CN103060217B - Recombinant yeast strain capable of efficiently metabolizing xylose and application thereof - Google Patents
Recombinant yeast strain capable of efficiently metabolizing xylose and application thereof Download PDFInfo
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- CN103060217B CN103060217B CN201210507117.XA CN201210507117A CN103060217B CN 103060217 B CN103060217 B CN 103060217B CN 201210507117 A CN201210507117 A CN 201210507117A CN 103060217 B CN103060217 B CN 103060217B
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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
Technical field
The invention belongs to technical field of bioengineering, being specifically related to a strain can the restructuring yeast strains of xylose fermentation ethanol.
Background technology
Utilize lignocellulosic materials for fuel ethanol, the production cost of alcohol fuel can not only be reduced, and all play a very important role in environmental protect and refuse process etc.China is as a large agricultural country, and the agricultural waste material resources such as stalk are quite abundant, and this provides advantageous condition for Fuel Alcohol Development industry.The general bacterial strain being used as industrial production ethanol is yeast saccharomyces cerevisiae, although natural yeast saccharomyces cerevisiae can be good at utilizing the hexose in lignocellulose hydrolysate to produce ethanol, but lacks the pentose to rich content, the metabolic capacity of such as wood sugar.Therefore build and the engineering strain of efficiency utilization wood sugar can have critical significance to reducing the production cost of alcohol fuel.
Current China utilize metabolic engineering method build can utilize wood sugar produce ethanol saccharomyces cerevisiae engineered yeast strain in make some progress, but still can not meet the requirement of production, be mainly manifested in xylose utilization slowly, the accumulation of by product Xylitol too much and the aspect such as alcohol getting rate is low.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, the restructuring yeast strains of a plant height effect fermenting xylose is provided.
Second object of the present invention is to provide the purposes of the restructuring yeast strains of an above-mentioned plant height effect fermenting xylose.
Technical scheme of the present invention is summarized as follows:
The restructuring yeast strains of one plant height effect fermenting xylose, Classification And Nomenclature: yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) called after 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.
The purposes of above-mentioned bacterial strains fermenting xylose producing and ethanol.
Advantage of the present invention:
Restructuring yeast strains SyBE005 of the present invention can effectively utilize wood-sugar fermentation to produce ethanol.Alcohol yied reaches 64% of theoretical yield, and the productive rate of by product Xylitol, below 12%, can converting xylose be ethanol efficiently, is the excellent bacterial strain utilizing cellulosic hydrolysate.
Restructuring yeast strains (Saccharomyces cerevisiae) the called after SyBE005 of efficient fermenting xylose of the present invention, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 27th, 2012, be called for short CGMCC, deposit number is CGMCC No.6634.Address in Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Accompanying drawing explanation
Fig. 1 is the plasmid map containing Xylose reductase gene encoder block, xylose dehydrogenase gene encoder block and xylulokinase gene encoder block.
Fig. 2 is the plasmid pRS305-XDH collection of illustrative plates containing xylose dehydrogenase gene encoder block.
Fig. 3 is the plasmid pRS-RKI1-TKL1 collection of illustrative plates containing ribose phosphate isomerase gene encoder block, tkt gene encoder block.
Fig. 4 is the plasmid pAUR-RPE1-TAL1 collection of illustrative plates containing pentose phosphate epimerase genes encoding frame, transaldolase genes encoding frame.
Fig. 5 is bacterial strain L2612PR, L2612PR-DPPP, SyBE005 xylose utilization situation when fermenting in the fermention medium containing 20g/L wood sugar.
Fig. 6 is bacterial strain L2612PR, L2612PR-DPPP, SyBE005 ethanol production when fermenting in the fermention medium containing 20g/L wood sugar.
Fig. 7 is bacterial strain L2612PR, L2612PR-DPPP, SyBE005 Xylitol production when fermenting in the fermention medium containing 20g/L wood sugar.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated, and embodiments of the invention understand the present invention better to enable those skilled in the art to, but do not do any restriction to the present invention.
Embodiment 1
The structure of restructuring yeast strains SyBE005 and screening
Starting strain: starting strain L2612, be that Thomas professor Jeffries of University of Wisconsin at Madison grants, genotype is MAT alpha, leu2, ura3, trp1.Leucine, uridylic and tryptophane defect.
Substratum
Screening culture medium A: synthesis nitrogenous source YNB6.7g/L, kilnitamin powder 2g/L(specifically fills a prescription with reference to yeast genetics method test guides such as [U.S.] D.C. Ambergs), leucine 100mg/L, tryptophane 100mg/L, glucose 20g/L.
Screening culture medium B: synthesis nitrogenous source YNB6.7g/L, kilnitamin powder 2g/L, tryptophane 100mg/L, glucose 20g/L.
Screening culture medium C: synthesis nitrogenous source YNB6.7g/L, kilnitamin powder 2g/L, glucose 20g/L.
Screening culture medium D: synthesis nitrogenous source YNB6.7g/L, kilnitamin powder 2g/L, short stalk mycin A0.5mg/L, glucose 20g/L.Domestication substratum: synthesis nitrogenous source YNB6.7g/L, kilnitamin powder 2g/L, wood sugar 50g/L.
In solid medium, add the agar of 20g/L.
Set out the structure of recombinant bacterial strain L2612PR-DPPP
With bacterial strain L2612 genome for template, clone promotor TDH1p, TDH3p, PGK1p, xylulokinase gene XKS1(is containing terminate self), pentose phosphate epimerase gene RPE1, ribose phosphate isomerase gene RKI1, tkt gene TKL1, transaldolase gene TAL1, terminator sequence PGK1t.
Be connected with promotor PGK1, terminator PGK1t respectively by Xylose reductase gene XR, the xylose dehydrogenase gene XDH of fusion DNA vaccine technology by synthesis, promotor PGK1 is connected with xylulokinase gene XKS1, then three gene expression frames are connected to carrier YIplac211(in turn and are purchased from ATCC) on, form expression plasmid YIplac211-XRXDHXK(Fig. 1).
Xylose reductase gene XR sequence is with shown in SEQ ID NO:1.
Xylose dehydrogenase gene XDH sequence is with shown in SEQ ID NO:2.
The xylose dehydrogenase gene expression cassette built above is cut out from enzyme plasmid YIplac211-XRXDHXK, is again cloned into carrier pRS305(and is purchased from ATCC) on, form plasmid pRS305-XDH (Fig. 2).
Utilize fusion DNA vaccine technique construction pentose phosphate epimerase expression casette TDH1p-RPE1-PGK1t, transaldolase expression casette PGK1p-TAL1-PGK1t, ribose phosphate isomerase gene expression cassette PGK1p-RKI1-PGK1t, tkt gene expression cassette TDH3p-TKL1-PGK1t.Expression casette PGK1p-RKI1-PGK1t and TDH3p-TKL1-PGK1t is connected to carrier pRS304(and is purchased from ATCC) on, form carrier pRS-RKI1-TKL1 (Fig. 3).Expression casette TDH1p-RPE1-PGK1t and expression casette PGK1p-TAL1-PGK1t is connected to successively the precious biotech firm in carrier pAUR101(Dalian to buy), carrier construction pAUR-RPE1-TAL1 (Fig. 4).
With restriction enzyme A pa I linearizing YIplac211-XRXDHXK, transform bacterial strain L2612 by Li-acetate method, on screening culture medium A, screening obtains recombinant bacterial strain L2612PR.And on the basis of this recombinant bacterial strain, with Li-acetate method transform plastids pRS305-XDH, screening culture medium B obtains recombinant bacterial strain.With restriction enzyme EcoRI linearization plasmid pRS-RKI1-TKL1, transform recombinant bacterial strain obtained in the previous step with Li-acetate method, screening culture medium C expresses obtaining recombinant bacterial strain.And on the basis of this bacterial strain, the linearizing plasmid pAUR-RPE1-TAL1 of conversion StuI, screening culture medium D obtains the recombinant bacterial strain L2612PR-DPPP that sets out further.
The structure of mutant strain SyBE005 and screening
By bacterial strain L2612PR-DPPP with initial OD
600=0.2 receives in the 250mL containing 50mL domestication substratum, and 30 DEG C, 200 revs/min aerobics cultivate 3 days.Measure the OD of culture
600, and with initial OD
600=0.2 is transferred in fresh domestication substratum, repeats the OD of bacterium liquid after cultivation 10 times
600remain unchanged, continue to repeat cultivation 10 times.The bacterium liquid taken a morsel, after dilution, on the solid plate of domestication substratum, coating is cultivated, and obtains single bacterium colony bacterial strain.From wherein choosing 20 maximum bacterium colonies of bacterium colony size, being inoculated in the 15mL test tube containing 2mL liquid screening medium C respectively, cultivating 2 days under the condition of 30 DEG C, 200 revs/min.Getting 100 μ L bacterium liquid is transferred in the fresh fermention medium A of 3mL, and under the condition of 30 DEG C, 200 revs/min, anaerobic is cultivated.With the rubber stopper seal test tube mouth with exhaust needle.After anaerobic cultivates 24 hours, measure the OD of bacterium liquid
600, remaining Xylose Content and meta-bolites ethanol, glycerine, Determination of Xylitol.The bacterial strain SyBE005 that the remaining wood sugar of a strain is minimum is obtained from 20 strain list bacterium colonies.
Analytical procedure:
Thalline light absorption value (the OD measured at 600nm place with 722 type spectrophotometers
600) characterize cell concentration.The concentration of wood sugar, ethanol, Xylitol, glycerine measures with high performance liquid chromatography (Waters1515).Chromatographic column is Aminex HPX-87H, column temperature: 65 DEG C; Detector: Waters Composition distribution 2421, detector temperature is 40 DEG C.Moving phase is 5mM sulphuric acid soln, flow velocity 0.6mL/min, and sample size is 10 μ L.
The wood-sugar fermentation of embodiment 2 bacterial strain L2612PR, L2612PR-DPPP and SyBE005 is compared
1. test materials: bacterial strain L2612PR, L2612PR-DPPP, SyBE003
2. test method:
Seed culture medium 1: synthesis nitrogenous source YNB6.7g/L, kilnitamin powder 2g/L, glucose 20g/L, leucine 100mg/L, tryptophane 100mg/L.
Seed culture medium 2: synthesis nitrogenous source YNB6.7g/L, kilnitamin powder 2g/L, glucose 20g/L.
Fermention medium: yeast leaching powder 10g/L, peptone 20g/L, wood sugar 20g/L.
A ring L2612PR bacterium colony is connect in 50mL seed culture medium 1 from fresh inclined-plane.Similar, connect in bacterium colony L2612PR-DPPP and SyBE005 to 50mL seed culture medium 2 from fresh inclined-plane.Cultivate 24 hours under the condition of 30 DEG C, 200 revs/min.With initial cell concentration OD
600=1.0 are inoculated in 100mL fermention medium, cultivate under 30 DEG C, 150 revs/min conditions, ferment 60 hours.Detect the concentration of cell concentration, xylose concentration and product ethanol, Xylitol, glycerine.
3. analytical procedure:
With embodiment 1.
4. test-results:
Shown in Fig. 5, in 48 hours, bacterial strain SyBE005 make use of wood sugar completely, and bacterial strain L2612PR, L2612PR-DPPP only make use of the wood sugar of about 60%, and the maximum wood sugar specific consumption rate of SyBE005 reaches 0.301g/g dry weight/h; After fermentation ends, the ethanol production of L2612PR, L2612PR-DPPP, SyBE005 reaches 2.02g/L, 2.97g/L, 6.85g/L respectively.The alcohol getting rate that L2612PR, L2612PR-DPPP are corresponding is respectively 0.15g/g wood sugar, 0.20g/g wood sugar, the fermenting alcohol yield of SyBE005 reaches 0.33g/g wood sugar, 120% and 65% is improve respectively, as shown in Figure 6 relative to L2612PR, L2612PR-DPPP; After fermentation ends, the Determination of Xylitol of L2612PR, L2612PR-DPPP, SyBE005 reaches 6.24g/L, 6.29g/L, 2.43g/L respectively.The xylitol yield of L2612PR, L2612PR-DPPP reaches 0.48g/g wood sugar, 0.42g/g wood sugar, and the xylitol yield of SyBE005 is reduced to 0.12g/g wood sugar, reduces 3,2.5 times relative to L2612PR, L2612PR-DPPP, 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.
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| CN105567745A (en) * | 2016-01-21 | 2016-05-11 | 天津大学 | Method for improving utilization of xylose in lignocellulose hydrolysate |
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