CN101463328B - Metabolic Engineering yeast using xylose fermentation for producing ethanol - Google Patents
Metabolic Engineering yeast using xylose fermentation for producing ethanol Download PDFInfo
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- CN101463328B CN101463328B CN200710179863XA CN200710179863A CN101463328B CN 101463328 B CN101463328 B CN 101463328B CN 200710179863X A CN200710179863X A CN 200710179863XA CN 200710179863 A CN200710179863 A CN 200710179863A CN 101463328 B CN101463328 B CN 101463328B
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Abstract
Xylose fermentation ethanol is one of the key technologies utilizing biomass to produce ethanol. A saccharomyces cerevisiae engineering bacterial strain is set up by a method of metabolic engineering. The bacterial strain not only can ferment the ethanol produced by the xylose, but also can automatically balance oxido-reduction cofactor, so as to be a metabolic engineering microzyme which has little by-product and can effectively use xylose fermentation for producing the ethanol. The conversion rate of xylose fermentation ethanol of bacterial strain reaches 88%, thus laying the foundation for utilizing the biomass to produce fuel ethanol.
Description
Technical field
The invention belongs to biological technical field, relate in particular to a plant height and imitate yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) engineering bacterial strain E33 and the application thereof that utilizes xylose fermentation for producing ethanol.
Background technology
Along with the exhaustion day by day of petroleum resources, ethanol has substituted part gasoline as clean fuel, and demand increases year by year.Production alcoholic acid raw material mainly is the starch from grain at present, and raw materials cost accounts for the over half of whole production cost, becomes a bottleneck of alcohol fuel large-scale promotion.Biomass are renewable resourcess the abundantest on the earth.Utilizing biomass to produce alcohol fuel is hot of research and development competitively in the world.Carbohydrate thoroughly is degraded into and has behind the monose closely 1st/3rd in the biomass, and wood sugar efficiently transforms the not only environmental protection but also help reducing cost of xylose production ethanol.Yeast saccharomyces cerevisiae is widely used in extensive ethanol fermentation industry, but it can not utilize wood sugar, and therefore many scientists attempt to make up the saccharomyces cerevisiae engineered yeast of energy xylose-fermenting.Common construction process is with external source Xylose reductase, xylitol dehydrogenase (from pichia spp (Pichia stipitis)) gene heterogenous expression in yeast saccharomyces cerevisiae, simultaneously the endogenous xylulokinase of overexpression (from yeast saccharomyces cerevisiae).Such recombinant Saccharomyces cerevisiae can less efficiently xylose fermentation ethanol (Kuyper etc., FEMS YeastResearch, 4:655-664,2004), secretes a large amount of by product Xylitols simultaneously.Xylitol dehydrogenase can only specificity utilize oxidized form of nicotinamide-adenine dinucleotide because the Xylose reductase that pichia spp is originated in the recombinant Saccharomyces cerevisiae cell in the wood-sugar fermentation process preferentially utilizes DPNH I, has caused the accumulation of DPNH I exhaustion and DPNH.The imbalance of redox coenzyme has caused the accumulation of the low and Xylitol of xylose utilization rate, the engineering bacteria that makes up both at home and abroad is when xylose fermentation ethanol at present, wood sugar is to the lower (Gnansounou etc. of ethanol conversion, Bioresource Technology, 96:985-1002,2005), this problem has become the key problem in technology of biomass ferment producing and ethanol.
Summary of the invention
The purpose of this invention is to provide and efficiently to utilize the engineering bacteria of xylose fermentation for producing ethanol, and utilize the application of biomass ferment producing and ethanol.
In one aspect of the invention, the new yeast saccharomyces cerevisiae that utilizes xylose fermentation for producing ethanol of one strain (Saccharomyces cerevisiae) E33 engineering bacterial strain is provided, described bacterial strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (address is the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City) on June 28th, 2006, and preserving number is CGMCC No.1746.
Aspect another, provide described yeast saccharomyces cerevisiae E33 engineering bacterial strain to utilize the application of xylose fermentation for producing ethanol of the present invention.
Compared with the prior art, the saccharomycetic characteristics of this project are the coenzyme II specificity Xylose reductase activity that not only have from candiyeast, and also have solubility transhydrogenase activity.Can keep the coenzyme balance by the autoregeneration coenzyme in the wood-sugar fermentation process, therefore can efficiently utilize xylose fermentation for producing ethanol, wood sugar reaches 88% to ethanol conversion, lays a good foundation for utilizing biomass to produce alcohol fuel.
Description of drawings
Fig. 1 is the physical property collection of illustrative plates of yeast vector pY2 (a) and pGH3 (b).
Fig. 1 (a) pADH1: alcoholdehydrogenase (ADH1) promotor; The tADH1:ADH1 terminator; PGPD1: glycero-3-phosphate dehydrogenase enzyme promotor; TCYC1: cytochrome c equipotential 1 terminator; XDH: from the gene of the xylitol dehydrogenase of pichia spp; URA3 (uridylic) and LEU2 (leucine): yeast selective marker:
Fig. 1 (b) ADH1p: alcoholdehydrogenase (ADH1) promotor; The ADH1t:ADH1 terminator; GPD1p: glycero-3-phosphate dehydrogenase enzyme promotor; CYC1t: cytochrome c equipotential 1 terminator; CmXYL1: from the gene of the coding Xylose reductase of maltose candiyeast (Candida maltosa); XKS1: from the gene of the coding xylulokinase of yeast saccharomyces cerevisiae; UdhA: from the gene of the coding transhydrogenases of intestinal bacteria (E.coli); LEU2: yeast selective marker
Constructed yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) the engineering bacterial strain E33 of the present invention on June 28th, 2006 by China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, the preservation centre address is the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City.Preserving number is: CGMCC No.1746.
Below be several embodiment, the present invention may be better understood with reference to the following example.These embodiment mean the embodiment of specific embodiments of the present invention, but do not limit the scope of the invention.
Embodiment
Among the embodiment used carrier unless otherwise indicated outside, all available from Invitrogene company.
The structure of embodiment one yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) E33 engineering bacteria
Yeast saccharomyces cerevisiae W303-1B α (Pearce, A.K. etc.: Micobiology 147,391-401., 2001) is as the starting strain that makes up engineering bacteria.Used bacterial strain also comprises pichia spp CBS 6054 (Wahlbom in building process, C.F. etc.: FEMS Yeast Res 3,319-26., 2003), maltose candiyeast (Candida maltosa) XU316 (C.Guo, Deng: J.Appl.Microb.101,139-150,2006) and intestinal bacteria (E.coli) JM109 (Invitrogene).According to standard method (Ausubel, F.M., Deng: Current protocols in molecular biology.Wiley, Toronto, 1987) extract yeast pichia spp CBS 6054 respectively, yeast saccharomyces cerevisiae W303-1B α, maltose candiyeast XU316 and intestinal bacteria E.Coli JM109 genomic dna.With pichia spp CBS 6054 genomic dnas is template, utilizes primer
Pxdhs (5 '-GCAGGATCCATGACTGCTAACCCTTCCTTG-3 ') and
Pxdha(5′-GCACTCGAGTTACTCAGGG CCGTCAATGAG-3′)
Pcr amplification,
The PCR mixed solution:
(1) template DNA: 1 μ l (10ng)
(2) primer Pxdhs:1 μ l (10mM)
(3) primer Pxdha:1 μ l (10mM)
(4)dNTPs： 1μl(10mM)
(5) Taq archaeal dna polymerase: 0.5 μ l (5U/ μ l)
(6) 10 * damping fluids: 5 μ l
(7) redistilled water: 40.5 μ l
PCR condition: the following circulation of beginning behind 94 ℃ of sex change 5min, 94 ℃ of reaction of degeneration 30sec; 55 ℃ of annealing reaction 30sec; 72 ℃ of extension 1min; Carry out 30 circulations; Last 72 ℃ of reaction 5min; 4 ℃ of coolings are constant.Obtain its xylose dehydrogenase gene (PsXYL2).Xylose dehydrogenase gene (PsXYL2) is cloned into expression vector pRUL129 (van den Berg, M.A. and Steensma, H.Y.1997.Yeast 13, thereby 551-559.) upward make it obtain yeast saccharomyces cerevisiae composition type expression promoter GPD1 and CYC1 terminator.With this expression cassette: promotor GPD1, the ORF of PsXYL2 and CYC1 terminator further are cloned among the multi-copy integration plasmid pSAK068, obtain plasmid pY2.This plasmid is a selected marker with ura3 (uridylic).With maltose candiyeast XU316 genomic dna is template, utilizes primer
Pxrs (5 '-GCAGGATCCATGACTACTTCTTCTACTATT-3 ') and
Pxra(5′-GCACTCGAGTTAAACGAAAATTGGAATGTTGTC-3′)
Pcr amplification obtains its Xylose reductase gene (CmXYL1).With yeast saccharomyces cerevisiae W303-1B α genomic dna is template, utilizes primer Pxks (5 '-GCGGATCCATGTTGTGTTCAGTAATTCAGAGACAG-3 ') and Pxka (5 '-CGGTCGACGCTACAGTATACGAAACATACAAGG-3 ') PCR its xylulokinase gene (ScXKS1) that obtains increasing.Equally respectively the Xylose reductase gene (CmXYL1) and the endogenous xylulokinase gene (ScXKS1) of external source are cloned on the expression vector pRUL129, make it obtain needed promotor GPD1 of eukaryotic expression and CYC1 terminator.Make up the coexpression box (promotor GPD1, CmXYL1 and terminator CYC1, promotor GPD1, ScXKS1 and terminator CYC1) of two gene tandem expression by the DNA recombinant technology.2 novel μ type expression vector pGH are selected marker from cloning vector pbluescript SK (Invitrogene) and expression vector pAD-GAL4-4.1 (Invitrogene) and with leu2.With E.Coli JM109 genomic dna is template, utilizes primer Pths (5 '-AGGGATTCCATGCCACATTCCTACGATTAC-3 ') and Ptha (5 '-CCCTCGAGTTAAAACAGGCGGTTTAAACC-3 ') pcr amplification to obtain the transhydrogenase gene (udhA) of its solubility.Transhydrogenase gene (udhA) is cloned on the 2 μ type expression vector pGH.Insert above-mentioned Xylose reductase gene (CmXYL1) xylulokinase gene (ScXKS1) coexpression box simultaneously, form plasmid pGH3.With LiAc yeast conversion method (Gietz, R.D., etc., Yeast, 11:355-360,1995), with plasmid pY2 transformation receptor bacterium S.cerevisiae W303-1B α.The SC of uridylic defective selective medium (0.67%, the yeast nitrogen base; 2%, glucose; 1.5%, agar, an amount of VITAMIN B4 and indispensable amino acid (leucine, Histidine, tryptophane)) go up and screen the recombinant Saccharomyces cerevisiae bacterial strain E3 that obtains carrying xylose dehydrogenase gene.With recombinant Saccharomyces cerevisiae bacterial strain E3 is recipient bacterium, transform (LiAc yeast conversion method (and Gietz, R.D., etc., Yeast, 11:355-360,1995)) plasmid pGH3, the SC of uridylic and leucine auxotrophy selective medium (0.67%, the yeast nitrogen base; 2%, glucose; 1.5%, agar, an amount of VITAMIN B4 and indispensable amino acid (Histidine, tryptophane)) go up screening and obtain yeast saccharomyces cerevisiae E33 engineering bacterial strain.
Embodiment two, the Xylose reductase of yeast saccharomyces cerevisiae E33 engineering bacteria and transhydrogenase activity
Recombinant bacterial strain E33 and starting strain W303-1B α be inoculated in respectively 5ml the SC substratum (0.67%, the yeast nitrogen base; 2%, glucose; An amount of indispensable amino acid (Histidine, tryptophane)) in, cultivating (30 ℃, the 250rpm shaking table is cultivated) spends the night.Starting strain W303-1B α is as contrast.Centrifugal (5000rpm, 3 minutes) collect thalline.(high speed centrifugation (13000rpm, 40 minutes) is collected the albumen supernatant for " yeast genetics method experiment guide ", 87-88) lysing cell to utilize glass bead method.According to the described measuring method of Bruinenberg (Bruinenberg, P.M., etc., J.Gen.Microbiol., 129:953-964,1983) Xylose reductase and the transhydrogenase measured in the cell extract compare vigor.In starting strain W303-1B α, only detect faint coenzyme II relevant Xylose reductase activity, do not detect the transhydrogenase activity.Yeast saccharomyces cerevisiae E33 is owing to introduced the Xylose reductase gene (CmXYL1) of candiyeast and the transhydrogenase gene (udhA) of bacterium has had tangible Xylose reductase activity (0.251U mg
-1, specificity is coenzyme with coenzyme II, detects less than nadide involved enzyme activity) and transhydrogenase activity (6.288U mg
-1).Needed enzyme amount when Xylose reductase activity unit is defined as per minute catalytic reduction 1mmol wood sugar; Transhydrogenase activity unit is defined as the enzyme amount of per minute catalytic reduction 1nmol tNAD.
Embodiment three, yeast saccharomyces cerevisiae E33 engineering bacterial strain xylose fermentation ethanol
Recombined engineering bacteria strain E33 is inoculated in and is contained in 250ml and shakes 100ml YPD substratum (yeast extract 10gl in the bottle
-1Tryptones 20gl
-1Glucose 20gl
-1) in.30 ℃, the 250rpm shaking table was cultivated 24 hours.Centrifugal collection thalline (5000rpm, 5 minutes) is with twice of isopyknic 0.9% physiological saline washed cell.Cell is resuspended in and is contained in 250ml and shakes (yeast extract 10gl in the 100ml YPX substratum in the bottle
-1Tryptones 20gl
-1Wood sugar 30gl
-1), in 30 ℃, the cultivation of 100rpm shaking table.Taking a sample after 84 hours and recording the concentration that consumes wood sugar is 22.2gl
-1, concentration of ethanol is 9.0gl
-1, wood sugar is 0.405gg to ethanol yield
-1, transformation efficiency is 88%.
Claims (2)
1. the yeast saccharomyces cerevisiae of a strain xylose fermentation ethanol (Saccharomyces cerevisiae) engineering bacterial strain E33 is characterized in that preserving number is: CGMCC No.1746.
2. the application of saccharomyces cerevisiae engineered yeast bacterial strain E33 in xylose fermentation ethanol of claim 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103484388A (en) * | 2013-05-07 | 2014-01-01 | 大连理工大学 | Industrial saccharomyces cerevisiae bacterial strain realizing chromosome integrative expression of xylose metabolic pathways |
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| CN103354836B (en) * | 2010-11-15 | 2015-11-25 | 斯堪的纳维亚科技集团公司 | Novel Wine brewing yeast strain |
| CN102154136B (en) * | 2010-12-17 | 2012-07-18 | 云南大学 | Saccharomyces cerevisiae strain capable of using xylose directly |
| CN102634463B (en) * | 2012-03-23 | 2013-08-07 | 中国科学院微生物研究所 | Saccharomycete producing xylitol and applicaton of saccharomycete |
| TW201437367A (en) | 2013-03-29 | 2014-10-01 | Far Eastern New Century Corp | Yeast strain for lactic acid production by using pentose and hexose |
| CN103710396A (en) * | 2013-12-30 | 2014-04-09 | 南京工业大学 | Method for improving conversion rate of target product of anaerobic fermentation |
| WO2016088278A1 (en) * | 2014-12-05 | 2016-06-09 | 本田技研工業株式会社 | Highly efficient ethanol-fermentative bacteria |
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| WO2004099381A2 (en) * | 2003-05-02 | 2004-11-18 | Cargill Dow Llc | Genetically modified yeast species and fermentation processes using genetically modified yeast |
| CN1824767A (en) * | 2005-02-25 | 2006-08-30 | 中国科学院微生物研究所 | Method of producing xylosic alcohol and its special baterial strain |
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|---|---|---|---|---|
| CN1225125A (en) * | 1996-05-06 | 1999-08-04 | 普渡研究基金会 | Stable recombinant yeasts for fermenting xylose to ethanol |
| WO2004099381A2 (en) * | 2003-05-02 | 2004-11-18 | Cargill Dow Llc | Genetically modified yeast species and fermentation processes using genetically modified yeast |
| CN1824767A (en) * | 2005-02-25 | 2006-08-30 | 中国科学院微生物研究所 | Method of producing xylosic alcohol and its special baterial strain |
Non-Patent Citations (5)
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| Marie Jeppsson,et al.Reduced Oxidative Pentose Phosphate Pathway Flux inRecombinant Xylose-Utilizing Saccharomyces cerevisiaeStrains Improvesthe Ethanol Yield from Xylose.APPLIED AND ENVIRONMENTAL MICROBIOLOGY68 4.2002,68(4),1604-1609. |
| Marie Jeppsson,et al.Reduced Oxidative Pentose Phosphate Pathway Flux inRecombinant Xylose-Utilizing Saccharomyces cerevisiaeStrains Improvesthe Ethanol Yield from Xylose.APPLIED AND ENVIRONMENTAL MICROBIOLOGY68 4.2002,68(4),1604-1609. * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484388A (en) * | 2013-05-07 | 2014-01-01 | 大连理工大学 | Industrial saccharomyces cerevisiae bacterial strain realizing chromosome integrative expression of xylose metabolic pathways |
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