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Recombinant strain for high yield of butanol and construction method thereof

A technology of recombinant strains and construction methods, applied in the field of microbial engineering, can solve the problems of butanol fermentation lack of economic competitiveness, low selectivity of butanol, high cost of carbon source, etc., to achieve improved metabolic intensity, increased activity, yield and conversion rate-enhancing effect

Active Publication Date: 2012-10-03
GUANGZHOU INST OF ENERGY CONVERSION - CHINESE ACAD OF SCI
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Problems solved by technology

[0008] But at present, butanol fermentation lacks economic competitiveness, the main reasons are: 1) the cost of carbon source used for fermentation is high; 2) the concentration of butanol in the fermentation broth is low; 3) the selectivity of butanol in the fermentation process is not good. high

Method used

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  • Recombinant strain for high yield of butanol and construction method thereof
  • Recombinant strain for high yield of butanol and construction method thereof
  • Recombinant strain for high yield of butanol and construction method thereof

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Embodiment 1

[0043] 1. Preparation of expression cassette EC, operon Sol and butanol dehydrogenase gene bdh.

[0044] 1) Analysis of enzyme genes of butanol biosynthetic pathway in butanol-producing bacteria and preparation of expression cassettes

[0045] Schematic diagram of butanol biosynthetic pathway figure 1 As shown, the expression intensity of the genes of the enzymes in the butanol biosynthetic pathway is controlled by the promoters, and the promoters used in the present invention are shown in Table 1. The acquisition of the target gene and the fusion of the target gene and the promoter are achieved by PCR.

[0046] Table 1: Sequences of functional regions of the promoters used

[0047]

[0048] 50μL PCR reaction system includes: dNTP 4μL, 10×PS buffer (Mg 2+ plus) 5 μL, forward primer (20 μmol / L) 1 μL, reverse primer (20 μmol / L) 1 μL, DNA template 100 μg, PrimeSTAR TM HS DNAPolymerase 0.5μL, add dd H2O to 50μL. PCR reaction conditions: pre-denaturation at 94°C for 5 min;...

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Abstract

The invention discloses a recombinant strain for the high yield of butanol and a construction method thereof. The construction method is characterized in that an objective segment is connected with an expression vector to obtain a recombination expression plasmid, and then the recombination expression plasmid is transformed to enter clostridium acetobutylicum, thereby obtaining the recombinant strain for the high yield of the butanol; the objective segment is an expression cassette EC, butanol dehydrogenase gene bdh, or a combination of two or three of the expression cassette EC, the butanol dehydrogenase gene bdh or an operon Sol; the nucleotide sequence of the expression cassette EC is shown in SEQ ID NO.11; the nucleotide sequence of the operon Sol is shown in SEQ ID NO.16; and the nucleotide sequence of the butanol dehydrogenase gene bdh is shown in SEQ ID NO.20. The recombinant strain provided by the invention can be used for expressing butanol biosynthetic enzymes coded by butanol producing strain butanol biosynthetic genes, thereby enabling the activity of the butanol biosynthetic enzymes and the metabolism degree of the strain to be increased; and the yield and conversion rate of the butanol can be further increased so that the industrial production of the butanol is facilitated.

Description

Technical field: [0001] The invention belongs to the technical field of microbial engineering, and in particular relates to a high-butanol-producing recombinant bacterial strain and a construction method thereof. Background technique: [0002] Butanol is an important chemical raw material, mainly used in plasticizers, solvents, extractants, etc. In recent years, it has been found that the calorific value and octane number of butanol are equivalent to those of gasoline, and it can be mixed with gasoline in any ratio, and can be transported by existing oil pipelines. A promising new biofuel. [0003] Butanol is mainly produced by fermentation and petrochemical synthesis. Fermentation method is grain or other starchy agricultural and sideline products, which are hydrolyzed to obtain fermentation liquid, and then under the action of acetone and Clostridium butyricum, the mixture of acetone, butanol and ethanol is obtained through fermentation, the usual ratio is 3:6:1, And th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12P7/16C12R1/145
Inventor 陈新德侯小虎熊莲黄超张海荣陈雪芳
Owner GUANGZHOU INST OF ENERGY CONVERSION - CHINESE ACAD OF SCI
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