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Isoprene synthase variants for improved microbial production of isoprene

A technology of isoprene synthase and isoprene synthase amino group, applied in the field of mutant plant isoprene synthase, can solve the problem of insufficient isoprene level for commercial use

Inactive Publication Date: 2011-08-31
DANISCO US INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the levels of isoprene obtainable by state-of-the-art bacterial systems are not sufficient for commercial use

Method used

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  • Isoprene synthase variants for improved microbial production of isoprene
  • Isoprene synthase variants for improved microbial production of isoprene
  • Isoprene synthase variants for improved microbial production of isoprene

Examples

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Embodiment approach

[0259] In some embodiments, the glucose isomerase promoter is used (see, e.g., U.S. Pat. No. 7,132,527 and references cited therein, each of which is hereby incorporated by reference in its entirety, particularly for expression of a polypeptide of interest). promoters and plasmid systems). Reported glucose isomerase promoter mutants can be used to alter the expression level of a polypeptide encoded by a nucleic acid operably linked to the glucose isomerase promoter (US Pat. No. 7,132,527). In various embodiments, the glucose isomerase promoter is contained in a low, medium or high copy plasmid (US Patent No. 7,132,527).

[0260] In various embodiments, the isoprene synthase nucleic acid, DXS nucleic acid, IDI nucleic acid, and / or MVA pathway nucleic acid is contained on a low copy plasmid (e.g., a plasmid maintained at about 1 to about 4 copies per cell), A medium copy plasmid (eg, a plasmid maintained at about 10 to about 15 copies per cell) or a high copy plasmid (eg, a pla...

Embodiment 1

[0339] Cloning of kudzu isoprene synthase for expression in recombinant bacteria

[0340] In this example, a method for producing kudzu isoprene synthase (IspS) in E. coli is described. The protein sequence of the kudzu (Gamum) isoprene synthase gene (IspS) was obtained from GenBank (AAQ84170). The kudzu isoprene synthase gene optimized for E. coli codon usage was purchased from DNA2.0 (Menlo Park, CA) and described in SEQ ID NO: 1 ( figure 1 ). The isoprene synthase gene was removed from the provided plasmid by restriction endonuclease digestion with BspLU11I / PstI, gel purified and ligated into pTrcHis2B (Invitrogen) which had been digested with NcoI / PstI. The construct was designed such that the stop codon in the isoprene synthase gene was located 5' to the PstI site. Therefore, when this construct is expressed, the His tag is not attached to the isoprene synthase protein. The resulting plasmid pTrcKudzu was verified by sequencing.

[0341] The isoprene synthase gene w...

Embodiment 2

[0345] Cloning of poplar isoprene synthase for expression in recombinant bacteria

[0346] In this example, a method for producing poplar isoprene synthase (IspS) in E. coli is described. The protein sequence of poplar (Pulus alba x Populus alba) isoprene synthase (Schnitzler et al., Planta 222:777-786, 2005) was obtained from GenBank (CAC35696). A gene optimized for codon pair E. coli was purchased from DNA2.0 and described in SEQ ID NO: 6 ( image 3 ). The isoprene synthase gene was removed from the provided plasmid (p9796-poplar) by restriction endonuclease digestion with BspLU11I / PstI, gel purified and ligated into pTrcHis2B which had been digested with NcoI / PstI. The construct was cloned such that the stop codon in the inserted sequence preceded the PstI site, which resulted in a construct in which the His tag was not attached to the isoprene synthase protein. The resulting plasmid pTrcPoplar was verified by sequencing using commercially available primers (forward and ...

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Abstract

The present invention provides methods and compositions comprising at least one isoprene synthase enzyme with improved catalytic activity and / or solubility. In particular, the present invention provides variant plant isoprene synthases for increased isoprene production in microbial host cells. Biosynthetically produced isoprene of the present invention finds use in the manufacture of rubber and elastomers.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application Serial No. 61 / 125,336, filed April 23, 2008, which is hereby incorporated by reference in its entirety. technical field [0003] The present invention provides methods and compositions comprising at least one isoprene synthase with improved catalytic activity and / or solubility. Specifically, the present invention provides variant plant isoprene synthases for use in increasing isoprene production in microbial host cells. The isoprene of the invention produced biosynthetically is useful in the manufacture of rubbers and elastomers. Background of the invention [0004] Isoprenoids are isoprene polymers used in pharmaceuticals, nutraceuticals, flavours, fragrances and rubber products. However, the supply of natural isoprenoids is limited by ecological concerns. For this reason and to provide isoprenoid compositions with fewer impurities and greater unifor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/52C12N5/10
CPCC12N9/88C12P5/007C12N5/10C12N15/52C12Q1/25C12Y402/03027
Inventor M·A·卡文G·M·怀特德A·米亚斯尼科夫C·M·派瑞斯W·韦勒D·H·韦尔斯R·R·博特C·L·莱福J·C·麦考利夫J·凯利斯
Owner DANISCO US INC
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