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A kind of butadiene producing bacteria and its method for producing butadiene

A technology for producing bacteria and butadiene, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of high cost, low and efficient synthesis of butadiene, and no cost, and achieve the effect of cost reduction

Inactive Publication Date: 2020-11-06
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, microbial synthesis of high-value-added products mainly relies on six-carbon sugars as carbon sources. The cost of butadiene is still too high, and there is currently no low-cost and efficient production method for synthesizing butadiene

Method used

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  • A kind of butadiene producing bacteria and its method for producing butadiene
  • A kind of butadiene producing bacteria and its method for producing butadiene
  • A kind of butadiene producing bacteria and its method for producing butadiene

Examples

Experimental program
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Effect test

Embodiment 1

[0054] The butadiene synthesis pathway consists of 4 steps (e.g. figure 2 shown):

[0055] 1) With crotonyl coenzyme A as a precursor, coenzyme A reductase (coenzyme A reductase Far or butyraldehyde dehydrogenase-butanol dehydrogenase BLD-BDH or aldol dehydrogenase ADHE2) of the present invention converts crotonyl coenzyme A Coenzyme A is reduced to crotyl alcohol;

[0056] 2) crotyl alcohol is phosphorylated into crotyl alcohol monophosphate by crotyl alcohol kinase (THK or MK or TK);

[0057] 3) Crotyl alcohol monophosphate kinase IPK catalyzes the phosphorylation of crotyl alcohol monophosphate to generate crotyl alcohol pyrophosphate;

[0058] 4) Butadiene synthase MTS or ISPS catalyzes crotyl alcohol pyrophosphate to generate butadiene.

[0059] (1) For the first step reaction, according to the structural characteristics of the substrate crotonyl-CoA and the mechanism of the enzyme-catalyzed reaction, the present invention screens the bld-bdh from Clostridium saccharo...

Embodiment 2

[0067] Following the experimental operation of Example 1, the construction of a one-pot reaction from crotonyl-CoA to butadiene in vitro.

[0068] Select the enzyme with the highest catalytic efficiency in each step to build a one-pot reaction system, which are FAR (from Marinobactermanganoxydans), THK, IPK and MTS. First, optimize the pH of the four enzyme reactions, and the pH gradient is 6, 6.5, 7, 7.5, 8, 8.5 and 9, it was found that most of the enzymes catalyzed to generate the highest target products at pH 8.0. The temperature of the four enzymatic reactions was optimized with a temperature gradient of: 29.5°C, 32°C, 34.5°C, 37°C, 39.5°C, 42°C and 44.5°C. It was found that most of the enzymes catalyzed the highest target products at 39.5°C.

[0069] Under the conditions of the optimal temperature of 39.5°C and pH of 8.0, the optimal enzyme amount of each enzyme in the one-pot reaction was optimized, and the enzyme amount gradient was 0.25mg / mL, 0.5mg / mL, 1mg / mL and 1.5m...

Embodiment 3

[0073] The four genes far+thim+ipk+mts are expressed in Methylobacterium extorquens AM1, and each two genes are connected by RBS. Construct the target gene fragment of BamHI restriction site+far+RBS+thim+RBS+ipk+RBS+mts+SacI restriction site, and carry out step-by-step digestion on the target gene fragment and pCM80, and combine the target gene and plasmid After the backbone was ligated through restriction sites BamHI and SacI, plasmid pYJ was obtained. Then point-transfer into M. extorquens AM1 to construct strain M. extorquensAM1-pYJ. Inoculate strain AM1-pYJ to prepare seed liquid, and then carry out vial culture. The specific vial culture condition is to use 10mL Hypho medium with 10ug / mL tetracycline in a 50mL anaerobic vial at 30°C, 200rpm shaker, and seal it away from light. Cultivate for 70h. The standard of inoculum volume is OD 600 =0.8 seed liquid inoculation 1mL, the thalline growth status of above-mentioned culture uses absorbance OD 600 To characterize; butad...

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Abstract

The invention relates to a butadiene producing strain taking methyl alcohol as a raw material and a method for producing butadiene, and belongs to the technical field of construction of engineering bacteria. The butadiene producing strain belongs to methylobacterium, the producing strain belongs to methylobacterium, and the producing strain is obtained by that foreign genes of the following enzymes are expressed by methylobacterium and then transferred into modification and expression, wherein the following enzymes include butadiene synthetase MTS or ISPS, crotyl alcohol mono-phosphate kinaseIPK, crotyl alcohol kinase THK or MK or TK an coenzyme A reductase FAR or BLD-BDH or ADHE2; and methylobacterium contains a serine circulation route and an acetyl malonyl-CoA route. The butadiene producing strain provided by the invention can take methyl alcohol as the raw material to produce butadiene, and can sufficiently utilize the raw material of methyl alcohol which has the competitive priceand is sufficiently supplied, and a butadiene product is directly synthesized through microbial metabolism. The cost is greatly reduced, and achievement of industrialized production of the procedurethat butadiene is synthesized by methyl alcohol is facilitated.

Description

technical field [0001] The invention relates to the technical field of engineering bacteria construction, in particular to a butadiene-producing bacteria using methanol as a raw material and a method for producing butadiene. Background technique [0002] 1,3-butadiene (hereinafter referred to as butadiene) is the molecular formula C 4 h 6 organic compound, consisting of two vinyl groups, it is the simplest conjugated diene ( figure 1 ). It is an easily liquefied, colorless gas with a characteristic odour. Slightly soluble in water, soluble in ethanol, methanol, easily soluble in acetone, ether and chloroform, etc. (https: / / en.wikipedia.org / wiki / 1,3-Butadiene) Butadiene is an important platform compound used in the production of synthetic rubbers including styrene-butadiene rubber (SBR), poly Butadiene (PB), styrene-butadiene latex (SBL), acrylonitrile-butadiene-styrene resin (ABS), nitrile rubber and adiponitrile), which are also used in the manufacture of Nylon -66 (W...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12R1/01
CPCC12N9/00C12N9/0004C12N9/12C12P5/026
Inventor 杨松杨靖张长太莫旭华朱丽萍
Owner QINGDAO AGRI UNIV
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