CN101631869A - Method for preparing butanol through butyryl-CoA as an intermediate using bacteria - Google Patents
Method for preparing butanol through butyryl-CoA as an intermediate using bacteria Download PDFInfo
- Publication number
- CN101631869A CN101631869A CN200780046237.1A CN200780046237A CN101631869A CN 101631869 A CN101631869 A CN 101631869A CN 200780046237 A CN200780046237 A CN 200780046237A CN 101631869 A CN101631869 A CN 101631869A
- Authority
- CN
- China
- Prior art keywords
- butanols
- gene
- coding
- coa
- recombinant bacteria
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to a method for producing butanol using a bacterium capable of biosynthesizing butanol from butyryl-CoA as an intermediate. More particularly, a method for producing butanol, the method comprising generating bytyryl-CoA in a bacterium which contains a gene coding for AdhE (an enzyme responsible for the conversion of butyryl-CoA to butanol) using various methods, and converting the butyryl-CoA into butanol.
Description
Technical field
The method that the present invention relates to use butyryl coenzyme A (butyryl-CoA) in can the bacterium of biosynthesizing butanols, to produce butanols as intermediate.
Background technology
Along with oil price sharp rise and for the worry that increases day by day to Global warming and Greenhouse effect, biofuel is using microorganism that it is carried out being subjected to aspect the production concern that increases day by day in recent years.Particularly, compare with bio-ethanol, the advantage that biological butanol has is to have higher easy Combination because its oxygen level is low with fossil oil.Emerging in recent years conduct is used for the alternative fuel of gasoline, and the market share of biological butanol increases sharply.The biological butanol amount of American market reaches 37,000 ten thousand gals every year, and price is 3.75$/gal.As to the substituting of petroleum gasoline, butanols is better than ethanol.Owing to have high-energy-density, low-steam pressure, be similar to the octane rating and the low impurity content of gasoline, its can with existing gasoline with the mixed higher than ethanol, and do not damage performance, mileage or organic pollutant criteria.Can reach economy and the environmental advantages that reduces crude oil import and greenhouse gas emission by microorganism mass production butanols.
Butanols can pass through clostridium (Clostridial) bacterial strain anaerobism ABE (acetone-butanols-ethanol) fermentation (Jones, D.T. and Woods, D.R., Microbiol.Rev., 50:484,1986; Rogers, P., Adv.Appl.Microbiol., 31:1,1986; Lesnik, people such as E.A., Necleic Acids Research, 29:3583,2001).This biological method is to be used for the major technique that butanols and acetone are produced over more than 40 year, up to eighties of last century fifties.It is because its complicated growth conditions and to be used for its biology tool and the learn a skill development of (omics technology) of its group not mature enough that but clostridium (Clostridial) bacterial strain is difficult to further improve.
Therefore, suggestion utilizes microorganism such as growing fast under normal operation and can using the intestinal bacteria of the multiple group of operation that learns a skill to develop into the production of butanol bacterial strain.Particularly, be used for a small amount of metabolic engineering of production of butanol bacterial strain development and group and learn a skill and be employed thereon intestinal bacteria bacterial classification, make it have the very big potential that develops into the production of butanol bacterial strain.
Clostridium acetobutylicum (Clostridium acetobutylicum) produces butanols (Jones, D.T. and Woods, D.R., Microbiol.Rev., 50:484,1986 by the butanols biosynthetic pathway that shows among Fig. 1; Desai, people such as R.P., J.Biotechnol., 71:191,1999).In wild-type e. coli, ethanol is synthetic through the similar approach that plays a crucial role under anaerobic derivable adhE wherein (coding is responsible for producing alcoholic acid enzyme AdhE by acetyl-CoA through acetaldehyde).Intestinal bacteria can contain for the necessary gene of the biosynthesizing of butyryl coenzyme A and butanols, but different with its corresponding gene in clostridium (Clostridia) are, the too low and effectively corresponding enzyme reaction of catalysis of its expression level.
Simultaneously, the derivative therein method that can produce the recombinant bacteria of butanols and use described bacterium to produce butanols of butanols biosynthetic pathway is disclosed (US 2007/0259410 A1; But production efficiency general (modest) US2007/0259411 A1).
The present inventor has paid and has made great efforts to utilize bacterium (particularly intestinal bacteria) greatly, its body contain be responsible for coding can the multiple butyryl coenzyme A that will in bacterium, produce as intermediate and convert it into the gene of the enzyme (AdhE) of butanols, and definite butyryl coenzyme A can change into butanols by AdhE.
Summary of the invention
Therefore, the object of the present invention is to provide the method for multiple production as the butyryl coenzyme A of the biosynthesizing important intermediate of butanols and analogue thereof.
Another object of the present invention is to provide use can produce the method for butanols as the bacterium of intermediate biosynthesizing butanols by butyryl coenzyme A.
To achieve these goals, the invention provides the method for producing butanols, described method comprises: cultivate to contain in the substratum that contains butyric acid or etheric acid and be responsible for coding and butyryl coenzyme A changed into the recombinant bacteria of gene of enzyme (AdhE) of butanols to produce butanols, coding CoAT (acetyl-CoA: the butyryl coenzyme A transferring enzyme) be introduced in the described bacterium; And from nutrient solution, reclaim butanols.
The present invention also provides the method that produces butyryl coenzyme A, and described method comprises: (acetyl-CoA: the butyryl coenzyme A transferring enzyme) gene is introduced in wherein the recombinant bacteria to cultivate coding CoAT in the substratum that contains butyric acid or etheric acid.
In addition, the invention provides the method that produces butyryl coenzyme A, described method comprises: cultivate coding AtoDA (acetyl-CoA: the etheric acid thiophorase) be introduced in wherein the recombinant bacteria in containing butyro-substratum.
In addition, the invention provides the method that produces butanols, described method comprises: cultivate gene that contains the AtoDA that encodes and the gene bacterium of encoding AdhE to produce butanols in containing butyro-substratum; From nutrient solution, reclaim butanols.
In addition, the invention provides the method that produces butyryl coenzyme A, described method comprises: cultivate in the substratum that contains butyric acid or etheric acid and contain coding AtoDA (acetyl-CoA: the etheric acid thiophorase), FadB or PaaH (3-glycoloyl-CoA desaturase), the microorganism of PaaFG (enoyl-CoA hydratase) and FadE (acetyl-CoA desaturase) gene.
In addition, the invention provides the method for producing butanols, described method comprises: in the substratum that contains butyric acid or etheric acid, cultivate coding AtoDA, FadB or PaaH, PaaFG and FadE gene with the microorganism of the gene of coding AdhE to produce butanols; From nutrient solution, reclaim butanols.
The present invention also provides recombinant microorganism with butanols generation ability and the method that is used to produce butanols, coding thiolase (THL), 3-maloyl group coa dehydrogenase (BHBD), enoyl-CoA hydratase (CRO), be introduced in the described microorganism with the gene of functional BCD (butyryl-CoA dehydrogenase), described method comprises: cultivate recombinant microorganism in substratum; And from nutrient solution, reclaim butanols.
The present invention also provides the method that produces acetyl-CoA, and described method comprises: cultivate coding THL, BHBD, the gene of enoyl-CoA hydratase and functional BCD are introduced in wherein the recombinant bacteria.
The present invention also provides butanols to produce the recombinant bacteria of ability and the method for producing butanols, coding THL, BHBD, the gene of enoyl-CoA hydratase and functional BCD and chaperone is introduced in the described microorganism, and the gene of lacI gene (coding lac operon aporepressor) and the enzyme that relates in lactic biological is synthetic is disallowable, and described method comprises: cultivate recombinant bacteria to produce butanols in substratum; And from nutrient solution, reclaim butanols.
By following detailed description and appending claims, other features of the present invention and aspect will be conspicuous.
Description of drawings
Fig. 1 is the synoptic diagram that shows butanols biosynthetic pathway in the clostridium acetobutylicum (Clostridium acetobutylicum).
Fig. 2 is the synoptic diagram that shows the butanols biosynthetic pathway of inferring in recombination bacillus coli according to the present invention.
Fig. 3 is presented at the synoptic diagram that causes producing through butyryl coenzyme A the biosynthetic pathway of butanols in ato system and/or the fad system.
Fig. 4 has shown that in clostridium acetobutylicum (Clostridium acetobutylicum) acetyl-CoA changes into the approach of butyryl coenzyme A.
Fig. 5 has shown the construction process and the gene mapping of pKKhbdthiL carrier.
Fig. 6 has shown the construction process and the gene mapping of pTrc184bcdcrt carrier.
Fig. 7 has shown the construction process and the gene mapping of pKKhbdadhEthiL (pKKHAT) carrier.
Fig. 8 has shown the construction process and the gene mapping of pKKhbdadhEatoB (pKKHAA) carrier.
Fig. 9 has shown the construction process and the gene mapping of pKKhbdadhEphaA (pKKHAP) carrier.
Figure 10 has shown the construction process and the gene mapping of pKKhbdydbMadhEphaA (pKKHYAP) carrier.
Figure 11 has shown the construction process and the gene mapping of pKKhbdbcdPA01adhEphaA (pKKHPAP) carrier.
Figure 12 has shown the construction process and the gene mapping of pKKhbdbcdKT2440adhEphaA (pKKHKAP) carrier.
Figure 13 has shown the construction process and the gene mapping of pTrc184bcdbdhABcrt (pTrc184BBC) carrier.
Figure 14 has shown the butanols biosynthetic pathway under the following situation: a part of gene from clostridium acetobutylicum (C.acetobutylicum) that wherein relates in the butanols biosynthetic pathway is replaced from colibacillary gene.
Figure 15 has shown the construction process and the gene mapping of pKKmhpFpaaFGHatoB (pKKMPA) carrier.
Embodiment
In the present invention, check the gene that contains from the following enzyme of coding of clostridium acetobutylicum (Clostridiumacetobutylicum): thiolase (THL; Gene: thl or thiL); Acetyl-CoA: butyryl coenzyme A-transferring enzyme (CoAT; Gene: ctfA and ctfB); And E.C. 4.1.1.4 (AADC; Gene: whether recombination bacillus coli adc) [ATCC 11303 (pACT)] can pass through its endogenous enzyme (AdhE under anaerobic expresses) produces butanols by butyryl coenzyme A.This recombination bacillus coli [ATCC 11303 (pACT)] is fabricated so that produce acetone (Bermejo, people such as L.L., Appl.Environ.Microbiol., 64:1079,1998) by acetyl-CoA through the etheric acid coenzyme A.
Predictably, when using the CoAT enzyme from clostridium acetobutylicum (Clostridium acetobutylicum) (be responsible for butyric acid (BA) or acetate and change into butyryl coenzyme A or acetyl-CoA) by expression of recombinant e. coli when replacing the coenzyme A residue of etheric acid coenzyme A, butyryl coenzyme A can be produced (Fig. 2).And predictably, the AdhE enzyme catalysis butyryl coenzyme A of under anaerobic expressing and be responsible for acetyl-coenzyme A is changed into alcoholic acid recombination bacillus coli (ATCC11303 (pACT)) changes into butanols to produce butanols (Fig. 2).
In order to confirm above-mentioned prediction, recombination bacillus coli is cultivated in containing butyro-substratum, the result as can be seen, butyric acid is converted to butanols through butyryl coenzyme A, show that this is because by being introduced in the ctfA in the recombinant chou and the CoAT enzyme of ctfB genes encoding, this AdhE enzyme with expression under anaerobic is consistent.
In the following embodiments, when cultivating in recombination bacillus coli is containing the substratum of butyric acid and/or etheric acid, it is verified the generation butanols.
Therefore, in one aspect, the present invention relates to produce the method for butanols, described method comprises: cultivate to contain in the substratum that contains butyric acid or etheric acid and be responsible for coding and butyryl coenzyme A changed into the recombinant bacteria of gene of enzyme (AdhE) of butanols to produce butanols, coding CoAT (acetyl-CoA: the butyryl coenzyme A transferring enzyme) be introduced in the described bacterium; And from nutrient solution, reclaim butanols.
The invention still further relates to the method that produces butyryl coenzyme A, described method comprises: (acetyl-CoA: the gene butyryl coenzyme A transferring enzyme) is introduced in wherein the recombinant bacteria to cultivate coding CoAT in the substratum that contains butyric acid or etheric acid.
In the present invention, preferably, the gene of coding thiolase (THL) and E.C. 4.1.1.4 (AADC) is introduced in the recombinant bacteria in addition.
Preferably, in the present invention useful CoAT (acetyl-CoA: the butyryl coenzyme A transferring enzyme) ctfA and ctfB genes encoding from clostridium (Clostridium) can be arranged, but the invention is not restricted to this.And, the THL that expresses in recombinant microorganism of the present invention is preferably by belonging to the phaA of (Ralstonia sp.) or by the atoB coding from intestinal bacteria from the thl of fusobacterium (Clostridium sp.) or thiL or from Ralstonia solanacearum, but is not limited thereto.Preferably, in recombinant microorganism of the present invention, express by adc genes encoding from fusobacterium (Clostridium sp.), but be not limited thereto.As long as it is expressed as the active enzyme that has with identical in the host bacterium, any foreign gene all can use in the present invention ad lib.
In the present invention, the host bacterium is preferably intestinal bacteria.But as long as it holds the gene of the AdhE that encodes, the host bacterium is not limited thereto.
In an embodiment of the present invention, the wild-type e. coli when not having pACT to be introduced in is wherein containing that butanols is detected when cultivating in the substratum of butyric acid and/or etheric acid.
Producing butanols by the wild-type e. coli of cultivating in containing butyro-substratum is considered to owing to the AtoDA of ato system butyric acid be changed into butyryl coenzyme A (Lioliou and Kyriakidis, Microbial Cell Factories, 3:8,2004) become the result (Fig. 3) of butanols then by colibacillary AdhE enzymatic conversion.AtoDA, wherein AtoD represents acetyl-CoA: etheric acid thiophorase alpha subunit and AtoA represent acetyl-CoA: etheric acid thiophorase β subunit is an enzyme of being responsible for following reaction
Aa-coenzyme A+formic acid (perhaps butyric acid) ← → aa+ acetyl (butyryl)-coenzyme A
Therefore, on the other hand, the present invention relates to produce the method for butanols, described method comprises: cultivate in the substratum that contains butyric acid or etheric acid and contain coding CoAT (acetyl-CoA: the gene butyryl coenzyme A transferring enzyme) and be responsible for coding and butyryl coenzyme A changed into the bacterium of gene of AdhE of butanols to produce butanols; And from nutrient solution, reclaim butanols.
The present invention also provides the method that produces butyryl coenzyme A, and described method comprises: (acetyl-CoA: the butyryl coenzyme A transferring enzyme) gene is introduced in wherein the recombinant bacteria to cultivate coding AtoDA in the substratum that contains butyric acid or etheric acid.
In the present invention, the bacterium that contains the gene of the gene of the AtoDA that encodes and/or the AdhE that encodes is preferably intestinal bacteria, but is not limited thereto, as long as it can hold said gene.
In addition, produce the method for butanols and be assumed that it is etheric acid to be changed into the etheric acid coenzyme A then by the FadB (perhaps PaaH) of fad system by in containing the substratum of etheric acid, cultivating wild-type e. coli by the ato system, PaaFG and FadE change into result (Park and the Lee of butyryl coenzyme A, Biotechnol.Bioeng., 86:681,2004), and finally become butanols (Fig. 3) by colibacillary AdhE enzymatic conversion.
Known FadB has four kinds of functions: 3-hydroxyacyl-CoA dehydrogenase; 3-maloyl group coenzyme A epimerase; δ (3)-suitable-δ (2)-anti--alkene acyl-coenzyme A isomerase and alkene acyl-CoA hydratase, and in following reaction, related to FadA:
Acyl group-coenzyme A+acetyl-coenzyme A ← → coenzyme A+3-oxygen acyl group-coenzyme A
FadB (perhaps PaaH) plays the effect that the etheric acid coenzyme A is changed into the beta-hydroxy butyryl coenzyme A.PaaFG is an enoyl-CoA hydratase of being responsible for the beta-hydroxy butyryl coenzyme A is changed into crotonyl-CoA.FadE is the acyl group-coa dehydrogenase that crotonyl-CoA is changed into butyryl coenzyme A that relates in below the reaction:
Butyryl coenzyme A+FAD ← → the FADH2+ crotonyl-CoA
Therefore, on the other hand, the present invention relates to produce the method for butanols, described method comprises: cultivate in the substratum that contains butyric acid or etheric acid and contain coding AtoDA, FadB or PaaH, PaaFG and FadE gene change into butyryl coenzyme A the bacterium of gene of AdhE of butanols to produce butanols with being responsible for coding; And from nutrient solution, reclaim butanols.
The invention still further relates to the method that produces butyryl coenzyme A, described method comprises: cultivate in the substratum that contains butyric acid or etheric acid and contain coding AtoDA (acetyl-CoA: the etheric acid thiophorase), FadB or PaaH (3-glycoloyl-coa dehydrogenase), the bacterium of PaaFG (enoyl-CoA hydratase) and FadE (acetyl-CoA desaturase) gene.
In the present invention, intestinal bacteria are the genes that preferably hold coding FadB or PaaH, the gene of the gene of coding PaaFG and coding FadE, and the bacterium of the gene of coding AdhE.But as long as it contains described gene, any bacterium all can be used in the present invention.
As mentioned above, when the approach that acetyl-CoA is changed into butyryl coenzyme A is introduced in bacterium when containing in the intestinal bacteria of gene that coding plays the enzyme (AdhE) that butyryl coenzyme A is changed into butanols, bacterium can produce butanols.
Fusobacterium (Clostridium sp.) approach is known as the approach (Fig. 4) that acetyl-CoA is changed into butyryl coenzyme A.In the approach of Fig. 4, from the gene thl of fusobacterium (Clostridium sp.) be accredited as in intestinal bacteria can effective expression THL (Bermejo, L.L. wait people, Appl.Environ.Microbiol., 64:1079,1998). except that thl, gene thiL is known as THL (Nolling, people such as J., the J.Bacteriol. of coding from fusobacterium (Clostridium sp.), 183:4823,2001).THL plays the effect that acetyl-CoA is changed into the etheric acid coenzyme A.And, in an embodiment of the present invention, except that thl or thiL from fusobacterium (Clostridium sp.), belong to the phaA of (Ralstonia sp.) or also be found from Ralstonia solanacearum and demonstrate bacterium THL activity, as using production of butanol to detect from colibacillary atoB.Therefore, belong to the phaA of (Ralstonia sp.) or can be used to replace thl or thiL from Ralstonia solanacearum from colibacillary atoB.In addition, as long as it is expressed in host cell showing the THL activity, any gene of coding THL, even foreign gene all can be used and without limits.
And people such as Bennett have reported except that BCD and have been expressed (Boynton, people such as Z.L., J.Bacteriol., 178:3015,1996) at the required enzyme of the production butyryl coenzyme A of etheric acid coenzyme A, BHBD and CRO in intestinal bacteria.But, according to this paper, report, because the expression of BCD or its cofactor (inferring the electron transfer flavoprotein by the coding of the gene (etfB and eftA) in the clostridium acetobutylicum (Clostridium acetobutylicum)) is very weak, perhaps because the very low stability of BCD or its cofactor and in external its activity of not observing, so intestinal bacteria do not have the function of BCD.
In the present invention, the low expression level of butyryl-CoA dehydrogenase can solve by a gene (groESL) of being responsible for the coding chaperone is introduced together with the bcd that comes from clostridium acetobutylicum (Clostridiumacetobutylicum).In an embodiment of the present invention, when the bcd that derives from clostridium acetobutylicum (Clostridium acetobutylicum) and chaperone encoding gene (groESL) are introduced in the intestinal bacteria, prove that e. coli host cell is observed butanols output and increases.
In another embodiment, the low expression level of butyryl-CoA dehydrogenase can overcome by bcd that will derive from Pseudomonas aeruginosa (Pseudomonas aeruginosa) or pseudomonas putida (Pseudomonasputida) or the ydbM that derives from Bacillus subtilus (Bacillus subtilis).Therefore, as long as it express to be shown BCD activity in host cell, although the BCD gene is all can being used of external source and without limits.
In an embodiment of the present invention, determine to contain thiL from fusobacterium (Clostridium sp.), hbd, bcd, the intestinal bacteria of groESL and crt produce butanols by glucose through butyryl coenzyme A.In another embodiment of the present invention, determine Rhodopseudomonas (Pseudomonas sp.) bcd from or replace producing butanols through butyryl coenzyme A as intermediate by glucose from the ydbM of Bacillaceae (Bacillus sp.) from bcd and the groESL of fusobacterium (Clostridium sp.).
Therefore, on the other hand, the present invention relates to have butanols and produce the recombinant bacteria of ability and the method that is used to produce butanols, coding thiolase (THL), 3-maloyl group coa dehydrogenase (BHBD), the gene of enoyl-CoA hydratase (CRO) and functional BCD (butyryl-CoA dehydrogenase) is introduced in the described bacterium, and described method comprises: cultivate recombinant bacteria in substratum; And from nutrient solution, reclaim butanols.
The present invention also provides the method that produces butyryl coenzyme A, and described method comprises: the cultivation THL that will encode, and BHBD, the gene of enoyl-CoA hydratase and functional BCD is introduced into recombinant bacteria wherein.
Consequent intermediate butyryl coenzyme A is converted to butanols by the AdhE enzyme of being encoded by the native gene of coding AdhE in the bacterium.Intestinal bacteria have been carried to encode and butyryl coenzyme A have been changed into the gene of the enzyme (AdhE) of butanols.Under the situation of the host cell that does not carry the AdhE encoding gene, when the gene of coding AAD (butyraldehyde desaturase) and BHD (butanols desaturase) was introduced into, host cell can be produced butanols by butyryl coenzyme A.Even if hold at host cell itself under the situation of gene of the AdhE that encodes, when the gene of coding AAD (butyraldehyde desaturase) and BHD (butanols desaturase) was introduced into, butyryl coenzyme A changed into butanols and can promote by expressed enzyme AdhE, AAD and BDH.
According to an aspect of the present invention, preferably, the gene of the gene of coding AAD (butyraldehyde desaturase) and/or coding BDH (butanols desaturase) is introduced in the recombinant bacteria in addition.The gene of coding AAD is preferably from the adhE of fusobacterium (Clostridium sp.) or from colibacillary mhpF, but is not limited thereto.For example, also can be used, just be used restriction as long as they express to show that identical AAD is active from the ADD-encoding gene of the microorganism outside the fusobacterium (Clostridium sp.).And the gene of coding BDH is preferably the bdhAB from fusobacterium (Clostridiumsp.), but is not limited thereto.For example, the gene from the microorganism outside the fusobacterium (Clostridium sp.) also can be used and without limits, need only them and expressed to show identical BDH activity.
In the present invention, the gene of coding THL can be preferably from thl or the thiL of fusobacterium (Clostridium sp.), from the phaA of Ralstonia solanacearum genus (Ralstonia sp.) or from colibacillary atoB.The gene of coding BHBD and enoyl-CoA hydratase can be preferably respectively from hbd and the crt of fusobacterium (Clostridium sp), but be not limited thereto.For example, any foreign gene all can be used and without limits, need only it and expressed in host cell to show active and enoyl-CoA hydratase (PaaFG in the intestinal bacteria) activity of BHBD (FadB in the intestinal bacteria or PaaH).Be replaced by in an embodiment of the present invention, even therein that butanols is determined under the situation from the hbd of fusobacterium (Clostridium sp) and crt still is detected from the gene of colibacillary paaH (coding 3-hydroxyacyl-CoA dehydrogenase) and paaFG (coding enoyl-CoA hydratase).
The meaning of the term of Shi Yonging " functional BCD " is to be introduced in host cell such as colibacillary bcd gene to be expressed to show the BCD activity in this article.The example of encoding function BCD gene comprises from the bcd of Rhodopseudomonas (Pseudomonas sp.) or from the ydbM of Bacillaceae (Bacillus sp.), but is not limited thereto.Because it shows faint activity in intestinal bacteria, also can be included in from the bcd of fusobacterium (Clostridium sp.) and to contain in the functional BCD-encoding gene, as the BCD activity is enhanced when being introduced into the gene (groESL) of coding chaperone when it.
According to an aspect of the present invention, preferably, the gene of coding chaperone is introduced in the recombinant bacteria in addition.The gene of coding chaperone is preferably groESL.
In order to increase the genetic expression of being responsible for the biosynthetic enzyme of coding butanols, recombinant bacteria can preferably disallowable lacI (coding lac operon aporepressor).Preferably, the gene that is coded in the enzyme that lactic biological relates in synthetic is rejected in addition.The gene that is coded in the enzyme that lactic biological relates in synthetic is preferably ldhA (coding serum lactic dehydrogenase).
At last, in the present invention, by with THL, BHBD, enoyl-CoA hydratase, functional BCD, AAD, BDH and chaperone are incorporated into intestinal bacteria, and reject wherein lacI gene (coding lac operon aporepressor) and the gene of the responsible lactic biological synthetic enzyme of coding, made up the intestinal bacteria that produce butanols thus, thereby determined that production of butanol power increases significantly in described recombination mutation microorganism through recombination mutation.
The meaning of the term " rejecting " relevant with gene used herein is that gene can not be expressed, if perhaps it is expressed, the blocking-up that causes wherein relating to by the biosynthetic pathway of the enzyme of genes encoding owing to sudden change, replacement, disappearance or the insertion of the Nucleotide of the arbitrary number from single base to whole gene fragment does not have enzymic activity.
Embodiment
Enumerate to explain the present invention but be not interpreted as the EXAMPLE Example of restriction of the present invention by following, can obtain the present invention is better understood.
Though intestinal bacteria W3110 is used as host microorganism in the following embodiments, but it will be apparent to those skilled in the art that, by rejecting target gene to be rejected and be introduced in the gene that relates in the butanols biosynthesizing so that produce butanols, other coli strains, bacterium, yeast and fungi also can be used as host cell.
In addition, though the gene from specific bacterial strain is used as the example of waiting to introduce gene in the following embodiments, but it will be apparent to those skilled in the art that as long as their are expressed showing identical activity any gene all may be utilized and without limits in host cell.
And, should be noted that, though in the following embodiments only as an example with defined medium and cultural method, saccharification liquid, such as whey, CSL (corn leaching solution) etc., and other substratum and multiple cultural method such as (people such as Lee, Bioprocess Biosyst.Eng. such as (fed-batch) cultivation, cultured continuously in batches, 26:63,2003; People such as Lee, Appl.Microbiol.Biotechnol., 58:663,2002; People such as Lee, Biotechnol.Lett., 25:111,2003; People such as Lee, Appl.Microbiol.Biotechnol., 54:23,2000; People such as Lee, Biotechnol.Bioeng., 72:41,2001) also fall within the scope of the present invention.
Embodiment 1: produce butanols by adding butyric acid
Attempt to produce butanols by cultivating recombination bacillus coli [ATCC 11303 (pACT)].In order to carry out this cultivation, use following substratum: contain 10g/L NaCl, 10g/L Bacto Tryptones and 5g/L yeast extract paste+20g/L glucose and 1g/L NaHCO
3The LB substratum.
For the 12ml substratum in the 15ml culture tube, add 50 μ g/ml Ampicillin Trihydrates and recombination bacillus coli [ATCC 1103 (pACT)] is inoculated in the substratum under aerobic conditions and cultivated 1 hour, then under anaerobic cultivated 2 hours.
Afterwards, bacterium is cultivated in substratum, and every two hours with 50 μ l, 100 μ l, the 0.8mM butyric acid of 200 μ l or 300 μ l amount joins wherein.Before adding, with the pH value of butyro-pH regulator to substratum.
After inoculation 24,48,72,96,140 and 164 hours, the composition (table 1) that uses high performance liquid chromatography (HPLC) analysis to cultivate.In limit 1, the meaning of ' L-200-48 ' is to cultivate in having added the butyro-substratum of 0.8mM of 200 μ l 48 hours, and ' C ' is illustrated in does not have the control group cultivated in the butyro-substratum.
As a result, as shown in table 1, in negative control, LB are cultivated, do not detect any in ethanol, butanols, acetic acid or the butyric acid, and in not adding butyro-positive control, only produced acetic acid and ethanol.A kind of exception is, detects low-level butanols after cultivating 164 hours, and this may be from butyryl coenzyme A, also may not be.In contrast,, show that the AdhE enzyme under anaerobic expressed and produce the expression that acetone shows the CoAT enzyme, thereby finally determine to produce butanols when recombination bacillus coli [ATCC 1103 (pACT)] when being cultivated and producing butyric acid, ethanol.
Table 1: use the HPLC analysis of investigating (mM) inductive recombination bacillus coli [ATCC 1103 (pACT)] culture supernatants from butyric acid
Embodiment 2: prepare butanols by adding etheric acid
Identical among culturing process and the embodiment 1 contained etheric acid and butyro-LB and M9 substratum but use.That is to say that recombination bacillus coli [ATCC 11303 (pACT)] is cultivated in the LB that contains etheric acid (10mM) and/or butyric acid (20mM or 40mM) (30g/L glucose) substratum, then analyze culture (table 2) by HPLC.In table 2, ' 10-M9-200-2-72h ' refers in containing the butyro-M9 substratum of 10mM etheric acid and 20mM and to cultivate 72 hours, ' 400 ' expression 40mM butyric acid, and ' C ' is illustrated in and do not have the control group cultivated in the butyro-substratum.
As a result, as shown in table 2, butanols is detected with containing in etheric acid and the butyro-substratum at the substratum that contains etheric acid, and irrelevant with the kind of substratum.
Table 2: analyze all concentration from the HPLC that uses etheric acid (10mM) and/or butyric acid (20mM or 40mM) inductive recombination bacillus coli [ATCC 1103 (pACT)] culture supernatants and all provide with mM
| Sample | Glucose | Acetic acid | Acetoin | Ethanol | Butyric acid | Acetone | Butanols |
| ??10-M9-C-2-24h | ??401.5648 | ??131.163 | ??67.34 | ??25.693 | |||
| ??10-M9-200-2-24h | ??380.6775 | ??287.421 | ??500.036 | ??5.722 | |||
| ??10-M9-400-2-24h | ??156.5175 | ??33.422 | ??17.776 | ??39.739 | ??7.661 | ??0.476 | |
| ??10-LB-C-2-24h | ??158.4865 | ??17.207 | ??3.575 | ??1.794 | ??0.492 | ||
| ??10-LB-200-2-24h | ??160.5093 | ??16.512 | ??4.531 | ??2.061 | ??0.503 | ||
| ??10-LB-400-2-24h | ??125.2173 | ??16.382 | ??5.232 | ??2.941 | ??0.59 | ||
| ??10-M9-C-2-72h | ??396.3895 | ??148.93 | ??76.658 | ??29.12 | ??0.548 | ||
| ??10-M9-200-2-72h | ??365.218 | ??141.408 | ??49.579 | ??22.871 | ??35.363 | ??0.746 | |
| ??10-M9-400-2-72h | ??124.3028 | ??17.975 | ??9.3 | ??3.628 | ??0.899 | ||
| ??10-LB-C-2-72h | ??156.8248 | ??19.419 | ??7.158 | ??2.794 | ??0.812 | ||
| ??10-LB-200-2-72h | ??159.6945 | ??19.127 | ??8.539 | ??3.151 | ??0.89 | ||
| ??10-LB-400-2-72h | ??154.3995 | ??34.977 | ??19.454 | ??39.304 | ??7.176 | ??0.645 | |
| ??10-M9-C-2-96h | ??286.3553 | ??118.666 | ??59.421 | ??17.483 | ??0.55 | ||
| ??10-M9-200-2-96h | ??271.9225 | ??107.956 | ??34.066 | ??25.704 | ??8.192 | ??0.772 | |
| ??10-M9-400-2-96h | ??114.623 | ??26.804 | ??16.284 | ??50.433 | ??5.754 | ??0.67 |
| ??10-LB-C-2-96h | ??108.9373 | ??19.842 | ??11.458 | ??2.342 | ??0.782 | ||
| ??10-LB-200-2-96h | ??107.5158 | ??17.616 | ??12.413 | ??14.501 | ??2.916 | ??0.769 | |
| ??10-LB-400-2-96h | ??94.365 | ??14.925 | ??11.088 | ??3.086 | ??0.927 |
Embodiment 3: produce butanols by adding etheric acid or butyric acid in wild-type e. coli
With pre-the cultivation 24 hours during wild-type e. coli is in the culture tube that contains 15ml substratum (LB that contains 30g/L glucose).With the OD value is that 2.02 culture is inoculated in the flask of the substratum that contains 500ml.The OD value arrives after 0.4 after inoculation culture, and the culture that obtains is distributed in the bottle of two 250ml.When the OD value reaches 0.42, with culturing bottle with centrifugal 10 minutes of the speed of 5000rpm with abandoning supernatant.Then culturing bottle is placed on the fresh culture that also adds the 30ml that places anaerobic room in the aerobic chamber respectively.With 300 μ l, and the etheric acid lithium of 0.108g/ml (Sigma, A-8509) solution joins in each pipe, and final concentration is 10mM, and the adding butyric acid is 0.8mM to final concentration.The pH value of substratum is adjusted to 6.25, i.e. the pH value of the substratum before adding butyric acid.After cell was suspended and cultivates, final nutrient solution used HPLC to analyze (table 3)
In table 3, ' L8-200-72h ' is illustrated in to contain in 10mM etheric acid and the butyro-LB substratum of 0.8mM and cultivated 72 hours, and ' LC8 ' is illustrated in and only contains etheric acid and do not have to cultivate in the butyro-substratum group in contrast.
As a result, as shown in table 3, can determine to produce butanols when wild-type e. coli (ATCC11303) is cultivated in containing etheric acid and butyro-substratum, as what in embodiment 1, can be shown.In addition, butanols also is detected in the substratum that only contains etheric acid.
Table 3: analyze all concentration from the HPLC that uses etheric acid (10mM) and/or butyric acid (0.8mM) inductive wild-type e. coli ATCC 11303 (w/0pACT) culture supernatants and all provide with mM
| Sample | Glucose | Acetic acid | Acetoin | Ethanol | Butyric acid | Acetone | Butanols |
| ??LC8-24h | ??160.4175 | ??13.426 | ??4.585 | ??6.803 |
| ??LC8-48h | ??159.9535 | ??12.81 | ??5.034 | ??5.842 | |||
| ??LC8-72h | ??159.5753 | ??11.948 | ??5.943 | ??4.812 | |||
| ??LC8-96h | ??144.644 | ??11.74 | ??6.575 | ??3.767 | |||
| ??LC8-120h | ??145.9135 | ??11.399 | ??7.499 | ??3.167 | |||
| ??LC8-144h | ??158.6893 | ??13.765 | ??9.988 | ??3.012 | ??0.067 | ||
| ??LC8-168h | ??148.1663 | ??7.301 | ??12.385 | ??2.718 | ??0.114 | ||
| ??LC8-192h | ??149.9288 | ??7.003 | ??12.764 | ??2.285 | ??0.088 | ||
| ??LC8-264h | ??155.7643 | ??18.58 | ??15.3 | ??1.389 | ??0.544 | ||
| ??LC8-288h | ??155.3868 | ??16.797 | ??17.72 | ??0.991 | ??0.459 | ||
| ??L8-200-24h | ??155.5293 | ??12.876 | ??5.607 | ??24.055 | ??7.712 | ||
| ??L8-200-48h | ??155.6293 | ??12.086 | ??4.834 | ??21.089 | ??6.045 | ||
| ??L8-200-72h | ??155.1658 | ??12.701 | ??5.854 | ??20.407 | ??5.578 | ||
| ??L8-200-96h | ??141.744 | ??12.934 | ??6.426 | ??18.852 | ??4.661 | ??0.021 | |
| ??L8-200-120h | ??147.2203 | ??13.557 | ??7.486 | ??19.539 | ??3.922 | ??0.069 | |
| ??L8-200-144h | ??150.471 | ??7.1 | ??10.369 | ??19.975 | ??4.047 | ??0.093 | |
| ??L8-200-192h | ??147.4355 | ??11.333 | ??14.416 | ??19.954 | ??2.574 | ??0.147 | |
| ??L8-200-264h | ??151.9705 | ??17.761 | ??16.967 | ??22.17 | ??1.107 | ??0.543 | |
| ??L8-200-288h | ??150.526 | ??16.271 | ??18.951 | ??22.319 | ??0.846 | ??0.516 |
Embodiment 4: produce butanols in the approach that is produced butyryl coenzyme A by acetyl-coenzyme A is introduced into wherein intestinal bacteria
The structure of 4-1:pKKhbdthiL carrier
For the essential gene of butanols biosynthetic pathway, comprise hbd (coding 3-maloyl group coa dehydrogenase) and thiL (coding thiolase) is amplified and be cloned in the pKK223-3 expression vector (Pharmacia Biotech) in proper order, thereby the structure recombinant expression vector, called after pKKhbdthiL (Fig. 5).
Use clostridium acetobutylicum (Clostridium acetobutylicum) (KCTC 1724) chromosomal DNA to use primer SEQ ID NOS:1 and 2 to carry out PCR as template, described PCR carries out 24 circulations, condition is: 95 ℃ of following sex change 20 seconds, annealed 30 seconds down for 55 ℃, 72 ℃ were extended 1 minute down.The PCR product that obtains (hbd gene) uses EcoRI and PstI digestion and is inserted in the pKK223-3 expression vector (PharmaciaBiotech) that uses the same restrictions enzymic digestion, thereby makes up pKKhbd expression vector (Fig. 5)
In order to make up the pKKhbdthiL carrier, at first use primer SEQ ID NOS:3 and 4 to carry out PCR.The PCR product that obtains (thiL gene) uses SacI to handle, and is inserted into then in the pKKhbd carrier that uses same restrictions enzyme (SacI) digestion, thereby makes up the pKKhbdthiL carrier (Fig. 5) that contains hbd gene and thiL gene.
[SEQ?ID?NO：1]hbdf：5′-acgcgaattcatgaaaaaggtatgtgttat-3′
[SEQ?ID?NO：2]hbdr：5′-gcgtctgcaggagctcctgtctctagaatttga
taatggggattctt-3′
[SEQ?ID?NO：3]thiLf：5′-acgcgagctctatagaattggtaaggatat-3′
[SEQ?ID?NO：4]thiLr：5′-gcgtgagctcattgaacctccttaataact-3′
In order to make up the pKKhbdgroESLthiL carrier, use primer SEQ ID NOS:5 and 6 to carry out PCR as template with the chromosomal DNA of clostridium acetobutylicum (Clostridiumacetobutylicum).The PCR product that obtains (groESL gene) uses the XbaI cracking, is inserted into then in the pKKhbdthiL carrier that uses same restrictions enzyme (XbaI) digestion, thereby makes up pKKhbdgroESLthiL carrier (Fig. 5).
[SEQ?ID?NO：5]groESLf：5’-agcttctagactcaagattaacgagtgcta-3’
[SEQ?ID?NO：6]groESLr：5’-tagctctagattagtacattccgcccattc-3’
The structure of 4-2:pTrc184bcdcrt carrier
Use acetone clostridium butylicum (Clostridium acetobutylicum) chromosomal DNA to use primer SEQ ID NOS:7 and 8 to carry out PCR as template.The PCR product that obtains (bcd gene) uses NcoI and KpnI to digest and it is cloned in the pTrc99A expression vector (AmershamPharmacia Biotech), thereby makes up the pTrc99Abcd carrier.To from the pTrc99Abcd carrier, be inserted among the pACYC184 (New England Biolabs) that uses same restrictions enzyme (BspHI and EcoRV) digestion by the dna fragmentation by BspHI and EcoRV digestion cutting, thereby make up the pTrc184bcd carrier (Fig. 6) that contains the bcd gene.
[SEQ?ID?NO：7]bcdf：5′-agcgccatggattttaatttaacaag-3′
[SEQ?ID?NO：8]bcdr：5′-agtcggtacccctccttaaattatctaaaa-3′
In order to make up the pTrc184bcdcrt carrier, use primer SEQ ID NOS:9 and 10 to carry out PCR.The PCR product that obtains (crt gene) uses BamHI and PstI digestion and is inserted among the pTrc184bcd that uses same restrictions enzyme (BamHI and PstI) digestion, thereby makes up the pTrc184bcdcrt carrier (Fig. 6) that contains bcd gene and crt gene.
[SEQ?ID?NO：9]crt1：5′-atacggatccgagattagtacggtaatgtt-3′
[SEQ?ID?NO：10]crt2：5′-gtacctgcagcttacctcctatctattttt-3′
The rejecting of 4-3:lacI gene
LacI gene on the chromosomal DNA is disallowable, the tac promotor and the trc promotor that are included in the recombinant vectors for preparing among embodiment 4-1 and the 4-2 can be worked by composing type, thereby cause being cloned into gene (hbd, thiL in the respective carrier, groESL, bcd and crt) constitutive expression.Be responsible among the intestinal bacteria W3110 (ATTC 39936) of gene of enzyme (AdhE) that coding changes into butyryl coenzyme A in butanols containing, use primer SEQ ID NOS:11 and 12 by a step inactivation method (Warner et al., PNAS, 6:97 (12): 6640,2000) reject a coding lac operon aporepressor and an inhibition lacI gene for the Transcription of the metabolic required lac operon of lactose, then from bacterium, remove antibiotics resistance gene, thereby prepare new WL bacterial strain.
[SEQ?ID?NO：11]lacI_1stup：5′-gtgaaaccagtaacgttatacgat
gtcgcagagtatgccggtgtctcttagattgcagcattacacgtcttg-3′
[SEQ?ID?NO：12]lacI_1stdo：5′-tcactgcccgctttccagtcgg
gaaacctgtcgtgccagctgcattaatgcacttaacggctgacatggg-3′
4-4: the preparation of producing the butanols microorganism
PKKhbdgroESLthiL carrier for preparing in embodiment 4-1 and 4-2 and pTrc184bcdcrt carrier are introduced in the WL bacterial strain among the embodiment 4-3, thereby prepare new production butanols recombinant microorganism (WL+pKKhbdgroESLthiL+pTrc184bcdcrt).
4-5: the mensuration of production of butanol power
The production butanols microorganism for preparing in embodiment 4-4 is containing on the LB flat board of 50 μ g/ml penbritins and 30 μ g/ml paraxin selected.Recombinant chou is 37 ℃ of pre-down cultivations 12 hours in the LB of 10ml nutrient solution.Then, behind autoclaving, in the 100mL LB nutrient solution that in the 250mL flask, keeps 80 ℃ or higher temperature, add glucose (10g/L) and it is being used cool to room temperature in the anaerobic room of nitrogen purge.Be inoculated into the pre-culture of 2mL in the flask and cultivation under 37 ℃.When the glucose of substratum exhausts fully, as use glucose analyser (STAT, Yellow Springs Instrument, Yellow Springs, Ohio, that USA) measures is such, get nutrient solution and use and be equipped with packed column (Supelco CarbopackTM B AW/6.6%PEG 20M, 2m * 2mm ID, Bellefonte, PA, USA) gas chromatograph (Agillent6890N GC System, Agilent Technologies Inc., CA, USA) butanol concentration in the analysis substratum.
The result summarizes in following table 4.As shown in table 4, wild-type e. coli W3110 does not produce butanols, on the contrary according to recombination mutation microorganisms butanols of the present invention.With the digital proof that obtains the above consideration that combines, because hbd, thiL, bcd, groESL and the mistake of crt gene are expressed butyryl coenzyme A and are successfully produced by acetyl-CoA and become butanols by the AdhE enzymatic conversion that itself exists in the intestinal bacteria.
Table 4
| Bacterial strain | Butanols (mg/L) |
| ??W3110 | ??ND 1 |
| ??WL+pKKhbdgroESLthiL+pTrc?184bcdcrt | ??0.85 |
1
Be not detected
Embodiment 5: produce butanols by the recombinant microorganism of having introduced foreign gene
The rejecting of 5-1:ldhA gene
In the intestinal bacteria W3110 that the lacI-of embodiment 4-3 knocks out, ldhA (coding serum lactic dehydrogenase) uses primer SEQ ID NOS:13 to 14 further disallowable by a step inactivation method.Prepare the WLL bacterial strain thus.
[SEQ?ID?NO：13]ldhA1stup：5′-atgaaactcgccgtttatagcacaaaa
cagtacgacaagaagtacctgcagattgcagcattacacgtcttg-3′
[SEQ?ID?NO：14]ldhA1stdo：5′-ttaaaccagttcgttcgggcaggtttcgc
ctttttccagattgcttaagtcacttaacggctgacatggga-3′
The structure of 5-2:pKKhbdadhEthiL (pKKHAT) carrier
For the required gene of butanols biosynthetic pathway, comprise hbd (coding 3-maloyl group coa dehydrogenase), adhE (coding butyraldehyde desaturase: spell identical but the function difference with the adhE (coding ethanol dehydrogenase) of 1-2) and thiL (coding thiolase) use clostridium acetobutylicum (Clostridiumacetobutylicum) (KCTC 1724) chromosomal DNA to use primer SEQ IDNOS:15 to 20 amplification as template, and be cloned into successively in the pKK223-3 expression vector (PharmaciaBiotech), thereby the structure recombinant expression vector, called after pKKhbdadhEthiL (pKKHAT) (Fig. 7).
[SEQ?ID?NO：15]hbdf：5′-acgcgaattcatgaaaaaggtatgtgttat-3′
[SEQ?ID?NO：16]hbdr：5′-gcgtctgcaggagctcctgtctctagaatttga
taatggggattctt-3′
[SEQ?ID?NO：17]adhEf：5′-acgctctagatataaggcatcaaagtgtgt-3′
[SEQ?ID?NO：18]adhEr：5′-gcgtgagctccatgaagctaatataatgaa-3′
[SEQ?ID?NO：19]thiLf：5′-acgcgagctctatagaattggtaaggatat-3′
[SEQ?ID?NO：20]thiLr：5′-gcgtgagctcattgaacctccttaataact-3′
The structure of 5-3:pKKhbdadhEatoB (pKKHAA) carrier
For the atoB (coding acetyl-CoA Transacetylase) of intestinal bacteria W3110 is cloned into (Fig. 7) in the pKKhbdadhE expression vector, use primer SEQ ID NOS:21 and 22 at the enterprising performing PCR of the chromosomal DNA of intestinal bacteria W3110, described PCR carries out 24 circulations, condition is: 95 ℃ of following sex change 20 seconds, annealed 30 seconds down for 55 ℃, 72 ℃ were extended 90 seconds down.The PCR product (atoB) that obtains uses SacI digestion and is inserted in the pKKhbdadhE carrier that uses same restrictions enzyme (SacI) digestion, thereby makes up new recombinant vectors, and called after pKKhbdadhEatoB (pKKHAA) (Fig. 8).
[SEQ?ID?NO：21]atof：5′-atacgagctctacggcgagcaatggatgaa-3’
[SEQ?ID?NO：22]ator：5′-gtacgagctcgattaattcaaccgttcaat-3’
The structure of 5-4:pKKhbdadhEphaA (pKKHAP) carrier
For the phaA (coding thiolase) of Ralstonia eutropha (Ralstonia eutropha) (KCTC 1006) is cloned in the pKKhbdadhE carrier, use the chromosomal DNA of Ralstonia eutropha (Ralstoniaeutropha) to use primer SEQ ID NOS:23 and 24 to carry out PCR as template.The PCR product (phaA) that obtains uses the SacI cracking and is inserted in the pKKhbdadhE carrier that uses same restrictions enzyme (SacI) digestion, thereby makes up new recombinant vectors, and called after pKKhbdadhEphaA (pKKHAP) (Fig. 9).
[SEQ?ID?NO：23]phaAf：5′-agtcgagctcaggaaacagatgactg?acgt
tgtcatcgt-3′
[SEQ?ID?NO：24]phaAr：5′-atgcgagctcttatttgcgctcgactgcca-3′
The structure of 5-5:pKKhbdydbMadhEphaA (pKKHYAP) carrier
For the ydbM (coding putative protein) of subtilis (Bacillus subtilis) (KCTC 1022) is cloned in the pKKhbdadhE carrier, use the chromosomal DNA of subtilis (Bacillussubtilis) to use primer SEQ ID NOS:25 and 26 to carry out PCR as template.The PCR product (ydbM) that obtains uses the XbaI cracking and is inserted in the pKKhbdadhEphaA carrier that uses same restrictions enzyme (XbaI) digestion, thereby makes up new recombinant vectors, and called after pKKhbdydbMadhEphaA (pKKHYAP) (Figure 10).
[SEQ?ID?NO：25]ydbMf：5′-agcttctagagatgggttacctgacatata-3′
[SEQ?ID?NO：26]ydbMr：5′-agtctctagattatgactcaaacgcttcag-3′
The structure of 5-6:pKKhbdbcdPA01adhEphaA (pKKHPAP) carrier
For the bcd (coding butyryl-CoA dehydrogenase) of Pseudomonas aeruginosa (Pseudomonas aeruginosa) PA01 (KCTC 1637) is cloned in the pKKhbdadhEphaA carrier, use the chromosomal DNA of Pseudomonas aeruginosa (Pseudomonas aeruginosa) PA01 to use primer SEQ ID NOS:27 and 28 to carry out PCR as template.The PCR product (bcd) that obtains uses the XbaI cracking and is inserted in pKKhbdadhEphaA (pKKHAP) carrier that uses same restrictions enzyme (XbaI) digestion, thereby make up new recombinant vectors, called after pKKhbdbcdPA01adhEphaA (pKKHPAP) (Figure 11).
[SEQ?ID?NO：27]bcdPA01f：5′-agcttctagaactgctccttggacagcgcc-3′
[SEQ?ID?NO：28]bcdPA01r：5′-agtctctagaggcaggcaggatcagaacca-3′
The structure of 5-7:pKKhbdbcdKT2440adhEphaA (pKKHKAP) carrier
For the bcd (coding butyryl-CoA dehydrogenase) of pseudomonas putida (Pseudomonas putida) KT2440 (KCTC 1134) is cloned in the pKKhbdadhEphaA carrier, use the chromosomal DNA of pseudomonas putida (Pseudomonas putida) KT2440 to use primer SEQ ID NOS:29 and 30 to carry out PCR as template.The PCR product (bcd) that obtains uses the XbaI cracking and is inserted in the pKKhbdadhEphaA carrier that uses same restrictions enzyme (XbaI) digestion, thereby make up new recombinant vectors, called after pKKhbdbcdKT2440adhEphaA (pKKHKAP) (Figure 12).
[SEQ?ID?NO：29]bcdKT2440f：5′-agcttctagaactgttccttggacagcgcc-3′
[SEQ?ID?NO：30]bcdKT2440r：5′-agtctctagaggcaggcaggatcagaacca-3′
The structure of 5-8:pTrc184bcdbdhABcrt carrier
Use the chromosomal DNA of clostridium acetobutylicum (Clostridium acetobutylicum) to use primer SEQ ID NOS:31 and 32 to carry out PCR as template.The PCR product (bcd) that obtains uses NcoI and KpnI to digest and it is cloned in the pTrc99A expression vector (AmershamPharmacia Biotech), thereby makes up new recombinant vectors, called after pTrc99Abcd.After using BspHI and EcoRV digestion pTrc99Abcd, cut thus dna fragmentation is inserted among the pACYC184 (New England Biolabs) that uses identical restriction enzyme (BspHI and EcoRV) processing, thereby the recombinant expression vector of construction expression bcd gene, called after pTrc184bcd (Figure 13).
[SEQ?ID?NO：31]bcdf：5′-agcgccatggattttaatttaacaag-3′
[SEQ?ID?NO：32]bcdr：5′-agtcggtacccctccttaaattatctaaaa-3′
Use the chromosomal DNA of clostridium acetobutylicum (Clostridium acetobutylicum) to use primer SEQ ID NOS:33 and 34 to carry out PCR as template.The PCR product (bdhAB) that obtains uses BamHI and PstI digestion and is inserted among the pTrc184bcd that uses same restrictions enzyme (BamHI and PstI) digestion, thereby make up new recombinant vectors, called after pTrc184bcdbdhAB (pTrc184BB), it contains bcd and bdhAB.
[SEQ?ID?NO：33]bdhABf：5′-acgcggatccgtagtttgcatgaaatttcg-3′
[SEQ?ID?NO：34]bdhABr：5′-agtcctgcagctatcgagctctataatggct
acgcccaaac-3′
Use the chromosomal DNA of clostridium acetobutylicum (Clostridium acetobutylicum) to use SEQ ID NOS:35 and 36 to carry out PCR as template.The PCR product (crt) that obtains uses SacI and PstI digestion and is inserted among the pTrc184bcdbdhAB that uses same restrictions enzyme (SacI and PstI) digestion, thereby make up new recombinant vectors, called after pTrc184bcdbdhABcrt (pTrc184BBC), it contains bcd gene and bdhAB gene and crt gene (Figure 13).
[SEQ?ID?NO：35]crtf：5′-actcgagctcaaaagccgagattagtacgg-3′
[SEQ?ID?NO：36]crtr：5′-gcgtctgcagcctatctatttttgaagcct-3′
5-9: the preparation of producing the butanols microorganism
The intestinal bacteria W3110 (WLL) of shortage lacI for preparing in embodiment 5-1 and ldhA uses pTrc184bcdbdhABcrt (pTrc184BBC) carrier among the embodiment 5-8 and is selected from the carrier conversion of group down, be included in the pKKhbdadhEthiL (pKKHAT) that embodiment 5-2 makes up in the 5-7, pKKhbdadhEatoB (pKKHAA), pKKhbdydbMadhEphaA (pKKHYAP), pKKhbdadhEphaA (pKKHAP), pKKhbdbcdPA01adhEphaA (pKKHPAP) and pKKhbdbcdKT2440adhEphaA (pKKHKAP), thereby preparation can produce the recombination mutation microorganism (WLL+pKKHAT+pTrc184BBC of butanols, WLL+pKKHAA+pTrc184BBC, WLL+pKKHAP+pTrc184BBC, WLL+pKKHYAP+pTrc184BBC, WLL+pKKHPAP+pTrc184BBC and WLL+pKKHKAP+pTrc184BBC).
5-10: the mensuration of production of butanol power
Production butanols microorganism among in embodiment 5-9 is containing on the LB flat board of 50 μ g/ml penbritins and 30 μ g/ml paraxin selected.In order to select the WLLPA+pKKHPAP+pTrc184BBC bacterial strain, the amount of kantlex with 30 μ g/ml is added in the LB flat board.Recombinant chou is 37 ℃ of pre-down cultivations 12 hours in the LB of 10ml nutrient solution.Behind the autoclaving, to 80 ℃ down or add glucose (5g/L) in the 100mL LB nutrient solution of allotting under the higher temperature and it is being used cool to room temperature in the anaerobic room of nitrogen purge in the 250mL flask.The pre-culture of 2mL is inoculated in the flask and at 37 ℃ to descend to cultivate 10 hours.Then, at 5L fermentor tank (LiFlus GX, Biotron Inc., Korea) in 2L is contained the substratum autoclaving of following ingredients and use speed the supply of nitrogen 10 hours of 0.5vvm, with it from 80 ℃ or higher temperature cool to room temperature: contain glucose 20g, KH every liter of distilled water of this substratum
2PO
42g, (NH
4)
2SO
47H
2O 15g, MnSO
45H
2O 20mg, MgSO
47H
2O 2g, yeast extract 3g and every liter of distilled water trace-metal solution 5ml (FeSO in every liter of distilled water
47H
2O 10g, CaCl
21.35g, ZnSO
47H
2O 2.25g, MnSO
44H
2O 0.5g, CuSO
45H
2O 1g, (NH
4)
6Mo
7O
244H
2O 0.106g, Na
2B
4O
710H
2O 0.23g and 35%HCl 10ml).In fermentor tank, at 37 ℃, under the 200rpm with the 200rpm shake-flask culture.In culturing process, by automatic supply 25% (v/v) NH
4OH remains on 6.8 with pH, and supplies with nitrogen with the speed of 0.2vvm (gas volume/working volume/minute).
When using glucose analyser (STAT, Yellow Springs Instrument, YellowSprings, Ohio, when USA) glucose that records substratum exhausts fully, use to be equipped with packed column (Supelco CarbopackTM B AW/6.6%PEG 20M, 2m * 2mm ID, Bellefonte, PA, gas-chromatography USA) (Agillent 6890N GC System, AgilentTechnologies Inc., CA, USA) butanol concentration in the analysis substratum.
As a result, as shown in table 5, butanols is by thiL (WLL+pKKHAT+pTrc184BBC), and the cell that the gene of phaA (WLL+pKKHAP+pTrc184BBC) or atoB (WLL+pKKHAA+pTrc184BBC) coding THL is introduced into wherein produces.By this result, the foreign gene of the THL that can determine to encode also can be expressed in such as intestinal bacteria at host cell demonstrates the THL activity.
And, the data presentation that butanols produces, compare the situation (WLL+pKKHYAP+pTrc184BBC) that the butyryl-CoA dehydrogenase activity is introduced into the ydbM from subtilis (Bacillus subtilis) from the bcd of clostridium acetobutylicum (Clostridium acetobutylicum) therein (WLL+pKKHPAP+pTrc184BBC down or under the situation that the bcd from Pseudomonas aeruginosa (Pseudomonasaeruginosa) or pseudomonas putida (Pseudomonas putida) is introduced into the situation (WLL+pKKHAP+pTrc184BBC) that wherein only is introduced into from the bcd of clostridium acetobutylicum (Clostridium acetobutylicum); WLL+pKKHKAP+pTrc184BBC) obtain increasing.By this result, the foreign gene of the BCD that can determine to encode also can be expressed in such as intestinal bacteria at host cell shows the butyryl-CoA dehydrogenase activity.
Table 5
| Bacterial strain | The gene that contains | Butanols (mg/L) |
| ??W3110 | ??- | ??ND 1 |
| ??WLL+pKKHAT+pTrc184BBC | ??hbd，adhE，thiL，bcd，bdhAB，crt | ??1.2 |
| ??WLL+pKKHAA+pTrc184BBC | ??hbd，adhE，atoB，bcd，bdhAB，crt | ??1.3 |
| ??WLL+pKKHAP+pTrc184BBC | ??hbd，adhE，phaA，bcd，bdhAB，crt | ??1.4 |
| ??WLL+pKKHYAP+pTrc184BBC | ??hbd，adhE，ydbM，phaA，bcd，bdhAB，crt | ??1.7 |
| ??WLL+pKKHPAP+pTrc184BBC | ??hbd，adhE，bcdPA01，phaA，bcd，bdhAB， ??crt | ??3.1 |
| ??WLL+pKKHKAP+pTrc184BBC | ??hbd，adhE，bcdKT2440，phaA，bcd， ??bdhAB，crt | ??9.1 |
1Do not detect
Embodiment 6: by having introduced from intestinal bacteria and clostridium acetobutylicum (C.
Acetobutylicum) recombination mutation microorganisms producing butanols
In this embodiment, when from
Clostridium acetobutylicum (When the Gene Partial of responsible butanols biosynthetic pathway C.acetobutylicum) is replaced from colibacillary gene, measure the productivity (Figure 14) of butanols.Be well known that from colibacillary mhpF be the gene (Ferrandez, people such as A., J.Bacteriol., 179:2573,1997) of being responsible for the coding acetaldehyde dehydrogenase.In this embodiment, as the adhE from fusobacterium (Clostridium sp.), crt, hbd and thiL are respectively by from colibacillary gene [mhpF (acetaldehyde dehydrogenase encoding gene), paaFG, paaH and atoB] when replacing, the production of butanol power of the recombinant microorganism that obtains is determined.
The structure of 6-1:pKKmhpFpaaFGHatoB carrier
Use the chromosomal DNA of intestinal bacteria W3110 to use primer SEQ IDNOS:37 to 42 to carry out PCR as template, to increase for the necessary gene of butanols biosynthetic pathway, comprise mhpF (coding acetaldehyde dehydrogenase), paaFG (coding enoyl-CoA hydratase), paaH (coding 3-hydroxyl-acetyl-coa dehydrogenase) and atoB (coding acetyl-CoA Transacetylase).These genes are cloned in the pKK223-3 expression vector (Pharmacia Biotech) successively, thereby make up new recombinant expression vector, and called after pKKmhpFpaaFGHatoB (pKKMPA) (Figure 15).
[SEQ?ID?NO：37]mhpFf：5′-atgcgaattcatgagtaagcgtaaagtcgc-3′
[SEQ?ID?NO：38]mhpFr：5′-tatcctgcaggagctctctagagctagcttac
cgttcatgccgcttct-3′
[SEQ?ID?NO：39]paaFGHf：5′-atacgctagcatgaactggccgcaggt?tat-3′
[SEQ?ID?NO：40]paaFGHr：5′-tatcgagctcgccaggccttatgactcata-3′
[SEQ?ID?NO：41]atoBf：5′-atacgagctctgcatcactgccctgctctt-3′
[SEQ?ID?NO：42]atoBr：5′-tgtcgagctccgctatcgggtgtttttatt-3′
6-2: the preparation of producing the butanols microorganism
PTrc184bcdbdhAB (pTrc184BB) carrier among intestinal bacteria W3110 (WLL) the use embodiment 6-1 of shortage lacI for preparing in embodiment 5-1 and ldhA among pKKMPA carrier and the embodiment 5-8 transforms, thereby preparation can produce the recombination mutation microorganism (WLL+pKKMPA+pTrc184BB) of butanols.
The mensuration of 6-3 production of butanol power
The production butanols microorganism for preparing in embodiment 6-2 cultivates and measures under the same conditions production of butanol power in mode identical among the embodiment 5-10.
The result, as shown in table 6, with when only using the butanols biosynthetic pathway of clostridium acetobutylicum (C.acetobutylicum), compare, when from the corresponding enzyme (adhE → mhpF of colibacillary predicted coding, crt → paaFG, hbd → paaH, the gene of thiL → atoB) and be combined when using from the bcd of clostridium acetobutylicum (C.acetobutylicum) and bdhAB gene, the output of butanols improves.That is to say, from can using by mhpF of clostridium acetobutylicum (Clostridiumacetobutylicum) from intestinal bacteria for producing the necessary four kinds of enzymes (butyraldehyde desaturase, enoyl-CoA hydratase, BHBD and THL) of butanols in the intestinal bacteria, paaFG, paaH and atoB gene replace, and find that these have higher activity from the corresponding enzyme that colibacillary enzyme has recently from clostridium acetobutylicum (C.acetobutylicum), proved as increasing by production of butanol.
Table 6
| Bacterial strain | The gene that contains | Butanols (mg/L) |
| ??WLL+pKKMPA+pTrc184BB | ??mhpF，paaFGH，atoB，bcd，bdhAB | ??18.4 |
Industrial applicibility
As described in detail above, the present invention has the effect that the method for producing butanols is provided, and it comprises and produces in many ways butyryl coenzyme A and use butyryl coenzyme A to produce butanols as intermediate.
Although with reference to specific features the present invention is described in detail, it will be understood to those of skill in the art that this description only is preferred embodiment, rather than limit the scope of the invention. Therefore, actual range of the present invention will be limited by appending claims and equivalent thereof.
Sequence table
＜110〉Biofuelchem Co. Ltd
＜120〉use bacterium by the method for butyryl coenzyme A as the intermediate preparation butanols
<130>FP09KR839
<140>PCT/KR2007/006524
<141>2007-12-14
<150>60/875,145
<151>2006-12-15
<150>60/899,201
<151>2007-02-02
<160>42
<170>PatentIn?version?3.2
<210>1
<211>30
<212>DNA
<213>Artificial
<220>
<223>hbdf?primer
<400>1
acgcgaattc?atgaaaaagg?tatgtgttat????????????????????????????????30
<210>2
<211>47
<212>DNA
<213>Artificial
<220>
<223>hbdr?primer
<400>2
gcgtctgcag?gagctcctgt?ctctagaatt?tgataatggg?gattctt?????????????47
<210>3
<211>30
<212>DNA
<213>Artificial
<220>
<223>thiLf?primer
<400>3
acgcgagctc?tatagaattg?gtaaggatat????????????????????????????????30
<210>4
<211>30
<212>DNA
<213>Artificial
<220>
<223>thiLr?primer
<400>4
gcgtgagctc?attgaacctc?cttaataact????????????????????????30
<210>5
<211>30
<212>DNA
<213>Artificial
<220>
<223>groESLf?primer
<400>5
agcttctaga?ctcaagatta?acgagtgcta????????????????????????30
<210>6
<211>30
<212>DNA
<213>Artificial
<220>
<223>groESLr?primer
<400>6
tagctctaga?ttagtacatt?ccgcccattc????????????????????????30
<210>7
<211>26
<212>DNA
<213>Artificial
<220>
<223>bcdf?primer
<400>7
agcgccatgg?attttaattt?aacaag????????????????????????????26
<210>8
<211>30
<212>DNA
<213>Artificial
<220>
<223>bcdr?primer
<400>8
agtcggtacc?cctccttaaa?ttatctaaaa????????????????????????30
<210>9
<211>30
<212>DNA
<213>Artificial
<220>
<223>crt1?primer
<400>9
atacggatcc?gagattagta?cggtaatgtt????????????????????????30
<210>10
<211>30
<212>DNA
<213>Artificial
<220>
<223>crt2?primer
<400>10
gtacctgcag?cttacctcct?atctattttt?????????????????????????????????????30
<210>11
<211>72
<212>DNA
<213>Artificial
<220>
<223>lacl_1st?up?primer
<400>11
gtgaaaccag?taacgttata?cgatgtcgca?gagtatgccg?gtgtctctta?gattgcagca????60
ttacacgtct?tg????????????????????????????????????????????????????????72
<210>12
<211>70
<212>DNA
<213>Artificial
<220>
<223>lacl_1st?do?primer
<400>12
tcactgcccg?ctttccagtc?gggaaacctg?tcgtgccagc?tgcattaatg?cacttaacgg????60
ctgacatggg???????????????????????????????????????????????????????????70
<210>13
<211>72
<212>DNA
<213>Artificial
<220>
<223>ldhA?1st?up?primer
<400>13
atgaaactcg?ccgtttatag?cacaaaacag?tacgacaaga?agtacctgca?gattgcagca????60
ttacacgtct?tg????????????????????????????????????????????????????????72
<210>14
<211>71
<212>DNA
<213>Artificial
<220>
<223>ldhA?1st?do?primer
<400>14
ttaaaccagt?tcgttcgggc?aggtttcgcc?tttttccaga?ttgcttaagt?cacttaacgg????60
ctgacatggg?a?????????????????????????????????????????????????????????71
<210>15
<211>71
<212>DNA
<213>Artificial
<220>
<223>ldhA1stdo?primer
<400>15
ttaaaccagt?tcgttcgggc?aggtttcgcc?tttttccaga?ttgcttaagt?cacttaacgg????60
ctgacatggg?a?????????????????????????????????????????????????????????71
<210>16
<211>47
<212>DNA
<213>Artificial
<220>
<223>hbdr?primer
<400>16
gcgtctgcag?gagctcctgt?ctctagaatt?tgataatggg?gattctt??????????????????47
<210>17
<211>30
<212>DNA
<213>Artificial
<220>
<223>adhEf?primer
<400>17
acgctctaga?tataaggcat?caaagtgtgt?????????????????????????????????????30
<210>18
<211>30
<212>DNA
<213>Artificial
<220>
<223>adhEr?primer
<400>18
gcgtgagctc?catgaagcta?atataatgaa?????????????????????????????????????30
<210>19
<211>30
<212>DNA
<213>Artificial
<220>
<223>thiLf?primer
<400>19
acgcgagctc?tatagaattg?gtaaggatat?????????????????????30
<210>20
<211>30
<212>DNA
<213>Artificial
<220>
<223>thiLr?primer
<400>20
gcgtgagctc?attgaacctc??cttaataact????????????????????30
<210>21
<211>30
<212>DNA
<213>Artificial
<220>
<223>atof?primer
<400>21
atacgagctc?tacggcgagc??aatggatgaa????????????????????30
<210>22
<211>30
<212>DNA
<213>Artificial
<220>
<223>ator?primer
<400>22
gtacgagctc?gattaattca?accgttcaat?????????????????????30
<210>23
<211>39
<212>DNA
<213>Artificial
<220>
<223>phaAf?primer
<400>23
agtcgagctc?aggaaacaga?tgactgacgt?tgtcatcgt???????????39
<210>24
<211>30
<212>DNA
<213>Artificial
<220>
<223>phaAr?primer
<400>24
atgcgagctc?ttatttgcgc?tcgactgcca?????????????????????30
<210>25
<211>30
<212>DNA
<213>Artificial
<220>
<223>ydbMf?primer
<400>25
agcttctaga?gatgggttac?ctgacatata????????????????30
<210>26
<211>30
<212>DNA
<213>Artificial
<220>
<223>ydbMr?primer
<400>26
agtctctaga?ttatgactca?aacgcttcag????????????????30
<210>27
<211>30
<212>DNA
<213>Artificial
<220>
<223>bcdPA01f?primer
<400>27
agcttctaga?actgctcctt?ggacagcgcc????????????????30
<210>28
<211>30
<212>DNA
<213>Artificial
<220>
<223>bcdPA01r?primer
<400>28
agtctctaga?ggcaggcagg?atcagaacca????????????????30
<210>29
<211>30
<212>DNA
<213>Artificial
<220>
<223>bcdKT2440f?primer
<400>29
agcttctaga?actgttcctt?ggacagcgcc????????????????30
<210>30
<211>30
<212>DNA
<213>Artificial
<220>
<223>bcdKT2440r?primer
<400>30
agtctctaga?ggcaggcagg?atcagaacca????????????????30
<210>31
<211>26
<212>DNA
<213>Artificial
<220>
<223>bcdf?primer
<400>31
agcgccatgg?attttaattt?aacaag????????????????????26
<210>32
<211>30
<212>DNA
<213>Artificial
<220>
<223>bcdr?primer
<400>32
agtcggtacc?cctccttaaa?ttatctaaaa????????????????30
<210>33
<211>30
<212>DNA
<213>Artificial
<220>
<223>bdhABf?primer
<400>33
acgcggatcc?gtagtttgca?tgaaatttcg????????????????30
<210>34
<211>41
<212>DNA
<213>Artificial
<220>
<223>bdhABr?primer
<400>34
agtcctgcag?ctatcgagct?ctataatggc?tacgcccaaa?c???41
<210>35
<211>30
<212>DNA
<213>Artificial
<220>
<223>crtf?primer
<400>35
actcgagctc?aaaagccgag?attagtacgg????????????????????????30
<210>36
<211>30
<212>DNA
<213>Artificial
<220>
<223>crtr?primer
<400>36
gcgtctgcag?cctatctatt?tttgaagcct????????????????????????30
<210>37
<211>30
<212>DNA
<213>Artificial
<220>
<223>mhpFf?primer
<400>37
atgcgaattc?atgagtaagc?gtaaagtcgc????????????????????????30
<210>38
<211>48
<212>DNA
<213>Artificial
<220>
<223>mhpFr?primer
<400>38
tatcctgcag?gagctctcta?gagctagctt?accgttcatg?ccgcttct????48
<210>39
<211>30
<212>DNA
<213>Artificial
<220>
<223>paaFGHf?primer
<400>39
atacgctagc?atgaactggc?cgcaggttat????????????????????????30
<210>40
<211>30
<212>DNA
<213>Artificial
<220>
<223>paaFGHr?primer
<400>40
tatcgagctc?gccaggcctt?atgactcata????????????????????????30
<210>41
<211>30
<212>DNA
<213>Artificial
<220>
<223>atoBf?primer
<400>41
atacgagctc?tgcatcactg?ccctgctctt????????????????30
<210>42
<211>30
<212>DNA
<213>Artificial
<220>
<223>atoBr?primer
<400>42
tgtcgagctc?cgctatcggg?tgtttttatt????????????????30
Claims (37)
1, a kind of method of producing butanols, described method comprises: cultivate to contain in the substratum that contains butyric acid or etheric acid and be responsible for coding and butyryl coenzyme A changed into the recombinant bacteria of gene of enzyme (AdhE) of butanols to produce butanols, coding CoAT (acetyl-CoA: the butyryl coenzyme A transferring enzyme) be introduced in the described bacterium; And from nutrient solution, reclaim butanols.
2, according to the method for the production butanols of claim 1, it is characterized in that: the gene of the gene of coding thiolase (THL) and coding E.C. 4.1.1.4 (AADC) is incorporated in the recombinant bacteria in addition.
3, according to the method for the production butanols of claim 1 or 2, it is characterized in that: described bacterium is intestinal bacteria.
4, according to the method for the production butanols of claim 1, it is characterized in that: (acetyl-CoA: the gene butyryl coenzyme A transferring enzyme) is ctfA and ctfB to coding CoAT.
5, according to the method for the production butanols of claim 4, it is characterized in that: described ctfA and ctfB are from fusobacterium.
6, according to the method for the production butanols of claim 2, it is characterized in that: the gene of coding THL is thl or the thiL from fusobacterium, from the phaA of Bacillaceae or from colibacillary atoB.
7, according to the method for the production butanols of claim 2, it is characterized in that: the gene of coding AADC is the adc from fusobacterium.
8, according to the method for the production butanols of claim 1, it is characterized in that: described cultivation is under anaerobic carried out.
9, a kind of method of producing butyryl coenzyme A, described method comprises: (acetyl-CoA: the gene butyryl coenzyme A transferring enzyme) is introduced in recombinant bacteria wherein to cultivate coding CoAT in the substratum that contains butyric acid or etheric acid.
10, according to the method for the production butyryl coenzyme A of claim 9, it is characterized in that: the gene of the gene of coding thiolase (THL) and coding E.C. 4.1.1.4 (AADC) is incorporated in the recombinant bacteria in addition.
11, according to the method for the production butyryl coenzyme A of claim 9 or 10, it is characterized in that: described bacterium is intestinal bacteria.
12, a kind of method of producing butyryl coenzyme A, described method comprises: cultivate coding AtoDA (acetyl-CoA: the etheric acid thiophorase) be introduced in wherein recombinant bacteria in containing butyro-substratum.
13, according to the method for the production butyryl coenzyme A of claim 12, it is characterized in that: described bacterium is intestinal bacteria.
14, a kind of method of producing butanols, described method comprises: cultivate gene that contains the AtoDA that encodes and the gene bacterium of encoding AdhE to produce butanols in containing butyro-substratum; From nutrient solution, reclaim butanols.
15, according to the method for the production butyryl coenzyme A of claim 14, it is characterized in that: described bacterium is intestinal bacteria.
16, a kind of method of producing butyryl coenzyme A, described method comprises: cultivate in the substratum that contains butyric acid or etheric acid and contain coding AtoDA (acetyl-CoA: the etheric acid thiophorase), FadB or PaaH (3-glycoloyl coa dehydrogenase), the bacterium of PaaFG (enoyl-CoA hydratase) and FadE (acetyl-CoA desaturase) gene.
17, according to the method for the production butyryl coenzyme A of claim 16, it is characterized in that described bacterium is intestinal bacteria.
18, a kind of method of producing butanols, described method comprises: in the substratum that contains butyric acid or etheric acid, cultivate coding AtoDA, FadB or PaaH, PaaFG and FadE gene with the microorganism of the gene of coding AdhE to produce butanols; From nutrient solution, reclaim butanols.
19, according to the method for the production butanols of claim 18, it is characterized in that: described bacterium is intestinal bacteria.
20, a kind of recombinant bacteria with production butanols ability, coding thiolase (THL), 3-maloyl group coa dehydrogenase (BHBD), the gene of enoyl-CoA hydratase (CRO) and functional BCD (butyryl-CoA dehydrogenase) is introduced into wherein.
21, according to the recombinant bacteria of producing the butanols ability that has of claim 20, it is characterized in that having the gene that coding changes into butyryl coenzyme A the enzyme (AdhE) of butanols.
According to the recombinant bacteria of producing the butanols ability that has of claim 20 or 21, it is characterized in that 22, the gene of the gene of coding AAD (butyraldehyde desaturase) and/or coding BDH (butanols desaturase) is introduced wherein in addition.
23, according to the recombinant bacteria with production of butanol ability of claim 22, it is characterized in that: the gene of coding AAD be from fusobacterium (adhE or from colibacillary mhpF, the gene of coding BDH is bdhAB from fusobacterium.
24, according to the recombinant bacteria with production of butanol ability of claim 20 or 21, it is characterized in that: the gene of coding THL is thl or the thiL from fusobacterium, from the phaA of Bacillaceae or from colibacillary atoB.
25, according to the recombinant bacteria of producing the butanols ability that has of claim 20 or 21, it is characterized in that: coding BHBD gene is from the hbd of fusobacterium or from colibacillary paaH.
26, according to the recombinant bacteria of producing the butanols ability that has of claim 20 or 21, it is characterized in that: the gene of coding CRO is from the crt of fusobacterium or from colibacillary paaFG.
27, produce the recombinant bacteria of butanols ability according to having of claim 20 or 21, it is characterized in that: the gene of encoding function BCD is from the bcd of Rhodopseudomonas or from the ydbM of Bacillaceae.
28, according to the recombinant bacteria of producing the butanols ability that has of claim 27, it is characterized in that: the gene of coding chaperone is introduced into wherein in addition.
29, according to the recombinant bacteria of producing the butanols ability that has of claim 20 or 21, it is characterized in that: the gene of coding chaperone is introduced into wherein in addition, and wherein the gene of encoding function BCD is the bcd from Rhodopseudomonas.
30, according to the recombinant bacteria of producing the butanols ability that has of claim 20 or 21, it is characterized in that: lacI gene (coding lac operon aporepressor) is rejected the expression of gene of being responsible for the biosynthetic enzyme of coding butanols to increase.
31, produce the recombinant bacteria of butanols ability according to having of claim 30, it is characterized in that: the gene that further is coded in the enzyme that lactic biological relates in synthetic is rejected in addition.
32, produce the recombinant bacteria of butanols ability according to having of claim 31, it is characterized in that: the gene that is coded in the enzyme that lactic biological relates in synthetic is ldhA (a coding serum lactic dehydrogenase).
33, a kind of recombinant bacteria with production butanols ability, coding THL, BHBD, enoyl-CoA hydratase, functional BCD, AAD, the gene of BDH and chaperone is introduced into wherein, and the gene of lacI gene (coding lac operon aporepressor) and the enzyme that relates in lactic biological is synthetic is disallowable.
34, a kind of method of producing butanols, described method comprises: the recombinant bacteria of cultivating claim 20 or 21 in substratum is to produce butanols; And from nutrient solution, reclaim butanols.
35, a kind of method of producing butanols, described method comprises: the recombinant bacteria of cultivating claim 22 in substratum is to produce butanols; And from nutrient solution, reclaim butanols.
36, a kind of method of producing acetyl-CoA, described method comprises the recombinant bacteria of cultivating claim 20.
37, a kind of method of producing butanols, described method comprises: the recombinant bacteria of cultivating claim 33 in substratum is to produce butanols; And from nutrient solution, reclaim butanols.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US87514506P | 2006-12-15 | 2006-12-15 | |
| US60/875,145 | 2006-12-15 | ||
| US89920107P | 2007-02-02 | 2007-02-02 | |
| US60/899,201 | 2007-02-02 | ||
| PCT/KR2007/006524 WO2008072920A1 (en) | 2006-12-15 | 2007-12-14 | Method for preparing butanol through butyryl-coa as an intermediate using bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101631869A true CN101631869A (en) | 2010-01-20 |
| CN101631869B CN101631869B (en) | 2013-06-19 |
Family
ID=41576362
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200780046310A Pending CN101668852A (en) | 2006-12-15 | 2007-12-14 | Enhanced butanol producing microorganisms and method for preparing butanol using the same |
| CN200780046237.1A Active CN101631869B (en) | 2006-12-15 | 2007-12-14 | Method for preparing butanol through butyryl-CoA as an intermediate using bacteria |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200780046310A Pending CN101668852A (en) | 2006-12-15 | 2007-12-14 | Enhanced butanol producing microorganisms and method for preparing butanol using the same |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN101668852A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104789487A (en) * | 2014-01-17 | 2015-07-22 | 逢甲大学 | Bacterial strain capable of respectively producing butyric acid and n-butanol and method for producing n-butanol |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206615B (en) * | 2011-04-27 | 2013-06-12 | 珠海市御品堂生物科技有限公司 | Process for preparing alcohol dehydrogenase by enzymolysis method |
| CN102399736A (en) * | 2011-10-18 | 2012-04-04 | 江南大学 | Genetic engineering bacterium for producing monophosphoryl lipid A as well as construction method and application thereof |
| CN102618569B (en) * | 2012-03-15 | 2013-09-18 | 南京工业大学 | Construction of butanol producing genetic engineering bacteria, strain and application thereof |
| CN103865951B (en) * | 2012-12-18 | 2018-06-26 | 上海市农业科学院 | Saccharomyces cerevisiae expression and its structure and application |
-
2007
- 2007-12-14 CN CN200780046310A patent/CN101668852A/en active Pending
- 2007-12-14 CN CN200780046237.1A patent/CN101631869B/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104789487A (en) * | 2014-01-17 | 2015-07-22 | 逢甲大学 | Bacterial strain capable of respectively producing butyric acid and n-butanol and method for producing n-butanol |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101668852A (en) | 2010-03-10 |
| CN101631869B (en) | 2013-06-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100971790B1 (en) | Method for preparing butanol through butyryl-coa as an intermediate using bacteria | |
| CN105051179B (en) | Recombinant microorganism and its purposes | |
| KR101229525B1 (en) | Advanced microorganism for producing 1,2-propanediol | |
| Lehmann et al. | Switching Clostridium acetobutylicum to an ethanol producer by disruption of the butyrate/butanol fermentative pathway | |
| CN100506991C (en) | Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism | |
| CN101058799B (en) | Method of producing polyhydroxyalkanoates and special-purpose engineering bacterium for the same | |
| CN101952430B (en) | Enhanced ethanol and butanol producing microorganisms and method for preparing ethanol and butanol using the same | |
| CN104508136A (en) | Recombinant microorganisms and uses therefor | |
| CN101679940A (en) | Metabolically engineered microorganism useful for the production of 1,2-propanediol | |
| CN101437936A (en) | Solvent tolerant microorganisms and methods of isolation | |
| CN101473027A (en) | Solvent tolerant microorganisms and methods of isolation | |
| JP2012511908A (en) | Biosynthesis of commodity chemicals | |
| CA2915524A1 (en) | Method for producing organic compositions from oxyhydrogen and co2 via acetoacetyl-coa as intermediate product | |
| CN101631869B (en) | Method for preparing butanol through butyryl-CoA as an intermediate using bacteria | |
| AU2017202804B2 (en) | Microbial production of n-butyraldehyde | |
| KR20080077080A (en) | Method for preparing butanol through butyryl-coa as an intermediate using yeast | |
| Hölscher et al. | Production of the chiral compound (R)-3-hydroxybutyrate by a genetically engineered methylotrophic bacterium | |
| JP2020536520A (en) | Production of polyhydroxybutyrate in WOOD-LJUNGDAHL microorganisms | |
| RU2375451C1 (en) | RECOMBINANT PLASMID DNA, CONTAINING GENES OF BUTANOL SYNTHESIS FROM Clostridium acetobutylicum (VERSIONS), RECOMBINANT STRAIN Lactobacillus brevis - PRODUCER OF N-BUTANOL (VERSIONS) AND METHOD FOR MICROBIOLOGICAL SYNTHESIS OF N-BUTANOL | |
| JP2009247217A (en) | Method for producing 2-propanol using aerobic recombinant microorganism | |
| US20160138049A1 (en) | OXYGEN-TOLERANT CoA-ACETYLATING ALDEHYDE DEHYDROGENASE CONTAINING PATHWAY FOR BIOFUEL PRODUCTION | |
| WO2017035141A2 (en) | Microbial organisms for converting acetyl-coa into crotyl alcohol and methods for producing crotyl alcohol | |
| CN116783289A (en) | Method and cell for producing volatile compounds | |
| AS | Molecular Characterization and Enzyme Analysis of Butanol Tolerant Bacterium Paenibacillus sp | |
| CN101522889A (en) | Thermophilic microorganisms for ethanol production |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20160225 Address after: Seoul, South Kerean Patentee after: GS Caltex Corp. Address before: South Korea Patentee before: Biofuelchem Co. Ltd |