CN116179382A

CN116179382A - Genetic engineering bacterium for high-yield erythritol, construction method and application

Info

Publication number: CN116179382A
Application number: CN202211301011.4A
Authority: CN
Inventors: 柳志强; 肖博文; 黄良刚; 张博; 周俊平; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2022-10-24
Filing date: 2022-10-24
Publication date: 2023-05-30

Abstract

The invention relates to a genetic engineering strain of erythritol, a construction method thereof and application thereof in preparing erythritol by microbial fermentation. According to the invention, xylose reductase, xylitol dehydrogenase and xylitol kinase are introduced into yarrowia lipolytica to remodel a xylose metabolism path, the expression of transketolase TKL1, transaldolase TAL and erythritol reductase ER is enhanced in an erythritol biological generation path, the generation of byproducts mannitol and arabitol is reduced by knocking out mannitol dehydrogenase MDH and arabitol dehydrogenase ArDH, the conversion of erythritol into other substances is reduced by knocking out erythritol dehydrogenase EYD, the accumulation of erythritol is weakened, the expression of hexokinase HK, transporter Stp1 and Stp2 is enhanced, and finally, the high-yield erythritol of glucose and xylose mixed carbon sources can be utilized, so that the yield is improved from 50.17g/L to 195.56g/L, and the industrial application potential is provided for synthesizing erythritol by taking xylose as a substrate.

Description

Genetic engineering bacterium for high-yield erythritol, construction method and application

Field of the art

The invention relates to a genetic engineering bacterium for high-yield erythritol, a construction method and application thereof.

(II) background art

Erythritol is a four-carbon sugar alcohol with a molecular formula of C ₄ H ₁₀ O ₄ Is widely available in nature. With the importance of health in recent years, erythritol has been one of the hot spots for sweetener research due to its excellent properties such as zero calories, non-caries nature, safety for eating, and not being absorbed by human metabolism, and its unique properties in the research of sugar substitutes. Currently, erythritol is widely used as a natural sweetener and a food additive in ingredients of foods, is increasingly used in the food, chemical and pharmaceutical industries, and market demands are increasing year by year.

At present, the microorganism for synthesizing erythritol in China is mainly yarrowia lipolytica, and the genetic background is clear. Aiming at different carbon utilization ways, related works in the fields of gene editing, metabolic engineering, synthetic biology and the like are developed, and the strain is widely applied to development of various chemicals and is a good industrial production strain. Although studies have been conducted to develop metabolic engineering of erythritol-producing strains of yarrowia lipolytica, there have been few reports of metabolic synthesis of erythritol using xylose, focusing mainly on the use of glucose and glycerol. Although yarrowia lipolytica itself has a xylose metabolic pathway, the amount of gene expression in its pathway is extremely low, resulting in its inability to utilize xylose for growth and production. There have also been studies on engineering yarrowia lipolytica cells by genetic engineering to allow them to grow on xylose, but this has not been associated with commercial production, particularly with erythritol production.

In yarrowia lipolytica, the synthesis of erythritol is complex and is divided into three pathways depending on the type of carbon source.

Glucose metabolic pathway (6C) is a skeletal pathway: glucose (Glucose) is first phosphorylated by Hexokinase (HK) to Glucose-6-phosphate (G-6-P) and taken into the cell, and then converted to ribulose-5-phosphate (5-P-Ru) by Glucose-6-phosphate dehydrogenase and Glucose-6-phosphate dehydrogenase catalysis. Ribulose-5-phosphate can be converted to an arabitol by-product by the action of an arabitol dehydrogenase. The ribulose-5-phosphate is respectively isomerised into ribose-5-phosphate (5-P-R) and xylulose-5-phosphate (5-P-Xylu), then converted into sedoheptulose-7-phosphate (Sep-7-P) and glyceraldehyde-3-phosphate (3-P-GA) under the catalysis of transketolase (TKL 1), and then converted into fructose-6-phosphate (F-6-P) and erythrose-4-phosphate (Ery-4-P) under the action of transaldolase. The 4-phosphoerythrulose is dephosphorylated to Erythrose (Erythrose) by the action of phosphatase (EryPase) and then converted to Erythritol (Erythrose) by reduction by Erythrose reductase. Meanwhile, glucose-6-phosphate is converted into fructose-6-phosphate under the catalysis of glucose phosphate isomerase (PGI), fructose is converted into fructose under the action of phosphorylase, and fructose is converted into byproduct mannitol under the catalysis of mannitol dehydrogenase.

The glycerol pathway is a secondary pathway (3C): after glycerol enters cells, 1 molecule of ATP is consumed to produce 3-phosphoglycerate under the action of Glycerol Kinase (GK), dihydroxyacetone phosphate (DHAP) is formed by dehydrogenating 3-phosphoglycerate dehydrogenase (GPD), glyceraldehyde phosphate isomerase (TPI) is used for isomerizing the glyceraldehyde phosphate to form 3-phosphoglycerate aldehyde (G3P), xylulose 5-phosphate and erythrose 4-phosphate are formed together with fructose 6-phosphate under the action of transketolase TKL1, and the glyceraldehyde phosphate enters a pentose phosphate pathway.

Xylose pathway is a low level expression pathway (5C): xylose enters cells through a transport protein, is reduced into xylitol under the action of NAD (P) H dependent xylose reductase XR, then NAD+ dependent xylitol dehydrogenase XDH oxidizes the xylitol into xylulose, the xylulose is converted into 5-P xylulose under the action of xylulophosphate kinase XK, enters a pentose phosphate pathway in a non-oxidation stage, and is catalyzed by transketolase (TKL 1), transaldolase (TAL), phosphatase (EryP) and erythrose reductase (Erth) to complete the synthesis of erythritol. Although yarrowia lipolytica has a xylose pathway, it is affected by carbon catabolism repression, resulting in extremely low XK gene expression and failure to utilize xylose.

Among the metabolic pathways mentioned above, it is common to modify the glucose pathway and the glycerol pathway. The synthesis of erythritol by metabolic engineering has been reported in a number of documents (Zhang L, et al multiple gene integration to promote erythritol production on glycerol in Yarrowia lipolytica [ J ]. Biotechnology Letters,2021 (17); carly F, et al identification and characterization of EYK1, a key gene for erythritol catabolism in Yarrowia lipolytica [ J ]. Applied Microbiology & Biotechnology,2017;Wang N,et al.Metabolic engineering of Yarrowia lipolytica for thermoresistance and enhanced erythritol productivity[J ]. Biotechnology for Biofuels,2020,13 (1)) and patents (CN 202010069250.6 and CN 202011516582.0). These more mature erythritol is produced by microbial plants fermentation using glucose or glycerol as a substrate. The fermentation production with industrial crude glycerol as a carbon source has low cost, but the yield of erythritol is not high; the fermentation production with glucose as a carbon source has high productivity, consumes a large amount of starchy raw materials, and faces the problem of high price of the carbon source. The lignocellulose biomass is used as renewable resources with the largest reserve on the earth, the sugar component of the lignocellulose biomass is mainly glucose (accounting for 60% -70%), and the sugar component of the lignocellulose biomass is xylose (accounting for 30% -40%), and particularly a large amount of waste starch is generated in the processing process of Chinese wheat starch. The carbon content is classified into B starch and C starch according to their different carbon contents, and the carbon content ratio is shown in Table 1 below. If the chassis cells utilizing glucose and xylose double carbon can be developed, not only the development of the erythritol industry in China can be promoted, but also the waste carbon source of the starch processing industry can be fully and deeply processed.

Table 1: wheat starch production intermediate attributes

Intermediate products

Starch content

Proteins

Dextran

Xylan (xylan)

Arabinose (Arabic sugar)

Others

B starch (Dry basis)

63.2％

8.9％

2.7％

13％

2.4％

9.8％

C starch (Dry basis)

48.2％

12.9％

1.7％

15.6％

2.2％

19.3％

B+C starch (Dry basis)

67.8％

7.5％

3.1％

15.2％

2.9％

3.5％

Based on the research background, the utilization of carbon sources in the erythritol production process becomes a difficult problem to overcome in the industrialization process. The method can improve the waste starchiness raw material generated in the wheat processing process based on the classical glucose metabolism pathway modification and the reconstructed xylose pathway as a key carbon source for fermentation production, thereby promoting the deep processing of the wheat starch and achieving the purpose of reducing the cost. At present, no chassis cell integrating glucose and xylose co-metabolism exists, so that the industry is urgent to design and construct chassis cells capable of utilizing waste starchiness raw materials, reducing resource waste and improving the utilization efficiency of biological energy sources.

(III) summary of the invention

The invention aims to provide a yarrowia lipolytica engineering strain with a xylose metabolism pathway (XR-XDH pathway) and capable of rapidly metabolizing glucose and xylose to produce erythritol, and application of the genetically engineered strain in preparing erythritol by microbial fermentation.

The technical scheme adopted by the invention is as follows:

the genetic engineering bacteria for producing erythritol at high yield are constructed and obtained by the following method:

(1) The engineering bacteria Yarrowia lipolytica XR, XDH: XK, were obtained by over-expressing a gene encoding Xylose Reductase (XR), a gene encoding xylitol dehydrogenase (Xylitol dehydrogenase, XDH) and a gene encoding Xylulokinase (XK) with yarrowia lipolytica (Yarrowia lipolytica) as Chassis bacteria;

(2) Knocking out erythritol dehydrogenase (Erythritol dehydrogenase, EYD) genes in engineering bacterium ERY1 genome to obtain engineering bacterium Yarrowia lipolytica XR, wherein XDH is XKdelta EYD and is marked as ERY2;

(3) Knocking out mannitol dehydrogenase genes (Mannitol dehydrogenase, MDH) in engineering bacteria ERY2 genome to obtain engineering bacteria Yarrowia lipolytica XR, wherein XDH is XKdelta EYD delta MDH and is marked as ERY3;

(4) Knocking out arabitol dehydrogenase gene (Arabitol dehydrogenase, arDH) in engineering bacterium ERY3 genome to obtain engineering bacterium Yarrowia lipolytica XR, wherein XDH is XKdelta EYD delta MDHdelta ArDH and is marked as ERY4;

(5) Engineering bacteria ERY4 is taken as chassis bacteria, and non-oxidized module genes of pentose phosphate pathway, namely transketolase TKL1, transaldolase TAL and erythrose reductase ER are overexpressed to obtain engineering bacteria Yarrowia lipolytica XR, wherein XDH is XKΔ EYD ΔMDHΔArDH, TKL1, TAL is ER and is marked as ERY5;

(6) And (3) taking engineering bacteria ERY5 as chassis bacteria, and over-expressing HK genes, stp1 genes and Stp2 genes to obtain engineering bacteria Yarrowia lipolytica XR, namely, the engineering bacteria with the high erythritol yield, wherein the engineering bacteria comprise XDH, XKΔ EYD ΔMDHΔArDH, TKL1, TAL, ER, HK, stp1 and Stp2, and are marked as ERY6.

The invention improves the yarrowia lipolytica by means of metabolic engineering and genetic engineering, so that the recombinant yarrowia lipolytica can better utilize a glucose xylose mixed culture medium to efficiently synthesize the erythritol, and the xylose metabolism module of the erythritol synthesis pathway of the yarrowia lipolytica is strengthened by (1) enhancing the utilization capacity of the xylose; (2) Reducing the influence of carbon diversion effect of byproducts and erythritol degradation on yield; (3) Enhancing the non-redox module of pentose phosphate pathway, and improving the erythritol conversion efficiency; (4) The sugar uptake module is enhanced, and thus the yarrowia lipolytica erythritol genetic engineering strain capable of metabolizing glucose and xylose mixed sugar is obtained, and the strain can efficiently synthesize erythritol by using glucose and xylose mixed carbon sources, and does not synthesize byproducts (such as mannitol, arabitol and the like).

The invention also relates to a method for constructing the genetically engineered bacterium, which comprises the following steps:

(1) The yarrowia lipolytica (Yarrowia lipolytica) is taken as chassis fungus, 26s rDNA multi-site integration plasmid is used for carrying out three-gene tandem multi-copy expression on xylose metabolism gene XR, XDH, XK, and a promoter is selectedRespectively P _TEF1 、P _GPD2 、P _hp4d Obtaining engineering bacteria Yarrowia lipolytica XR, wherein XDH and XK are marked as ERY1;

(2) Knocking out erythritol dehydrogenase (Erythritol dehydrogenase, EYD) genes in an engineering bacterium ERY1 genome by using a homologous recombination technology to obtain engineering bacterium Yarrowia lipolytica XR, wherein XDH is XK delta EYD, URA3d, and simultaneously, removing a screening mark URA3d by using a knocking-out frame inner self-weight technology by adopting a reverse screening function of pentafluororotic acid (5-FOA) and Uridine (U), and marking the engineering bacterium Yarrowia lipolytica XR, XDH is XK delta EYD as ERY2;

(3) Knocking out mannitol dehydrogenase genes (Mannitol dehydrogenase, MDH) in an engineering bacterium ERY2 genome by using a homologous recombination technology to obtain engineering bacterium Yarrowia lipolytica XR, wherein XDH is XK delta EYD delta MDH, URA3d, and simultaneously removing a screening mark URA3d by using a knocking-out in-frame self-weight technology by adopting a reverse screening function of pentafluororotic acid and uridine to obtain engineering bacterium Yarrowia lipolytica XR, wherein XDH is XK delta EYD delta MDH and is marked as ERY3;

(4) Knocking out arabitol dehydrogenase genes (Arabitol dehydrogenase, arDH) in engineering bacteria ERY3 genome by using homologous recombination technology to obtain Yarrowia lipolytica XR XDH XK delta EYD delta MDH delta ArDH which is URA3d, and removing a screening mark URA3d by using a knocking-out in-frame self-weight technology by adopting the reverse screening function of pentafluororotic acid and uridine to obtain engineering bacteria Yarrowia lipolytica XR XDH XK delta EYD delta MDH delta ArDH which is marked as ERY4;

(5) The engineering bacterium ERY4 is taken as chassis fungus, a ZETA sequence multi-site integration plasmid is used for carrying out three-gene multi-copy expression on a Transketolase gene (TKL 1), a Transaldolase gene (TAL) and an erythrose reductase gene (Erythrose Reductase, ER), and the promoters are respectively P _TEF1 、P _GPD2 、 P _hp4d Obtaining engineering bacteria Yarrowia lipolytica XR, wherein XDH is XKΔ EYD delta MDHdelta ArDH is TKL1 is TAL is ER, and the engineering bacteria is ERY5;

(6) Engineering bacterium ERY5 is taken as chassis fungus, and 26S rDNA sequence multi-site integrating plasmid is used for integrating Hexokinase gene (Hexokinase, HK) and Sugar transporter 1 gene (SugarTransporter 1, stp 1) and sugar transporter 2 genes (Sugar transporter, stp 2) are over expressed, and the promoters are selected as P respectively _TEF1 、P _GPD2 、 P _hp4d Obtaining engineering bacteria Yarrowia lipolytica XR, wherein XDH is XKΔ EYD delta MDHdelta ArDH, TKL1, TAL is ER, HK is Stp1, stp2 is marked as ERY6, namely the high-yield erythritol genetic engineering bacteria.

Specifically, the promoter P _TEF1 The sequence is shown as SEQ ID NO.1, and the promoter P _GPD2 The sequence is shown as SEQ ID NO.2, and the promoter P _hp4d The sequence is shown as SEQ ID NO.3.

Specifically, the NCBI accession numbers of the XR, XDH, XK, TKL, TAL, ER, HK, stp and Stp2 gene sequences are respectively: XP_502540.1, XP_503864.1, XP_505266.1, XP_503628.1, XP_505460.1, XP_505585.1, XP_501216.1, XP_002548209.1 and XP_002545887.1.

The invention also relates to application of the genetically engineered bacterium in preparation of erythritol by microbial fermentation.

Specifically, the application is as follows: inoculating the genetically engineered strain into a fermentation culture medium with glucose and/or xylose as a carbon source, performing fermentation culture for 96-200 h at the temperature of 25-32 ℃ and the rpm of 180-300, and taking a fermentation liquor supernatant after fermentation, and separating and purifying to obtain the erythritol. Before fermentation, the genetically engineered strain is inoculated into YPD culture medium, cultured overnight at 28-30 deg.c and rotation speed of 180-250 rpm in a shaking table, and inoculated into fermentation culture medium for culture in 10 vol% concentration.

Further, the application is: and streaking the genetically engineered strain to a YPD solid culture medium, standing and culturing at 30 ℃ for 2 days, picking single bacterial colony, inoculating to 50mL of liquid YPD culture medium, culturing for 24 hours at 28-30 ℃ and 180-250 rpm, transferring to 30mL of 300mLYPD culture medium, fermenting for 24 hours at 28-30 ℃ and 180-250 rpm, and transferring to a 5L fermentation tank, wherein the total volume of fermentation liquor is 3L. The method adopts a mode of constant culture, and controls the fermentation conditions as follows: the rotation speed is 450-600 rpm, the pH=6.0, the fermentation ventilation rate is 0.5-1 vvm, the fermentation is carried out for 200 hours, and the erythritol is obtained by separation and purification after the fermentation is finished.

Preferably, the fermentation medium is composed of: 100-300 g/L of glucose, 50-200 g/L of xylose, 1-10 g/L of yeast powder, 1-10 g/L of peptone, 0.5-2.0 g/L of triammonium citrate, 0.05-0.2 g/L of magnesium sulfate heptahydrate, 0.05-0.2 g/L of zinc sulfate heptahydrate, 0.01-0.05 g/L of manganese chloride tetrahydrate, water as a solvent and natural pH.

The invention modifies erythritol biosynthesis network of yarrowia lipolytica by strong promoter P _TEF1 、 P _GPD2 、P _hp4d The XR, XDH, XK genes are respectively expressed strongly, the biosynthesis path of the erythritol taking xylose as a carbon source is remodeled, and the P _TEF1 、P _GPD2 、P _hp4d The promoters correspond to SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively. The accumulation of erythritol is increased by knocking out the erythritol degradation pathway gene EYD, the carbon shunt pathway genes mannitol dehydrogenase MDH and the arabitol dehydrogenase ArDH; through over-expression of TKL1 gene, TAL gene and ER gene, the precursor conversion capability of a pentose phosphate pathway non-oxidation module in the process of erythritol biosynthesis is enhanced, and accumulation is increased; further, the HK gene, the Stp1 gene and the Stp2 gene are overexpressed, so that the sugar uptake capacity is enhanced, and the erythritol yield is further improved.

To sum up, in order to further optimize the way of synthesizing erythritol by glucose and xylose mixed carbon source, the invention selects wild type yarrowia lipolytica strain as an original strain, introduces a new way into yarrowia lipolytica by means of metabolic engineering and genetic engineering, knocks out related genes synthesized by byproducts, expresses the related genes synthesized by erythritol, constructs a method for synthesizing the recombinant yarrowia lipolytica strain of erythritol by fermenting glucose and xylose mixed carbon source more efficiently, and obtains a strain Yarrowia lipolytica ERY with obviously improved yield and yield of synthesized erythritol.

The beneficial effects of the invention are mainly as follows: the invention remodels the xylose metabolism path in yarrowia lipolytica by adopting a multi-copy expression mode, strengthens the expression of TKL1, TAL and ER in the erythritol biological generation path, reduces the generation of byproducts mannitol and arabitol by knocking out MDH and ArDH, reduces the accumulation of erythritol by converting the erythritol into other substances by knocking out EYD, and finally obtains the erythritol high-yield strain capable of utilizing glucose and xylose mixed carbon sources by strengthening the expression of genes HK, stp1 and Stp2 in the sugar uptake path, and the yield is improved from 50.17g/L to 195.56g/L.

(IV) description of the drawings

FIG. 1 is the shake flask fermentation yield of erythritol from a wild-type strain in glucose, xylose, and glucose xylose mixed medium;

FIG. 2 is a 5L tank fermentation substrate and product content determination of xylose glucose mixed carbon source;

FIG. 3 is a 5L tank fermentation substrate and product content determination of wheat B starch as carbon source.

(fifth) detailed description of the invention

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

in the examples, the concentration of pentafluororotic acid (5-FOA) in the medium was 0.1% (w/w), the concentration of Uridine (U) in the medium was 10mM, the concentration of ampicillin in the medium was 0.10mg/L, and the concentration of kanamycin in the medium was 0.05mg/L.

Table 2: gene engineering related genes and related functions

Gene name	NCBI numbering	Function of
			XR	XP_502540.1	Xylose reductase
XDH	XP_503864.1	Xylitol dehydrogenase
			XK	XP_505266.1	Xylulokinase
EYD	YALI0_F01650g	Erythritol dehydrogenase
			MDH	YALI0_B16192g	Mannitol dehydrogenase
ArDH	YALI0_F02211g	Arabitol dehydrogenase
			TKL1	XP_503628.1	Transketolase
TAL	XP_505460.1	Transaldolase
			ER	XP_505585.1	Erythrose reductase
HK	XP_501216.1	Hexokinase
			Spt1	XP_002548209.1	Sugar transporter 1
Spt2	XP_002545887.1	Sugar transporter 2

Table 3: primer sequences for use in the present invention

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Example 1: wild strain erythritol shake flask fermentation and erythritol production performance test

Culturing strains: yarrowia lipolytica po1f (ATCC 20260) deposited at-80℃was streaked onto YPD medium and incubated at 28℃for 2 days, single colonies were picked up to 50mL YPD liquid medium and incubated at 200rpm for 24h at 28 ℃.

Shaking and fermenting: inoculating 5mL of the cultured seed liquid into a 250 mL convex bottom shake flask containing 50mL of fermentation medium, wherein the fermentation medium comprises three groups of components:

a: 350g/L of glucose, 10g/L of yeast powder, 5g/L of peptone, 1g/L of triammonium citrate, 0.1g/L of magnesium sulfate heptahydrate, 0.1g/L of zinc sulfate heptahydrate and 0.02g/L of manganese chloride tetrahydrate;

b: 250g/L of glucose, 100g/L of xylose, 10g/L of yeast powder, 5g/L of peptone, 1g/L of tri-ammonium citrate, 0.1g/L of magnesium sulfate heptahydrate, 0.1g/L of zinc sulfate heptahydrate and 0.02g/L of manganese chloride tetrahydrate;

c: 100g/L of xylose, 10g/L of yeast powder, 5g/L of peptone, 1g/L of triammonium citrate, 0.1g/L of magnesium sulfate heptahydrate, 0.1g/L of zinc sulfate heptahydrate and 0.02g/L of manganese chloride tetrahydrate.

The culture was continued in a shaker at 28℃and 200 rpm. After 5 days, the fermentation is finished, and the fermentation liquor is taken for erythritol HPLC detection.

HPLC detection: centrifuging 1mL of fermentation liquor to obtain supernatant, diluting 100 times, and filtering with 0.22 μm water phase membrane to obtain liquid phase bottle for use; 1L of 5mM sulfuric acid was prepared as a mobile phase, and after filtration with a 0.22 μm filter, the solution was sonicated for 30min. Chromatographic conditions: the chromatographic column is an AminexHPX-87H sugar analysis column (300 mM ×7.8mm), the column temperature is 35 ℃, and the mobile phase is 5mM H ₂ SO ₄ The flow rate is 0.6mL/min, the sample injection amount is 10 mu L, the detector is a differential refraction detector, and the detector temperature is 30 ℃.

Example 2: construction of xylose metabolizing Strain Y.lipolytica ERY1

The yarrowia lipolytica XR, XDH, XK gene sequence was retrieved at the NCBI website, and the XR, XDH and XK three gene coexpression nucleic acid sequences pUC19-RDK were designed and synthesized: SEQ ID NO.4. Using P respectively _TEF1 、P _GPD2 、P _hp4d Driving transcription of XR, XDH and XK to make them strongly expressed; while flanking 26S rDNA sequences serve as multiple copy insertion sites. Extracting plasmid containing expression vector pUC19-RDK, and carrying out double enzyme digestion by using endonuclease KpnI to obtain a linearization expression frame: upstream 26S rDNA sequence → P _TEF1 XR Gene sequence Tleu P _GPD2 XDH gene sequence Tmig1P _hp4d XK gene sequence, txpr2, 26S rDNA sequence.

The expression cassette was introduced into the erythritol producing strain yarrowia lipolytica (AJD) by lithium acetate mediated chemical transformation in SD medium containing xylose (YNB 6.7g/L, (NH) ₄ ) ₂ SO ₄ 5g/L, agar powder 15 g/L) on a sieve (wild type strain cannot grow in xylose medium). The components of the culture medium are 20g/L xylose and 6.7g/L YNB, (NH) ₄ ) ₂ SO ₄ 5g/L, 15g/L of agar powder and pH6.0. Since yarrowia lipolytica is not able to utilize xylose by itself, a transformant that can grow in a xylose-containing medium is a transformant that can utilize xylose metabolismThereby obtaining a Yarrowia lipolytica ERY strain.

Example 3: construction of erythritol degradation gene and carbon shunt branch gene knockout bacterium and shake flask fermentation

Using the primers listed in Table 2, a knock-out box (SEQ ID NO. 7) of erythritol dehydrogenase gene EYD was constructed by PCR and one-step cloning, the knock-out box structure being EYD upstream homology arm 1 kb-downstream homology arm 500bp (down) -URA3d marker-EYD downstream homology arm 1kb, wherein the down sequence was capable of self-recombination with the downstream homology arm under selective pressure conditions of 0.1% pentafluororotic acid (5-FOA) and 10mM Uridine (U), thereby completing self-recombination removal of the URA3d marker for comparative recycling. The EYD gene is knocked out on the basis of engineering bacterium ERY1, and the EYD gene knocked out strain is subjected to positioning verification by using PCR, so that the positioning knockout of homologous recombination is confirmed. Subsequently, the correct transformants were subjected to dilution plating onto SD plates containing 0.1% of pentafluororotic acid (5-FOA) and 10mM of Uridine (U), subjected to resistance pressure screening, and subjected to self-recombination to effect removal of the markers and recycling, thereby obtaining a URA-marker-free EYD knockout strain Yarrowia lipolytica XR:XDH: XK.DELTA. EYD, the transformant being designated ERY2.

Further, the knock-out boxes (SEQ ID NO. 9) of the mannitol dehydrogenase gene MDH (SEQ ID NO. 8) and the arabitol dehydrogenase gene ArDH were constructed in the same manner. MDH and ArDH knockout vectors treated by double enzyme digestion with the endonuclease XbaI were sequentially introduced into ERY2 engineering strains according to the method described above, and Yarrowia lipolytica XR: XDH: XKΔ EYD. DELTA. MDH (ERY 3) and Yarrowia lipolytica XR: XDH: XKΔ EYD. DELTA. MDHΔArDH (ERY 4) strains were obtained, respectively, by marker removal and transformant identification.

The obtained ERY4 engineering strain was subjected to an erythritol synthesis experiment, and the fermentation medium and fermentation conditions were the same as in example 2. The results show that compared with the strain ERY1, the knockout of the degradation gene and the byproduct gene can significantly improve the yield of erythritol (8.24 g/L) and reduce the yield of byproducts, wherein mannitol (0.023 g/L) and arabitol (0.002 g/L) substantially eliminate the accumulation of byproducts.

Example 4: erythritol precursor conversion module enhances enhanced metabolic flux

The expression frame containing the transketolase TKL1, the transaldolase TAL and the erythrose reductase gene ER is designed and synthesized, and the sequence of the expression frame is SEQ ID NO.5, and the expression frame comprises DNA elements expressed by three genes of the transketolase TKL1, the transaldolase TAL and the erythrose reductase. The expression vector linearized by using the endonuclease NotI is introduced into an engineering strain ERY4 after DNA transformation, and a three-gene co-expression strain Yarrowia lipolytica XR is obtained by PCR identification, wherein XDH is XKΔ EYD delta MDHdelta ArDH, TKL1, TAL is ER and is marked as ERY5. The obtained correct transformant was subjected to fermentation synthesis erythritol test, and the fermentation medium and fermentation conditions were the same as in example 2. The result shows that compared with the original strain, the synthesis efficiency of erythritol can be greatly improved by strengthening the metabolic flux through the precursor transformation module, and the shake flask yield is improved to 19.8 g/L.

Example 5: the sugar intake module is reinforced to further improve the erythritol production capacity of the engineering strain

The expression frame containing hexokinase HK and sugar transport proteins Stp1 and Stp2 genes is designed and synthesized, and the sequence of the expression frame is SEQ ID NO.6. The expression vector linearized with the endonuclease EcoRI was introduced into the engineering strain ERY5 after DNA transformation. PCR identification shows that three gene co-expression strains Yarrowia lipolytica XR of HK, stp1 and Stp2 are obtained, wherein XDH is XKΔ EYD ΔMDHΔArDH, TKL1, TAL is ER, HK is Stp1 and Stp2. The obtained strain was designated as Yarrowia lipolytica ERY, and the obtained correct transformant was subjected to fermentation synthesis erythritol test, and the fermentation medium and fermentation conditions were the same as in example 2. The results show that sugar uptake module fortification can significantly increase yield to 23.5g/L.

Example 6: application of engineering strain ERY6 in fermentation production of erythritol

The recombinant yarrowia lipolytica ERY6 strain cultured by YPD is inoculated into a 2L triangular flask containing 300mL of fermentation medium according to the inoculation amount of 10%, the initial thallus concentration (OD 600) is controlled between 0.8 and 1, and the fermentation medium comprises the following components: 250g/L of glucose, 100g/L of xylose, 3g/L of yeast powder, 2g/L of peptone, 1g/L of tri-ammonium citrate, 0.1g/L of magnesium sulfate heptahydrate, 0.1g/L of zinc sulfate heptahydrate, 0.02g/L of manganese chloride tetrahydrate and natural initial pH. Culturing at 30deg.C and 220rpm for 24 hr, transferring to a fermenter containing 2.7L fermentation medium, fermenting at 30deg.C and pH=6.0 with aeration rate of 1vvm at 500rpm/min for 204 hr, and sampling at fixed time to determine glucose, xylose and erythritol contents (FIG. 2). The consumption of glucose and xylose in the fermenter was completed up to 168 hours (day 7), at which time the erythritol content was 195.56g/L and the erythritol synthesis efficiency was 1.16g/L/h.

Example 7: 5L tank chemostat fermentation test for producing erythritol by fermenting engineering strain ERY6 with B starch

The ERY6 engineering strain cultured by YPD is inoculated into a 2L triangular flask containing 300mL of fermentation medium according to 10% of inoculation amount, the initial thallus concentration (OD 600) is controlled between 0.8 and 1, and the fermentation medium comprises the following components: b500 mL/L of starch, 3g/L of yeast powder, 2g/L of peptone, 1g/L of triammonium citrate, 0.1g/L of magnesium sulfate heptahydrate, 0.1g/L of zinc sulfate heptahydrate, 0.02g/L of manganese chloride tetrahydrate and an initial pH value of 6.0. Wherein, the starch B is subjected to hydrolysis treatment: 350mL of B starch is slowly heated in a boiling water bath until the B starch becomes starch milk, the boiling is continued, 0.5 g of high temperature alpha-amylase is added, the mixture is stirred until the starch is liquefied into clear and transparent, the temperature is reduced to 55 ℃, 0.5 g of medium temperature beta-amylase, 0.2g of pullulanase and 0.5 g of xylanase are added for saccharification, and the mixture is used for fermenting raw materials after 5h of heat preservation. At 30 ℃, ph=6.0, fermentation was performed for 144 hours at 500rpm/min, and glucose, xylose and erythritol content were measured by regular sampling (fig. 3). After the consumption of glucose and xylose in the fermentation tank is completed for 96 hours (the fourth day), the erythritol content is measured to be 151.83g/L, and the erythritol synthesis efficiency is measured to be 1.58g/L/h.

The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that the inventors may make various changes or modifications within the scope of the claims without affecting the essential content of the invention.

Claims

1. The genetic engineering bacteria for producing erythritol at high yield are constructed and obtained by the following method:

2. A method of constructing the genetically engineered bacterium of claim 1, the method comprising:

(1) The yarrowia lipolytica (Yarrowia lipolytica) is taken as chassis fungus, 26s rDNA multi-site integration plasmid is used for carrying out three-gene tandem multi-copy expression on xylose metabolism gene XR, XDH, XK, and promoters are selected as P respectively _TEF1 、P _GPD2 、P _hp4d Obtaining engineering bacteria Yarrowia lipolytica XR, wherein XDH and XK are marked as ERY1;

(5) The engineering bacterium ERY4 is taken as chassis fungus, a ZETA sequence multi-site integration plasmid is used for carrying out three-gene multi-copy expression on a Transketolase gene (TKL 1), a Transaldolase gene (TAL) and an erythrose reductase gene (Erythrose Reductase, ER), and the promoters are respectively P _TEF1 、P _GPD2 、P _hp4d Obtaining engineering bacteria Yarrowia lipolytica XR, wherein XDH is XKΔ EYD delta MDHdelta ArDH is TKL1 is TAL is ER, and the engineering bacteria is ERY5;

(6) The engineering bacterium ERY5 is taken as chassis fungus, a 26S rDNA sequence multi-site integrating plasmid is used for over-expressing Hexokinase gene (Hexokunase, HK), sugar transporter 1 gene (Sugar transporter, stp 1) and sugar transporter 2 gene (Sugar transporter, stp 2), and promoters are selected as P respectively _TEF1 、P _GPD2 、P _hp4d Obtaining engineering bacteria Yarrowia lipolytica XR, namely the engineering bacteria with high erythritol yield, wherein the engineering bacteria comprise XDH, XKDelta EYD, MDHDeltaArDH, TKL1, TAL, ER, HK, stp1, stp2 and ERY6.

3. The method of claim 2, wherein the chassis fungus is yarrowia lipolytica (Yarrowia lipolytica AJD).

4. The method of claim 2, wherein promoter P _TEF1 The nucleotide sequence of (a) is shown as SEQ ID NO.1, and the promoter P _GPD2 The nucleotide sequence of (2) is shown as SEQ ID NO.2, and the promoter P _hp4d The nucleotide sequence of (2) is shown as SEQ ID NO.3.

5. The use of the genetically engineered bacterium of claim 1 in the preparation of erythritol by microbial fermentation.

6. The application according to claim 5, characterized in that the application is: inoculating the genetically engineered strain into a fermentation culture medium with glucose and/or xylose as a carbon source, performing fermentation culture for 96-200 h at the temperature of 25-32 ℃ and the rpm of 180-300, and taking a fermentation liquor supernatant after fermentation, and separating and purifying to obtain the erythritol.

7. The use according to claim 5, characterized in that the fermentation medium consists of: 100-300 g/L of glucose, 50-200 g/L of xylose, 1-10 g/L of yeast powder, 1-10 g/L of peptone, 0.5-2.0 g/L of triammonium citrate, 0.05-0.2 g/L of magnesium sulfate heptahydrate, 0.05-0.2 g/L of zinc sulfate heptahydrate, 0.01-0.05 g/L of manganese chloride tetrahydrate, water as a solvent and natural pH.