CN106434700B - A kind of saccharomyces cerevisiae spt15 fixed point saturation gene mutation method improving alcohol yied - Google Patents
A kind of saccharomyces cerevisiae spt15 fixed point saturation gene mutation method improving alcohol yied Download PDFInfo
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Abstract
The invention proposes a kind of saccharomyces cerevisiae spt15 fixed point saturation gene mutation methods for improving alcohol yied.It is the spt15 fixed point saturation mutation gene that saccharomyces cerevisiae is obtained using genetic engineering means, and it is connected on expression vector pYES2NTc, construct mutation library, 12 simple point mutation genes have been obtained, with wild type gene spt15, spt3 and the gene constructed recombinant plasmid of crt gene Neo, are transferred in saccharomyces cerevisiae INVSC1 with Li-acetate method respectively, obtain the Wine brewing yeast strain of 15 recombinations.Using glucose as substrate, by recombinant Saccharomyces cerevisiae inoculation fermentation, its alcohol yied is measured, wherein recombinant Saccharomyces cerevisiae bacterium INVSC1-SPT15-M, INVSC1-SPT15-N, alcohol yied increase substantially, respectively 43.0 ± 0.9g/L and 43.7 ± 0.2g/L.17.8% and 19.7% are increased separately than control strain INVSC1-Neo.2 mutated genes are respectively methionine and asparagine by lysine mutation at 127.The method of the present invention is simple, and operation is easy, and alcohol yied improvement effect is obvious.With good economic benefit and social benefit.
Description
Technical field
The present invention relates to the technology for being Ethanol Production by Biomass Fermentation, especially a kind of saccharomyces cerevisiae for improving alcohol yied
Spt15 fixed point saturation gene mutation method
Background technique
Based on traditional bio-ethanol production is mainly fermented with grain, world food phenomenon in short supply seriously limits raw material
Source.Lignocellulosic is biomass resource the most abundant in the world, and amount is maximum, valence is most honest and clean, and annual total output accounts for about all
The 50% of biomass resource[1], but most of such substances are not utilized efficiently at present.Using biomass as waste
Industrial alcohol be the inexorable trend for substituting the fossil fuels such as petroleum, be included into perhaps as a kind of clean renewable energy
The strategic planning of development of multinational family.
Saccharomyces cerevisiae (S.cerevisiae) is eukaryotic microorganisms, and cell wall thickness, sterol content are high, to ethyl alcohol and wooden fibre
The tolerance for tieing up plain hydrolysate poisoning sex factor is higher [3]；Can under the conditions of low pH, strictly anaerobic Rapid Fermentation glucose production
Ethyl alcohol, by-product are few；It is not easy by bacterium and virus pollution, and related industries technology maturation.Saccharomyces cerevisiae genome sequencing is
It completes [4], is to study most thorough eukaryotic microorganisms so far, using the ways and means of bioinformatics, Yi Ji
The Protocols in Molecular Biology for becoming perfect carries out genetic manipulation and the reconstruct of metabolism network to it.It is the research emphasis of alcohol fermentation
With primary goal microorganism.The alcohol resistance of saccharomycete is related with multiple genes, thus knocked out by traditional single-gene or
Overexpression is extremely difficult to improve saccharomycete alcohol patience and improves the purpose of alcohol yied.Alper in 2006 etc. reports logical
The method for crossing gTME improves the pionerring research of saccharomycete alcohol yied and patience, is that saccharomycete alcohol yied and patience are metabolized work
Journey operation provides new thinking.
Transcriptional level control is the highest link of efficiency in gene expression regulation.Global transcription mechanism engineering
(Global transcription machinery engineering, gTME) is that one kind is optimized by genetic transcription rearrangement
The technology of cell phenotype[6], it is reorganized by molecular biology method, such as fallibility PCR, DNA, it is established that beginning transcription factor is prominent
Become library, global transcription regulatory factor is transformed, is oriented screening for purpose product or desired phenotype, makes entire transcriptional control mistake
Journey changes to change or improve the transcription and expression of target gene, obtains the enhancing of purpose metabolic fluxes or particular phenotype enhancing
Strain.Transcription is executed by RNA polymerase, and RNA polymerase II is responsible for the most of function of transcription generation in eucaryote
The mRNA of gene, and II transcriptional efficiency of RNA polymerase is determined by hansen initiation transcription factor and promoter binding ability.Wine brewing ferment
The spt15 of one of hansen initiation transcription factor belongs to transcription initiation complex part in mother, it is a kind of TATA binding protein[7], participate in
The formation of transcription initiation complex controls gene expression efficiency.It changes with related gene promoter region TATA binding ability
Become, related gene expression efficiency is influenced, so as to cause the character mutation of strain.Its mutation makes related gene overexpression,
Yeast is improved in phenotype to ethanol tolerance ability.
Chinese patent: the saccharomyces cerevisiae hansen initiation transcription factor and its encoding gene of a kind of mutation and application
(ZL200810024036.8) it utilizes random mutation technology revulsion transcription factor spt15 gene, obtains spt15's by screening
One mutant spt15-6；Another Chinese patent: the saccharomyces cerevisiae hansen initiation transcription factor gene and its expression vector of mutation and
Using (ZL201310008539.7) also with random mutation technology revulsion transcription factor spt15 gene, obtained by screening
A mutant spt15-10 of spt15.The comparison of they and former saccharomyces cerevisiae spt15 gene order changes significantly.It will
They construct recombinant Saccharomyces cerevisiae using technique for gene engineering, and fermentation detection alcohol yied all changes, but comes to engineer application
It says, all undesirable, i.e., alcohol yied should also further increase.
The present invention is expanded from wild type yeast strain obtains starting transcription regulatory factor gene spt15, is saturated with fixed point
Mutation method obtains mutated gene, and is building up to pYES2NTc carrier, is transformed into table in saccharomyces cerevisiae INVSC1 by Li-acetate method
It reaches, the recombinant yeast ethanol production of acquisition is measured.2 point mutation spt15 genes are obtained through screening.It is recombinated after mutation
The metabolic characteristic of recombinant Saccharomyces cerevisiae greatly improve, that is, substantially increase its alcohol yied.
Bibliography
[1]Liu HM,Xu L,Yan M,et al.gTME for construction of recombinant yeast
co-fermenting xylose and glucose.Chin J Biotech,2008,24(6):1 6.
Liu Hongmei, Xu Lin, it is strict and impartial, wait the recombinant Saccharomyces cerevisiae bioengineering of .gTME building common fermentation xylose and glucose
Journal, 2008,24 (6): 1 6.
[2]Hal A,Gregory S.Global transcription machinery engineering:A new
approach for improving cellular phenotype.Metab Eng,2007,9:258 267.
Summary of the invention
The present invention proposes a kind of spt15 fixed point saturation gene mutation using genetic engineering means to saccharomyces cerevisiae, improves
The method of alcohol yied.
The invention is realized in this way.A kind of saccharomyces cerevisiae spt15 fixed point saturation gene mutation improving alcohol yied
Method the steps include:
1, the amplification of Saccharomyces Cerevisiae in S .cerevisiae INVSC1 spt15 gene and construction of recombinant plasmid
A) genome extraction kit is used, S.cerevisiae INVSC1 genome is extracted.It is total with S.cerevisiae
DNA is template, expands saccharomyces cerevisiae hansen initiation transcription factor using Spt15_Forward and Spt15_Reverse primer PCR
Spt15 gene.PCR cycle parameter are as follows: 94 DEG C, 5min；94 DEG C of 1min, 56 DEG C of 1min, 72 DEG C of 2min, 30 circulations.Use glue
QIAquick Gel Extraction Kit (TaKaRa company) carries out purification and recovery to the gene amplified.
Wherein primer: Spt15_Forward 5'-ATGGCCGATGAGGAACGTTTAAAGGAGTTTA-3'
Spt15_Reverse 5'-TCACATTTTTCTAAATTCACTTAGCACAGGGTATATAG-3'
Original spt15 gene order is shown in sequence table SEQ ID No.1
B) spt15 gene after the recovery is connect with pMD18-T (TaKaRa company) carrier, and Calcium Chloride Method will connect carrier and turn
Change to bacillus coli DH 5 alpha competent cell, extract monoclonal recombinant plasmid pMD18-spt15, obtains gene order through sequencing
Spt15 is correct.Then using on Spt15_yes2ntF and Spt15_yes2ntR primer PCR amplification pMD18-spt15 plasmid
Spt15 gene.PCR cycle parameter are as follows: 94 DEG C, 5min；94 DEG C of 1min, 56 DEG C of 1min, 72 DEG C of 2min, 30 circulations.Through
Purification and recovery is carried out to the gene amplified with plastic recovery kit (TaKaRa company) after I double digestion of Not I, BamH.
Wherein primer: Spt15_yes2ntF 5'-GGATCCGCCGATGAGGAACGTTTA-3'
Spt15_yes2ntR 5'-GCGGCCGCTCACATTTTTCTAAATTCAC-3'
C) spt15 gene after the recovery connects with through Not I, I double digestion of BamH and pYES2NTc linear carrier after the recovery
It connects, Calcium Chloride Method is transformed into bacillus coli DH 5 alpha competent cell for carrier is connected, and extracts monoclonal recombinant plasmid pYES2NTc-
It is correct to obtain gene order spt15 through sequencing by spt15.
2, the acquisition of saturation mutation gene pool and recombinant Saccharomyces cerevisiae is pinpointed
A) by the homologous comparison of spt15 sequence and three-dimensional structural analysis, the discovery site Lys127 is located at symmetrical structure
The mutation of intermediate binding site, this site may influence the combination of spt15 and spt3, therefore selecting the site is mutational site.
B) according to reversed Overlap extension PCR (overlap extension PCR) principle, a pair of of mutant primer is designed
Spt15_mutF and Spt15_mutR, wherein small letter scribing line letter part is mutational site, corresponding 127 amino acids passwords
Son.
Firstly, using recombinant plasmid pYES2NTc-spt15 as template, using primer Spt15_mutF and Spt15_mutR into
Row plasmid amplification, PCR amplification loop parameter are 94 DEG C of 50sec, 62 DEG C of 45sec, 72 DEG C of 7min, 30 circulations, finally, 72
DEG C extend 10min, reversed amplification obtains the linear fragment comprising carrier sequence and gene order, by I enzymic digestion template of Dpn
Afterwards, glue recycles, and from connecting, converts bacillus coli DH 5 alpha, kalamycin resistance plate screening transformant, sequencing identifies whether be prominent
Become gene.Mutant gene sequence and former saccharomyces cerevisiae spt15 gene order[10]Comparison result show, altogether obtain at the 127th
12 different point mutation of lysine, other sites do not mutate.12 point mutation the results are shown in Table 1.
Wherein primer: Spt15_mutF 5'-ATGGTTGTTACCGGTGCAnnkAGTGAGGATGACTCA-3'
Spt15_mutR 5'-TGCACCGGTAACAACCATTTTCCCTGAGGCAAAAATTAAAGC-3'
C) by containing mutation and unmutated spt15 gene recombination plasmid, Li-acetate method is utilized[9]It is transformed into wine brewing ferment
Female INVSC1.Transformant is screened using SX screening and culturing medium, obtains a series of recombinant Saccharomyces cerevisiaes containing spt15 mutated gene
(totally 12) are respectively designated as serial recombinant Saccharomyces cerevisiae INVSC1-spt15-X by mutated gene serial number, wherein X be F, V, R,
M, L, G, T, S, Q, D, N, I (being shown in Table 1).
Table 1 pinpoints saturation mutation site codon and amino acid
3, recombinant Saccharomyces cerevisiae INVSC1-spt15-X fermenting experiment, interpretation of result and screening.
12 recombinant Saccharomyces cerevisiaes obtained above and control group are inoculated in respectively in 50mL seed culture medium, 30 DEG C,
200r/min is cultivated for 24 hours, is inoculated in the 500mL triangular flask of the fermentation medium containing 100mL with 10% (V/V), and bacterium solution is appropriate
Absorbance value is measured after dilution at 600nm, selects to be inoculated into respectively in 100mL fermentation medium in the bacterial strain of logarithmic growth phase
30 DEG C, 200r/min anaerobic fermentation culture.Measure ethanol content and residual sugar amount.Fermentation broth sample is through 0.45 μm of acetate fiber filter membrane
Filtering, using SBA-40C type bio-sensing analyzer and reagent (Shandong Province academy sciences Biology Research Institute) carry out concentration of alcohol and
The detection of concentration of glucose.It analyzes and screens after measured, recombinant bacterium recombinant Saccharomyces cerevisiae INVSC1-SPT15-M, INVSC1-
SPT15-N is improved largely using glucose producing and ethanol yield, and the alcohol yied in dextrose culture-medium is respectively 43.0
± 0.9g/L and 43.7 ± 0.2g/L.Increase by 17.8% and 19.7% than control strain respectively.
Preferred spt15-M mutant gene sequence is shown in SEQ ID No.2, and the mutated gene is at 127 by lysine mutation
For methionine.
Preferred spt15-N mutant gene sequence is shown in SEQ ID No.3, and the mutated gene is at 127 respectively by lysine
Sport asparagine.
The principle of the present invention is such.The phenotype of cell is determined by numerous genes synthesis, and an ideal table is constructed
The bacterial strain of type needs while carrying out polygenic modification, however the ability for introducing these modifications is usually very limited.Gene table
Occur in each level that hereditary information is transmitted up to regulation, and transcriptional control is a most important ring in gene expression regulation
Section.Global transcription mechanism engineering method allows to change the expression of many terminal genes.Pass through the change for transcription factor response
More so that entire transcripton generates big disturbance.Transcription regulatory factor spt15 is originated by transformation, utilizes particular screen condition
The desired phenotype optimized exactly realizes polygenic change simultaneously to adjust whole by the change of hansen initiation transcription factor
A metabolism network.
The present invention selectes the site Lys127 as catastrophe point and carries out fixed point saturation mutation, obtains apparent technical effect.
The present invention has clear advantage.The method of the present invention is simple, and operation is easy, and alcohol yied improvement effect is obvious.Tool
There are good economic benefit and social benefit.
Detailed description of the invention
The clone of Fig. 1 .spt15 gene and the electrophoresis detection verifying of pYES2NTc-spt15 recombinant plasmid
The wherein PCR product of (A) 1:spt15；M:DNA marker DL2000
(B) the double digestion verifying of 1:pMD18-spt15 plasmid；M:DNA marker DL10000
(C) the PCR verifying of 1:pYES2NTc-spt15 plasmid；2:pYES2NTc-spt15 plasmid；
3:pYES2NTc-spt15 single endonuclease digestion product；M:DNA marker DL5000
The sugared utilization power of Fig. 2 mutated gene recombination yeast INVSC1-spt15-X
The alcohol yied of Fig. 3 mutated gene recombination yeast INVSC1-spt15-X and control strain
Fig. 4 mutated gene restructuring yeast strains INVSC1-SPT15-M, INVSC1-SPT15-N and control strain recombinant bacterium
The ethanol production and glucose utilization curve of INVSC1-Neo；
Three-dimensional structure and the mutational site of transcription regulatory factor gene spt15 are originated in Fig. 5 wild type yeast strain；
Specific embodiment
With example, the present invention is further described below:
The clone of embodiment 1:spt15 gene and its building of recombinant plasmid
Using the genomic DNA of S.cerevisiae INVSC1 as template, PCR reaction, PCR are carried out using the primer of design
Product electrophoresis detection has an obvious band (Fig. 1) in 0.75kb or so.It will be after the purified recycling of PCR product and cloning vector
PMD18-T connection, connection product convert E. coli DH5 α, filter out positive transformant pMD18- using blue hickie
Spt15, plasmid order-checking the result shows that obtained spt15 sequence is correct and gene pool in gene homology 100%
(GenBank gene number M29459.1, protein number AAA34458.1).
Through PCR amplification on plasmid pMD18-spt15, through Not I, I double digestion of BamH, agarose gel electrophoresis recycling is pure
Change.Spt15 gene after the recovery is connected with through Not I, I double digestion of BamH and pYES2NTc linear carrier after the recovery, is passed through
PCR and digestion verification obtain recombinant plasmid pYES2NTc-spt15, obtain gene order spt15 through sequencing.
Embodiment 2 pinpoints the acquisition of saturation mutation gene pool and recombinant Saccharomyces cerevisiae
The recombinant plasmid pYES2NTc-spt15 obtained using embodiment 1 carries out reversed Overlap extension PCR as template, passes through
From connecting after I enzymic digestion of Dpn, recycling, bacillus coli DH 5 alpha, kalamycin resistance plate screening transformant, sequencing identification are converted
It whether is mutated gene.Mutant gene sequence and former saccharomyces cerevisiae spt15 gene order[10]Comparison result show, altogether obtain
In 12 different point mutation of the 127th lysine, other sites do not mutate.12 point mutation the results are shown in Table 1.
The screening of 3 mutated gene recombinant Saccharomyces cerevisiae of embodiment
By containing mutation and unmutated spt15 gene recombination plasmid, Li-acetate method is utilized[9]It is transformed into saccharomyces cerevisiae
INVSC1.Transformant is screened using the screening and culturing medium for lacking uracil, obtains a series of recombinations containing spt15 mutated gene
Saccharomyces cerevisiae.Be respectively designated as serial recombinant Saccharomyces cerevisiae INVSC1-spt15-X by mutated gene serial number, wherein X be K, F, V,
R, M, L, G, T, S, Q, D, N, I (being shown in Table 1).Wherein K is unmutated.
The sugared utilization power of 4 INVSC1-spt15-X mutated gene recombination yeast of embodiment
Most gene all shows that glucose consumption rate has different degrees of reduction.Especially SPT15-N, SPT15-K.
But control strain and mutated gene the recombinant bacterial strain glucose in 12 hours consume completely, show glucose consumption rate simultaneously
Its ethanol production is not influenced.(see Fig. 3)
The detection of 5 mutated gene recombinant yeast INVSC1-spt15-X anaerobic fermentation ethanol production of embodiment
Control strain INVSC1-Neo and recombinant bacterial strain INVSC1-spt15-X are in the dextrose culture-medium of 100g/L, and 30
DEG C, the ethanol production that obtains after the 48h that ferments under conditions of 200r/min it is as shown in table 2.
Compared with compareing bacterium INVSC1-Neo, INVSC1-SPT15-L, INVSC1-SPT15-G, INVSC1-SPT15-K,
INVSC1-SPT15-I, INVSC1-SPT15-S ethanol production do not change substantially, and top is all for 24 hours.And INVSC1-
SPT15-T, INVSC1-Spt3, INVSC1-SPT15-Q, INVSC1-SPT15-F, INVSC1-SPT15-V, INVSC1-
SPT15-D alcohol yied is decreased obviously, and alcohol yied is in 25-29g/L or so.It is the 68.5%-79.5% of control strain.And
Although the highest alcohol yied of SPT15-R changes less compared with compareing bacterium, the time that ethyl alcohol reaches maximum output pushes away from for 24 hours
Late 48h.
Recombinant yeast INVSC1-SPT15-M, INVSC1-SPT15-N have significantly using glucose producing and ethanol yield
It improves, the alcohol yied in dextrose culture-medium is respectively 43.0 ± 0.9g/L and 43.7 ± 0.2g/L.Respectively than compareing bacterium
Strain increases by 17.8% and 19.7%.Effective mutation that the result tentatively discloses hansen initiation transcription factor spt15 gene makes ferment of making wine
Great change has occurred in female metabolic pathway and metabolic fluxes.
2 recombinant bacterium of table is compared with the producing and ethanol under control strain the same terms
The material explanation that the present invention uses.
Escherichia coli Escherichia coli DH5, Saccharomyces cerevisiae host bacterial strain are Saccharomyces
Cerevisiae INVSC1, Yeast expression carrier pYES2NTc can be bought.
It tests genome DNA extracting reagent kit used and is purchased from Shanghai Hua Shun bioengineering Co., Ltd, restriction enzyme
Not I, BamH I is purchased from NEB company, and pMD18-T vector, plastic recovery kit are Dalian treasured bioengineering Co., Ltd
(TaKaRa) it produces, primer synthesis, plasmid extraction kit, Taq polymerase, dNTP Mixture ammonia benzyl antibiotic are raw for Shanghai
The production of work bio-engineering corporation, TransTaq DNA Polymerase High Fidelity High fidelity PCR polymerase are full formula
The measurement of King Company's production, gene order is completed by the biological Co., Ltd of English fine horse (Invitrogen).Other reagents are that analysis is pure.
Culture medium
50 μ g/mL ampicillins are added in Escherichia coli LB culture medium culture.Saccharomyces cerevisiae is trained with YPAD culture medium
It supports.
Minimal medium (YPAD) (g/L): yeast powder 10, peptone 20, glucose 20, adenine sulfate 0.075.
Screening and culturing medium (SX) (g/L): amino acid yeast nitrogen (YNB) 6.7 is free of, it is necessary to which ispol (lacks urine
Pyrimidine) 1.3, glucose 20, agar powder 20.
Seed culture medium (g/L): yeast powder 10, peptone 20, glucose 20.
Fermentation medium (g/L): yeast powder 10, peptone 20, glucose 100.
Claims (4)
1. a kind of saccharomyces cerevisiae spt15 for improving alcohol yied pinpoints saturation mutation gene, it is characterised in that sequence is SEQ ID
Spt15-M mutated gene shown in No.2, the mutated gene are methionine by lysine mutation at 127.
2. a kind of recombinant expression carrier for the saccharomyces cerevisiae spt15 fixed point saturation mutation gene for improving alcohol yied, feature exist
Containing sequence in the recombinant expression carrier is spt15-M mutated gene shown in SEQ ID No.2.
3. a kind of saccharomyces cerevisiae spt15 for improving alcohol yied pinpoints saturation mutation genetic recombination saccharomyces cerevisiae, it is characterised in that
It is spt15-M mutated gene shown in SEQ ID No.2 that the recombinant Saccharomyces cerevisiae, which contains sequence, is named as INVSC1-
SPT15-M。
4. a kind of application of the recombinant Saccharomyces cerevisiae for the saccharomyces cerevisiae fixed point saturation mutation gene spt15-M for improving alcohol yied,
It is characterized in that recombinant Saccharomyces cerevisiae as claimed in claim 3 is used to produce ethyl alcohol by fermenting substrate of glucose.
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| CN101270156A (en) * | 2008-04-25 | 2008-09-24 | 南京工业大学 | Mutant saccharomyces cerevisiae initiation transcription factor and coding gene and application thereof |
| CN103122351A (en) * | 2013-01-10 | 2013-05-29 | 刘红梅 | Mutational saccharomyces cerevisiae starting transcription factor gene as well as expression vector and application thereof |
| KR20130078049A (en) * | 2011-12-30 | 2013-07-10 | 이화여자대학교 산학협력단 | Furfural-tolerant yeast strains |
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| US20100291648A1 (en) * | 2006-12-07 | 2010-11-18 | Massachusetts Institute Of Technology | Global transcription machinery engineering |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270156A (en) * | 2008-04-25 | 2008-09-24 | 南京工业大学 | Mutant saccharomyces cerevisiae initiation transcription factor and coding gene and application thereof |
| KR20130078049A (en) * | 2011-12-30 | 2013-07-10 | 이화여자대학교 산학협력단 | Furfural-tolerant yeast strains |
| CN103122351A (en) * | 2013-01-10 | 2013-05-29 | 刘红梅 | Mutational saccharomyces cerevisiae starting transcription factor gene as well as expression vector and application thereof |
Non-Patent Citations (2)
| Title |
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| Mot1 Regulates the DNA Binding Activity of Free TATA-binding Protein in an ATP-dependent Manner;Russell P. Darst;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20031231;第278卷(第15期);13216–13226 |
| SPT3定向进化提高酿酒酵母乙醇耐性的研究;姜如娇;《中国优秀硕士学位论文全文数据库 基础科学辑》;20091015(第10期);A006-74 |
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| CN109825516A (en) | 2019-05-31 |
| CN109825516B (en) | 2020-01-14 |
| CN106434700A (en) | 2017-02-22 |
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