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Genetically engineered bacterium for co-generating butanol and 2,3-butanediol and construction method and application thereof

A technology of genetically engineered bacteria and construction methods, applied in the field of genetically engineered bacteria co-producing butanol and 2,3-butanediol and its construction, can solve the problem of no increase in the production of 3-hydroxybutanone, and achieve improved product quality. Value, improve product yield, improve the effect of economic benefits

Active Publication Date: 2016-04-20
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, neither the expression of its own ALS (acetolactate synthase) nor the ALS of Bacillus subtilis in Clostridium acetobutylicum showed an increase in 3-hydroxybutanone production (Wardwell, S.A. MetabolismofacetoininC.acetobutylicumATCC824.RiceUniversity.1999)

Method used

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  • Genetically engineered bacterium for co-generating butanol and 2,3-butanediol and construction method and application thereof
  • Genetically engineered bacterium for co-generating butanol and 2,3-butanediol and construction method and application thereof
  • Genetically engineered bacterium for co-generating butanol and 2,3-butanediol and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction of Clostridium acetobutylicum containing expression plasmids of acetolactate decarboxylase genes CAC2967, BSU36000, BL02479, A3UG_05860, KPN2242-13255, EL23_18310 and CK00_RS0123920.

[0036] Table 1 Gene name and source of acetolactate decarboxylase

[0037] Acetolactate decarboxylase gene name

source

serial number

CAC2967

C. acetobutylicum ATCC 824

SEQ ID NO: 1

BSU36000

B. Subtilis 168

SEQ ID NO: 2

BL02479

B. licheniformis ATCC14580

SEQ ID NO: 3

A3UG_05860

E. cloacae SDM

SEQ ID NO: 4

KPN2242—13255

K. pneumoniae KCTC 2242

SEQ ID NO: 5

EL23_18310

P. polymyxa DSM 365

SEQ ID NO: 6

CK00_RS0123920

S. marcescens ATCC 14041

SEQ ID NO: 7

[0038] The bacterial genome kit was used to extract the genomic DNA of Clostridium acetobutylicum ATCC824 in the middle and late logarithmic growth period, and the acetolactate decarboxylase...

Embodiment 2

[0056] Example 2: Construction of Clostridium acetobutylicum containing expression plasmids of acetoin reductase BSU06240, Cbei_1464, KPN_02061, CAETHG_0385.

[0057] Table 3 Acetoin reductase gene name and its source

[0058]

[0059] Bacillus subtilis B.Subtilis168 and Clostridium beijerinckii (Clostridium beijerinckii) NCIMB8052 were extracted by bacterial genome kit. The acetoin reductase genes BSU06240 and Cbei_1464 were amplified by PCR with the following primers:

[0060] BSU06240-s (SEQ ID NO: 16): AAAAGGGAGTGTCGA CATATG AAGGCAGCAAGATG (the underlined part is the NdeI recognition site);

[0061] BSU06240-as (SEQ ID NO: 17): GTACTGAGAGTGCAC CATATG TTAGTTAGGTACAAGGA (the underlined part is the NdeI recognition site). The amplified product was constructed according to the one-step cloning method in Example 1 to obtain vector pIMP1-BSU06240. After sequencing, its sequence is determined as shown in SEQ ID No:8.

[0062] Cbei_1464-s (SEQ ID NO: 18): AAAAGGGAGTGTCG...

Embodiment 3

[0068] Example 3: Construction of Clostridium acetobutylicum containing expression plasmids linking acetolactate decarboxylase gene BSU36000 and acetoin reductase BSU06240, Cbei_1464, KPN_02061, CAETHG_0385 respectively. In the above four gene sequences, the stop codon of the acetolactate decarboxylase gene BSU36000 was immediately followed by the start codon of the acetoin reductase BSU06240, Cbei_1464, KPN_02061, and CAETHG_0385.

[0069] Use the following primers to amplify the gene fragments that can connect the acetolactate decarboxylase gene BSU36000 and the acetoin reductase gene BSU06240, Cbei_1464, KPN_02061 and CAETHG_0385:

[0070] BSU36000BSU06240-s (SEQ ID NO: 20): AAAAGGGAGTGTCGA CATATG AAACGAGAAAGCAACATTC (the underlined part is the NdeI recognition site);

[0071] BSU36000BSU06240-as (SEQ ID NO: 21): TGCCATCTTGCTGCCTTCATTTATTCAGGGCTTCCTTCAG;

[0072] BSU06240-s (SEQ ID NO: 22): CTGAAGGAAGCCCTGAATAAATGAAGGCAGCAAGATG;

[0073] BSU06240-as (SEQ ID NO: 23): GTA...

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Abstract

The invention discloses a genetically engineered bacterium for co-generating butanol and 2,3-butanediol. The genetically engineered bacterium is a butanol generating clostridium to which an acetoin reductase gene is introduced or a butanol generating clostridium to which an acetolactic acid decarboxylase gene and the acetoin reductase gene are introduced. The nucleotide sequence of the acetolactic acid decarboxylase gene is any one of nucleotide sequences shown in SEQ ID NO:1-7, and the nucleotide sequence of the acetoin reductase gene is any one of nucleotide sequences shown in SEQ ID NO:8-11. An acetolactic acid decarboxylic reaction is enhanced in a clostridium acetobutylicum, the yield of 3-hydroxy butanone is greatly increased to about 6.4 g / L from initial about 1.8 g / L, and then acetoin is converted into 2,3-butanediol reaching up to 8.05 g / L under the effect of acetoin reductase; acetone is reduced to 2.2 g / L from about 4.5 g / L, and economic benefits of fermentation of the clostridium acetobutylicum are remarkably increased.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered bacterium for co-producing butanol and 2,3-butanediol, a construction method and application thereof. Background technique [0002] 2,3-Butanediol is a colorless, odorless liquid with strong hygroscopicity. It is liquid at room temperature, viscosity is 30.6mPa·s, boiling point is 178-182°C, freezing point is -60°C, miscible with water, soluble in ether and ethanol, and used as monomer in polymer synthesis. 2,3-Butanediol is an important bio-based four-carbon platform compound, which is widely used in food, chemical industry, aerospace fuel and other fields. [0003] The production methods of 2,3-butanediol mainly include chemical synthesis method and microbial fermentation method. The chemical synthesis method uses fossil raw materials to produce 2,3-butanediol with high cost, harsh conditions and cumbersome process, which is eas...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P7/16C12P7/18C12R1/145
CPCC12N9/0006C12N9/88C12P7/16C12P7/18C12Y101/01005C12Y401/01005Y02E50/10Y02T50/678
Inventor 应汉杰沈晓宁柳东王岩岩许佳慧刘俊郭亭
Owner NANJING UNIV OF TECH
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