CN102127512A - Saccharomyces cerevisiae engineering bacterium capable of fermenting xylose - Google Patents
Saccharomyces cerevisiae engineering bacterium capable of fermenting xylose Download PDFInfo
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Abstract
The invention discloses a Saccharomyces cerevisiae engineering bacterium capable of fermenting xylose. The bacterium contains a host cell and recombinant vector. The engineering bacterium is characterized in that the recombinant vector contains a gene expression box formed by connecting a first promoter, xylose reductase genes, a second promoter, xylitol dehydrogenase genes and resistant marker genes in turn. The Saccharomyces cerevisiae engineering bacterium of the invention can be used to express xylose reductase and xylitol dehydrogenase simultaneously, can use xylose as the only carbon source to grow and can lay a foundation for the subsequent construction of the engineering bacterial strain which can produce ethanol through the co-fermentation of xylose and glucose; and on the basis of the engineering bacterial strain, further screening and transforming can be performed to ensure that ethanol is produced by using lignocellulose as raw material, thus the production cost of ethanol can be greatly reduced.
Description
Technical field
The present invention relates to biology field, but relate in particular to a kind of saccharomyces cerevisiae engineered yeast of metabolism wood sugar.
Background technology
The exhaustion day by day of petroleum resources makes production of fuel ethanol and is applied on Economic development and the strategic security to demonstrate important meaning.Simultaneously, constantly rising violently of international provision price becomes the bottleneck of alcohol fuel scale operation and popularization.In the vegetable fibre resource that nature enriches, have only 3-4% to be used effectively at present.Wood sugar content in the lignocellulose hydrolyzate is a kind of monose that is only second to glucose up to 30%, makes full use of wood-sugar fermentation and produces ethanol, and (J.N.Nigam, 2001) greatly can reduce production costs.
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is as unicellular eukaryote, and easily cultivation, growth cycle weak point have been widely used in expression of heterologous genes.Though yeast saccharomyces cerevisiae can high-yield ethanol and the inhibition in the ligno-cellulose hydrolysate is had strong resistance (Tang YQ et al., 2006), but can not utilize wood sugar, and can only utilize its isomer xylulose, make yeast saccharomyces cerevisiae can't become the dominant strain of lignocellulose alcohol production.So one of important channel that makes up the saccharomyces cerevisiae engineered yeast strain that can utilize the wood sugar producing and ethanol is natural saccharomycetic two key genes that utilize wood sugar of clone, be Xylose reductase gene XYL1 (xylose reductase) and xylose dehydrogenase gene XYL2 (xylitoldehydrogenase), and it is expressed in brewing yeast cell.
Producing and ethanol ability and stronger alcohol resistance become the important production bacterial strain of ethanol and food service industry to yeast saccharomyces cerevisiae efficiently with it.But its suitableeest leavening temperature is 25 ℃ to 30 ℃, often needs in process of production to spray a large amount of water of condensation (Kiran Sree.et al., 2000a to fermentor tank; Sridhar etal., 2002) or use other means to be lowered the temperature, this has increased production cost and Pollution risk undoubtedly.In the ethanol fermentation industry, the cost that stock liquid is cooled to before the yeast fermentation 30 ℃ accounts for about 30% of total cost of production, and if only reduce to about 37 ℃, then can reduce by 10% production cost.
Summary of the invention
But the invention provides a kind of saccharomyces cerevisiae engineered yeast of metabolism wood sugar, this saccharomyces cerevisiae engineered yeast can wood sugar be a sole carbon source, and fermentation produces ethanol.
A kind of saccharomyces cerevisiae engineered yeast, comprise host cell and recombinant vectors, it is characterized in that: comprise the expression casette that connects to form successively by first promotor, Xylose reductase gene, second promotor, xylose dehydrogenase gene and resistant maker gene in the described recombinant vectors.
Described first promotor is a yeast saccharomyces cerevisiae triosephosphate isomerase promotor (pTPI), and described second promotor is a yeast saccharomyces cerevisiae phosphoglycerokinase promotor (pPGK).These two kinds of promotors are constitutive promoter, not suppressed by glucose, need not wood sugar and induce.
The base sequence of described Xylose reductase gene is shown in SEQ ID NO.7, and the base sequence of described xylose dehydrogenase gene is shown in SEQ ID NO.9.Why adopt P.stipitis as gene source, be that mainly the enzyme when it uses the different auxiliary factor is alive than being 0.65 (NADH/NADPH), higher than this ratio of other zymic, therefore when its Xylose reductase gene and wood sugar desaturase coexpression are in recipient cell, help to alleviate the uneven situation of redox in the born of the same parents, thereby reduce byproducts build-up.
Described resistant maker gene is the G418 resistant gene, is convenient to the screening of yeast saccharomyces cerevisiae positive transformant.
Described host cell is yeast saccharomyces cerevisiae CBS 1200, and this cell can be high temperature resistant.
Described recombinant vectors comprises the homologous sequence of yeast saccharomyces cerevisiae, and recombinant vectors can be incorporated in host's the karyomit(e), improves genetic stability.Described homologous sequence is shown in SEQ ID NO.13.Gene can be integrated on the yeast saccharomyces cerevisiae rDNA tumor-necrosis factor glycoproteins with high copy number.
Saccharomyces cerevisiae engineered yeast of the present invention can be expressed Xylose reductase and xylitol dehydrogenase simultaneously, can be the sole carbon source growth with the wood sugar, for ferment the altogether engineering strain of producing and ethanol of subsequent builds wood sugar and glucose is laid a good foundation, and can this project bacterial strain be further screening and transformation of basis, making it is raw material production ethanol with the lignocellulose, thereby reduces the alcoholic acid production cost greatly.
Description of drawings
Fig. 1 is the physical map of the P-R-K of expression vector;
Fig. 2 is the physical map of the pB-D of expression vector;
Fig. 3 is the physical map of expression vector P-RD-K;
Fig. 4 is the physical map of expression vector PK-rDNA;
Fig. 5 is the electrophoretogram of the positive bacterial strain of check recombinant bacterial strain.
Embodiment
The structure of embodiment 1 promotor, Xylose reductase gene and G418 resistant gene expression cassette
1, the extraction of pichia stipitis genomic dna
(Pichia stipitis CBS 5774) is inoculated in the YPD liquid nutrient medium with pichia stipitis, and 30 ℃, 180r/min are cultivated 16h, enrichment of cell.
Collect the yeast cell of incubated overnight, after the STES damping fluid suspends, washs, re-suspended cell; In the yeast suspension liquid, add certain proportion pickling glass pearl and phenol/chloroform, thorough mixing concussion, upper strata, centrifugal back water ethanol sedimentation; The nucleic acid precipitation is collected in centrifugal back, is genomic dna.
2, pcr amplification Xylose reductase gene (XYL1)
Going up the XYL1 sequence (X59465.1) of issue according to GeneBank, is template with the pichia stipitis genomic dna of said extracted, utilizes upstream primer A1 and downstream primer A2 to carry out pcr amplification.
Upstream primer A1, its nucleotide sequence wherein contain BamH I restriction enzyme site shown in SEQ ID NO.1;
Downstream primer A2, its nucleotide sequence wherein contain Apa I restriction enzyme site shown in SEQ ID NO.2.
The PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 1min30s, 30 circulations; 72 ℃ of 10min.Sequencing result shows that pichia stipitis (CBS 5774) Xylose reductase gene XYL1 (X59465.1) sequence that the base sequence of PCR product and GeneBank go up issue has 100% homology.
3, pcr amplification G418 gene (KanMX)
Going up the KanMX sequence (S78175.1) of issue according to GeneBank, is template with plasmid pUG6, utilizes upstream primer K1 and downstream primer K2 to carry out pcr amplification.
Upstream primer K1, its nucleotide sequence wherein contain Sac I restriction enzyme site shown in SEQ ID NO.3;
Downstream primer K2, its nucleotide sequence wherein contain Nhe I restriction enzyme site shown in SEQ ID NO.4.
The PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 1min20s, 30 circulations; 72 ℃ of 10min.Sequencing result shows that the KanMX sequence (S78175.1) that the base sequence of PCR product and GeneBank go up issue has 100% homology.
4, the structure of promotor, Xylose reductase gene and G418 resistant gene expression cassette
Yeast saccharomyces cerevisiae triosephosphate isomerase promotor (pTPI) goes up existing for carrier pYX212.
With restriction enzyme BamH I, Apa I enzyme is cut XYL1 fragment and pYX212 plasmid, obtains the P-R plasmid after connecting through the T4 ligase enzyme; Use restriction enzyme Sac I again, Nhe I enzyme is cut KanMX and P-R plasmid, obtains P-R-K after connecting through the T4 ligase enzyme.
The above-mentioned enzyme system of cutting is:
DNA?50μl
Dd water 35 μ l
buffer10μl
Enzyme I 2.5 μ l
Enzyme II2.5 μ l
37℃3h
Linked system is:
Dna fragmentation: 4 μ l
Carrier segments: 16 ℃ of connections of 4 μ l are spent the night
T4 ligase enzyme: 0.4 μ l
Buffer：2μl
Dd water 9.6 μ l
1, the extraction of genes of brewing yeast group DNA
Yeast saccharomyces cerevisiae CBS 1200 is inoculated in the YPD liquid nutrient medium, and 40 ℃, 180r/min are cultivated 16h, enrichment of cell.Extraction step reference example 1
2, the clone of phosphoglycerokinase promotor (pPGK)
According to phosphoglycerokinase promotor (pPGK) sequence (FJ415226.1) of GeneBank issue, upstream and downstream primer P1, the P2 of design pPGK are that template is carried out pcr amplification with genes of brewing yeast group DNA.
Upstream primer P1, its nucleotide sequence wherein contain Apa I restriction enzyme site shown in SEQ ID NO.5;
Downstream primer P2, its nucleotide sequence wherein contains the EcoRV restriction enzyme site shown in SEQ ID NO.6.
The PCR reaction conditions is 94 ℃ of 5min; 94 ℃ of 30s, 60 ℃ of 30s, 70 ℃ of 1min, 30 circulations; 72 ℃ of 10min.The product sequencing result that pcr amplification obtains shows that the sequence among this PCR product and the GeneBank relatively has the sudden change of a base, and this gene order is shown in SEQ ID NO.7.
3, the clone of pichia stipitis Xylose reductase gene (XYL2)
Going up the XYL2 sequence (X55392.1) of issue according to GeneBank, is template with the pichia stipitis genomic dna of said extracted, utilizes upstream primer D1 and downstream primer D2 to carry out pcr amplification.
Upstream primer D1, its nucleotide sequence wherein contains the EcoRV restriction enzyme site shown in SEQ ID NO.8;
Downstream primer D2, its nucleotide sequence wherein contain Sac I restriction enzyme site shown in SEQ ID NO.9.
The PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 30s, 52 ℃ of 30s, 72 ℃ of 2min10s, 30 circulations; 72 ℃ of 10min.Sequencing result shows that the sequence among this PCR product and the GeneBank relatively has the sudden change of a base, and this gene order is shown in SEQ ID NO.10.
4, the structure that contains promotor and xylose dehydrogenase gene expression cassette
At first use restriction enzyme A pa I, the EcoRV enzyme is cut pPGK fragment and pBlueScript IISK (-) cloning vector, through obtaining the p-P plasmid after the connection of T4 ligase enzyme, use restriction enzyme EcoRV again, Sac I enzyme is cut Xylose reductase gene (XYL2) and p-P plasmid, obtains pB-D (enzyme is cut system and linked system with embodiment 1) after connecting through the T4 ligase enzyme.
The structure (P K-rDNA) of embodiment 3 yeast saccharomyces cerevisiae high effective integration type expression vectors
1, the structure of yeast saccharomyces cerevisiae additive type expression vector (P-RD-K)
With restriction enzyme A pa I, Sac I digested plasmid P-R-K and pB-D fragment obtain big fragment P-R-K (A-S) and small segment pPGK-XYL2 (A-S).Obtain yeast saccharomyces cerevisiae additive type expression vector P-RD-K (enzyme is cut system and linked system with embodiment 1) after connecting through the T4 ligase enzyme.
2, the clone of yeast saccharomyces cerevisiae rDNA sequence
According to the 18s-5s site in the sequence (Z73326.1) of the last issue of GeneBank, be template with yeast saccharomyces cerevisiae CBS 1200 genomes, utilize upstream primer R1 and downstream primer R2 to carry out pcr amplification.
Upstream primer R1, its nucleotide sequence wherein contain Not I restriction enzyme site shown in SEQ ID NO.11;
Downstream primer R2, its nucleotide sequence wherein contain Not I restriction enzyme site shown in SEQ ID NO.12.
The PCR reaction conditions is: 94 ℃ of 5min; 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 1min40s, 30 circulations; 72 ℃ of 10min.The PCR fragment is connected into pMD18-T carrier (Takara company), obtains the TA-rDNA order-checking.Sequencing result shows that the middle 18s-5s site sequence of sequence (Z73326.1) that the base sequence of PCR product and GeneBank go up issue has 100% homology, and this gene order is shown in SEQ ID NO.13.
3, the structure of yeast saccharomyces cerevisiae high effective integration type expression vector (P K-rDNA)
With restriction enzyme Not I digested plasmid P-RD-K and TA-rDNA, obtain big fragment P-RD-K (NotI) and small segment rDNA (NotI).Obtain the integrated expression vector P of yeast saccharomyces cerevisiae K-rDNA (enzyme is cut system and linked system with embodiment 1) after connecting through the T4 ligase enzyme.
1, the preparation of brewing yeast cell
Inoculation 20mL liquid YEPD substratum is cultivated 16-18h for 40 ℃; Inoculate 40 ℃ of cultivation 7-8h in the fresh liquid YEPD substratum of incubated overnight thalline 50 μ l to 50mL, make thalli growth to logarithmic growth middle and later periods (OD
600=0.8-1.0);
2, the linearizing of the integrated expression vector PK-rDNA of yeast saccharomyces cerevisiae
Use HpaI (Takara) restriction endonuclease, with plasmid P K-rDNA linearization process, and recovery and purifying linear fragment.
3, the conversion of yeast saccharomyces cerevisiae
The method that electricity consumption is transformed experimentizes, and step is as follows: with the thalline 3 of above-mentioned logarithmic growth middle and later periods, and the centrifugal 10min of 000r/min; Collect thalline and outwell supernatant, thalline suspends with the ice-cold sterilized water of 20mL, and washing once; Centrifugal collection thalline, the sterilized water ice-cold with 20mL washs once again; Centrifugal collection thalline, with the ice-cold sorbyl alcohol of the 1mL 1mol/L thalline that suspends again, washing once is transferred in the 5mL centrifuge tube; Centrifugal collection thalline, pipettor exhaustion supernatant adds the ice-cold sorbyl alcohol mixing thalline of 0.2mL1mol/L, makes bacterium liquid be the underflow shape; Get the centrifuge tube (every pipe 60 μ l) of the dense thick bacteria suspension packing 1.5mL of 0.2～0.4mL, in every pipe, add 10 μ L (≤1 μ g) plasmid DNA, mixing, ice bath 10min then; The transformed bacteria suspension is all changed in the conversion pool of precooling, 1.5KV, 25 μ F, 200 Ω shock by electricity under the 5ms condition; In the transformed bacteria suspension, add 1mL YPD liquid at once, hatch 1h for 40 ℃; 100 μ L bacterium liquid coating YEPX-G418 flat board is got with the resuspended thalline of 500 μ L physiological saline in centrifugal back.Cultivate 3-5d, select G418 resistance transformant for 40 ℃.Shock by electricity in the same way simultaneously, be coated on the YEPX flat board that does not contain G418 and contain G418 respectively observation by identical amount as original strain regeneration situation and negative control for the brewing yeast cell that does not add plasmid.Transformant finally can obtain about 6 transformant/micrograms of DNA, totally 30 transformants through the regeneration of 5d.
4, the phenotypic evaluation of transformant
It is good to select growing state, individual big transformant is to fresh YEPX-G418 liquid nutrient medium, 180rpm/min, 24h, extract genomic dna, with the transformant genomic dna is template, by primer A1 and A2, D1 and D2, P1 and D2 carry out PCR, and be that template is done contrast with unconverted original yeast saccharomyces cerevisiae CBS 1200 genomic dnas, the result shows, (getting a positive transformant is example) as shown in Figure 2, the PCR product of positive transformant obtains size through agarose gel electrophoresis and is about 1.2kb (XYL1), 1.8kb (XYL2), 2.6kb purpose fragment the (the 3rd (pPGK-XYL2), 5,7 roads), and negative control does not have PCR product the (the 2nd, 4,6 roads), confirm that goal gene has changed in the karyomit(e) of yeast saccharomyces cerevisiae CBS 1200.
Recombinant bacterial strain after continuous passage on the YEPX-G418 flat board is more than 7 times, is extracted single bacterium colony genomic dna and carries out the PCR checking.The result shows that PCR checking result and no change illustrate that the transformant that obtains can genetic stability.
1, the preparation of CBS 1200-P K-rDNA seed
Picking transformant list bacterium colony inserts in the triangular flask of the 250mL that the 50mL liquid seed culture medium is housed, places under 40 ℃, shaking speed 180r/min condition and cultivates 36h.Seed liquor after the cultivation is centrifugal 5min under 4000r/min, and the cell precipitation of collecting is used for the inoculation fermentation substratum.
2, meta-bolites detects
Meta-bolites detects test to carry out in the 250mL triangular flask of 100mL fermention medium is housed, fermented liquid inoculation back initial cell concentration is 1.2g/L (dry cell weight meter), after the inoculation triangular flask is put 40 ℃, shaking culture in the 120r/min constant temperature oscillator, soft rubber ball on the bottle plug (is inserted with syringe needle No. 7, to discharge the CO that generates
2) with control anaerobic condition, sampling and measuring in the culturing process.Residual sugar is measured by high performance liquid chromatography (HPLC) in tunning (ethanol, Xylitol etc.) and the fermented liquid.Fermented liquid to be measured is high speed centrifugation 5min under the 10000r/min condition earlier, and supernatant liquor sample introduction behind 0.45 μ m filtering with microporous membrane is measured, and external standard method is quantitative.Chromatographic condition is: 60 ℃ of column temperatures, dilute sulphuric acid is made moving phase, flow velocity 0.4mL/min, sample size 20 μ L.
After testing, in the fermented liquid, the xylose utilization rate is about 25% during 96h, and alcohol yied is 9.594%, and yield is about 20.86%, and Xylitol and glycerine are the by products of fermentation, and more Xylitol accumulation is arranged.
Claims (9)
1. saccharomyces cerevisiae engineered yeast, comprise the recombinant vectors of host cell and this host cell of importing, it is characterized in that: comprise the expression casette that connects to form successively by first promotor, Xylose reductase gene, second promotor, xylose dehydrogenase gene and resistant maker gene in the described recombinant vectors.
2. saccharomyces cerevisiae engineered yeast according to claim 1 is characterized in that: described first promotor is a yeast saccharomyces cerevisiae triosephosphate isomerase promotor.
3. saccharomyces cerevisiae engineered yeast according to claim 1 is characterized in that: second promotor is a yeast saccharomyces cerevisiae phosphoglycerokinase promotor.
4. saccharomyces cerevisiae engineered yeast according to claim 1 is characterized in that: described resistant maker gene is the G418 resistant gene.
5. saccharomyces cerevisiae engineered yeast according to claim 1 is characterized in that: the base sequence of described Xylose reductase gene is shown in SEQ ID NO.7.
6. saccharomyces cerevisiae engineered yeast according to claim 1 is characterized in that: the base sequence of described xylose dehydrogenase gene is shown in SEQ ID NO.9.
7. saccharomyces cerevisiae engineered yeast according to claim 1 is characterized in that: described host cell is yeast saccharomyces cerevisiae CBS 1200.
8. saccharomyces cerevisiae engineered yeast according to claim 1 is characterized in that: described recombinant vectors comprises the homologous sequence of yeast saccharomyces cerevisiae.
9. saccharomyces cerevisiae engineered yeast according to claim 8 is characterized in that: described homologous sequence is shown in SEQ ID NO.13.
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