A
Authority: CN
Inventors: 诸葛斌; 刘芳; 王玉芹; 陆信曜; 宗红
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-11-09
Filing date: 2020-11-09
Publication date: 2022-03-25
Anticipated expiration: 2040-11-09
Also published as: CN112322514A

Abstract

The invention discloses a method for enhancing the tolerance and the yield of glycerol-producing candida 2-phenethyl alcohol, belonging to the technical field of genetic engineering. The invention obtains a recombinant strain CgSLC1 by over-expressing a 1-acyl-sn-glycerol-3-phosphoryl transferase gene SLC1 in a self phospholipid synthesis pathway in Candida glycerinogenes (Candida glycerinogenes) CCTCC M93018. Compared with the no-load control strain, the recombinant strain has obvious growth advantage in the external environment with high concentration of 2-phenethyl alcohol (3.5g/L), and the biomass of the strain is improved by 18.3 percent. In the fermentation culture medium, the biomass of the recombinant strain and the yield of the 2-phenethyl alcohol are enhanced, which are respectively improved by 18.0 percent and 21.6 percent, and the yield reaches 4.07 g/L.

Description

Method for enhancing tolerance and yield of glycerol-producing candida 2-phenethyl alcohol

Technical Field

The invention relates to a method for enhancing the tolerance and the yield of glycerol-producing candida 2-phenethyl alcohol, belonging to the technical field of genetic engineering.

Background

2-phenethyl alcohol (2-PE) is a high-grade alcohol, has rose fragrance, and can be widely applied to industries such as tobacco, food, cosmetics and the like. In recent years, the market demand for 2-phenylethyl alcohol has been increasing. The existing production method of 2-phenethyl alcohol mainly comprises a chemical synthesis method, a plant extraction method and a biological method, the 2-phenethyl alcohol prepared by the chemical synthesis method is forbidden to be used in the fields of cosmetics, foods and the like because of containing carcinogens (such as styrene and the like), and the natural 2-phenethyl alcohol obtained by the plant extraction method has high supply price of raw materials (such as roses and the like) and the yield can not meet the market supply; therefore, the biological method for preparing the 2-phenethyl alcohol has wide application prospect. In biological methods, 2-phenylethyl alcohol is produced by yeast biotransformation, which has great potential value; however, the high concentration of 2-phenylethyl alcohol has a great toxicity to yeast itself, and can inhibit the growth of yeast, thereby reducing the yield of 2-phenylethyl alcohol.

The fermentation method based natural 2-phenylethyl alcohol production has wide application prospect, so that research and development of 2-phenylethyl alcohol resistant yeast strains become a research with great industrial application value.

To solve this bottleneck, researchers mostly adopt the following solutions: 1. the in-situ extraction technology is adopted to reduce the concentration of the 2-phenethyl alcohol in the fermentation liquor, further weaken the inhibition function of the product on the physiological metabolism of cells and improve the synthesis rate of the product. However, due to the addition of the extractant, the difficulty and cost of downstream product separation are increased, and in addition, the quality of the product is seriously influenced because the extractant is difficult to clean. 2. The mutagenesis obtains a high-tolerance strain, but the method has low efficiency and long period.

Therefore, the high-efficiency and high-yield 2-phenylethyl alcohol cannot be realized by the methods, and the inventor previously aims to improve the 2-phenylethyl alcohol tolerance of the candida glycerinogenes by over-expressing erg4 genes in the candida glycerinogenes by using a genetic engineering means; however, the over-expression of the gene does not significantly improve the yield of the 2-phenylethyl alcohol, and probably because the ergosterol content in cell membranes is improved after the erg4 gene is over-expressed, so that the cell membrane strength is increased, and the 2-phenylethyl alcohol is not discharged outside, therefore, the great research significance is provided for further improving the tolerance of the Candida glycerinogenes to the 2-phenylethyl alcohol and further improving the yield of the 2-phenylethyl alcohol.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention over-expresses 1-acyl-sn-glycerol-3-phosphate acyltransferase (1-acyl-sn-glycerol-3-phosphate acyltransferase) in Candida glycerinogenes, and the 1-acyl-sn-glycerol-3-phosphate acyltransferase SLC1 catalyzes 3-phosphoglycerol to synthesize phosphatidic acid, which is a key enzyme in the phospholipid synthesis pathway. Phospholipids are important constituents of cell membranes. The strain can resist the external environment damage by adjusting the types (PA, PE, PG and the like), the structures (the length, the saturation and the like of fatty acid carbon chains) and the abundance of phospholipid molecules. Therefore, the invention changes the expression level of the SLC1 gene through gene engineering, and further adjusts the phospholipid molecular composition to resist external damage, so as to further improve the tolerance of the candida glycerinogenes to 2-phenethyl alcohol and the yield of the 2-phenethyl alcohol.

Candida glycerinogenes CCTCC M93018 (disclosed in a patent with the publication number of CN 105713933B) is an industrial strain with excellent fermentation performance, has high-efficiency 2-phenethyl alcohol synthesis capacity and a relatively complete genetic modification tool, and provides a favorable basis for further molecular modification of the strain to improve the tolerance and yield of the 2-phenethyl alcohol.

The first purpose of the invention is to provide a candida glycerinogenes engineering bacterium, which uses pGAPa as a vector (described in the application text with the publication number of CN 110079468A) to overexpress a 1-acyl-sn-glycerol-3-phosphate acyltransferase gene, wherein the amino acid sequence of the 1-acyl-sn-glycerol-3-phosphate acyltransferase is shown as SEQ ID NO. 5.

In one embodiment of the present invention, Candida glycerinogenes (Candida glycerinogenes) CCTCC M93018 is used as a host. In one embodiment of the invention, the glycerol producing Candida is Candida glycerinogenes (Candida glycerinogenes) CCTCC M93018.

In one embodiment of the present invention, the nucleotide sequence encoding the 1-acyl-sn-glycerol-3-phosphate acyltransferase gene is set forth in SEQ ID No. 1.

In one embodiment of the invention, the 1-acyl-sn-glycerol-3-phosphate acyltransferase is derived from Candida glycerinogenes.

The second purpose of the invention is to provide a method for constructing the candida glycerinogenes engineering bacteria, which comprises the following steps:

(1) using a Candida glycerinogenes genome as a template, and carrying out PCR amplification on an SLC1 gene for coding 1-acyl-sn-glycerol-3-phosphate acyltransferase;

(2) inserting the SLC1 gene between the GAP strong promoter and the AOXI terminator of the plasmid pGAPa to obtain a recombinant plasmid pGAPa-SLC1 for over-expressing the SLC1 gene;

(3) the recombinant plasmid pGAPa-SLC1 was transformed into Candida glycerinogenes.

In one embodiment of the invention, the transformation is performed by lithium acetate transformation.

In one embodiment of the invention, the nucleotide sequence of the upstream primer SLC 1F amplified by PCR is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer SLC1R is shown as SEQ ID NO. 3.

In one embodiment of the invention, the method comprises the steps of:

(1) using Candida glycerinogenes as a template, and utilizing upstream and downstream primers SLC 1F (SEQ ID No.2) and SLC1R (SEQ ID No.3) to carry out PCR amplification on an SLC1 gene;

(2) inserting the plasmid into a GAP strong promoter of plasmid pGAPa and an AOXI terminator by using a one-step cloning method to obtain an over-expression recombinant plasmid pGAPa-SLC 1;

(3) after linearization by restriction endonuclease Sac I, transferring the strain into Candida glycerinogenes CCTCC M93018 by a lithium acetate (LiAC) conversion method, integrating linearized recombinant plasmid pGAPa-SLC1 into a 5.8S sequence of the Candida glycerinogenes by homologous recombination, screening by an MM plate to obtain a single colony, extracting a single colony genome, and performing PCR verification to obtain the recombinant strain CgSLC1 of the Candida glycerinogenes, wherein the recombinant strain is over-expressing SLC1 genes.

The third purpose of the invention is to provide a method for enhancing the 2-phenethyl alcohol tolerance of candida glycerinogenes, which uses pGAPa as a vector and overexpresses a 1-acyl-sn-glycerol-3-phosphate acyltransferase gene in the candida glycerinogenes, wherein the amino acid sequence of the 1-acyl-sn-glycerol-3-phosphate acyltransferase is shown as SEQ ID NO. 5.

The fourth purpose of the invention is to provide a method for producing phenethyl alcohol, which comprises the step of inoculating the candida glycerinogenes engineering bacteria into a reaction system containing L-phenylalanine for reaction.

In one embodiment of the invention, the method comprises the steps of selecting the engineering bacteria, inoculating the engineering bacteria into a seed culture medium, and performing shake culture for 12-20h at 28-30 ℃ and 200-220rpm to obtain liquid seeds; inoculating the obtained liquid seed into a fermentation culture medium containing L-phenylalanine according to the inoculation amount of 1-5% (v/v), controlling the fermentation temperature at 28-30 ℃, the rotation speed at 200-220rpm, and the fermentation time at 40-60 h.

The fifth purpose of the invention is to provide the application of the candida glycerinogenes engineering bacteria in preparing food, cosmetics or essence.

Advantageous effects

According to the invention, by constructing the engineered strain of the Candida glycerinogenes which overexpresses the SLC1 gene encoding the 1-acyl-sn-glycerol-3-phosphate acyltransferase, the tolerance of the Candida glycerinogenes to 2-phenethyl alcohol is improved, and thus the yield of the 2-phenethyl alcohol is improved. In 2-phenylethyl alcohol with the concentration of more than 3.5g/L, the tolerance of the candida glycerinogenes engineering bacteria CgSLC1 constructed by the method to the 2-phenylethyl alcohol is obviously improved, and the biomass of the bacteria is improved by 18.3 percent; the Candida glycerinogenes engineering bacteria CgSLC1 constructed by the invention are fermented to synthesize 2-phenethyl alcohol, after the fermentation is finished, the biomass of the engineering bacteria and the yield of the 2-phenethyl alcohol are respectively improved by 18.0 percent and 21.6 percent, so that the final yield can reach 4.07 g/L.

Drawings

FIG. 1: the recombinant plasmid pGAPa-SLC1 is constructed schematically.

FIG. 2: a schematic diagram of construction of a strain of Candida glycerinogenes overexpressing SLC 1.

FIG. 3: PCR verification of strain SLC1 over-expressed in Candida glycerinogenes.

FIG. 4: the tolerance of the recombinant strain 2-phenylethyl alcohol was observed on a gradient dilution dot plate.

FIG. 5: the tolerance of the recombinant strain 2-phenylethyl alcohol was observed in a shake flask.

FIG. 6: schematic representation of biomass change during shake flask fermentation of recombinant strains.

FIG. 7: the recombinant strain is used for synthesizing 2-phenethyl alcohol by shaking flask fermentation.

Detailed Description

The media involved in the following examples are as follows:

seed culture medium: 10g/L of yeast powder, 20g/L of peptone and 20g/L of glucose.

Fermentation medium: 7g/L of L-phenylalanine, 90g/L of glucose, KH₂PO₄5g/L, yeast powder 4g/L, MgSO₄·7H₂O0.5g/L。

MM plate medium: glucose 20g/L, amino-free yeast nitrogen source (YNB)6.7 g/L.

YPD liquid medium: 10g/L of yeast powder, 20g/L of peptone and 20g/L of glucose.

The detection methods referred to in the following examples are as follows:

the method for measuring the 2-phenethyl alcohol in the fermentation liquor comprises the following steps:

high Performance Liquid Chromatography (HPLC) analysis was used, as follows: the solution to be tested was centrifuged at 10,000rpm, and then filtered through a 0.45 μm microporous membrane using a high performance liquid chromatograph (Agilent, USA) as a measuring apparatus using a C18 column (250 mm. times.4.6 mm,10 μm; Ailite, China) as a mobile phase methanol/water 50:50(v/v), a flow rate of the mobile phase 0.8mL/min, a column temperature 30 ℃, a detection wavelength of 260nm, and a sample injection of 10 μ L.

The present invention is described in further detail below by way of examples.

Example 1: construction of recombinant plasmid pGAPa-SLC1

(1) The construction method of the vector pGAPa comprises the following steps:

chemically synthesizing ura5 gene with nucleotide sequence shown in SEQ ID No.4, cutting with restriction enzymes Sal I and sph I, and connecting to vector pUC-5.8S rDNA-P_CgGAP(pUC-5.8S rDNA-P_CgGAPThe method of construction of (2) is described in the patent document CN 103173483B), the vector pGAPa is obtained.

(2) Construction of recombinant plasmid pGAPa-SLC 1:

PCR amplifying SLC1 gene coding 1-acyl-sn-glycerol-3-phosphate acyltransferase by using upstream and downstream primers SLC 1F (SEQ ID No.2) and SLC1R (SEQ ID No.3) by taking Candida glycerinogenes as a template; T-Vector pMD19simple (Ts) was ligated and sent to the Shanghai Biotech sequencing.

The SLC1 gene after the sequencing is correctly connected with the expression vector pGAPa constructed in the step (1) through a one-step cloning method kit (purchased from Novowed Biotechnology Co., Ltd., product number: C115-01) to obtain a recombinant plasmid pGAPa-SLC1 (see FIG. 1).

Example 2: construction of glycerol-producing candida engineering bacteria

(1) Selecting a Candida glycerinogenes strain CCTCC M93018, inoculating the strain in a YPD liquid culture medium, and culturing at 30 ℃ for 12h to obtain a culture solution; transferring 100 μ L of culture medium into YPD liquid culture medium, and culturing at 30 deg.C for 4-6 hr to make culture medium OD₆₀₀And when the concentration reaches 0.8-1.2, centrifugally collecting thalli, and preparing the thalli centrifugally collected into the competent cells of the glycerol-producing candida strains.

(2) After the recombinant plasmid pGAPa-SLC1 obtained in example 1 was linearized with Sac I, it was transferred into competent cells of the glycerol-producing Candida strain obtained in step (1) by lithium acetate transformation, spread on MM plate medium, and cultured at 30 ℃ for 2-3 days to obtain single colonies.

(3) And (3) selecting the single colony obtained in the step (2), extracting a genome, and performing PCR verification (see figure 3), wherein after the verification is correct, the recombinant strain Candida glycerinogenes engineering bacterium CgSLC1 (see figure 2) is obtained.

According to the steps, the Sac I linearized recombinant plasmid pGAPa is transformed into a glycerol-producing Candida strain competent cell to obtain an empty load control strain.

Example 3: tolerance detection of recombinant strain CgSLC1 in high-concentration 2-phenylethyl alcohol

The specific implementation steps are as follows:

(1) respectively selecting the no-load control strain and the recombinant strain obtained in the 1-ring example 2, inoculating the no-load control strain and the recombinant strain into a seed culture medium, and performing shaking culture for 18h at the temperature of 30 ℃ and the speed of 200rpm to obtain liquid seeds. Measure its OD₆₀₀And diluted to OD with sterile water₆₀₀Is 1. Obtaining OD by 10-fold dilution method₆₀₀Is 10⁰、10^-1、10^-2、10^-3、10^-4The bacterial liquid of (4).

(2) And (2) taking 2 mu L of the bacterial liquid obtained in the step (1), sequentially spotting the bacterial liquid on YPD plates containing 2-phenethyl alcohol (0g/L and 3.5g/L) with different concentrations, culturing the bacterial liquid at the temperature of 30 ℃ for 48h, and observing the growth conditions of the control strain and the recombinant strain on the plates.

(3) Liquid seeds (initial biomass OD) of the same amount of the empty load control strain and the recombinant strain were inoculated₆₀₀0.2) were inoculated into 50mL of YPD liquid medium containing 2-phenylethyl alcohol (0g/L, 3.5g/L) at different concentrations, and cultured at 30 ℃ and 200 rpm. Sampling at different time to determine the biomass change.

The results show that: in high-concentration 2-phenylethyl alcohol (3.5g/L), the tolerance of the recombinant strain CgSLC1 is obviously improved, and the biomass of the strain is improved by 18.3 percent (as shown in figures 4-5).

Example 4: production of 2-phenylethyl alcohol by recombinant strain

(1) Respectively selecting the no-load control strain and the recombinant strain obtained in the 1-ring example 2, inoculating the no-load control strain and the recombinant strain into a seed culture medium, and performing shaking culture for 18h at the temperature of 30 ℃ and the speed of 200rpm to obtain liquid seeds.

(2) Inoculating the obtained liquid seeds into a 250mL conical flask containing 30mL of fermentation medium according to the inoculation amount of 5% (v/v), controlling the fermentation temperature to be 30 ℃, the rotation speed to be 200rpm, and the time to be 60h, and finishing the fermentation.

And (3) detecting the growth condition of the strain and the yield of the 2-phenethyl alcohol in the fermentation process.

Compared with the no-load control strain, the biomass and the yield of the recombinant strain are respectively increased by 18.0 percent and 21.6 percent at the end of fermentation, and the yield reaches 4.07g/L (the biomass and the yield are shown in figures 6-7).

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

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Claims

1. A method for enhancing the 2-phenethyl alcohol tolerance of candida glycerinogenes is characterized in that pGAPa is used as an expression vector in the candida glycerinogenes (C)Candida glycerinogenes) A1-acyl-sn-glycerol-3-phosphate acyltransferase gene is overexpressed in CCTCC M93018, and the nucleotide sequence of the 1-acyl-sn-glycerol-3-phosphate acyltransferase gene is shown as SEQ ID NO. 1.

2. A method for producing 2-phenethyl alcohol is characterized in that candida glycerinogenes engineering bacteria are inoculated into a reaction system containing L-phenylalanine for reaction; the engineering strain for producing the glycerol candida takes pGAPa as a carrier and produces the glycerol candida (Candida glycerinogenes) A1-acyl-sn-glycerol-3-phosphate acyltransferase gene is overexpressed in CCTCC M93018, and the nucleotide sequence of the 1-acyl-sn-glycerol-3-phosphate acyltransferase gene is shown as SEQ ID NO. 1.

3. The method as claimed in claim 2, wherein the seed liquid of the engineering strain Candida glycerinogenes is inoculated into the fermentation medium containing L-phenylalanine at an inoculum size of 1-5%, and the fermentation is carried out for 40-60h at a temperature of 28-30 ℃ and a rotation speed of 200-220 rpm.