Isoprene producing bacterial and isoprene producing method

A technology of prenyl diphosphate and isoprene, applied in the direction of microorganism-based methods, biochemical equipment and methods, botany equipment and methods, etc., can solve the problems of expensive glucose, biomass raw material supply and price Volatility and other issues

Active Publication Date: 2015-07-22
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the price of glucose from food sources is expensive, and there is a problem of competing with others for food, while the technical bottleneck of obtaining monosaccharides from cellulose sources has not yet been broken through; the supply and price of biomass raw materials will show large fluctuations due to seasonal and climate changes. fluctuation

Method used

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  • Isoprene producing bacterial and isoprene producing method
  • Isoprene producing bacterial and isoprene producing method
  • Isoprene producing bacterial and isoprene producing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0188] Example 1 Expression of isoprene synthase (IspS) in M.extorquens AM1

[0189] The selected isoprene synthase should have higher enzymatic activity after expression in the Methylobacterium strain, so as to ensure high utilization rate of DMAPP and high yield of isoprene. At present, there is no precedent for expressing isoprene synthase in Methylobacterium bacteria, but there is a precedent for heterologous expression of part of isoprene synthase in Escherichia coli and measurement of its enzyme activity. The results of all enzyme activities known so far are shown in the table below:

[0190] Table 4 Enzyme activity of each species of isoprene synthase (IspS) when expressed heterologously in Escherichia coli

[0191]

[0192] In a comparable range, it can be seen that the isoprene synthase of Populus alba has the highest enzyme activity when expressed heterologously in Escherichia coli. Therefore, the isoprene synthase (IspS) of Populus alba was chosen...

Embodiment 2

[0200] Example 2 Expression of isoprene synthase (IspS) and isoprene in M. extorquens AM1 diphosphate isomerase (IDI)

[0201] Isopentenyl diphosphate isomerase (IDI) can convert IPP into DMAPP, the precursor of isoprene. In order to ensure that isoprene synthase can obtain sufficient substrate supply, IDI enzyme is introduced from an exogenous source. . Select the IDI enzyme derived from Escherichia coli str.K-12substr.MG1655, and use the same method as (1) to optimize the codon of the IDI enzyme gene to obtain the IDI enzyme gene that is conducive to the expression in AM1.

[0202] SacI restriction site (5'-3'GAGCTC)+IDI gene+RBS(pCM110)+MIspS gene+KpnI restriction site (5'-3'GGTACC) operon gene was chemically synthesized in Shanghai Sangon Bioengineering Company fragment. The synthesized fragment and plasmid pCM110 were double-enzyme-digested with SacI and KpnI enzymes. After purification, they were ligated with ligase to obtain plasmid pIDI&MIspS. The plasmid...

Embodiment 3

[0204] Example 3 Expression in M.extorquens AM1 HMGS+HMGR+MK+PMK+MPD+IspS

[0205] As mentioned above, the metabolic pathway from acetyl-CoA to isoprene involves six enzymes of the mevalonate pathway, isopentenyl diphosphate isomerase (IDI) and isoprene synthase ( IspS) total eight enzymes, because there are acetoacetyl CoA thiolase gene and isopentenyl diphosphate isomerase gene (IDI) in the Methylobacterium bacterium itself in this paper, so introduce remaining six enzymes, can The pathway for the production of isoprene by the MEV pathway was constructed. In this example, the enzymes HMGS and HMGR of the upper mevalonate pathway of E.faecalis and the lower mevalonate pathway MK of Streptococcus pneumoniae (S.pneumoniae) were selected under the same conditions. , PMK, MPD enzymes, combined with isoprene synthase of poplar (P. canescens), constitute the isoprene synthesis pathway to be introduced. The basic information of these 6 genes is listed in Table 6 below. ...

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Abstract

The invention relates to isoprene producing bacterial and an isoprene producing method, specifically to bacterial for producing isoprene by adopting methanol as a raw material and belonging to Methylobacterium, wherein the bacterial is modified so as to express isoprene synthetase (IspS).

Description

technical field [0001] This article relates to an isoprene-producing bacterium and a method for producing isoprene using the isoprene-producing bacterium. Background technique [0002] Isoprene, or 2-methyl-1,3-butadiene, is a reactive olefin with a conjugated double bond ( figure 1 ). Isoprene is insoluble in water, but miscible with common organic solvents such as ethanol, ether, acetone and benzene. The boiling point of isoprene is only 34°C at 100kPa, so it is very volatile. It is also prone to self-polymerization and copolymerization with other unsaturated compounds. Polymerization over a Ziegler-Natta catalyst produces polyisoprene, the natural rubber (http: / / en.wikipedia.org / wiki / Isoprene). [0003] Due to the limitation of natural conditions, my country's natural rubber resources have been in short supply, and the import dependence has continued to exceed 70%. The import areas are concentrated in Thailand, Indonesia and Malaysia, the three major rubber-producing ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/54C12N15/60C12P5/02C12R1/26
Inventor 张翀朱文亮邢新会梁伟凡
Owner TSINGHUA UNIV
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