CN101631869B - 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
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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) to produce butanols as intermediate in can the bacterium of biosynthesizing butanols.
Background technology
Along with oil price sharp rise and for the worry that day by day increases to Global warming and Greenhouse effect, biofuel is using microorganism to carry out to it the concern that day by day increased aspect production 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 conduct in recent years 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 having high-energy-density, low-steam pressure, being similar to octane rating and the low impurity content of gasoline, it can mix with the ratio higher than ethanol with existing gasoline, and do not damage performance, mileage or organic pollutant criteria.Produce in a large number butanols by microorganism and can reach the economy and environment advantage that reduces crude oil import and greenhouse gas emission.
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, the people such as E.A., Necleic Acids Research, 29:3583,2001).This biological method is the major technique of producing for butanols and acetone over more than 40 year, until eighties of last century fifties.It is because its complicated growth conditions and to be used for its biology tool and the development of its omics technology (omics technology) not mature enough that but clostridium (Clostridial) bacterial strain is difficult to further improve.
Therefore, suggestion utilizes microorganism such as Fast Growth under normal operation and can develop into the production of butanol bacterial strain with the intestinal bacteria of multiple omics technology operation.Particularly, be used for a small amount of metabolic engineering of production of butanol bacterial strain development and omics technology and be employed thereon intestinal bacteria bacterial classification, make it have the very big potential that develops into the production of butanol bacterial strain.
Butanols biosynthetic pathway generation butanols (Jones, D.T. and Woods, D.R., Microbiol.Rev., 50:484,1986 of clostridium acetobutylicum (Clostridium acetobutylicum) by showing in Fig. 1; Desai, the people such as R.P., J.Biotechnol., 71:191,1999).In wild-type e. coli, ethanol is through to wherein under anaerobic the similar approach that plays a crucial role of derivable adhE (coding is responsible for being produced the enzyme AdhE of ethanol through acetaldehyde by acetyl-CoA) is synthetic.Intestinal bacteria can contain for the necessary gene of the biosynthesizing of butyryl coenzyme A and butanols, but different from its corresponding gene in clostridium (Clostridia) are, the too low and effectively corresponding enzyme reaction of catalysis of its expression level.
Simultaneously, the butanols biosynthetic pathway is derivative therein can produce the recombinant bacteria of butanols and use the method for described bacterium production butanols to be disclosed (US 2007/0259410 A1; But production efficiency general (modest) US2007/0259411 A1).
The present inventor has paid and has made great efforts greatly to utilize bacterium (particularly intestinal bacteria), its body contain be responsible for coding can the multiple butyryl coenzyme A that will produce in bacterium 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 multiple production as the method for 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 be produced by butyryl coenzyme A the method for butanols as the bacterium of intermediate biosynthesizing butanols.
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 described bacterium; And reclaim butanols from nutrient solution.
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 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 recombinant bacteria in containing the substratum of butyric acid.
In addition, the invention provides the method that produces butanols, described method comprises: cultivate the gene that contains the AtoDA that encodes and the gene bacterium of encoding AdhE to produce butanols in containing the substratum of butyric acid; Reclaim butanols from nutrient solution.
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: cultivate coding AtoDA in the substratum that contains butyric acid or etheric acid, the microorganism together with FadB or PaaH, the gene of PaaFG and FadE gene and coding AdhE is to produce butanols; Reclaim butanols from nutrient solution.
The present invention also provides has that butanols produces the recombinant microorganism of ability and for the production of the method for butanols, coding thiolase (THL), 3-maloyl group coa dehydrogenase (BHBD), enoyl-CoA hydratase (CRO), be introduced in described microorganism with the gene of functional BCD (butyryl-CoA dehydrogenase), described method comprises: cultivate recombinant microorganism in substratum; And reclaim butanols from nutrient solution.
The present invention also provides the method that produces acetyl-CoA, and described method comprises: cultivate coding THL, BHBD is in the recombinant bacteria that the gene of enoyl-CoA hydratase and functional BCD is introduced.
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 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 reclaim butanols from nutrient solution.
By following detailed description and appending claims, other features of the present invention and aspect will be apparent.
Description of drawings
Fig. 1 is the schematic diagram that shows butanols biosynthetic pathway in clostridium acetobutylicum (Clostridium acetobutylicum).
Fig. 2 is the schematic diagram that shows the butanols biosynthetic pathway of inferring in recombination bacillus coli according to the present invention.
Fig. 3 is presented at the schematic diagram that causes producing through butyryl coenzyme A the biosynthetic pathway of butanols in ato system and/or 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 construction process and the gene mapping of pKKhbdthiL carrier.
Fig. 6 has shown construction process and the gene mapping of pTrc184bcdcrt carrier.
Fig. 7 has shown construction process and the gene mapping of pKKhbdadhEthiL (pKKHAT) carrier.
Fig. 8 has shown construction process and the gene mapping of pKKhbdadhEatoB (pKKHAA) carrier.
Fig. 9 has shown construction process and the gene mapping of pKKhbdadhEphaA (pKKHAP) carrier.
Figure 10 has shown construction process and the gene mapping of pKKhbdydbMadhEphaA (pKKHYAP) carrier.
Figure 11 has shown construction process and the gene mapping of pKKhbdbcdPA01adhEphaA (pKKHPAP) carrier.
Figure 12 has shown construction process and the gene mapping of pKKhbdbcdKT2440adhEphaA (pKKHKAP) carrier.
Figure 13 has shown construction process and the gene mapping of pTrc184bcdbdhABcrt (pTrc184BBC) carrier.
Figure 14 has shown the butanols biosynthetic pathway under the following situations: 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 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 in order to produce acetone (Bermejo, the people such as L.L., Appl.Environ.Microbiol., 64:1079,1998) by acetyl-CoA through the etheric acid coenzyme A.
Predictably, when replacing the coenzyme A residue of etheric acid coenzyme A, butyryl coenzyme A can be produced (Fig. 2) when using the CoAT enzyme from clostridium acetobutylicum (Clostridium acetobutylicum) (be responsible for butyric acid (BA) or acetic acid and change into butyryl coenzyme A or acetyl-CoA) by expression of recombinant e. coli.And predictably, the AdhE enzyme catalysis butyryl coenzyme A of under anaerobic expressing and be responsible for acetyl-coenzyme A is changed into the recombination bacillus coli (ATCC11303 (pACT)) of ethanol changes into butanols to produce butanols (Fig. 2).
In order to confirm above-mentioned prediction, recombination bacillus coli is cultivated in containing the substratum of butyric acid, result can be found out, butyric acid is converted to butanols through butyryl coenzyme A, show that this is due to by being introduced in ctfA in recombinant chou and the CoAT enzyme of ctfB genes encoding, this is consistent with the AdhE enzyme of under anaerobic expressing.
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 described bacterium; And reclaim butanols from nutrient solution.
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 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 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 to this.Preferably, express in recombinant microorganism of the present invention by the adc genes encoding from fusobacterium (Clostridium sp.), but be not limited to this.As long as it is expressed as the enzyme that has with activity identical in Host Strains, any foreign gene all can use in the present invention ad lib.
In the present invention, Host Strains is preferably intestinal bacteria.But as long as it holds the gene of the AdhE that encodes, Host Strains is not limited to this.
In an embodiment of the present invention, the wild-type e. coli when not having pACT to be introduced in is wherein containing that when cultivating in the substratum of butyric acid and/or etheric acid, butanols is detected.
Producing butanols by the wild-type e. coli cultivated in containing the substratum of butyric acid is considered to due to the AtoDA of ato system, butyric acid be changed into butyryl coenzyme A (Lioliou and Kyriakidis, Microbial Cell Factories, 3:8,2004) then become the result (Fig. 3) of butanols 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 the 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 reclaim butanols from nutrient solution.
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 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 to this, as long as it can hold said gene.
In addition, the method of producing butanols by cultivate wild-type e. coli in containing the substratum of etheric acid is assumed that it is by the ato system, etheric acid to be changed into the etheric acid coenzyme A then by the FadB (perhaps PaaH) of fad 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 related to together with FadA in following reaction:
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 the enoyl-CoA hydratase of being responsible for the beta-hydroxy butyryl coenzyme A is changed into crotonyl-CoA.FadE be relate in below reaction crotonyl-CoA is changed into the acyl group-coa dehydrogenase of butyryl coenzyme A:
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 the FadE gene bacterium together with the gene of AdhE of being responsible for coding butyryl coenzyme A is changed into butanols is to produce butanols; And reclaim butanols from nutrient solution.
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 be introduced in bacterium such as the intestinal bacteria of gene that contain coding and play the enzyme (AdhE) that butyryl coenzyme A is changed into butanols in the time, 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 thl, gene thiL is known as coding from THL (Nolling, the people such as J., the J.Bacteriol. of 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 thl or thiL from fusobacterium (Clostridium sp.), belong to the phaA of (Ralstonia sp.) or also be found to demonstrate bacterium THL from colibacillary atoB active from Ralstonia solanacearum, as the detection of use production of butanol.Therefore, belong to the phaA of (Ralstonia sp.) or can be used to replace thl or thiL from colibacillary atoB from Ralstonia solanacearum.In addition, as long as it is expressed in host cell to show that THL is active, any gene of coding THL, even foreign gene all can be used and not restriction.
And the people such as Bennett have reported and be expressed (Boynton, the 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 except BCD 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 etfA) in clostridium acetobutylicum (Clostridium acetobutylicum)) is very weak, perhaps due to the very low stability of BCD or its cofactor 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 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 the 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 being expressed, it shows that BCD is active in host cell, although the BCD gene is all can being used of external source and not restriction.
In an embodiment of the present invention, determine to contain the 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 bcd and the groESL of fusobacterium (Clostridium sp.) from the ydbM of Bacillaceae (Bacillus sp.).
Therefore, on the other hand, the present invention relates to have that butanols produces the recombinant bacteria of ability and for the production of the method for butanols, coding thiolase (THL), 3-maloyl group coa dehydrogenase (BHBD), enoyl-CoA hydratase (CRO), and the gene of functional BCD (butyryl-CoA dehydrogenase) is introduced in described bacterium, described method comprises: cultivate recombinant bacteria in substratum; And reclaim butanols from nutrient solution.
The present invention also provides the method that produces butyryl coenzyme A, and described method comprises: the cultivation THL that will encode, BHBD, the recombinant bacteria that the gene of enoyl-CoA hydratase and functional BCD is introduced.
Consequent intermediate butyryl coenzyme A is converted to butanols by the AdhE enzyme of being encoded by the native gene of coding AdhE in bacterium.Intestinal bacteria have been carried coding and butyryl coenzyme A have been changed into the gene of the enzyme (AdhE) of butanols.In the situation that do not carry the host cell of 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 in the situation that host cell itself holds the 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 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 to this.For example, also can be used from the ADD-encoding gene of the microorganism outside fusobacterium (Clostridium sp.), as long as they are expressed to show that identical AAD is active just uses restriction.And the gene of coding BDH is preferably the bdhAB from fusobacterium (Clostridiumsp.), but is not limited to this.For example, the gene from the microorganism outside fusobacterium (Clostridium sp.) also can be used and less than restriction, need only them and be expressed to show that identical BDH is active.
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 to this.For example, any foreign gene all can be used and not restriction, as long as it is expressed to show BHBD (FadB in intestinal bacteria or PaaH) activity and enoyl-CoA hydratase (PaaFG in intestinal bacteria) activity in host cell.In an embodiment of the present invention, be replaced by from the gene of colibacillary paaH (coding 3-hydroxyacyl-CoA dehydrogenase) and paaFG (coding enoyl-CoA hydratase) even therein that in situation from the hbd of fusobacterium (Clostridium sp) and crt, butanols is determined still is detected.
The meaning of the term " functional BCD " that uses in this article is to be introduced in host cell such as colibacillary bcd gene to be expressed to show that BCD is active.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 to this.Because it shows faint activity in intestinal bacteria, also can be included in from the bcd of fusobacterium (Clostridium sp.) and contain in functional BCD-encoding gene, as BCD activity when it is introduced into together with the gene (groESL) of coding chaperone be enhanced.
According to an aspect of the present invention, preferably, the gene of coding chaperone is introduced in 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 be 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 lacI gene (coding lac operon aporepressor) wherein and encode and be responsible for the gene of the synthetic enzyme of lactic biological, built thus the intestinal bacteria through recombination mutation that produce butanols, thereby determined that production of butanol power increases significantly in described recombination mutation microorganism.
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 due 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
By the following EXAMPLE Example of enumerating to explain the present invention but being not interpreted as restriction of the present invention, can obtain the present invention is better understood.
Although 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 being introduced in the gene that relates in the butanols biosynthesizing in order to produce butanols, other coli strains, bacterium, yeast and fungi also can be used as host cell.
In addition, although be used as in the following embodiments the example of gene to be introduced from the gene of specific bacterial strain, but it will be apparent to those skilled in the art that as long as they are expressed to show identical activity in host cell, any gene all may be utilized and not restriction.
And, should be noted that, although in the following embodiments only with defined medium and cultural method as an example, saccharification liquid, such as whey, CSL (corn leaching solution) etc., and other substratum and multiple cultural method such as (people such as Lee, the Bioprocess Biosyst.Eng. such as (fed-batch) cultivation, cultured continuously in batches, 26:63,2003; The people such as Lee, Appl.Microbiol.Biotechnol., 58:663,2002; The people such as Lee, Biotechnol.Lett., 25:111,2003; The people such as Lee, Appl.Microbiol.Biotechnol., 54:23,2000; The 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 substratum and cultivated 1 hour under aerobic conditions, 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 regulator of the butyric acid pH value 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 48 hours in the substratum of the 0.8mM butyric acid that has added 200 μ l, and ' C ' is illustrated in the control group of cultivating in the substratum that there is no butyric acid.
As a result, as shown in table 1, any ethanol, butanols, acetic acid or butyric acid do not detected in negative control, LB are cultivated in, and only produced acetic acid and ethanol in the positive control that does not add butyric acid.A kind of exception is, low-level butanols detected after cultivating 164 hours, and this may be from butyryl coenzyme A, may not be also.In contrast, when being cultivated and producing butyric acid, ethanol, show that the AdhE enzyme under anaerobic is expressed and produces the expression that acetone shows the CoAT enzyme when recombination bacillus coli [ATCC 1103 (pACT)], thus final definite butanols that produces.
Table 1: use the HPLC of recombination bacillus coli [ATCC 1103 (the pACT)] culture supernatants of inducing from butyric acid investigation (mM) to analyze
Embodiment 2: prepare butanols by adding etheric acid
Identical in culturing process and embodiment 1, but use LB and the M9 substratum that contains etheric acid and butyric acid.That is to say, 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 analyzes culture (table 2) by HPLC.In table 2, ' 10-M9-200-2-72h ' refers in the M9 substratum that contains 10mM etheric acid and 20mM butyric acid and cultivated 72 hours, ' 400 ' expression 40mM butyric acid, and ' C ' is illustrated in the control group of cultivating in the substratum that there is no butyric acid.
As a result, as shown in table 2, butanols is detected at the substratum that contains etheric acid with in containing the substratum of etheric acid and butyric acid, and irrelevant with the kind of substratum.
Table 2: the HPLC from recombination bacillus coli [ATCC 1103 (the pACT)] culture supernatants of using etheric acid (10mM) and/or butyric acid (20mM or 40mM) to induce analyzes
All concentration 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 preculture 24 hours during wild-type e. coli is in the culture tube that contains 15ml substratum (LB that contains 30g/L glucose).Be that 2.02 culture is inoculated in the flask of the substratum that contains 500ml with the OD value.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 be placed in the aerobic chamber and add respectively the fresh culture of the 30ml that is placed in anaerobic room.With 300 μ l, etheric acid lithium (Sigma, the A-8509) solution of 0.108g/ml joins in each pipe, and final concentration is 10mM, and to add butyric acid be 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 the LB substratum that contains 10mM etheric acid and 0.8mM butyric acid and cultivated 72 hours, and ' LC8 ' is illustrated in and only contains etheric acid and do not have to cultivate as a control group in the substratum of butyric acid.
As a result, as shown in table 3, can determine to produce butanols when wild-type e. coli (ATCC11303) is cultivated in the substratum that contains etheric acid and butyric acid, as can being shown in embodiment 1.In addition, butanols also is detected in the substratum that only contains etheric acid.
Table 3: the HPLC from wild-type e. coli ATCC 11303 (w/0 pACT) culture supernatants of using etheric acid (10mM) and/or butyric acid (0.8mM) to induce analyzes
All concentration 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 intestinal bacteria that the approach that is produced butyryl coenzyme A by acetyl-coenzyme A is introduced
The 4-1:pKKhbdthiL Vector construction
For the essential gene of butanols biosynthetic pathway, comprise hbd (coding 3-maloyl group coa dehydrogenase) and thiL (coding thiolase) is amplified and sequentially be cloned in pKK223-3 expression vector (Pharmacia Biotech), 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 lower sex change 20 seconds, under 55 ℃, annealing is 30 seconds, 72 ℃ of downward-extensions 1 minute.The PCR product that obtains (hbd gene) uses EcoRI and PstI to digest and is inserted in the pKK223-3 expression vector (PharmaciaBiotech) that uses the same restrictions enzymic digestion, thereby builds pKKhbd expression vector (Fig. 5)
In order to build 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 process, and then is inserted in the pKKhbd carrier that uses same restrictions enzyme (SacI) digestion, thereby builds 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′-gcgtctgcaggagctcctgtctctagaatttgataatggggattctt-3′
[SEQ ID NO：3]thiLf：5′-acgcgagctctatagaattggtaaggatat-3′
[SEQ ID NO：4]thiLr：5′-gcgtgagctcattgaacctccttaataact-3′
In order to build the pKKhbdgroESLthiL carrier, use primer SEQ ID NOS:5 and 6 to carry out PCR with the chromosomal DNA of clostridium acetobutylicum (Clostridiumacetobutylicum) as template.The PCR product that obtains (groESL gene) uses the XbaI cracking, then is inserted in the pKKhbdthiL carrier that uses same restrictions enzyme (XbaI) digestion, thereby builds pKKhbdgroESLthiL carrier (Fig. 5).
[SEQ ID NO：5]groESLf：5’-agcttctagactcaagattaacgagtgcta-3’
[SEQ ID NO：6]groESLr：5’-tagctctagattagtacattccgcccattc-3’
The 4-2:pTrc184bcdcrt Vector construction
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 digestion and it is cloned in pTrc99A expression vector (AmershamPharmacia Biotech), thereby builds the pTrc99Abcd carrier.To be inserted in the pACYC184 (New England Biolabs) that uses same restrictions enzyme (BspHI and EcoRV) digestion by the DNA fragmentation by BspHI and EcoRV digestion cutting from the pTrc99Abcd carrier, thereby build 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 build 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 to digest and is inserted in the pTrc184bcd that uses same restrictions enzyme (BamHI and PstI) digestion, thereby builds 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 chromosomal DNA is disallowable, the tac promotor and the trc promotor that are included in the recombinant vectors for preparing in embodiment 4-1 and 4-2 can be worked by composing type, thereby cause being cloned into the gene (hbd in respective carrier, thiL, groESL, bcd and crt) constitutive expression.Be responsible for 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 coding lac operon aporepressor and an inhibition for the lacI gene of the Transcription of the metabolic required lac operon of lactose, then remove antibiotics resistance gene from bacterium, thereby prepare new WL bacterial strain.
[SEQ ID NO：11]lacI_1stup：5′-gtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttagattgcagcattacacgtcttg-3′
[SEQ ID NO：12]lacI_1stdo：5′-tcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgcacttaacggctgacatggg-3′
4-4: the preparation of producing the butanols microorganism
The pKKhbdgroESLthiL carrier for preparing in embodiment 4-1 and 4-2 and pTrc184bcdcrt carrier are introduced in WL bacterial strain in 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 37 ℃ of lower precultures 12 hours in the LB of 10ml nutrient solution.Then, after autoclaving, add glucose (10g/L) in the 100mL LB nutrient solution that keeps 80 ℃ or higher temperature in the 250mL flask and with its cool to room temperature in the anaerobic room that uses nitrogen purge.Be inoculated into 2mL preculture thing in flask and cultivate 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) analyze the butanol concentration in substratum.
Result is summarized 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 obtained above consideration that combines, due to hbd, thiL, bcd, groESL expresses butyryl coenzyme A with the mistake of crt gene and is successfully produced by acetyl-CoA and become butanols by the AdhE enzymatic conversion that itself exists in intestinal bacteria.
Table 4
| Bacterial strain | Butanols (mg/L) |
| W3110 | ND 1 |
| WL+pKKhbdgroESLthiL+pTrc184bcdcrt | 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 thus the WLL bacterial strain.
[SEQ ID NO：13]ldhA1stup：5′-atgaaactcgccgtttatagcacaaaacagtacgacaagaagtacctgcagattgcagcattacacgtcttg-3′
[SEQ ID NO：14]ldhA1stdo：5′-ttaaaccagttcgttcgggcaggtttcgcctttttccagattgcttaagtcacttaacggctgacatggga-3′
5-2:pKKhbdadhEthiL (pKKHAT) Vector construction
for the required gene of butanols biosynthetic pathway, comprise hbd (coding 3-maloyl group coa dehydrogenase), adhE (coding butyraldehyde desaturase: identical from adhE (coding ethanol dehydrogenase) spelling of 1-2 but function is different) and thiL (coding thiolase) use clostridium acetobutylicum (Clostridiumacetobutylicum) (KCTC 1724) chromosomal DNA to increase as template use primer SEQ IDNOS:15 to 20, and be cloned into successively in pKK223-3 expression vector (PharmaciaBiotech), thereby structure recombinant expression vector, called after pKKhbdadhEthiL (pKKHAT) (Fig. 7).
[SEQ ID NO：15]hbdf：5′-acgcgaattcatgaaaaaggtatgtgttat-3′
[SEQ ID NO：16]hbdr：5′-gcgtctgcaggagctcctgtctctagaatttgataatggggattctt-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′
5-3:pKKhbdadhEatoB (pKKHAA) Vector construction
For the atoB (coding acetyl-CoA Transacetylase) with 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 lower sex change 20 seconds, under 55 ℃, annealing is 30 seconds, 72 ℃ of downward-extensions 90 seconds.The PCR product (atoB) that obtains uses SacI to digest and is inserted in the pKKhbdadhE carrier that uses same restrictions enzyme (SacI) digestion, thereby build new recombinant vectors, called after pKKhbdadhEatoB (pKKHAA) (Fig. 8).
[SEQ ID NO：21]atof：5′-atacgagctctacggcgagcaatggatgaa-3’
[SEQ ID NO：22]ator：5′-gtacgagctcgattaattcaaccgttcaat-3’
5-4:pKKhbdadhEphaA (pKKHAP) Vector construction
For the phaA (coding thiolase) with 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 build new recombinant vectors, called after pKKhbdadhEphaA (pKKHAP) (Fig. 9).
[SEQ ID NO：23]phaAf：5′-agtcgagctcaggaaacagatgactg acgttgtcatcgt-3′
[SEQ ID NO：24]phaAr：5′-atgcgagctcttatttgcgctcgactgcca-3′
5-5:pKKhbdydbMadhEphaA (pKKHYAP) Vector construction
For the ydbM (coding putative protein) with 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 build new recombinant vectors, called after pKKhbdydbMadhEphaA (pKKHYAP) (Figure 10).
[SEQ ID NO：25]ydbMf：5′-agcttctagagatgggttacctgacatata-3′
[SEQ ID NO：26]ydbMr：5′-agtctctagattatgactcaaacgcttcag-3′
5-6:pKKhbdbcdPA01adhEphaA (pKKHPAP) Vector construction
For the bcd (coding butyryl-CoA dehydrogenase) with 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 build 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′
5-7:pKKhbdbcdKT2440adhEphaA (pKKHKAP) Vector construction
For the bcd (coding butyryl-CoA dehydrogenase) with 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 build 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 5-8:pTrc184bcdbdhABcrt Vector construction
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 digestion and it is cloned in pTrc99A expression vector (AmershamPharmacia Biotech), thereby builds new recombinant vectors, called after pTrc99Abcd.After using BspHI and EcoRV digestion pTrc99Abcd, cut DNA fragmentation is inserted in the pACYC184 (New England Biolabs) that uses identical restriction enzyme (BspHI and EcoRV) processing thus, 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 to digest and is inserted in the pTrc184bcd that uses same restrictions enzyme (BamHI and PstI) digestion, thereby build 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′-agtcctgcagctatcgagctctataatggctacgcccaaac-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 to digest and is inserted in the pTrc184bcdbdhAB that uses same restrictions enzyme (SacI and PstI) digestion, thereby build 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 the shortage lacI for preparing in embodiment 5-1 and ldhA uses pTrc184bcdbdhABcrt (pTrc184BBC) carrier in embodiment 5-8 and is selected from the carrier conversion of lower group, be included in the pKKhbdadhEthiL (pKKHAT) that embodiment 5-2 builds 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 37 ℃ of lower precultures 12 hours in the LB of 10ml nutrient solution.After autoclaving, add glucose (5g/L) under 80 ℃ in the 250mL flask or in the 100mL LB nutrient solution of allotting under higher temperature and with its cool to room temperature in using the anaerobic room of nitrogen purge.Be inoculated into 2mL preculture thing in flask and cultivated 10 hours under 37 ℃.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 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 the glucose that USA) records substratum exhausts fully, use to be equipped with packed column (Supelco CarbopackTM B AW/6.6%PEG 20M, 2m * 2mm ID, Bellefonte, PA, USA) gas-chromatography (Agillent 6890N GC System, AgilentTechnologies Inc., CA, USA) analyze the butanol concentration in 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 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 and demonstrate the THL activity.
and, the data presentation that butanols produces, compare with the situation (WLL+pKKHAP+pTrc184BBC) that wherein only is introduced into from the bcd of clostridium acetobutylicum (Clostridium acetobutylicum), the butyryl-CoA dehydrogenase activity is therein from clostridium acetobutylicum, the bcd of (Clostridium acetobutylicum) with from subtilis, the situation that the ydbM of (Bacillus subtilis) is introduced into together, (WLL+pKKHYAP+pTrc184BBC) lower or with from Pseudomonas aeruginosa, (Pseudomonasaeruginosa) or pseudomonas putida, in the situation that the bcd of (Pseudomonas putida) is introduced into together, (WLL+pKKHPAP+pTrc184BBC, WLL+pKKHKAP+pTrc184BBC) increased.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 and show that butyryl-CoA dehydrogenase is active.
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，bed， 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, the 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 6-1:pKKmhpFpaaFGHatoB Vector construction
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 pKK223-3 expression vector (Pharmacia Biotech) successively, thereby build 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′-tatcctgcaggagctctctagagctagcttaccgttcatgccgcttct-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 in intestinal bacteria W3110 (WLL) the use embodiment 6-1 of the shortage lacI for preparing in embodiment 5-1 and ldhA in pKKMPA carrier and 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 in embodiment 5-10.
Result, as shown in table 6, with compare when only using the butanols biosynthetic pathway of clostridium acetobutylicum (C.acetobutylicum), when from the corresponding enzyme (adhE → mhpF of colibacillary predicted coding, crt → paaFG, hbd → paaH, the gene of thiL → atoB) and being 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 the mhpF from intestinal bacteria for producing the necessary four kinds of enzymes (butyraldehyde desaturase, enoyl-CoA hydratase, BHBD and THL) of butanols in intestinal bacteria of clostridium acetobutylicum (Clostridiumacetobutylicum), 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), as increase by production of butanol prove.
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 is only preferred implementation, rather than limit the scope of the invention.Therefore, actual range of the present invention will be limited by appending claims and equivalent thereof.
FP09KR839.ST25
SEQUENCE LISTING
＜110〉Biofuelchem Co. Ltd
＜120〉use bacterium to prepare the method for butanols as intermediate by butyryl coenzyme A
<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
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ctgacatggg a 71
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tatcctgcag gagctctcta gagctagctt accgttcatg ccgcttct 48
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Claims (6)
1. produce the method for butanols as intermediate by butyryl coenzyme A for one kind, described method comprises: cultivate in the substratum that contains butyric acid or etheric acid and contain gene, the coding acetyl-CoA that coding is responsible for butyryl coenzyme A is changed into the enzyme AdhE of butanols: the recombinant bacteria of the foreign gene of the gene of butyryl coenzyme A transferring enzyme CoAT, coding thiolase THL and the foreign gene of coding E.C. 4.1.1.4 AADC is to produce butanols; And reclaim butanols from nutrient solution, wherein said bacterium is intestinal bacteria, described coding acetyl-CoA: the gene of butyryl coenzyme A transferring enzyme CoAT is ctfa and ctfB, described ctfA and ctfB are from fusobacterium, the gene of described coding thiolase THL is thl or the thiL from fusobacterium, from the phaA of Bacillaceae or from colibacillary atoB, and the gene of described coding E.C. 4.1.1.4 AADC is the adc from fusobacterium.
2. according to claim 1 the method for production butanols, it is characterized in that: described cultivation is under anaerobic carried out.
3. produce the method for butanols as intermediate by butyryl coenzyme A for one kind, described method comprises: cultivate in containing the substratum of butyric acid and contain the coding acetyl-CoA: the gene of etheric acid thiophorase AtoDA and coding change into butyryl coenzyme A the bacterium of gene of enzyme AdhE of butanols to produce butanols; And reclaim butanols from nutrient solution.
4. according to claim 3 the method for production butanols, it is characterized in that: described bacterium is intestinal bacteria.
5. produce the method for butanols as intermediate by butyryl coenzyme A for one kind, described method comprises: cultivate the coding acetyl-CoA in the substratum that contains butyric acid or etheric acid: etheric acid thiophorase AtoDA, 3-glycoloyl coa dehydrogenase FadB or PaaH, enoyl-CoA hydratase PaaFG and acetyl-CoA desaturase FadE gene also encode butyryl coenzyme A are changed into the bacterium of gene of enzyme AdhE of butanols to produce butanols; And reclaim butanols from nutrient solution.
6. according to claim 5 the method for production butanols, it is characterized in that: described bacterium is intestinal bacteria.
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| US60/899,201 | 2007-02-02 | ||
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| 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 |
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