CN103060217B - Recombinant yeast strain capable of efficiently metabolizing xylose and application thereof - Google Patents

Abstract

The invention discloses a recombinant yeast strain (Saccharomyces cerevisiae)) capable of efficiently metabolizing xylose, which is named SyBE005. The recombinant yeast strain has been collected in Common Microorganism Center of China Committee for Culture Collection of Microorganisms, the collection number is CGMCC No.6634, and the recombinant yeast strain has the capacity of efficiently metabolizing xylose to produce ethanol. The recombinant yeast strain SyBE005 can effectively ferment xylose to generate ethanol. The ethanol yield reaches 64% of the theoretical yield, and the yield of the byproduct xylitol is lower than 12%; and thus, the recombinant yeast strain can efficiently convert xylose into ethanol, and is an excellent strain utilizing cellulose hydrolysate.

Description

A recombinant yeast strain for efficiently metabolizing xylose and its application

technical field

The invention belongs to the technical field of bioengineering, in particular to a recombinant yeast strain capable of fermenting xylose to produce ethanol.

Background technique

Utilizing lignocellulose to produce fuel ethanol can not only reduce the production cost of fuel ethanol, but also play an important role in improving the environment and waste disposal. As a large agricultural country, our country is rich in crop waste resources such as straw, which provides unique conditions for the development of fuel ethanol industry. Saccharomyces cerevisiae is generally used for industrial production of ethanol. Although natural Saccharomyces cerevisiae can use the hexose sugar in lignocellulose hydrolyzate to produce ethanol, it lacks the ability to metabolize abundant pentose sugars, such as xylose. . Therefore, the construction of engineering strains that can efficiently utilize xylose is of key significance for reducing the production cost of fuel ethanol.

At present, my country has made some progress in the construction of Saccharomyces cerevisiae engineering strains that can use xylose to produce ethanol using metabolic engineering methods, but it still cannot meet the production requirements, mainly due to the slow utilization of xylose and excessive accumulation of by-product xylitol. Many and low ethanol yield.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a recombinant yeast strain that metabolizes xylose efficiently.

The second object of the present invention is to provide the use of the above-mentioned recombinant yeast strain that efficiently metabolizes xylose.

Technical scheme of the present invention is summarized as follows:

A recombinant yeast strain that efficiently metabolizes xylose, classified and named: Saccharomyces cerevisiae named SyBE005, has been preserved in the General Microbiology Center of China Committee for the Collection of Microbial Cultures, and the preservation number is CGMCC No.6634, which has high-efficiency metabolism The ability of xylose to produce ethanol.

The use of the above bacterial strains to metabolize xylose to produce ethanol.

Advantages of the present invention:

The recombinant yeast strain SyBE005 of the present invention can effectively utilize xylose to ferment and produce ethanol. The yield of ethanol reaches 64% of the theoretical yield, and the yield of by-product xylitol is below 12%. It can efficiently convert xylose into ethanol, and is an excellent strain for utilizing cellulose hydrolyzate.

The recombinant yeast strain (Saccharomyces cerevisiae) that efficiently metabolizes xylose of the present invention is named SyBE005, and it has been preserved in the General Microbiology Center of the China Committee for Microbial Culture Collection on September 27, 2012, referred to as CGMCC, and the preservation number is CGMCC No.6634 . The address is Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

Description of drawings

Fig. 1 is a plasmid map containing a xylose reductase gene coding frame, a xylitol dehydrogenase gene coding frame and a xylulokinase gene coding frame.

Fig. 2 is a map of the plasmid pRS305-XDH containing the coding frame of the xylitol dehydrogenase gene.

Fig. 3 is a map of plasmid pRS-RKI1-TKL1 containing ribose phosphate isomerase gene coding frame and transketolase gene coding frame.

Fig. 4 is a map of the plasmid pAUR-RPE1-TAL1 containing the coding frame of the pentose phosphate epimerase gene and the coding frame of the transaldolase gene.

Fig. 5 shows the utilization of xylose when the strains L2612PR, L2612PR-DPPP and SyBE005 were fermented in the fermentation medium containing 20 g/L xylose.

Figure 6 shows the production of ethanol by strains L2612PR, L2612PR-DPPP, and SyBE005 when they were fermented in a fermentation medium containing 20 g/L xylose.

Fig. 7 shows the production of xylitol when the strains L2612PR, L2612PR-DPPP, and SyBE005 were fermented in a fermentation medium containing 20 g/L xylose.

Detailed ways

The present invention will be further described below in conjunction with specific examples. The examples of the present invention are intended to enable those skilled in the art to better understand the present invention, but do not limit the present invention in any way.

Example 1

Construction and Screening of Recombinant Yeast Strain SyBE005

Starting strain: The starting strain L2612 was donated by Professor Thomas Jeffries of the University of Wisconsin-Madison in the United States, and the genotype is MAT alpha, leu2, ura3, trp1. It is deficient in leucine, uracil and tryptophan.

culture medium

Screening medium A: Synthetic nitrogen source YNB6.7g/L, mixed amino acid powder 2g/L (refer to [US] D.C. Amberg et al. Yeast Genetics Method Test Guide for specific formula), leucine 100mg/L, tryptophan 100mg/L, glucose 20g/L.

Screening medium B: synthetic nitrogen source YNB6.7g/L, mixed amino acid powder 2g/L, tryptophan 100mg/L, glucose 20g/L.

Screening medium C: synthetic nitrogen source YNB6.7g/L, mixed amino acid powder 2g/L, glucose 20g/L.

Screening medium D: synthetic nitrogen source YNB6.7g/L, mixed amino acid powder 2g/L, aureobasicin A0.5mg/L, glucose 20g/L. Acclimatization medium: synthetic nitrogen source YNB6.7g/L, mixed amino acid powder 2g/L, xylose 50g/L.

Add 20g/L agar to the solid medium.

Construction of Departure Recombinant Strain L2612PR-DPPP

Using the genome of strain L2612 as a template, the promoters TDH1p, TDH3p, and PGK1p, the xylulokinase gene XKS1 (including its own terminator), the pentose phosphate epimerase gene RPE1, the ribose phosphate isomerase gene RKI1, were cloned. Ketolase gene TKL1, transaldolase gene TAL1, terminator sequence PGK1t.

The synthetic xylose reductase gene XR and xylitol dehydrogenase gene XDH were connected with the promoter PGK1 and the terminator PGK1t respectively by fusion PCR technology, and the promoter PGK1 was connected with the xylulokinase gene XKS1, and then the three The gene expression cassette was sequentially connected to the vector YIplac211 (purchased from ATCC) to form the expression plasmid YIplac211-XRXDHXK (Figure 1).

The XR sequence of the xylose reductase gene is shown in SEQ ID NO:1.

The xylitol dehydrogenase gene XDH sequence is shown in SEQ ID NO: 2.

The xylitol dehydrogenase gene expression cassette constructed above was digested from the plasmid YIplac211-XRXDHXK, and re-cloned into the vector pRS305 (purchased from ATCC) to form the plasmid pRS305-XDH (Figure 2).

Construction of pentose phosphate epimerase gene expression cassette TDH1p-RPE1-PGK1t, transaldolase gene expression cassette PGK1p-TAL1-PGK1t, ribose phosphate isomerase gene expression cassette PGK1p-RKI1-PGK1t, transketone by fusion PCR technology Enzyme gene expression cassette TDH3p-TKL1-PGK1t. The gene expression cassettes PGK1p-RKI1-PGK1t and TDH3p-TKL1-PGK1t were linked to the vector pRS304 (purchased from ATCC) to form the vector pRS-RKI1-TKL1 (Figure 3). The gene expression cassette TDH1p-RPE1-PGK1t and the gene expression cassette PGK1p-TAL1-PGK1t were sequentially connected to the vector pAUR101 (purchased by Dalian Bao Biological Company) to construct the vector pAUR-RPE1-TAL1 (Figure 4).

YIplac211-XRXDHXK was linearized with restriction endonuclease Apa I, and strain L2612 was transformed by lithium acetate method, and recombinant strain L2612PR was obtained by screening on selection medium A. And on the basis of the recombinant strain, the plasmid pRS305-XDH was transformed by the lithium acetate method, and the recombinant strain was obtained on the screening medium B. The plasmid pRS-RKI1-TKL1 was linearized with the restriction endonuclease EcoRI, the recombinant strain obtained in the previous step was transformed by the lithium acetate method, and the recombinant strain was obtained by expressing on the screening medium C. And on the basis of this strain, the plasmid pAUR-RPE1-TAL1 linearized with StuI was further transformed, and the starting recombinant strain L2612PR-DPPP was obtained on the screening medium D.

Construction and Screening of Mutant Strain SyBE005

The strain L2612PR-DPPP was placed into 250 mL containing 50 mL acclimation medium at an initial OD ₆₀₀ =0.2, and aerobically cultured at 30°C and 200 rpm for 3 days. Measure the OD ₆₀₀ of the culture, and transfer it to the fresh acclimatization medium with the initial OD ₆₀₀ =0.2. After 10 repeated cultures, the OD ₆₀₀ of the bacterial liquid remains unchanged, and continue to repeat the culture 10 times. Take a small amount of bacterial solution, spread it on the solid plate of the acclimatization medium after dilution, and obtain a single colony strain. Select 20 colonies with the largest colony size, inoculate them into 15 mL test tubes containing 2 mL of liquid screening medium C, and culture them at 30°C and 200 rpm for 2 days. Take 100 μL of the bacterial liquid and transfer it to 3 mL of fresh fermentation medium A, and culture it anaerobically at 30°C and 200 rpm. Seal the mouth of the test tube with a rubber stopper with a vent needle. After 24 hours of anaerobic culture, the OD ₆₀₀ , residual xylose content and metabolite ethanol, glycerol and xylitol content of the bacterial solution were measured. A strain SyBE005 with the least residual xylose was obtained from 20 single colonies.

Analytical method:

The cell concentration was characterized by the cell absorbance value (OD ₆₀₀ ) measured at 600 nm by a 722-type spectrophotometer. The concentrations of xylose, ethanol, xylitol and glycerol were determined by high performance liquid chromatography (Waters1515). Chromatographic column is Aminex HPX-87H, column temperature: 65°C; detector: Waters differential detector 2421, detector temperature is 40°C. The mobile phase was 5 mM sulfuric acid solution, the flow rate was 0.6 mL/min, and the injection volume was 10 μL.

Example 2 Comparison of Xylose Fermentation of Bacterial Strains L2612PR, L2612PR-DPPP and SyBE005

1. Test materials: strains L2612PR, L2612PR-DPPP, SyBE003

2. Test method:

Seed medium 1: synthetic nitrogen source YNB6.7g/L, mixed amino acid powder 2g/L, glucose 20g/L, leucine 100mg/L, tryptophan 100mg/L.

Seed medium 2: synthetic nitrogen source YNB6.7g/L, mixed amino acid powder 2g/L, glucose 20g/L.

Fermentation medium: yeast extract powder 10g/L, peptone 20g/L, xylose 20g/L.

Pick up a loop of L2612PR colony from a fresh slant and put it into 50 mL seed medium 1. Similarly, colonies L2612PR-DPPP and SyBE005 were inoculated into 50 mL of seed medium 2 from a fresh slant. Cultivate for 24 hours at 30°C and 200 rpm. Inoculate 100 mL of fermentation medium with an initial cell concentration of OD ₆₀₀ =1.0, culture at 30°C and 150 rpm, and ferment for 60 hours. Detect bacterial cell concentration, xylose concentration and product ethanol, xylitol, glycerin concentration.

3. Analysis method:

With embodiment 1.

4. Test results:

As shown in Figure 5, the strain SyBE005 has fully utilized xylose within 48 hours, while the strains L2612PR and L2612PR-DPPP have only utilized about 60% of xylose, and the maximum xylose specific consumption rate of SyBE005 reached 0.301g/g dry weight/h; after fermentation, the ethanol yields of L2612PR, L2612PR-DPPP, and SyBE005 reached 2.02g/L, 2.97g/L, and 6.85g/L, respectively. The ethanol yields corresponding to L2612PR and L2612PR-DPPP are 0.15g/g xylose and 0.20g/g xylose respectively, and the fermentation ethanol yield of SyBE005 reaches 0.33g/g xylose, which is higher than that of L2612PR and L2612PR-DPPP respectively. 120% and 65%, as shown in Figure 6; after the fermentation, the xylitol content of L2612PR, L2612PR-DPPP, and SyBE005 reached 6.24g/L, 6.29g/L, and 2.43g/L, respectively. The xylitol yield of L2612PR and L2612PR-DPPP reached 0.48g/g xylose and 0.42g/g xylose, while the xylitol yield of SyBE005 decreased to 0.12g/g xylose, compared to L2612PR and L2612PR-DPPP Reduced by 3, 2.5 times, as shown in Figure 7.

Claims (2)

1. one plant height effect fermenting xylose recombinant Saccharomyces cerevisiae ( saccharomyces cerevisiae) SyBE005, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.6634, and it has the ability of efficient fermenting xylose producing and ethanol.

2. the purposes of recombinant Saccharomyces cerevisiae fermenting xylose producing and ethanol described in claim 1.