CN101429488A - Bacillus coli alcoholic fermentation engineering bacterium and uses thereof - Google Patents
Bacillus coli alcoholic fermentation engineering bacterium and uses thereof Download PDFInfo
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- CN101429488A CN101429488A CNA2007101770032A CN200710177003A CN101429488A CN 101429488 A CN101429488 A CN 101429488A CN A2007101770032 A CNA2007101770032 A CN A2007101770032A CN 200710177003 A CN200710177003 A CN 200710177003A CN 101429488 A CN101429488 A CN 101429488A
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- ethanol
- intestinal bacteria
- alcoholic acid
- engineering bacterium
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
The invention relates to a colon bacillus alcohol fermentation engineering bacterium resisting alcohol. A colon bacillus mutant strain highly resisting the alcohol is obtained through a plurality of generations of directional screening; and an alcohol dehydrogenase gene II and a pyruvate decarboxylase gene of zymomonas mobilis are transferred into the mutant strain to obtain the novel colon bacillus alcohol fermentation engineering bacterium. When the engineering bacterium uses pentose and hexose as substrates to ferment for producing the alcohol, the engineering bacterium can still maintain higher fermentation rate under high-concentration alcohol, and has higher transformation rate of the alcohol.
Description
Technical field
The present invention relates to a kind of bacillus coli alcoholic fermentation engineering bacterium of anti-alcoholic acid the and preparation method thereof, the invention still further relates to the application of described bacillus coli alcoholic fermentation engineering bacterium in fermentative production of ethanol.
Background technology
In agricultural crop straw and residue, the content of Mierocrystalline cellulose and hemicellulose can account for more than 75% of dry weight, and agricultural crop straw is carried out fermentative production of ethanol, is well to obtain the alcoholic acid mode.
Mierocrystalline cellulose mainly is the straight-chain molecule that is formed by connecting through β-1,4 glycosidic link by glucose molecule; Hemicellulose is the noncrystalline heterogeneous polymer that comprises multiple sugars such as wood sugar, pectinose, semi-lactosi, sweet dew and glucose, again with the content of wood sugar for the highest.
Intestinal bacteria contain all indispensable enzymes that utilize hexose and five-carbon sugar, but hexoses such as glucose fermentation, fructose, also can xylose-fermenting, five-carbon sugar such as pectinose.But utilize the wild-type e. coli fermentative production of ethanol, also infeasible on industry, reason has two:
The one, what wild-type e. coli carried out is mixed acid fermentation, and what obtain is multiple product, and ethanol only accounts for a seldom part in tunning.
Two to be wild-type e. colis very responsive to ethanol for it.Under higher concentration ethanol environment, the Bacillus coli cells film constitutes and will change, and growth is suppressed.Ethanol as 30g/L has obviously suppressed the intestinal bacteria growth, and when concentration was higher, restraining effect was more obvious.Therefore be not suitable for carrying out ethanol fermentation production.
To sum up, agricultural crop straw is carried out fermentative production of ethanol, and feasible in industry, need to solve above two difficult problems, that is: colibacillary alcohol metabolism approach is transformed, introduce ethanol fermentation approach efficiently, make ethanol become main tunning; The ethanol of the necessary resisting high-concentration of described bacterial strain still keeps higher fermentation rate under high alcohol concn simultaneously.
Summary of the invention:
The purpose of this invention is to provide a kind of escherichia coli alcoholic fermentation engineering bacterium, make it when making fermenting substrate production ethanol of five-carbon sugar and hexose, under high concentration ethanol, still can keep higher fermentation rate, and higher ethanol conversion is arranged.
The present invention has solved technical problem with following dual mode, has obtained a kind of novel bacillus coli alcoholic fermentation engineering bacterium:
1, screening has obtained high anti-alcoholic acid intestinal bacteria mutant strain;
To obtain alcohol patience behind the intestinal bacteria process high concentration ethanol polybasic directed screening, the mutant strain of anti-ethanol that obtains can be grown in the ethanol of 45g/L.
2, the anti-alcoholic acid intestinal bacteria of height mutant strain has been carried out genetic modification.
Alcohol dehydrogenase gene II and the Pyruvate Decarboxylase Gene of zymomonas mobilis in the anti-alcoholic acid intestinal bacteria of above-mentioned height mutant strain, have been changed over to, coexpression takes place in two genes under can being started by the strong promoter Ptac of e. coli rna polysaccharase efficient identification, obtain the bacillus coli alcoholic fermentation engineering bacterium of anti-the alcoholic acid.
Owing to introduce homotype ethanol fermentation approach, control its intracellular carbon metabolism and carry out to generating the alcoholic acid direction, make that its fermentation principal product is an ethanol.
The concrete concrete preparation method of bacillus coli alcoholic fermentation engineering bacterium of the present invention is:
1, utilize the e. coli jm109 bacterial strain to carry out anti-ethanol screening, through being applied on the solid LB substratum that contains Virahol after the screening several times, select the single rapidly bacterium colony of growth and proceed ethanol tolerance cultivation, through the mutant strain of the anti-ethanol ET1 that obtains behind the polybasic directed screening in the ethanol of 45g/L, growing.
Repeat the cultivation and the screening of bacterial strain according to above-mentioned screening process, available same result promptly can obtain the mutant strain of anti-ethanol of growing in the ethanol of 45g/L.
2, will include the zymomonas mobilis alcohol dehydrogenase gene of SD sequence and Pyruvate Decarboxylase Gene is connected on the pGEM-T easy carrier after through the PCR clone, cut through enzyme and to be placed on that contain can be by on the expression vector pZY507 of the strong promoter Ptac of e. coli rna polysaccharase efficient identification, make alcohol dehydrogenase gene and Pyruvate Decarboxylase Gene that corotation record and coexpression take place in intestinal bacteria, obtain bacillus coli alcoholic fermentation engineering bacterium.
Through experiment confirm, when ferment five-carbon sugar and hexose were produced ethanol, its glucose fermentation transformation efficiency reached 86% of theoretical yield with bacillus coli alcoholic fermentation engineering bacterium provided by the present invention, and the transformation efficiency of xylose-fermenting reaches 78% of theoretical yield.
Advantage of the present invention is:
1. make up first equal bacterial strain of ethanol fermentation engineering with the intestinal bacteria mutant strain of anti-ethanol ET1, make engineering bacteria under high concentration ethanol, still can keep higher fermentation rate;
2. the present invention has utilized and has contained alcohol dehydrogenase gene and Pyruvate Decarboxylase Gene common transcript and expression in the intestinal bacteria mutant strain of anti-ethanol ET1 that can be made zymomonas mobilis by the expression vector of the strong promoter Ptac of e. coli rna polysaccharase efficient identification.Make that its fermentation principal product is an ethanol, when improving alcohol yied, reduced the input in the follow-up still-process.
Embodiment
The preparation of embodiment 1 bacillus coli alcoholic fermentation engineering bacterium of anti-ethanol
A, intestinal bacteria ethanol tolerance screening mutant strains
E. coli jm109 is in the LB culture medium culturing, grow into to be transferred to after the logarithmic phase and contain in 10g/L, 20g/L, 30g/L, 40g/L and the 50g/L alcoholic acid LB culture medium culturing, (ethanol joins in the LB substratum through after the membrane filtration degerming of 0.45um), the alcohol concn of 30g/L is the growth of obvious suppression e. coli jm109.The switching of the JM109 process continuous multi-generation that contains among the 30g/L alcoholic acid LB will be grown in, revolution connects behind the three generations it is applied to and contains on the Virahol solid LB substratum of (the not volatile while can keep ethanol to select to press), selects the single rapidly bacterium colony of growth to continue switching.
With the JM109 that is grown in the no alcoholic acid LB substratum is contrast, is coated with by doubling dilution and dull and stereotypedly calculates colony number and measure OD in the liquid LB substratum
600Light absorption value measure the difference of the speed of growth between screening bacterial strain and the control strain, through 12 generations continuously after the switching screening, obtained with contrast do not have notable difference can be in 30g/L alcoholic acid LB substratum the JM109 bacterial strain of normal growth.This inoculation is proceeded orthoselection to containing in the 35g/L alcoholic acid LB substratum, measure its speed of growth with same method, to the last select to obtain the JM109 bacterial strain ET1 that can grow in the LB of 45g/L, continuing in high concentration ethanol that screening do not obtain can the alcoholic acid of anti-greater concn mutant strain.
The clone of B, zymomonas mobilis XW101 alcohol dehydrogenase gene and Pyruvate Decarboxylase Gene
With SEQ ID NO:1 (5 '-GGATCCGTGTTTTGAATATATGGA-3 ') and SEQ ID NO:2 (5 '-GCATGCTAGAGGAGCTTGTTAACAG-3 ') is primer, the Pyruvate Decarboxylase Gene of amplification zymomonas mobilis, its product size is 1800bp; With SEQ ID NO:3 (5 '-GCGAGCTCAGTATGTAGGGTGAGG-3 ') and SEQ ID NO:4 (5 '-GTCGACTTAGAAAGCGCTCAGGAA-3 ') is primer, the alcohol dehydrogenase gene of amplification zymomonas mobilis, its product size is 1200bp.And be cloned into respectively on pGEM-T easy (Promeg) carrier, behind sequencing analysis, with the homology of zymomonas mobilis alcohol dehydrogenase gene of having reported and Pyruvate Decarboxylase Gene be 99%, after cutting through enzyme pdc and adhB gene be connected to and obtain plasmid pZY507bc on the expression vector pZY507.
PCR reaction system (Z.mpdc):
10×PCR?buffer 2μl
2.5mMdNTP 2μl
10×BSA 2μl
50pM?gfol 2μl
50pM?gfo2 2μl
Template DNA 5ng
5U/ μ lTaq enzyme 0.25 μ l
Add sterilized water to 20 μ l
The PCR reaction conditions:
1,94 ℃ of pre-sex change 300s
2、94℃ 30s
56℃ 30s
72℃ 60s
(28 circulations)
3、72℃ 5min
PCR reaction system (Z.m adhB):
10×PCR?buffer 2μl
2.5mMdNTP 2μl
10×BSA 2μl
50pM?gfol 2μl
50pM?gfo2 2μl
Template DNA 5ng
5U/ μ lTaq enzyme 0.25 μ l
Add sterilized water to 20 μ l
The PCR reaction conditions:
1,94 ℃ of pre-sex change 300s
2、94℃ 30s
62℃ 30s
72℃ 40s
(28 circulations)
3、72℃ 5min
Linked system and ligation:
2×buffer 5μl
pGEM-T?easy 1μl
PCR product 1 μ l
T
4Ligase enzyme (3U/ μ l) 1 μ l
Add water to 10 μ l, 4 ℃ of connections of spending the night.
C, based on the structure of the intestinal bacteria ethanol engineering bacteria of ethanol tolerance mutant strain
Utilize calcium chloride that intestinal bacteria ethanol tolerance mutant strain ET1 is made competent cell, after heat shock (42 ℃ 90 seconds, 120 seconds on ice) transformed, engineering strain on the LB of paraxin that contains 25 μ g/ml and Virahol substratum obtained engineering strain ET1bc.
The checking of D, zymomonas mobilis XW101 alcohol dehydrogenase gene and Pyruvate Decarboxylase Gene
Adopt the primer among the B, plasmid with engineering strain ET1bc is template (described in reaction conditions and reaction system such as the B), the band of about 1.2kb of the size that can increase respectively and 1.8kb, utilize the RNA test kit to extract total RNA of ET1bc, the band of pcr amplification to one 3.0Kb after reverse transcription, the corotation record has taken place as a transcriptional units in account for motion fermentation single cell bacterium XW101 alcohol dehydrogenase gene and Pyruvate Decarboxylase Gene in intestinal bacteria.
Embodiment 2 engineering strain ET1bc fermentative production of ethanol
1. culture condition
1.1 medium component
Glucose 10%, yeast extract 0.5%, sodium-chlor 1.0%, peptone 1.0%, phosphoric acid buffer pH7.0;
Wood sugar 10%, yeast extract 0.5%, sodium-chlor 1.0%, peptone 1.0%, phosphoric acid buffer pH7.0;
1.2 cultivate and fermentation condition
ET1bc is inoculated in the LB substratum, cultivates OD
600The IPTG that adds 1.0mM during=0.2 left and right sides induces, when ET1bc grows into OD
600=1.0 o'clock centrifugal collection thalline are inoculated in the 1.1 described fermention mediums, make its OD after the inoculation
600Value is approximately about 1.0, and 37 degrees centigrade of anaerobism are cultivated.
2. the mensuration of residual sugar in the fermented liquid
Adopt the residual sugar content in the high performance liquid chromatography detection fermented liquid, condition determination is as follows, detector: the Waters410 differential refraction detector; Column temperature: 35 ℃; Chromatographic column: Dalian is according to the HypersilNH2 of Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S. (4.6mmi.d. * 250mm5 μ m); Moving phase: V (acetonitrile): V (water)=80: 20; Flow velocity: 1mlmin-1, sample size 10um.
3. alcoholic acid is measured
Adopt the ethanol content in the GC-17A island functional activities of the body fluid chromatography detection fermented liquid, fid detector.
Chromatographic column: CP-Wax57CB capillary column, long 50m, internal diameter 0.25mm, thickness 0.2um.
Chromatographic condition column temperature: 130 ℃.
Detector temperature: 180 ℃.
Sampler temperature: 180 ℃.
Carrier gas: high purity nitrogen; Hydrogen flow rate: 50ml/min; Air: 500ml/min; Tail blows: 30.0ml/min.
Sample size: 0.5ul.
5. testing data (50ml substratum) 10% sugared content
6. test-results
Five-carbon sugar with 10% or hexose are fermentation substrate, and under 37 degrees centigrade fermentation condition, after fermentation in 96 hours, xylose-fermenting and glucose productive rate reach 79% and 87% of theoretical yield respectively.
Illustrate: all technology of utilizing agricultural crop straw to carry out alcohol production all it are passed through enzymolysis or acidolysis is treated as five carbon or hexose ferments later on again.
7. conclusion (of pressure testing)
The engineering bacteria of the present invention's preparation has higher ethanol conversion when making fermenting substrate production ethanol of primary product five-carbon sugar after the decomposition such as agricultural crop straw and hexose.
Claims (7)
1. bacillus coli alcoholic fermentation engineering bacterium of anti-the alcoholic acid is characterized in that having changed over to alcohol dehydrogenase gene II and the Pyruvate Decarboxylase Gene of zymomonas mobilis in the anti-alcoholic acid intestinal bacteria of height mutant strain.
2, the preparation method of the bacillus coli alcoholic fermentation engineering bacterium of anti-alcoholic acid the according to claim 1, its step is as follows:
1) with having obtained alcohol patience behind the intestinal bacteria process high concentration ethanol polybasic directed screening, obtains high anti-alcoholic acid intestinal bacteria mutant strain;
2) in the anti-alcoholic acid intestinal bacteria of above-mentioned height mutant strain, change alcohol dehydrogenase gene II and the Pyruvate Decarboxylase Gene of zymomonas mobilis over to, coexpression takes place in two genes under can being started by the strong promoter Ptac of e. coli rna polysaccharase efficient identification, obtain the bacillus coli alcoholic fermentation engineering bacterium of anti-the alcoholic acid.
3, the preparation method of the bacillus coli alcoholic fermentation engineering bacterium of anti-alcoholic acid the according to claim 2, describedly through high concentration ethanol polybasic directed screening be: utilize the e. coli jm109 bacterial strain to carry out anti-ethanol screening with intestinal bacteria, through being applied on the solid LB substratum that contains Virahol after the screening several times, select the single rapidly bacterium colony of growth and proceed ethanol tolerance cultivation, through the mutant strain of anti-ethanol that obtains behind the polybasic directed screening in the ethanol of 45g/L, growing.
4, the preparation method of the bacillus coli alcoholic fermentation engineering bacterium of anti-alcoholic acid the according to claim 2, described alcohol dehydrogenase gene II and the Pyruvate Decarboxylase Gene that changes zymomonas mobilis over to is: will include the zymomonas mobilis alcohol dehydrogenase gene of SD sequence and Pyruvate Decarboxylase Gene and be connected on the pGEM-T easy carrier after through the PCR clone, the process enzyme is cut to be placed on and is contained and can be made alcohol dehydrogenase gene and Pyruvate Decarboxylase Gene that corotation record and coexpression take place in intestinal bacteria by on the expression vector pZY507 of the strong promoter Ptac of e. coli rna polysaccharase efficient identification.
5, with the application of the described bacillus coli alcoholic fermentation engineering bacterium of anti-alcoholic acid of claim 1 in make fermenting substrate production ethanol of five-carbon sugar and hexose.
6, a kind of high anti-alcoholic acid intestinal bacteria mutant strain is characterized in that can growing in the ethanol of 45g/L having obtained alcohol patience behind the intestinal bacteria process high concentration ethanol polybasic directed screening.
7, the preparation method of the anti-alcoholic acid intestinal bacteria of height according to claim 6 mutant strain, it is characterized in that utilizing the e. coli jm109 bacterial strain to carry out anti-ethanol screening, through being applied on the solid LB substratum that contains Virahol after the screening several times, select the single rapidly bacterium colony of growth and proceed ethanol tolerance cultivation, through obtaining behind the polybasic directed screening.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2007101770032A CN101429488A (en) | 2007-11-08 | 2007-11-08 | Bacillus coli alcoholic fermentation engineering bacterium and uses thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160544A (en) * | 2011-12-16 | 2013-06-19 | 逢甲大学 | Method for simultaneously fermenting pentose and hexose by microorganisms |
| CN104278043A (en) * | 2014-09-29 | 2015-01-14 | 天津大学 | Application of gene on short-chain alcohol tolerance target and method of using gene to improve tolerance of microorganism short-chain alcohol |
-
2007
- 2007-11-08 CN CNA2007101770032A patent/CN101429488A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160544A (en) * | 2011-12-16 | 2013-06-19 | 逢甲大学 | Method for simultaneously fermenting pentose and hexose by microorganisms |
| CN104403983A (en) * | 2011-12-16 | 2015-03-11 | 逢甲大学 | Method for simultaneously fermenting pentose and hexose by microorganisms |
| CN103160544B (en) * | 2011-12-16 | 2015-05-06 | 逢甲大学 | Method for simultaneously fermenting pentose and hexose by microorganisms |
| CN104278043A (en) * | 2014-09-29 | 2015-01-14 | 天津大学 | Application of gene on short-chain alcohol tolerance target and method of using gene to improve tolerance of microorganism short-chain alcohol |
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Open date: 20090513 |