CN104372031A - Method for synthesizing isoprene from acetic acid, and corresponding reconstituted cell and application thereof - Google Patents
Method for synthesizing isoprene from acetic acid, and corresponding reconstituted cell and application thereof Download PDFInfo
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- CN104372031A CN104372031A CN201410617430.8A CN201410617430A CN104372031A CN 104372031 A CN104372031 A CN 104372031A CN 201410617430 A CN201410617430 A CN 201410617430A CN 104372031 A CN104372031 A CN 104372031A
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Abstract
The invention provides a method for synthesizing isoprene from acetic acid, and a corresponding reconstituted cell and application thereof. The method aims to reconstruct the traditional mevalonic acid way and express the exogenous acetylcoenzyme A synthetase, acetylcoenzyme A carboxylase, acetyl acetylcoenzyme A synthetase, 3-hydroxy-3-methyl glutaryl coenzyme A synthase, hydroxymethyl glutaryl coenzyme A reductase, mevalonate kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-diphosphate decarboxylase, isopentenylpyrophosphoric acid isomerase and isoprene synthetase to finally establish a new metabolic way for synthesizing isoprene from acetic acid in the cell body.
Description
Technical field
The invention belongs to technical field of molecular biology, relate to the method utilizing Biological preparation isoprenoid, being specifically related to a kind of take acetic acid as the method for Material synthesis isoprene and corresponding reconstitution cell thereof and application.
Background technology
Isoprene is a kind of important chemical industry platform chemicals, and it is 95% for the synthesis of rubber.In addition, isoprene also can be used on the aspects such as medical pesticide intermediate, ucon oil additive, vulcanizer and aviation fuel, and its exploitation prospect is very wide.
At present, the source of isoprene is mainly by petroleum-based feedstock iso-pentane, dehydrogenation of isoamylene method, chemical synthesis (comprising iso-butylene-formaldehyde method, acetylene-acetone method, propylene dimerization) and pyrolysis C 5 extractive distillation method.But along with the exhaustion day by day of fossil resource, raw material sources are the important bottleneck problems utilizing petroleum-based feedstock to prepare isoprene.
The pathways metabolism that in organism, main existence two kinds is natural carries out the biosynthesizing of isoprene, i.e. mevalonic acid (MVA) approach and methyl E4P (MEP) approach.MVA approach is mainly present in the enchylema of eukaryote, archeobacteria and higher plant, and MEP approach is present in the plastid of plant, bacterium, algae.The final product of this two classes pathways metabolism is all the precursor substance dimethylallylpyrophosphate (dimethylallyl diphosphate, DMAPP) forming isoprene, afterwards through isoprenoid synthase catalysis DMAPP to isoprene.
Microorganism has fast growth, fermentation period is short, genetic background is clear, be easy to through engineering approaches operation, can utilize the features such as cheap renewable resources, and therefore microorganism has become the effective means of the chemical of production bio-based in recent years as biological catalyst.
Summary of the invention
It take acetic acid as the method for Material synthesis isoprene and corresponding reconstitution cell thereof and application that goal of the invention of the present invention there is provided a kind of, the present invention transforms utilizing the isoprene biosynthetic pathway of traditional mevalonate pathway, set up the method for the isoprene novel metabolic pathways that a synthesis can utilize acetic acid to be raw material, in reconstitution cell body, by engineered means process LAN acetyl-CoA-synthetase, acetyl-CoA carboxylase, acetoacetyl-CoA synthetic enzyme, by acetic acid synthesis of acetyl coenzyme A, traditional MVA part approach is utilized to be translated into target product isoprene after being translated into acetoacetyl-CoA again, thus the isoprene biosynthetic pathway utilizing acetic acid to be carbon source that structure one is new.
For achieving the above object, the present invention is achieved by the following technical solutions:
Take acetic acid as a method for Material synthesis isoprene, it comprises the following steps:
(1) build successively and contain respectively
acsgene,
accsthe carrier pGH-of gene
acs, pGH-
aacs;
(2) structure contains
ispSthe carrier pACY-of gene
ispS pa ; Structure contains
eRG12gene,
eRG8gene,
eRG19gene and
iDI1the carrier pTrc-of gene
eRG12-
eRG8-
eRG19-
iDI1;
Amplification
accgene
accADfragment, by pCOLADuet-1 carrier with
accADfragment is used respectively
pst Iwith
hind IIIcarry out double digestion, enzyme cut after carrier and two ends with corresponding restriction enzyme site
accADthe ratio of gene fragment 1:5 in molar ratio connects, and connect product conversion intestinal bacteria, the positive colony of screening is recombinant plasmid pCOL-
accAD;
Amplification
accgene
accBCfragment, by pCOL-
accADwith
accBCfragment is used respectively
bgl IIwith
xho Icarry out double digestion, enzyme cut after carrier with
accBCthe ratio of gene fragment 1:5 in molar ratio connects, and connect product conversion intestinal bacteria, the positive colony of screening is for containing
accADBCthe recombinant plasmid pCOL-of gene
accADBC;
Described pGH-
acswith described carrier pCOL-
accADBCuse respectively
nde Iwith
bgl IIcarry out double digestion, enzyme cut after carrier pCOL-
accADBCwith exogenous genetic fragment
acsthe ratio of 1:5 connects in molar ratio, and connect product conversion intestinal bacteria, the positive colony of screening is recombinant plasmid pCOL-
accADBC-
acs;
Described pGH-
aacswith described carrier pCOL-
accADBC-
acsuse respectively
xho Iwith
pac Icarry out double digestion, enzyme cut after carrier pCOL-
accADBC-
acswith exogenous genetic fragment
aacsthe ratio of 1:5 connects in molar ratio, and connect product conversion intestinal bacteria, the positive colony of screening is recombinant plasmid pCOL-
accADBC-
acs-
aacs;
(3) increase
hMGSgene, by described pACY-
ispS pa with gene
hMGSuse respectively
fseIwith
pvuIcarry out double digestion, enzyme cut after carrier and exogenous genetic fragment
hMGSthe ratio of 1:5 connects in molar ratio, and connect product conversion intestinal bacteria, the positive colony of screening is recombinant plasmid pACY-
ispS pa -
hMGS;
Amplification
hMGRgene, by pACY-
hMGS-
ispS pa with gene
hMGRuse respectively
nco Iwith
bamH Icarry out double digestion, enzyme cut after carrier and exogenous genetic fragment
hMGRthe ratio of 1:5 connects in molar ratio, and connect product conversion intestinal bacteria, the positive colony of screening is recombinant plasmid pACY-
hMGR-
ispS pa -
hMGS;
(4) by carrier pACY-
hMGR-
ispS pa -
hMGS, pTrc-
eRG12-
eRG8-
eRG19-
iDI1and pCOL-
accADBC-
acs-
aacscotransformation intestinal bacteria, coat and are added with the antibiotic LB solid plate of kantlex, paraxin and penbritin, obtain positive colony YJM57 engineering colon bacillus;
(5) the YJM57 engineering colon bacillus after activation is inoculated in the LB liquid medium containing kantlex, paraxin or ammonia benzyl mycin and cultivates, work as OD
600nmduring for 0.6-0.8, in bacterium liquid, add inductor to final concentration 0.5 mmolL
-1, then proceed to and continue inducing culture 24h at 30 DEG C, fermentation obtains isoprene.
Present invention also offers a kind of reconstitution cell synthesizing isoprene, described reconstitution cell comprises corresponding coding and produces acetyl-CoA-synthetase, acetyl-CoA carboxylase, the gene of acetoacetyl-CoA synthetic enzyme, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, isoprenoid synthase, and this reconstitution cell can synthesize isoprene from acetic acid.
Described reconstitution cell is intestinal bacteria, subtilis, yeast saccharomyces cerevisiae or micro-algae.
Present invention also offers described reconstitution cell and preparing the application in compound or composition, described compound and composition comprise: isoprene, polyisoprene, polyisoprene rubber, isoprene-isobutylene rubber, styrene isoprene styrene block copolymer (SIS) elastomerics, integrated rubber, CD glue, tackiness agent, agricultural chemicals, spices, lubricating oil additive, vulcanizer and catalyzer.
The new isoprene biosynthetic pathway utilizing acetic acid to be carbon source in the present invention its advantage compared with traditional MVA approach is: (1) traditional MVA approach can only be that carbon source synthesizes target product isoprene with glucose, can not take acetic acid as Material synthesis isoprene; And the present invention is by after genetic engineering means process LAN acetyl-CoA-synthetase, acetic acid can be utilized for Material synthesis isoprene; (2) after the present invention utilizes genetically engineered process LAN acetyl-CoA carboxylase and acetoacetyl-CoA synthetic enzyme, compared with traditional MVA approach, new way makes carbon source increase to isoprene metabolic pathway of synthesizing flux, acetyl-CoA changes to acetoacetyl-CoA path for transformation simultaneously: being by acetyl-CoA acyltransferase catalysis acetyl-CoA synthesis of acetyl acetyl-CoA in traditional MVA approach, is single step reaction; And new way is made up of two-step reaction: first, acetyl-CoA carboxylase catalysis acetyl-CoA synthesis malonyl CoA, secondly, acetoacetyl-CoA synthetic enzyme catalysis malonyl CoA synthesis of acetyl acetyl-CoA.
The present invention is mainly through engineered means, the reconstitution cell that the gene of process LAN acetyl-CoA-synthetase, acetyl-CoA carboxylase, acetoacetyl-CoA synthetic enzyme, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, isoprenoid synthase obtain in cell contains the gene of expressing above-mentioned enzyme, and this reconstitution cell can synthesize isoprene from acetic acid.Finally in Bacillus coli cells, successfully set up a kind of new isoprene biosynthetic metabolism approach that acetic acid can be utilized efficiently to be raw material, obtain the reconstitution cell that efficiently can synthesize isoprene, thus set up the method that isoprene produced by a new biological catalyst utilizing acetic acid to be raw material.
Accompanying drawing explanation
Fig. 1 is the schematic diagram utilizing acetic acid to synthesize the new route of synthesis of isoprene in the present invention.
Fig. 2 is pYJM52 (pCOL-in the present invention
accAD) plasmid map.
Fig. 3 is pYJM53 (pCOL-in the present invention
accADBC) plasmid map.
Fig. 4 is pYJM54 (pCOL-in the present invention
accADBC-
acs) plasmid map.
Fig. 5 is pYJM55 (pCOL-in the present invention
accADBC-
acs-
aacs) plasmid map.
Fig. 6 is pYJM56 (pACY-in the present invention
ispS pa -
hMGS) plasmid map.
Fig. 7 is pYJM57 (pACY-in the present invention
hMGR-
ispS pa -
hMGS) plasmid map.
Fig. 8 shows that in the present invention, engineering bacteria YJM57 produces the time curve of isoprene.
Embodiment
Further describe the present invention referring to specific embodiment, but it should be appreciated by those skilled in the art that the present invention is not limited to following specific embodiment.
embodiment 1
As shown in Figure 1, the present invention by overexpression common in intestinal bacteria derive from Salmonella typhimurium (
salmonell aenterica) acetyl-CoA-synthetase (ACS); Derive from intestinal bacteria (
escherichia coli) acetyl-CoA carboxylase (ACC); Derive from streptomycete (
streptomycessp. CL190) acetoacetyl-CoA synthase (AACS), derive from faecium (
enterococcus faecium) 3-Hydroxy-3-methylglutaryl CoA A synthase (HMGS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR); Derive from yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) Mevalonic kinase (ERG12), mevalonic acid-5-phosphokinase (ERG8), mevalonic acid-5-bisphosphate decarboxylase (ERG19), isopentenylpyrophosphate isomerase (IDI1) and isoprenoid synthase (IspS); Utilize acetic acid for Material synthesis intermediate product acetyl-CoA biosynthesizing isoprene.
Described acetyl coenzyme A synthetase gene (
acsgene) derive from: 1) intestinal bacteria (
escherichia colistr. K-12 substr. MG1655) (GenBank:AAC43163), or 2) derive from other bacterium, preferred Salmonella typhimurium (
salmonell typhimuriumlT2) (GenBank:16767525), or 3) Neuraspora crassa (
neurospora crassa) (GenBank:BAJ83612), yeast saccharomyces cerevisiae (
saccharomyces cerevisiaeyJM789) (GenBank:EDN59693), yeast saccharomyces cerevisiae (
saccharomyces cerevisiaes288c) (GenBank:NP_009347), Arabidopis thaliana (
arabidopsis thaliana) (GenBank:AED94121)
, or 4) derive from other organism, and acetyl coenzyme A synthetase gene does not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described acetyl-coA carboxylase gene (
accgene, namely
accADBCgene) be derive from: 1) derive from bacterium, preferred intestinal bacteria (
escherichia colistr. K-12 substr. MG1655) (GenBank:accA:110590387; AccB:110590390; AccC:110590391; AccD 110590392), or 2) Corynebacterium glutamicum (
corynebacterium glutamicumaTCC 14067) (GenBank:KEI22444); Acinetobacter calcoaceticus (
acinetobactersp. ADP1) (GenBank:accA:50086048; AccB:50084890; AccC:50086323; AccD:50083297); Or 3) derive from other organism, and acetyl-coA carboxylase gene does not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described acetoacetyl-CoA synthase gene (
aacsgene) be derive from: 1) streptomycete (
streptomycessp. CL190) (GenBank:AB272317.1); Or 2) Aspergillus fumigatus (
aspergillus fumigatusvar. RP-2014) (GenBank:KEY79579); Or 3) bacillus thuringiensis (
bacillus thuringiensisiBL 4222) (GenBank:EEN03006); Or 4) derive from other organism, and acetoacetyl-CoA synthase gene does not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described 3-Hydroxy-3-methylglutaryl CoA A synthase gene (
hMGSgene) can derive from: 1) yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) (GenBank:YM4987.09C); Or 2) derive from other bacterium, preferred faecium (
enterococcus faecium) (GenBank:AAG02443.1), enterococcus faecalis (
enterococcus faecalis) (GenBank:ESU74184.1), aurococcus (
staphylococcus aureus) (GenBank:YP_501316.1), Wei Shi Li Site bacterium (
listeria welshimeri) (GenBank:YP_849629.1), streptococcus pyogenes (
streptococcus pyogenes) (GenBank:AAL97584.1); Or 3) derive from other organism, and 3-Hydroxy-3-methylglutaryl CoA A synthase gene does not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (
hMGRgene) derive from: 1) yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) (GenBank:YLR450W); Or 2) derive from other bacterium, preferred faecium (
enterococcus faecium) (GenBank:YP_005354442.1), enterococcus faecalis (
enterococcus faecalis) (GenBank:AAG02438.2), aurococcus (
staphylococcus aureus) (GenBank:AAG02423.1), streptococcus pyogenes (
streptococcus pyogenes) (GenBank:WP_030125991.1); Or 3) derive from other organism, and 3-hydroxy-3-methylglutaryl coenzyme A reductase gene does not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described Mevalonic kinase gene (
eRG12gene) derive from: 1) yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) (GenBank:NP_013935.1), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, enterococcus faecalis (
enterococcus faecalis) (GenBank:EPI38248.1), aurococcus (
staphylococcus aureus) (GenBank:ABR51486.1), faecium (
enterococcus faecium) (GenBank:EFS06266.1), streptococcus pyogenes (
streptococcus pyogenes) (GenBank:AFV37808.1); Or 3) derive from other organism, and Mevalonic kinase gene does not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described mevalonic acid-5-phosphokinase gene (
eRG8gene) derive from: 1) yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) (GenBank:NP_013947.1), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, enterococcus faecalis (
enterococcus faecalis) (GenBank:WP_016626966.1), aurococcus (
staphylococcus aureus) (GenBank:AIA27148.1), faecium (
enterococcus faecium) (GenBank:WP_016629841.1), streptococcus pyogenes (
streptococcus pyogenes) (GenBank:WP_023613167.1); Or 3) derive from other organism, and mevalonic acid-5-phosphokinase gene does not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described mevalonic acid-5-bisphosphate decarboxylase gene (
eRG19gene) derive from: 1) yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) (GenBank:AY757921.1), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, enterococcus faecalis (
enterococcus faecalis) (GenBank:YP_005707688.1), faecium (
enterococcus faecium) (GenBank:EPI24610.1), streptococcus pyogenes (
streptococcus pyogenes) (GenBank:AAL97580.1); Or 3) derive from other organism, and mevalonic acid-5-bisphosphate decarboxylase gene does not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described isopentenylpyrophosphate isomerase gene (
iDI1gene) derive from: 1) yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) (GenBank:NP_015208.1), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, enterococcus faecalis (
enterococcus faecalis) (GenBank:NP_814639.1), aurococcus (
staphylococcus aureus) (GenBank:YP_501084.1), faecium (
enterococcus faecium) (GenBank:ERK34722.1), streptococcus pyogenes (
streptococcus pyogenes) (GenBank:YP_001128672.1) or 3) derive from other organism, and isopentenylpyrophosphate isomerase gene does not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described isoprenoid synthase gene (
ispSgene) derive from: 1) white poplar (
populus alba) (GenBank:AB198180), preferred white poplar, or 2) black poplar (
populus nigra) (GenBank:AM410988), or 3) white poplar x trembling poplar (
populus albax
populus tremula) (GenBank:AJ294819) or 4) and the nucleotide sequence of isoprenoid synthase genetic homology more than 70%, or 3) derive from other organism, there is no obvious homology with isoprenoid synthase gene, but with isoprenoid synthase gene, there is the nucleotide sequence of same or similar function.
1. the clone of foreign gene
the clone of 1.1 foreign genes
1.1.1 the clone of Salmonella typhimurium acetyl coenzyme A synthetase gene
Choose from Salmonella typhimurium (
salmonella typhimuriumacetyl coenzyme A synthetase gene LT2) (
acs), GenBank:16767525, by Shanghai, JaRa company is obtained by chemical synthesis process, afterwards with carrier pGH(Shanghai Jierui Biology Engineering Co., Ltd) be connected and obtain pGH-
acs.
1.1.2 the clone of intestinal bacteria acetyl-coA carboxylase gene
Extract
e. coligenomic dna, according to GenBank primers, pcr amplification acetyl coenzyme A synthetase gene (
accADBC), GenBank:accA 110590387; AccB 110590390; AccC 110590391; AccD 110590392.Recycling glue reclaims test kit and reclaims goal gene fragment.
Amplimer sequence is:
accAD-L:5’-AAAA
CTGCAGAGTCTGAATTTCCTTGATTTTG-3’
accAD-R:5’-CCC
AAGCTTTCAGGCCTCAGGTTCCTGA-3’
accBC-L: 5’-GGA
AGATCTATGGATATTCGTAAGATTAAAAAACT-3’
accBC-R: 5’-CCG
CTCGAGTTATTTTTCCTGAAGACCGAG-3’。
1.1.3 the clone of streptomycete acetoacetyl-CoA synthase gene
From streptomycete (
streptomycessp. CL190) acetoacetyl-CoA synthase gene (
aacs), Gene ID:325302226, by Shanghai, JaRa company is obtained by chemical synthesis process.Be connected with described carrier pGH afterwards and obtain pGH-
aacs.
1.1.4 the clone of faecium 3-Hydroxy-3-methylglutaryl CoA A synthase gene
Extract the genomic dna of faecium, according to GenBank primers, pcr amplification 3-Hydroxy-3-methylglutaryl CoA A synthase gene (GenBank:AAG02443.1), recycling glue reclaims test kit and reclaims goal gene fragment.
Amplimer sequence is:
HMGS-F: 5’-ATGC
GGCCGGCCATGAAAATAGGGATTGATCGTCTTT-3’；
HMGS-R: 5’-ATCG
GCGATCGCTTATATTTTGTAGTAACGA-3’。
1.1.5 the clone of faecium 3-hydroxy-3-methylglutaryl coenzyme A reductase gene
Extract the genomic dna of faecium, according to GenBank primers, pcr amplification 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (GenBank:YP_005354442.1), recycling glue reclaims test kit and reclaims goal gene fragment.
Amplimer sequence is:
HMGR-F: 5’-ATGC
CCATGGATGAAAGAAGTCGTTATGATAG-3’；
HMGR-R: 5’-ATTG
GGATCCTTATTTTTCCCGGATTTTTTCCA-3’。
1.1.6 the clone of yeast saccharomyces cerevisiae MVA downstream metabolic pathway gene
Yeast saccharomyces cerevisiae MVA downstream metabolic pathway gene comprises four genes:
eRG12,
eRG8,
eRG19with
iDI1.Clone's reference literature of four genes obtains (Jianming Yang, Guang Zhao, Yuanzhang Sun, Yanning Zheng, Xinglin Jiang, Wei Liu, Mo Xian*. Bio-isoprene production using exogenous MVA pathway and isoprene synthase in
e.coli.
bioresource Technology , 2012,104:642 – 647.)
1.1.7 the clone of white poplar isoprenoid synthase gene
Choose and come from
populus alba's
ispSgene (
ispS pa , GenBank No.AB198180) and gene order carries out rare codon analysis (http://www. genscript.com/ cgi-bin/tools/ rare_ codon_ analysis), and is optimized for by its rare codon
e. colithe codon (http://www.jcat.de/) of preference.Isoprenoid synthase gene after optimization (
ispS pa ) serve extra large JaRa company and carry out chemosynthesis, be connected on pGH carrier and form pGH-respectively
ispS pa carrier.
2. the structure of expression vector
the structure of 2.1 pYJM8 carriers
PYJM8 carrier (i.e. carrier pACY
-ispS pa ), it builds reference literature and obtains (Jianming Yang, Guang Zhao, Yuanzhang Sun, Yanning Zheng, Xinglin Jiang, Wei Liu, Mo Xian*. Bio-isoprene production using exogenous MVA pathway and isoprene synthase in
e.coli. Bioresource Technology, 2012,104:642 – 647.).
the structure of 2.2 pYJM14 carriers
PYJM14 carrier (i.e. carrier pTrc-
eRG12-
eRG8-
eRG19-
iDI1), it builds reference literature and obtains (Jianming Yang, Guang Zhao, Yuanzhang Sun, Yanning Zheng, Xinglin Jiang, Wei Liu, Mo Xian*. Bio-isoprene production using exogenous MVA pathway and isoprene synthase in
e .coli. Bioresource Technology, 2012,104:642 – 647.)
the structure of 2.3 pYJM52 carriers
Utilize following primer accAD-L(5 '-AAAA
cTGCAGaGTCTGAATTTCCTTGATTT TG-3 ') and accAD-R(5 '-CCC
aAGCTTtCAGGCCTCAGGTTCCTGA-3 ') and genome of E.coli be template amplification
accgene
accADfragment (two ends are with the corresponding restriction enzyme site connected to carrier).
By pCOLADuet-1 carrier (Novagen company) and gene
accADuse respectively
pst Iwith
hind IIIcarry out double digestion, enzyme cut after carrier and two ends with corresponding restriction enzyme site
accADthe ratio of gene 1:5 in molar ratio, 4 DEG C of connections spend the night or 16 DEG C connect 4 ~ 6 h, connect product conversion
e. colidH5 α, then coating is added with the LB solid plate of kantlex, and pcr amplification screening positive clone, extracts recombinant plasmid pYJM52 (pCOL-from positive colony
accAD) after, then by restriction enzyme digestion and order-checking qualification.Plasmid construction collection of illustrative plates as shown in Figure 2.
the structure of 2.4 pYJM53 carriers
Utilize following primer accBC-L(5 '-GGA
aGATCTaTGGATATTCGTAAGATTAA AAAACT-3 ') and accBC-R(5 '-CCG
cTCGAGtTATTTTTCCTGAAGACCGAG-3 ') and genome of E.coli be template amplification
accgene
accBCfragment (two ends are with the corresponding restriction enzyme site connected to carrier).
By pYJM52 carrier with
accBCfragment is used respectively
bgl IIwith
xho Icarry out double digestion, enzyme cut after carrier and exogenous genetic fragment
accBCthe ratio of 1:5 in molar ratio, 4 DEG C of connections are spent the night or 16 DEG C of connection 4 ~ 6 h, connect product conversion
e. colidH5 α, then coating is added with the LB solid plate of kantlex, and PCR screening positive clone, extracts recombinant plasmid pYJM53 (pCOL-from positive colony
accADBC) after, then by restriction enzyme digestion and order-checking qualification.Plasmid construction collection of illustrative plates as shown in Figure 3.
the structure of 2.5 pYJM54 carriers
As shown in Figure 4, by described carrier pGH-
acsuse respectively with described carrier pYJM53
nde Iwith
bgl IIcarry out double digestion, enzyme cut after carrier pYJM53 and exogenous genetic fragment
acsthe ratio of 1:5 in molar ratio, 4 DEG C of connections are spent the night or 16 DEG C of connection 4 ~ 6 h, connect product conversion
e. colidH5 α, then coating is added with the LB solid plate of kantlex, and PCR screening positive clone, extracts recombinant plasmid pYJM54 (pCOL-from positive colony
accADBC-
acs) after, then by restriction enzyme digestion and order-checking qualification.Plasmid construction collection of illustrative plates as shown in Figure 4.
the structure of 2.6 pYJM55 carriers
As shown in Figure 5, by described carrier pGH-
aacsuse respectively with described carrier pYJM54
xho Iwith
pac Icarry out double digestion, enzyme cut after carrier pYJM54 and exogenous genetic fragment
aacsthe ratio of 1:5 in molar ratio, 4 DEG C of connections are spent the night or 16 DEG C of connection 4 ~ 6 h, connect product conversion
e. colidH5 α, then coating is added with the LB solid plate of kantlex, and PCR screening positive clone, extracts recombinant plasmid pYJM55 (pCOL-from positive colony
accADBC-
acs-
aacs) after, then by restriction enzyme digestion and order-checking qualification.Plasmid construction collection of illustrative plates as shown in Figure 5.
the structure of 2.7 pYJM56 carriers
Utilize following primer HMGS-F:(5 '-ATGC
gGCCGGCCaTGAAAATAGGGATTGAT CGTCTTT-3 ') and HMGS-R:(5 '-ATCG
gCGATCGCtTATATTTTGTAGTAACGA-3 ') and faecium genome be template amplification
hMGSgene (two ends are with the corresponding restriction enzyme site connected to carrier).
By pYJM8 carrier and gene
hMGSuse respectively
fse Iwith
pvu Icarry out double digestion, enzyme cut after carrier pYJM8 and exogenous genetic fragment
hMGSthe ratio of 1:5 in molar ratio, 4 DEG C of connections are spent the night or 16 DEG C of connection 4 ~ 6 h, connect product conversion
e. colidH5 α, then coating is added with the LB solid plate of paraxin, and PCR screening positive clone, extracts recombinant plasmid pYJM56 (pACY-from positive colony
ispS pa -
hMGS) after, then by restriction enzyme digestion and order-checking qualification.Plasmid construction collection of illustrative plates as shown in Figure 6.
the structure of 2.8 pYJM57 carriers
Utilize following primer HMGR-F(5 '-ATGC
cCATGGaTGAAAGAAGTCGTTATGA TAG-3 ') and HMGR-R(5 '-ATTG
gGATCCtTATTTTTCCCGGATTTTTTCCA-3 ') and faecium genome be template amplification
hMGRgene (two ends are with the corresponding restriction enzyme site connected to carrier).
By pYJM56 carrier and gene
hMGRuse respectively
nco Iwith
bamH Icarry out double digestion, enzyme cut after carrier pYJM56 and exogenous genetic fragment
hMGRthe ratio of 1:5 in molar ratio, 4 DEG C of connections are spent the night or 16 DEG C of connection 4 ~ 6 h, connect product conversion
e. colidH5 α, then coating is added with the LB solid plate of paraxin, and PCR screening positive clone, extracts recombinant plasmid pYJM57 (pACY-from positive colony
hMGR-
ispS pa -
hMGS) after, then by restriction enzyme digestion and order-checking qualification.Plasmid construction collection of illustrative plates as shown in Figure 7.
3.
e. colithe structure of recombinant bacterial strain
By carrier pYJM57 (pACY-
hMGR-
ispS pa -
hMGS), pYJM14 (pTrc-
eRG12-
eRG8-
eRG19-
iDI1) and pYJM55 (pCOL-
accADBC-
acs-
aacs) the common thermal shock conversion of recombinant plasmid
e. colibL21 (DE3) competent cell, coats and is added with the antibiotic LB solid plate of kantlex, paraxin and penbritin, obtains positive colony, obtain YJM57 engineering colon bacillus thus by PCR screening.
The reconstitution cell that the present invention obtains comprises following gene fragment: acetyl-CoA-synthetase, acetyl-CoA carboxylase, acetoacetyl-CoA synthetic enzyme, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, isoprenoid synthase, this recombination bacillus coli can synthesize isoprene from acetic acid.
Described reconstitution cell is bacterial cell, as intestinal bacteria, and subtilis or fungal cell's (as yeast saccharomyces cerevisiae), or micro-algae etc. is built by genetic engineering technique.
Described reconstitution cell may be used for preparing compound or composition, and described compound and composition comprise: isoprene, polyisoprene, polyisoprene rubber, isoprene-isobutylene rubber, vinylbenzene-isoprene-styrene block copolymer elastomer, integrated rubber, CD glue, tackiness agent, agricultural chemicals, spices, lubricating oil additive, vulcanizer and catalyzer.
4. engineering bacterium fermentation experiment
By the recombinant plasmid transformed that builds in competent cell, by ferment tank, fermentation culture is carried out to recombinant bacterium, utilize chromatography of gases technology to carry out the detection of quantitative and qualitative analysis to tunning.
Picking mono-clonal contains M9 seed culture medium (1 L M9 salts:5 g NaAc, the 6 g Na of sodium-acetate to 50ml
2hPO
4, 3 g KH
2pO
4, 1 g NH
4cl, 0.5 g NaCl, 0.24 g MgSO
4, 121 ° of C high pressure steam sterilization 15min.) in, 37 DEG C, (18-24h) is spent the night in 180rpm activation.By seed by 10% inoculum size be seeded to containing 2L fermention medium (14.6 g/L K
2hPO
4, 4 g/L KH
2pO
4, 10 g/L (NH
4)
2sO
42 g/L Trisodium Citrates; 2.05 g/L sodium-acetate; 0.117 g/L trimethyl-glycine; 0.011 g/L calcium chloride; 0.72 g/L magnesium sulfate, 1 × VITAMIN (Sigma) and 1 × trace element solution. 1000 × trace element solution (often liter) is containing ammonium molybdate 0.123mg; Zinc sulfate 0.097mg; Boric acid 0.823mg; Copper sulfate 0.083mg; Manganous chloride tetrahydrate 0.527mg, 4ml 1M magnesium sulfate, 1900ml distilled water) 5L small-sized fermentation tank in, air flow 1.3 VVM, rotating speed 400rpm, 37 ° of C are cultured to OD
600when being about 12,0.25mM IPTG, 30 DEG C of abduction deliverings, adjust pH, control pH 7.0 with pure acetic acid, add an IPTG every 8h.The isoprene product obtained carries out qualitative and quantitative analysis by GC to it.Every 4h gets fermented liquid 5ml, measures cell OD
600, glucose concn; Every 15min gets tail gas 1ml, utilizes gas chromatographic detection product isoprene concentrations.Until OD no longer changes, till product no longer produces.
Testing conditions: GC system adopts Shandong Lunan auspicious rainbow SP-6890 type gas chromatograph, chromatographic column is HP-INNOWAX column (25 m × 250 μm × 0.2 μm), and detector is fid detector; Vaporizer temperature 200 DEG C, detector temperature 230 DEG C, flow rate of carrier gas: 1ml/min.
Post heating schedule is: 50 DEG C of insulation 0.5min,
4 DEG C/min rises to 70 DEG C,
25 DEG C/min rises to 250 DEG C, insulation 5min.
Fig. 8 shows, the isoprene output (■) of engineering bacteria YJM57 and Growth of Cells (▲).When engineering bacteria Growth of Cells 12h starts to carry out inducing culture.
Above embodiment only in order to technical scheme of the present invention to be described, but not is limited; Although with reference to previous embodiment to invention has been detailed description, for the person of ordinary skill of the art, still can modify to the technical scheme described in previous embodiment, or equivalent replacement is carried out to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.
Claims (10)
1. be a method for Material synthesis isoprene with acetic acid, it is characterized in that it comprises the following steps:
(1) build successively and contain respectively
acsgene,
accsthe carrier pGH-of gene
acs, pGH-
aacs;
(2) structure contains
ispSthe carrier pACY-of gene
ispS pa ; Structure contains
eRG12gene,
eRG8gene,
eRG19gene and
iDI1the carrier pTrc-of gene
eRG12-
eRG8-
eRG19-
iDI1;
Amplification
accgene
accADfragment, by pCOLADuet-1 carrier with
accADfragment is used respectively
pst Iwith
hind IIIcarry out double digestion, enzyme cut after carrier and two ends with corresponding restriction enzyme site
accADthe ratio of gene fragment 1:5 in molar ratio connects, and connect product conversion intestinal bacteria, the positive colony of screening is recombinant plasmid pCOL-
accAD;
Amplification
accgene
accBCfragment, by pCOL-
accADwith
accBCfragment is used respectively
bgl IIwith
xho Icarry out double digestion, enzyme cut after carrier with
accBCthe ratio of gene fragment 1:5 in molar ratio connects, and connect product conversion intestinal bacteria, the positive colony of screening is for containing
accADBCthe recombinant plasmid pCOL-of gene
accADBC;
Described pGH-
acswith described carrier pCOL-
accADBCuse respectively
nde Iwith
bgl IIcarry out double digestion, enzyme cut after carrier pCOL-
accADBCwith exogenous genetic fragment
acsthe ratio of 1:5 connects in molar ratio, and connect product conversion intestinal bacteria, the positive colony of screening is recombinant plasmid pCOL-
accADBC-
acs;
Described pGH-
aacswith described carrier pCOL-
accADBC-
acsuse respectively
xho Iwith
pac Icarry out double digestion, enzyme cut after carrier pCOL-
accADBC-
acswith exogenous genetic fragment
aacsthe ratio of 1:5 connects in molar ratio, and connect product conversion intestinal bacteria, the positive colony of screening is recombinant plasmid pCOL-
accADBC-
acs-
aacs;
(3) increase
hMGSgene, by described pACY-
ispS pa with gene
hMGSuse respectively
fseIwith
pvuIcarry out double digestion, enzyme cut after carrier and exogenous genetic fragment
hMGSthe ratio of 1:5 connects in molar ratio, and connect product conversion intestinal bacteria, the positive colony of screening is recombinant plasmid pACY-
ispS pa -
hMGS;
Amplification
hMGRgene, by pACY-
hMGS-
ispS pa with gene
hMGRuse respectively
nco Iwith
bamH Icarry out double digestion, enzyme cut after carrier and exogenous genetic fragment
hMGRthe ratio of 1:5 connects in molar ratio, and connect product conversion intestinal bacteria, the positive colony of screening is recombinant plasmid pACY-
hMGR-
ispS pa -
hMGS;
(4) by carrier pACY-
hMGR-
ispS pa -
hMGS, pTrc-
eRG12-
eRG8-
eRG19-
iDI1and pCOL-
accADBC-
acs-
aacscotransformation intestinal bacteria, coat and are added with the antibiotic LB solid plate of kantlex, paraxin and penbritin, obtain positive colony YJM57 engineering colon bacillus;
(5) the YJM57 engineering colon bacillus after activation is inoculated in the LB liquid medium containing kantlex, paraxin or ammonia benzyl mycin and cultivates, work as OD
600nmduring for 0.6-0.8, in bacterium liquid, add inductor to final concentration 0.5 mmolL
-1, then proceed to and continue inducing culture 24h at 30 DEG C, fermentation obtains isoprene.
2. according to claim 1 take acetic acid as the method for Material synthesis isoprene, it is characterized in that: described in
acsgene source is in GenBank:AAC43163 in GenBank:AED94121, intestinal bacteria in GenBank:NP_009347, Arabidopis thaliana in GenBank:EDN59693, yeast saccharomyces cerevisiae in GenBank:BAJ83612, yeast saccharomyces cerevisiae in GenBank:16767525, Neuraspora crassa in Salmonella typhimurium;
Described
accADBCgene source is in GenBank:accA:110590387 in GenBank:KEI22444, intestinal bacteria in Corynebacterium glutamicum; AccB:110590390; AccC:110590391; GenBank:accA:50086048 in accD 110590392, acinetobacter calcoaceticus; AccB:50084890; AccC:50086323; AccD:50083297.
3. according to claim 1 take acetic acid as the method for Material synthesis isoprene, it is characterized in that: described in
aacsgene source is in GenBank:EEN03006 in GenBank:KEY79579, bacillus thuringiensis in GenBank:AB272317.1, Aspergillus fumigatus in streptomycete;
Described
hMGSgene source is in GenBank:AAL97584.1 in GenBank:AAG02443.1 or streptococcus pyogenes in GenBank:YP_501316.1, faecium in GenBank:ESU74184.1, aurococcus in GenBank:YM4987.09C, enterococcus faecalis in yeast saccharomyces cerevisiae.
4. according to claim 1 take acetic acid as the method for Material synthesis isoprene, it is characterized in that: described in
hMGRgene source is in GenBank:WP_030125991.1 in GenBank:AAG02423.1, streptococcus pyogenes in GenBank:AAG02438.2, aurococcus in GenBank:YP_005354442.1, enterococcus faecalis in GenBank:YLR450W, faecium in yeast saccharomyces cerevisiae;
Described
eRG12gene source is in GenBank:AFV37808.1 in GenBank:EFS06266.1, streptococcus pyogenes in GenBank:ABR51486.1, faecium in GenBank:EPI38248.1, aurococcus in GenBank:NP_013935.1, enterococcus faecalis in yeast saccharomyces cerevisiae.
5. according to claim 1 take acetic acid as the method for Material synthesis isoprene, it is characterized in that: described in
eRG8gene source is in GenBank:WP_023613167.1 in GenBank:WP_016629841.1, streptococcus pyogenes in GenBank:AIA27148.1, faecium in GenBank:WP_016626966.1, aurococcus in GenBank:NP_013947.1, enterococcus faecalis in yeast saccharomyces cerevisiae;
Described
eRG19gene source is in GenBank:AAL97580.1 in GenBank:EPI24610.1, streptococcus pyogenes in GenBank:YP_005707688.1, faecium in GenBank:AY757921.1, enterococcus faecalis in yeast saccharomyces cerevisiae.
6. according to claim 1 take acetic acid as the method for Material synthesis isoprene, it is characterized in that: described in
iDI1gene source is in GenBank:YP_001128672.1 in GenBank:ERK34722.1, streptococcus pyogenes in GenBank:YP_501084.1, faecium in GenBank:NP_814639.1, aurococcus in GenBank:NP_015208.1, enterococcus faecalis in yeast saccharomyces cerevisiae;
Described
ispSgene source is in GenBank:AJ294819 in GenBank:AM410988, white poplar x trembling poplar in GenBank:AB198180, black poplar in white poplar.
7. according to claim 1 take acetic acid as the method for Material synthesis isoprene, it is characterized in that: in described step (5), inductor is IPTG.
8. one kind is synthesized the reconstitution cell of isoprene, it is characterized in that described reconstitution cell comprises corresponding coding and produces acetyl-CoA-synthetase, acetyl-CoA carboxylase, the gene of acetoacetyl-CoA synthetic enzyme, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, isoprenoid synthase, this reconstitution cell can synthesize isoprene from acetic acid.
9. reconstitution cell according to claim 8, is characterized in that described reconstitution cell is intestinal bacteria, subtilis, yeast saccharomyces cerevisiae or micro-algae.
10. reconstitution cell according to claim 8 is preparing the application in compound or composition, it is characterized in that described compound and composition comprise: isoprene, polyisoprene, polyisoprene rubber, isoprene-isobutylene rubber, styrene isoprene styrene block copolymer (SIS) elastomerics, integrated rubber, CD glue, tackiness agent, agricultural chemicals, spices, lubricating oil additive, vulcanizer and catalyzer.
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| CN109852725A (en) * | 2019-04-12 | 2019-06-07 | 山东农业大学 | A kind of method of fruit resistant storage properties that identifying apple plants and its primer pair that uses |
| CN110964681A (en) * | 2018-09-29 | 2020-04-07 | 中国石油化工股份有限公司 | Engineering strain and method for preparing farnesene by using cellulose |
| CN111363709A (en) * | 2018-12-26 | 2020-07-03 | 中国科学院青岛生物能源与过程研究所 | Genetic engineering bacterium for improving isoprene yield and construction method and application thereof |
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2014
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110964681A (en) * | 2018-09-29 | 2020-04-07 | 中国石油化工股份有限公司 | Engineering strain and method for preparing farnesene by using cellulose |
| CN111363709A (en) * | 2018-12-26 | 2020-07-03 | 中国科学院青岛生物能源与过程研究所 | Genetic engineering bacterium for improving isoprene yield and construction method and application thereof |
| CN109852725A (en) * | 2019-04-12 | 2019-06-07 | 山东农业大学 | A kind of method of fruit resistant storage properties that identifying apple plants and its primer pair that uses |
| CN109852725B (en) * | 2019-04-12 | 2021-11-02 | 山东农业大学 | Method for identifying fruit storage tolerance of apple plants and specific primer pair used by method |
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