CN105463008A - Construction method for producing isobutanol engineering bacterium through cellulose fermentation - Google Patents

Construction method for producing isobutanol engineering bacterium through cellulose fermentation Download PDF

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CN105463008A
CN105463008A CN201511029576.1A CN201511029576A CN105463008A CN 105463008 A CN105463008 A CN 105463008A CN 201511029576 A CN201511029576 A CN 201511029576A CN 105463008 A CN105463008 A CN 105463008A
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gene
sequence
fragment
plasmid
isopropylcarbinol
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CN105463008B (en
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曹广丽
王震宇
杨谦
傅德峰
丛华
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Harbin Institute of Technology
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Abstract

The invention provides a construction method for producing an isobutanol engineering bacterium through cellulose fermentation. The method comprises the steps that PCR amplification is performed on a phenylpyruvate decarboxylase gene aro10 and an ethanol dehydrogenase gene adh2, the amplified genes aro10 and adh2 are connected with an expression vector pMA5 through double enzyme digestion, a metabolic pathway for synthesizing isobutanol is constructed in bacillus cereus, the exogenous genes adh2 and aro10 are introduced, an endogenous gene alsS is over-expressed, and key genes pflB and pta in a compete pathway are knocked out. According to the construction method for producing the isobutanol engineering bacterium through cellulose fermentation, the isobutanol can be produced efficiently, and the final yield can reach 1.310 g/L. The method is applied to the field of construction for producing the isobutanol engineering bacterium.

Description

A kind of construction process utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria
Technical field
The present invention relates to a kind of construction process utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria.
Background technology
Isopropylcarbinol, as one of the strategic emphasis of future source of energy, is also one of content of our national biomass energy development.The domestic and international main Bian chemical synthesis of suitability for industrialized production isopropylcarbinol, such as, by higher alcohols synthesis isopropylcarbinol, produces isopropylcarbinol by synthetic gas at present, synthesizes isopropylcarbinol etc. by propenecarbonyl.The isopropylcarbinol technique of chemical process synthesis is loaded down with trivial details, and catalyzer price is high.Simultaneously, along with the surging and lasting minimizing of oil price in recent years and the growth of isopropylcarbinol demand, the profit profit of oxo alcohol device is not high in addition, and some manufacturers close down partial devices, and turn to other production developments, make isopropylcarbinol supply conditions very nervous.Although in recent years, building up and setting device of domestic oxo-alcohols device, makes isopropylcarbinol imbalance between supply and demand will have certain improvement, but due to the wretched insufficiency and catalyzer producing isopropylcarbinol raw material expensive, cause isopropylcarbinol supply can not meet current needs, market has openings is still very large.The raw material supply of production isopropylcarbinol is synthesized isopropylcarbinol in a large number to chemical method and is defined serious restriction, and the serious environmental problems caused in building-up process, make people have to find new isopropylcarbinol synthetic method, more recognize that biosynthesizing isopropylcarbinol has good development prospect simultaneously.But the isopropylcarbinol that biological synthesis process is produced is mainly by a small amount of by product in grain fermentative production propyl carbinol.Therefore, biological synthesis process direct fermentation production isopropylcarbinol is utilized to be regarded as the important directions of future development.
Alcohol fuel and butanols reach industrialized degree by yeast saccharomyces cerevisiae and clostridium acetobutylicum fermentative production, and up to the present, never finding that there is original strain directly can synthesize isopropylcarbinol.Along with the development of synthetic biology, utilizing the method for synthetic biology to transform other microorganisms not producing isopropylcarbinol becomes possibility to produce isopropylcarbinol.Professors James in 2008 etc. utilize metabolic engineering means to transform intestinal bacteria, can utilize glucose production isopropylcarbinol, providing new thinking, opening the gate of biosynthesizing isopropylcarbinol for building high yield isopropylcarbinol genetic engineering bacterium.After this, extensive concern is obtained by the method for artificial constructed engineering strain production isopropylcarbinol.Although utilize metabolic engineering means can realize the direct biological fermentation of isopropylcarbinol; but; the matrix that the isopropylcarbinol production bacterial strain of existing structure utilizes mainly concentrates on some simple carbohydrates as glucose, sucrose etc.; cause product isopropylcarbinol cost to remain high, seriously hinder the large-scale production of biological isopropylcarbinol technology.Fibrous material is organic carbon hydrate abundant, the most cheap in the world.It is a frontier with great potential that fibrous material direct bioconversion produces isopropylcarbinol, the sugar that it produces after referring to the cellulose degraded Mierocrystalline cellulose only utilizing a kind of microorganism to produce still by same strain bacterium to complete the process of fermentation, this method not only achieves changing waste into resources, innoxious, also achieve the simplification of fermenting process, economization, be that bio-transformation fiber wastes produces the ideal a kind of form of isopropylcarbinol simultaneously.But due to complicacy and the heterogeneity of tackified fiber preform, so, be that the genetic engineering bacterium of fermenting substrate production isopropylcarbinol rarely has report up to now with Mierocrystalline cellulose.American scholar Liao in 2011 etc. to the fine clostridium of solution of degraded cellulose can carrying out genetic modification and build the engineering strain that non-fermented approach produces isopropylcarbinol, but due to transgene and host strain gene GC content difference is larger, aerobic causes the output of isopropylcarbinol to only have 0.66g/L with the difference of anaerobic environment.Therefore, how can in the genetically engineered Host Strains with efficient degradation Mierocrystalline cellulose ability reasonable construction isopropylcarbinol route of synthesis, find in synthetic approach the key gene affecting isopropylcarbinol synthesis, specify associated regulatory mechanism, and had great importance by the production of genetic engineering technique means formation high-efficiency fiber isobutanol fermentation.
Summary of the invention
The invention provides a kind of construction process utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria.
A kind of construction process utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria of the present invention, carries out according to following steps:
One, the total serum IgE extracting yeast saccharomyces cerevisiae (S.cerevisiae) carries out reverse transcription and obtains cDNA, according to phenylpyruvate decarboxylase gene and alcohol dehydrogenase gene design primer aroF/R and adhF/R, then PCR obtains phenylpyruvate decarboxylase gene aro10 and alcohol dehydrogenase gene adh2;
Two, primer aroF and adhR is utilized to carry out pcr amplification phenylpyruvate decarboxylase gene aro10 and alcohol dehydrogenase gene adh2, and insert BamHI, BglII, XhoI and NheI tetra-restriction enzyme sites, be connected to form and merge fragment BamHI – BglII – aro10 – XhoI – adh2 – NheI, then with BamHI and NheI, double digestion is carried out to above-mentioned fusion fragment, double digestion is carried out to expression vector pMA5 simultaneously; Phenylpyruvate decarboxylase gene aro10 after double digestion is connected with the expression vector pMA5 after double digestion with the fusion fragment of alcohol dehydrogenase gene adh2, then heat shock proceeds in intestinal bacteria Top10 bacterial strain, after qualification is correct, obtain recombinant plasmid pMA5-RD;
Three, according to bacillus cereus (B.cereus) whole genome sequence, design primer alsF/alsR, then be the alsS gene fragment that template amplification obtains two ends and contains restriction enzyme site BamHI/BglII with bacillus cereus (B.cereus) complete genome DNA, and by BamHI and BglII respectively enzyme cut the recombinant plasmid pMA5-RD that alsS gene fragment and step 2 obtain, then the alsS gene fragment after being cut by enzyme is connected with recombinant plasmid pMA5-RD, construction expression plasmid pMA5-SRD; Expression plasmid pMA5-SRD electric shock proceeded in bacillus cereus (B.cereus) bacterial strain, coat and screen containing 50 μ g/mL kalamycin resistance LB flat boards, the positive transformant that empirical tests is correct is engineering strain;
Four, knock out two key gene pflB and pta in the engineering strain that step 3 obtains in metabolism of pyruvate collateral branch approach by homologous recombination method, finally obtain utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria.
Advantage of the present invention: 1. bacillus cereus has higher tolerance for isopropylcarbinol, and the output that isopropylcarbinol limits isopropylcarbinol for the toxicity of cell in the production process of isopropylcarbinol is all key issue all the time, bacillus cereus is therefore utilized to produce isopropylcarbinol significant; 2. the present invention constructs the pathways metabolism of synthesis isopropylcarbinol in bacillus cereus, introduce foreign gene adh2 and aro10, process LAN native gene alsS, and knocked out competition pathway key gene pflB and pta, build utilize cellulose fermentation to produce isopropylcarbinol engineering bacteria can High-efficient Production isopropylcarbinol, ultimate capacity can reach 1.310g/L; 3. the engineering strain that obtains of the present invention can decomposition of cellulose biosynthesizing isopropylcarbinol, utilization can degraded cellulose strain fermentation produce isopropylcarbinol, substrate cost in fermenting process is reduced greatly, show bacillus cereus and produce great potential and value in isopropylcarbinol in fermentation, be significant at the energy and environmental area.
Accompanying drawing explanation
Fig. 1 is gene aro10 nucleic acid electrophoresis figure, and wherein M is DL2000DNAMaker, and 1,2 is gene aro10;
Fig. 2 is gene adh 2 nucleic acid electrophoresis figure, and wherein M is DL2000DNAMaker, and 3,4 is gene adh 2;
Fig. 3 is the nucleic acid electrophoresis figure that gene aro10 and adh2 merges fragment RD, and wherein M is that DL10000DNAMaker, RD are for merging fragment;
Fig. 4 is gene alsS nucleic acid electrophoresis figure, and wherein M is DL2000DNAMaker, and 1,2 is gene alsS;
Fig. 5 is gene pflB upstream and downstream sequencing nucleic acid electrophorogram, and wherein M is DL2000DNAMaker, 1,2 on have fragment pflU, 3,4 is segments downstream pflD;
Fig. 6 is for merging fragment pflUD nucleic acid electrophoresis figure, and wherein M is DL2000DNAMaker, and 1 ~ 3 for merging fragment pflUD;
Fig. 7 is chloramphenicol resistance gene cam nucleic acid electrophoresis figure, and wherein M is DL2000DNAMaker, and 1,2 is chloramphenicol resistance gene cam;
Fig. 8 is the nucleic acid electrophoresis figure of gene pta upstream sequence, and wherein M1 is DL2000DNAMaker, ptaU is fragment upstream;
Fig. 9 is the nucleic acid electrophoresis figure of gene pta downstream sequence, and wherein M1 is DL2000DNAMaker, ptaD is segments downstream; And merge fragment ptaUD;
Figure 10 is the nucleic acid electrophoresis figure merging fragment ptaUD, and wherein M2 is that DL5000DNAMaker, ptaUD are for merging fragment;
Figure 11 is tetracycline resistance gene tet nucleic acid electrophoresis figure, and wherein M is DL2000DNAMaker, and 1,2 is tetracycline resistance gene tet;
Figure 12 is the measurement result that cellulose fermentation that embodiment one builds produces biomass in isopropylcarbinol engineering bacteria and bacillus cereus degraded cellulose Fermentive production of isobutanol process; Wherein a is bacillus cereus, and b is that cellulose fermentation produces isopropylcarbinol engineering bacteria;
Figure 13 is the measurement result that cellulose fermentation that embodiment one builds produces substrate utilization in isopropylcarbinol engineering bacteria and bacillus cereus degraded cellulose Fermentive production of isobutanol process; Wherein a is bacillus cereus, and b is that cellulose fermentation produces isopropylcarbinol engineering bacteria;
Figure 14 is that the cellulose fermentation that embodiment one builds produces the measurement result that in isopropylcarbinol engineering bacteria and bacillus cereus degraded cellulose Fermentive production of isobutanol process, reducing sugar accumulates; Wherein a is bacillus cereus, and b is that cellulose fermentation produces isopropylcarbinol engineering bacteria;
Figure 15 is the measurement result that cellulose fermentation that embodiment one builds produces isopropylcarbinol output in isopropylcarbinol engineering bacteria and bacillus cereus degraded cellulose Fermentive production of isobutanol process; Wherein a is bacillus cereus, and b is that cellulose fermentation produces isopropylcarbinol engineering bacteria.
Embodiment
Embodiment one: a kind of construction process utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria of present embodiment, carry out according to following steps:
One, the total serum IgE extracting yeast saccharomyces cerevisiae (S.cerevisiae) carries out reverse transcription and obtains cDNA, according to phenylpyruvate decarboxylase gene and alcohol dehydrogenase gene design primer aroF/R and adhF/R, then PCR obtains phenylpyruvate decarboxylase gene aro10 and alcohol dehydrogenase gene adh2;
Two, primer aroF and adhR is utilized to carry out pcr amplification phenylpyruvate decarboxylase gene aro10 and alcohol dehydrogenase gene adh2, and insert BamHI, BglII, XhoI and NheI tetra-restriction enzyme sites, be connected to form and merge fragment BamHI – BglII – aro10 – XhoI – adh2 – NheI, then with BamHI and NheI, double digestion is carried out to above-mentioned fusion fragment, double digestion is carried out to expression vector pMA5 simultaneously; Phenylpyruvate decarboxylase gene aro10 after double digestion is connected with the expression vector pMA5 after double digestion with the fusion fragment of alcohol dehydrogenase gene adh2, then heat shock proceeds in intestinal bacteria Top10 bacterial strain, after qualification is correct, obtain recombinant plasmid pMA5-RD;
Three, according to bacillus cereus (B.cereus) whole genome sequence, design primer alsF/alsR, then be the alsS gene fragment that template amplification obtains two ends and contains restriction enzyme site BamHI/BglII with bacillus cereus (B.cereus) complete genome DNA, and by BamHI and BglII respectively enzyme cut the recombinant plasmid pMA5-RD that alsS gene fragment and step 2 obtain, then the alsS gene fragment after being cut by enzyme is connected with recombinant plasmid pMA5-RD, construction expression plasmid pMA5-SRD; Expression plasmid pMA5-SRD electric shock proceeded in bacillus cereus (B.cereus) bacterial strain, coat and screen containing 50 μ g/mL kalamycin resistance LB flat boards, the positive transformant that empirical tests is correct is engineering strain;
Four, knock out two key gene pflB and pta in the engineering strain that step 3 obtains in metabolism of pyruvate collateral branch approach by homologous recombination method, finally obtain utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria.
In present embodiment, bacillus cereus (B.cereus) is bacillus cereus ACCC03288 bacterial strain, purchased from Chinese agriculture Microbiological Culture Collection administrative center.
PCR system involved in present embodiment is
The process of PCR is
Recombinant plasmid pMA5 described in present embodiment is shuttle plasmid, be included in intestinal bacteria and genus bacillus express needed for promotor and replication origin, and comprise some single restriction enzyme sites.
Present embodiment advantage: 1. bacillus cereus has higher tolerance for isopropylcarbinol, and the output that isopropylcarbinol limits isopropylcarbinol for the toxicity of cell in the production process of isopropylcarbinol is all key issue all the time, bacillus cereus is therefore utilized to produce isopropylcarbinol significant; 2. present embodiment constructs the pathways metabolism of synthesis isopropylcarbinol in bacillus cereus, introduce foreign gene adh2 and aro10, process LAN native gene alsS, and knocked out competition pathway key gene pflB and pta, build utilize cellulose fermentation to produce isopropylcarbinol engineering bacteria can High-efficient Production isopropylcarbinol, ultimate capacity can reach 1.310g/L; 3. the engineering strain that obtains of present embodiment can decomposition of cellulose biosynthesizing isopropylcarbinol, utilization can degraded cellulose strain fermentation produce isopropylcarbinol, substrate cost in fermenting process is reduced greatly, show bacillus cereus and produce great potential and value in isopropylcarbinol in fermentation, be significant at the energy and environmental area.
Embodiment two: present embodiment and embodiment one are unlike the NCBIGeneID:851987 of the phenylpyruvate decarboxylase gene described in step one.Other is identical with embodiment one.
Embodiment three: present embodiment and embodiment one or two are unlike the NCBIGeneID:855349 of the alcohol dehydrogenase gene described in step one.Other is identical with embodiment one or two.
Embodiment four: one of present embodiment and embodiment one to three unlike the primer aroF sequence described in: step one and step 2 are: 5'-CG gGATCCcGA aGATCTtCCATGGCACCTTGTACAATTGA-3'; AroR sequence is: 5'-TGAGTTTCTGGAATAGACATCCG cTCGAGcGGCTATTTTTTATTTCTTTTAAGTGC-3'; AdhF sequence is: 5'-GCACTTAAAAGAAATAAAAAATAGCCG cTCGAGcGGATGTCTATTCCAGAAACTCA-3'; AdhR sequence is: 5'-CTA gCTAGCtTATTTAGAAGTGTCAACAAC-3'.Other is identical with one of embodiment one to three.
Embodiment five: one of present embodiment and embodiment one to four unlike: the primer alsF sequence described in step 3 is: 5'-CGC gGATCCcACACATGATTAGTTTGAGTACAGGTG-3'; AlsR sequence is: 5'-GGA aGATCTcTGCCTCCATTAGTTTAATTG-3'.Other is identical with one of embodiment one to four.
Embodiment six: one of present embodiment and embodiment one to five unlike: the flow process knocking out pflB gene in step 4 is: according to bacillus cereus (B.cereus) full-length genome, design primer pflU1/pflU2 and pflD1/pflD2 respectively, be upstream and downstream sequence pflU and the pflD that template amplification goes out pflB gene with bacillus cereus (B.cereus) strain gene group DNA, recycling primer pflU1 and pflD2 inserts restriction enzyme site EcoRI by PCR, BglII, XhoI and HindIII, be connected to form and merge fragment EcoRI – pflU – BglII – XhoI – pflD – HindIII, be connected in plasmid pUC18 again and form pUC18-pflUD,
According to plasmid pSTV28 gene order, design primer camF/camR, with plasmid pSTV28 gene order for template, amplification chloramphenicol resistance gene fragment cam, and utilize restriction enzyme site BglII and XhoI respectively enzyme cut gene fragment cam and plasmid pUC18-pflUD, and the gene fragment cam after being cut by enzyme is connected with plasmid pUC18-pflUD, obtains pUC18-pflUD recombinant plasmid; Utilizing primer RV-M/M13 (-47), pcr amplification is carried out to pUC18-pflUD recombinant plasmid, obtain pflB gene knockout fragment pflU – cam – pflD, then electric shock proceeds in the engineering strain that step 3 obtains, resistance screening is carried out in containing the LB substratum of 25 μ g/mL paraxin, it is correct that screening obtains positive bacterium colony empirical tests, obtains pflB gene knock-out bacterial strain Δ B; Wherein the sequence of primer pflU1 is 5'-CG gAATTCcGTCTGATCAAACAGCAACGATTGC-3'; The sequence of pflU2 is 5'-CTCAGGATGTTCC cTCGAGcGGA aGATCTaCTTGAGTCATCCTAATCTC-3'; The sequence of pflD1 is 5'-CCG cTCGAGgGAACATCCTGAGAAATATCCAC-3'; The sequence of pflD2 is 5'-CCC aAGCTTgGTATGGTAGCACTTCAACTTTCT-3'; The sequence of camF is 5'-GA aGATCTcTAGCGCTGATGTCCGGCGGTG-3'; The sequence of camR is 5'-CCG cTCGAGgGCAGAATAAATGATCATATCG-3'; The sequence of RV-M is 5'-GAGCGGATAACAATTTCACACAGG-3'; The sequence of M13 (-47) is 5'-CGCCAGGGTTTTCCCAGTCACGAC-3'.Other is identical with one of embodiment one to five.
PCR system described in present embodiment and PCR program identical with embodiment one.
Embodiment seven: one of present embodiment and embodiment one to six unlike: described chloramphenicol resistance gene fragment cam derives from plasmid pSTV28, includes independently promoter sequence and chloramphenicol resistance gene encoding sequence.Other is identical with one of embodiment one to six.
Embodiment eight: one of present embodiment and embodiment one to seven unlike: in step 4, the flow process that knocks out of pta gene is: design primer ptaU1/ptaU2 and ptaD1/ptaD2 respectively according to bacillus cereus (B.cereus) full-length genome, be upstream and downstream sequence ptaU and the ptaD that template amplification goes out pta gene with bacillus cereus (B.cereus) strain gene group DNA, recycling primer ptaU1 and ptaD2 inserts restriction enzyme site EcoRI by PCR, BglII, XhoI and HindIII, be connected to form and merge fragment EcoRI – ptaU – BglII – XhoI – ptaD – HindIII, and be connected in plasmid pUC18 and form pUC18-ptaUD, according to pBR322 plasmid gene order, design primer tetF/tetR, with pBR322 plasmid gene order for template, amplification tetracycline resistance gene fragment tet, and utilize restriction enzyme site BglII and XhoI to carry out enzyme respectively to tetracycline resistance gene fragment tet and plasmid pUC18-ptaUD to cut, and the tetracycline resistance gene fragment tet after being cut by enzyme is connected with plasmid pUC18-ptaUD, obtains pUC18-ptaUD recombinant plasmid, utilizing primer RV-M/M13 (-47), pcr amplification is carried out to pUC18-ptaUD recombinant plasmid, obtain pta gene knockout fragment ptaU – tet – ptaD, then electric shock proceeds in pflB gene knock-out bacterial strain Δ B, resistance screening is carried out in containing the LB substratum of 12.5 μ g/mL tsiklomitsins, it is correct that screening obtains positive bacterium colony empirical tests, namely completes, wherein the sequence of primer ptaU1 is 5'-CG gAATTCgGGATGTTGTTACTCCTAAG-3', the sequence of ptaU2 is 5'-GATAGGTCGTTTACAGGCAT cTCGAGcGGA aGATCTtGCTCACGAATAAACCCTCC-3', the sequence of ptaD1 is 5'-CCG cTCGAGaTGCCTGTAAACGACCTATC-3', the sequence of ptaD2 is 5'-CCC aAGCTTaGAAACTACCGTTCCTGCTA-3', the sequence of tetF is 5'-GA aGATCTtTCTCATGTTTGACAGCTTA-3', the sequence of tetR is 5'-CCG cTCGAGtGGAGTGGTGAATCCGTTA-3', the sequence of RV-M is 5'-GAGCGGATAACAATTTCACACAGG-3', the sequence of M13 (-47) is 5'-CGCCAGGGTTTTCCCAGTCACGAC-3'.Other is identical with one of embodiment one to seven.
PCR system described in present embodiment and PCR program identical with embodiment one.
Embodiment nine: one of present embodiment and embodiment one to eight unlike: described tetracycline resistance gene fragment tet derives from pBR322 plasmid, includes independently promoter sequence and tetracycline resistance gene encoding sequence.Other is identical with one of embodiment one to eight.
Beneficial effect of the present invention is verified by following examples:
Embodiment 1: a kind of construction process utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria of the present embodiment, carry out according to following steps:
One, the total serum IgE extracting yeast saccharomyces cerevisiae (S.cerevisiae) carries out reverse transcription and obtains cDNA, according to phenylpyruvate decarboxylase gene and alcohol dehydrogenase gene design primer aroF/R and adhF/R, then PCR obtains phenylpyruvate decarboxylase gene aro10 and alcohol dehydrogenase gene adh2;
Two, primer aroF and adhR is utilized to carry out pcr amplification phenylpyruvate decarboxylase gene aro10 and alcohol dehydrogenase gene adh2, and insert BamHI, BglII, XhoI and NheI tetra-restriction enzyme sites, be connected to form and merge fragment BamHI – BglII – aro10 – XhoI – adh2 – NheI, then with BamHI and NheI, double digestion is carried out to above-mentioned fusion fragment, double digestion is carried out to expression vector pMA5 simultaneously; Phenylpyruvate decarboxylase gene aro10 after double digestion and alcohol dehydrogenase gene adh2 are merged fragment be connected with the expression vector pMA5 after double digestion, then heat shock proceeds in intestinal bacteria Top10 bacterial strain, after qualification is correct, obtain recombinant plasmid pMA5-RD;
Three, according to bacillus cereus (B.cereus) whole genome sequence, design primer alsF/alsR, then be the alsS gene fragment that template amplification obtains two ends and contains restriction enzyme site BamHI/BglII with bacillus cereus (B.cereus) complete genome DNA, and by BamHI and BglII respectively enzyme cut the recombinant plasmid pMA5-RD that alsS gene fragment and step 2 obtain, then the alsS gene fragment after being cut by enzyme is connected with recombinant plasmid pMA5-RD, construction expression plasmid pMA5-SRD; Expression plasmid pMA5-SRD electric shock proceeded in bacillus cereus (B.cereus) bacterial strain, coat and screen containing 50 μ g/mL kalamycin resistance LB flat boards, the positive transformant that empirical tests is correct is engineering strain;
Four, knock out two key gene pflB and pta in the engineering strain that step 3 obtains in metabolism of pyruvate collateral branch approach by homologous recombination method, finally obtain utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria.
The NCBIGeneID:851987 of the phenylpyruvate decarboxylase gene described in the present embodiment step one; The NCBIGeneID:855349 of alcohol dehydrogenase gene.Step one and the primer aroF sequence described in step 2 are: 5'-CG gGATCCcGA aGATCTtCCATGGCACCTTGTACAATTGA-3'; AroR sequence is: 5'-TGAGTTTCTGGAATAGACATCCG cTCGAGcGGCTATTTTTTATTTCTTTTAAGTGC-3'; AdhF sequence is: 5'-GCACTTAAAAGAAATAAAAAATAGCCG cTCGAGcGGATGTCTATTCCAGAAACTCA-3'; AdhR sequence is: 5'-CTA gCTAGCtTATTTAGAAGTGTCAACAAC-3'.
PCR system is
The process of PCR is
PCR system described in step 2 and course synchronization rapid;
Primer alsF sequence described in step 3 is: 5'-CGC gGATCCcACACATGATTAGTTTGAGTACAGGTG-3'; AlsR sequence is: 5'-GGA aGATCTcTGCCTCCATTAGTTTAATTG-3'; PCR system and course synchronization rapid;
The flow process knocking out pflB gene in step 4 is: according to bacillus cereus (B.cereus) full-length genome, design primer pflU1/pflU2 and pflD1/pflD2 respectively, be upstream and downstream sequence pflU and the pflD that template amplification goes out pflB gene with bacillus cereus (B.cereus) strain gene group DNA, recycling primer pflU1 and pflD2 inserts restriction enzyme site EcoRI by PCR, BglII, XhoI and HindIII, be connected to form and merge fragment EcoRI – pflU – BglII – XhoI – pflD – HindIII, be connected in plasmid pUC18 again and form pUC18-pflUD,
According to plasmid pSTV28 gene order, design primer camF/camR, with plasmid pSTV28 gene order for template, amplification chloramphenicol resistance gene fragment cam, and utilize restriction enzyme site BglII and XhoI respectively enzyme cut gene fragment cam and plasmid pUC18-pflUD, and the gene fragment cam after being cut by enzyme is connected with plasmid pUC18-pflUD, obtains pUC18-pflUD recombinant plasmid; Utilizing primer RV-M/M13 (-47), pcr amplification is carried out to pUC18-pflUD recombinant plasmid, obtain pflB gene knockout fragment pflU – cam – pflD, then electric shock proceeds in the engineering strain that step 3 obtains, resistance screening is carried out in containing the LB substratum of 25 μ g/mL paraxin, it is correct that screening obtains positive bacterium colony empirical tests, obtains pflB gene knock-out bacterial strain Δ B; Wherein the sequence of primer pflU1 is 5'-CG gAATTCcGTCTGATCAAACAGCAACGATTGC-3'; The sequence of pflU2 is 5'-CTCAGGATGTTCC cTCGAGcGGA aGATCTaCTTGAGTCATCCTAATCTC-3'; The sequence of pflD1 is 5'-CCG cTCGAGgGAACATCCTGAGAAATATCCAC-3'; The sequence of pflD2 is 5'-CCC aAGCTTgGTATGGTAGCACTTCAACTTTCT-3'; The sequence of camF is 5'-GA aGATCTcTAGCGCTGATGTCCGGCGGTG-3'; The sequence of camR is 5'-CCG cTCGAGgGCAGAATAAATGATCATATCG-3'; The sequence of RV-M is 5'-GAGCGGATAACAATTTCACACAGG-3'; The sequence of M13 (-47) is 5'-CGCCAGGGTTTTCCCAGTCACGAC-3'.
In step 4, the flow process that knocks out of pta gene is: design primer ptaU1/ptaU2 and ptaD1/ptaD2 respectively according to bacillus cereus (B.cereus) full-length genome, be upstream and downstream sequence ptaU and the ptaD that template amplification goes out pta gene with bacillus cereus (B.cereus) strain gene group DNA, recycling primer ptaU1 and ptaD2 inserts restriction enzyme site EcoRI by PCR, BglII, XhoI and HindIII, be connected to form and merge fragment EcoRI – ptaU – BglII – XhoI – ptaD – HindIII, and be connected in plasmid pUC18 and form pUC18-ptaUD, according to pBR322 plasmid gene order, design primer tetF/tetR, with pBR322 plasmid gene order for template, amplification tetracycline resistance gene fragment tet, and utilize restriction enzyme site BglII and XhoI to carry out enzyme respectively to tetracycline resistance gene fragment tet and plasmid pUC18-ptaUD to cut, and the tetracycline resistance gene fragment tet after being cut by enzyme is connected with plasmid pUC18-ptaUD, obtains pUC18-ptaUD recombinant plasmid, utilizing primer RV-M/M13 (-47), pcr amplification is carried out to pUC18-ptaUD recombinant plasmid, obtain pta gene knockout fragment ptaU – tet – ptaD, then electric shock proceeds in pflB gene knock-out bacterial strain Δ B, resistance screening is carried out in containing the LB substratum of 12.5 μ g/mL tsiklomitsins, it is correct that screening obtains positive bacterium colony empirical tests, namely completes, wherein the sequence of primer ptaU1 is 5'-CG gAATTCgGGATGTTGTTACTCCTAAG-3', the sequence of ptaU2 is 5'-GATAGGTCGTTTACAGGCAT cTCGAGcGGA aGATCTtGCTCACGAATAAACCCTCC-3', the sequence of ptaD1 is 5'-CCG cTCGAGaTGCCTGTAAACGACCTATC-3', the sequence of ptaD2 is 5'-CCC aAGCTTaGAAACTACCGTTCCTGCTA-3', the sequence of tetF is 5'-GA aGATCTtTCTCATGTTTGACAGCTTA-3', the sequence of tetR is 5'-CCG cTCGAGtGGAGTGGTGAATCCGTTA-3', the sequence of RV-M is 5'-GAGCGGATAACAATTTCACACAGG-3', the sequence of M13 (-47) is 5'-CGCCAGGGTTTTCCCAGTCACGAC-3'.
Chloramphenicol resistance gene fragment cam described above derives from plasmid pSTV28 (purchased from precious biological (Dalian) company limited), includes independently promoter sequence and chloramphenicol resistance gene encoding sequence; Described tetracycline resistance gene fragment tet derives from pBR322 plasmid (purchased from precious biological (Dalian) company limited), includes independently promoter sequence and tetracycline resistance gene encoding sequence.
Step one obtains the nucleic acid electrophoresis figure of phenylpyruvate decarboxylase gene aro10 and alcohol dehydrogenase gene adh2 as illustrated in fig. 1 and 2, and from Fig. 1 and 2, Successful amplification goes out phenylpyruvate decarboxylase gene aro10 and alcohol dehydrogenase gene adh2.
In step 2, phenylpyruvate decarboxylase gene aro10 and alcohol dehydrogenase gene adh2 merges the nucleic acid electrophoresis figure of amplification as shown in Figure 3, and wherein M is DL10000DNAMaker, RD is fusion fragment, and gene aro10 and adh2 merges fragment amplification success as shown in Figure 3;
As shown in Figure 4, wherein M is DL2000DNAMaker to gene alsS nucleic acid electrophoresis figure in step 3, and 1,2 is gene alsS, and as can be seen from Figure 4, gene alsS increases successfully.
As shown in Figure 5, wherein M is DL2000DNAMaker to gene pflB upstream and downstream sequencing nucleic acid electrophorogram in step 4, and 1,2 is fragment upstream pflU, and 3,4 is segments downstream pflD, as can be seen from Figure 5, and the success of gene pflB upstream and downstream sequence amplification.
As shown in Figure 6, wherein M is DL2000DNAMaker to fusion fragment pflUD nucleic acid electrophoresis figure in step 4, and 1 ~ 3 for merging fragment pflUD, as can be seen from Figure 6, merges fragment pflUD and increases successfully.
As shown in Figure 7, wherein M is DL2000DNAMaker to the chloramphenicol resistance gene cam nucleic acid electrophoresis figure of step 4, and 1,2 is chloramphenicol resistance gene cam.As can be seen from Figure 7, chloramphenicol resistance gene cam increases successfully.
The gene pta upstream and downstream sequence of step 4 and merge fragment ptaUD nucleic acid electrophoresis figure as shown in figs. 8-10, wherein M1 is DL2000DNAMaker, M2 be DL5000DNAMaker, ptaU is fragment upstream, and ptaD is segments downstream, and ptaUD is for merging fragment.Gene pta upstream and downstream sequence can be found out and merge fragment ptaUD from Fig. 8 ~ 10 and increase successfully.
As shown in figure 11, wherein M is DL2000DNAMaker to the tetracycline resistance gene tet nucleic acid electrophoresis figure of step 4, and 1,2 is tetracycline resistance gene tet; Tetracycline resistance gene tet increases successfully as can be seen from Figure 8.
It is ferment for sole carbon source with 20g/L filter paper fibre element that the engineering bacteria built utilizes cellulose fermentation to produce isopropylcarbinol, and inoculum size is 2% (v/v), and 37 DEG C, shaking culture under the condition of 60rpm, fermentation period is 12d.
The preparation of cellulose fermentation substratum: (NH 4) 2sO 410.0g/L, NaCl10.0g/L, KH 2pO 41.0g/L, K 2hPO 42.0g/L, MgSO 40.2g/L, 20g/L filter paper fibre element, pH7.0.
The preparation of inoculation liquid: the access of engineering strain that picking bacillus cereus (B.cereus) wild type strain successfully constructs containing in the LB liquid medium of final concentration 50 μ g/mL kalamycin resistance 37 DEG C, 160rpm carries out activation culture, gets the seed liquor of cultivation bacterium liquid as degraded cellulose fermentation high yield isopropylcarbinol process of 24h.
The cellulose fermentation that utilizes that Figure 12 ~ 15 build for the present embodiment produces biomass in isopropylcarbinol engineering bacteria (b) and commercially available bacillus cereus (a) degraded cellulose Fermentive production of isobutanol process, substrate utilization, reducing sugar accumulation and the measurement result of isopropylcarbinol output.As can be seen from the figure commercially available bacillus cereus cannot pass through self Fermentive production of isobutanol, and the engineering strain that the present embodiment builds can pass through degraded cellulose Fermentive production of isobutanol.
Produce isopropylcarbinol engineering bacteria production isopropylcarbinol with the cellulose fermentation that utilizes that the present embodiment builds, ultimate capacity can reach 1.310g/L.
In the present embodiment, bacillus cereus (B.cereus) is bacillus cereus ACCC03288 bacterial strain, purchased from Chinese agriculture Microbiological Culture Collection administrative center.
Recombinant plasmid pMA5 described in the present embodiment is shuttle plasmid, be included in intestinal bacteria and genus bacillus express needed for promotor and replication origin, and comprise some single restriction enzyme sites.
It can thus be appreciated that, bacillus cereus has higher tolerance for isopropylcarbinol, and the output that isopropylcarbinol limits isopropylcarbinol for the toxicity of cell in the production process of isopropylcarbinol is all key issue all the time, bacillus cereus is therefore utilized to produce isopropylcarbinol significant; The present embodiment constructs the pathways metabolism of synthesis isopropylcarbinol in bacillus cereus, introduce foreign gene adh2 and aro10, process LAN native gene alsS, and knocked out competition pathway key gene pflB and pta, build utilize cellulose fermentation to produce isopropylcarbinol engineering bacteria can High-efficient Production isopropylcarbinol, ultimate capacity can reach 1.310g/L; The engineering strain that the present embodiment obtains can decomposition of cellulose biosynthesizing isopropylcarbinol, utilization can degraded cellulose strain fermentation produce isopropylcarbinol, substrate cost in fermenting process is reduced greatly, show bacillus cereus and produce great potential and value in isopropylcarbinol in fermentation, be significant at the energy and environmental area.

Claims (9)

1. utilize cellulose fermentation to produce a construction process for isopropylcarbinol engineering bacteria, it is characterized in that it carries out according to following steps:
One, the total serum IgE extracting yeast saccharomyces cerevisiae (S.cerevisiae) carries out reverse transcription and obtains cDNA, according to phenylpyruvate decarboxylase gene and alcohol dehydrogenase gene design primer aroF/R and adhF/R, then PCR obtains phenylpyruvate decarboxylase gene aro10 and alcohol dehydrogenase gene adh2;
Two, primer aroF and adhR is utilized to carry out pcr amplification phenylpyruvate decarboxylase gene aro10 and alcohol dehydrogenase gene adh2, and insert BamHI, BglII, XhoI and NheI tetra-restriction enzyme sites, be connected to form and merge fragment BamHI – BglII – aro10 – XhoI – adh2 – NheI, then with BamHI and NheI, double digestion is carried out to above-mentioned fusion fragment, double digestion is carried out to expression vector pMA5 simultaneously; Phenylpyruvate decarboxylase gene aro10 after double digestion is connected with the expression vector pMA5 after double digestion with the fusion fragment of alcohol dehydrogenase gene adh2, then heat shock proceeds in intestinal bacteria Top10 bacterial strain, after qualification is correct, obtain recombinant plasmid pMA5-RD;
Three, according to bacillus cereus (B.cereus) whole genome sequence, design primer alsF/alsR, then be the alsS gene fragment that template amplification obtains two ends and contains restriction enzyme site BamHI/BglII with bacillus cereus (B.cereus) complete genome DNA, and by BamHI and BglII respectively enzyme cut the recombinant plasmid pMA5-RD that alsS gene fragment and step 2 obtain, then the alsS gene fragment after being cut by enzyme is connected with recombinant plasmid pMA5-RD, construction expression plasmid pMA5-SRD; Expression plasmid pMA5-SRD electric shock proceeded in bacillus cereus (B.cereus) bacterial strain, coat and screen containing 50 μ g/mL kalamycin resistance LB flat boards, the positive transformant that empirical tests is correct is engineering strain;
Four, knock out two key gene pflB and pta in the engineering strain that step 3 obtains in metabolism of pyruvate collateral branch approach by homologous recombination method, finally obtain utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria.
2. a kind of construction process utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria according to claim 1, is characterized in that the NCBIGeneID:851987 of the phenylpyruvate decarboxylase gene described in step one.
3. a kind of construction process utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria according to claim 1, is characterized in that the NCBIGeneID:855349 of the alcohol dehydrogenase gene described in step one.
4. a kind of construction process utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria according to claim 1, is characterized in that the primer aroF sequence described in step one and step 2 is: 5'-CG gGATCCcGA aGATCTtCCATGGCACCTTGTACAATTGA-3'; AroR sequence is: 5'-TGAGTTTCTGGAATAGACATCCG cTCGAGcGGCTATTTTTTATTTCTTTTAAGTGC-3'; AdhF sequence is: 5'-GCACTTAAAAGAAATAAAAAATAGCCG cTCGAGcGGATGTCTATTCCAGAAACTCA-3'; AdhR sequence is: 5'-CTA gCTAGCtTATTTAGAAGTGTCAACAAC-3'.
5. a kind of construction process utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria according to claim 1, is characterized in that the primer alsF sequence described in step 3 is: 5'-CGC gGATCCcACACATGATTAGTTTGAGTACAGGTG-3'; AlsR sequence is: 5'-GGA aGATCTcTGCCTCCATTAGTTTAATTG-3'.
6. a kind of construction process utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria according to claim 1, it is characterized in that the flow process knocking out pflB gene in step 4 is: according to bacillus cereus (B.cereus) full-length genome, design primer pflU1/pflU2 and pflD1/pflD2 respectively, be upstream and downstream sequence pflU and the pflD that template amplification goes out pflB gene with bacillus cereus (B.cereus) strain gene group DNA, recycling primer pflU1 and pflD2 inserts restriction enzyme site EcoRI by PCR, BglII, XhoI and HindIII, be connected to form and merge fragment EcoRI – pflU – BglII – XhoI – pflD – HindIII, be connected in plasmid pUC18 again and form pUC18-pflUD,
According to plasmid pSTV28 gene order, design primer camF/camR, with plasmid pSTV28 gene order for template, amplification chloramphenicol resistance gene fragment cam, and utilize restriction enzyme site BglII and XhoI respectively enzyme cut gene fragment cam and plasmid pUC18-pflUD, and the gene fragment cam after being cut by enzyme is connected with plasmid pUC18-pflUD, obtains pUC18-pflUD recombinant plasmid; Utilizing primer RV-M/M13 (-47), pcr amplification is carried out to pUC18-pflUD recombinant plasmid, obtain pflB gene knockout fragment pflU – cam – pflD, then electric shock proceeds in the engineering strain that step 3 obtains, resistance screening is carried out in containing the LB substratum of 25 μ g/mL paraxin, it is correct that screening obtains positive bacterium colony empirical tests, obtains pflB gene knock-out bacterial strain Δ B; Wherein the sequence of primer pflU1 is 5'-CG gAATTCcGTCTGATCAAACAGCAACGATTGC-3'; The sequence of pflU2 is 5'-CTCAGGATGTTCC cTCGAGcGGA aGATCTaCTTGAGTCATCCTAATCTC-3'; The sequence of pflD1 is 5'-CCG cTCGAGgGAACATCCTGAGAAATATCCAC-3'; The sequence of pflD2 is 5'-CCC aAGCTTgGTATGGTAGCACTTCAACTTTCT-3'; The sequence of camF is 5'-GA aGATCTcTAGCGCTGATGTCCGGCGGTG-3'; The sequence of camR is 5'-CCG cTCGAGgGCAGAATAAATGATCATATCG-3'; The sequence of RV-M is 5'-GAGCGGATAACAATTTCACACAGG-3'; The sequence of M13 (-47) is 5'-CGCCAGGGTTTTCCCAGTCACGAC-3'.
7. a kind of construction process utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria according to claim 6, it is characterized in that described chloramphenicol resistance gene fragment cam derives from plasmid pSTV28, include independently promoter sequence and chloramphenicol resistance gene encoding sequence.
8. a kind of construction process utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria according to claim 1 or 6, it is characterized in that in step 4, the flow process that knocks out of pta gene is: design primer ptaU1/ptaU2 and ptaD1/ptaD2 respectively according to bacillus cereus (B.cereus) full-length genome, be upstream and downstream sequence ptaU and the ptaD that template amplification goes out pta gene with bacillus cereus (B.cereus) strain gene group DNA, recycling primer ptaU1 and ptaD2 inserts restriction enzyme site EcoRI by PCR, BglII, XhoI and HindIII, be connected to form and merge fragment EcoRI – ptaU – BglII – XhoI – ptaD – HindIII, and be connected in plasmid pUC18 and form pUC18-ptaUD, according to pBR322 plasmid gene order, design primer tetF/tetR, with pBR322 plasmid gene order for template, amplification tetracycline resistance gene fragment tet, and utilize restriction enzyme site BglII and XhoI to carry out enzyme respectively to tetracycline resistance gene fragment tet and plasmid pUC18-ptaUD to cut, and the tetracycline resistance gene fragment tet after being cut by enzyme is connected with plasmid pUC18-ptaUD, obtains pUC18-ptaUD recombinant plasmid, utilizing primer RV-M/M13 (-47), pcr amplification is carried out to pUC18-ptaUD recombinant plasmid, obtain pta gene knockout fragment ptaU – tet – ptaD, then electric shock proceeds in pflB gene knock-out bacterial strain Δ B, resistance screening is carried out in containing the LB substratum of 12.5 μ g/mL tsiklomitsins, it is correct that screening obtains positive bacterium colony empirical tests, namely completes, wherein the sequence of primer ptaU1 is 5'-CG gAATTCgGGATGTTGTTACTCCTAAG-3', the sequence of ptaU2 is 5'-GATAGGTCGTTTACAGGCAT cTCGAGcGGA aGATCTtGCTCACGAATAAACCCTCC-3', the sequence of ptaD1 is 5'-CCG cTCGAGaTGCCTGTAAACGACCTATC-3', the sequence of ptaD2 is 5'-CCC aAGCTTaGAAACTACCGTTCCTGCTA-3', the sequence of tetF is 5'-GA aGATCTtTCTCATGTTTGACAGCTTA-3', the sequence of tetR is 5'-CCG cTCGAGtGGAGTGGTGAATCCGTTA-3', the sequence of RV-M is 5'-GAGCGGATAACAATTTCACACAGG-3', the sequence of M13 (-47) is 5'-CGCCAGGGTTTTCCCAGTCACGAC-3'.
9. a kind of construction process utilizing cellulose fermentation to produce isopropylcarbinol engineering bacteria according to claim 8, it is characterized in that described tetracycline resistance gene fragment tet derives from pBR322 plasmid, include independently promoter sequence and tetracycline resistance gene encoding sequence.
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CN103667163A (en) * 2012-09-04 2014-03-26 天津工业生物技术研究所 Recombinant microorganism for producing isobutanol and method thereof
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