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Isoprene synthase variants for improved production of isoprene

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

Inactive Publication Date: 2013-12-11
DANISCO US INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the isoprene content obtainable by prior art bacterial systems is insufficient for commercial use

Method used

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

Examples

Experimental program
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preparation example Construction

[0319] Exemplary Preparation of Isoprene

[0320] In some embodiments, the cells are cultured in culture medium under conditions that allow the cells to produce isoprene. In some embodiments, cells in culture produce greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500 , 1,750, 2,000, 2,500, 3,000, 4,000, 5,000 nanomoles or more of isoprene per gram of wet cell weight per hour (nmole / gwcm / hr). In some embodiments, the amount of isoprene is between about 2 and about 5,000 nmole / gwcm / hr, such as between about 2 and about 100 nmole / gwcm / hr, between about 100 and about 500 nmole / gwcm / hr hr, between about 150 to about 500 nmole / gwcm / hr, between about 500 to about 1,000 nmole / gwcm / hr, between about 1,000 to about 2,000 nmole / gwcm / hr, or about 2,000 to about 5,000 nmole / gwcm / hr between gwcm / hr. The amount of isoprene in nmole / gwcm / hr can be measured as disclosed in US Patent No. 5,849,970. For example, using a standard ...

example

[0362] The following examples are provided for illustrative purposes and are not intended to limit the invention in any way.

example 1

[0363] Example 1: Isoprene Synthase Growth Screen: Validation, Optimization and Limitations

[0364] This example describes the selection of improved variants of isoprene synthase by in vivo screening. The inventors found that an in vivo screen could be used to select cells with less isoprene synthase activity than a control (in our case, the best validated isoprene synthase MEA-Pulus alba). Alternatively, in vivo screens can be used to select cells with greater isoprene synthase activity than controls (in our case, the best validated isoprene synthase MEA-Pulus alba).

[0365] method

[0366] Strains: Strains were screened for variants containing constitutively expressed downstream pathway and isoprene synthase expressed from pET plasmids. The screening strain was DW425 positive control.

[0367] Assay conditions: Strains were grown overnight at 34°C in LB medium containing 50uM kanamycin. Containing 1% glucose, 0.1% yeast extract, 8mM MgSO 4 Dilute the overnight cul...

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Abstract

The present invention provides methods and compositions comprising at least one isoprene synthase enzyme with improved specific productivity. In particular, the present invention provides variant plant isoprene synthases for increased isoprene production in host cells.

Description

[0001] Cross references to related patent applications [0002] This patent application claims priority to US Provisional Patent Application No. 61 / 407,415, filed October 27, 2010, the entire contents of which are hereby incorporated by reference. technical field [0003] The present invention provides methods and compositions comprising isoprene synthase variants. In particular, the present invention provides variant plant isoprene synthases for use in increasing isoprene production in a host cell. Background technique [0004] Isoprenoids are isoprene polymers used in pharmaceuticals, nutraceuticals, flavors, fragrances and rubber products. However, the supply of natural isoprenoids is limited due to ecological concerns. Therefore, to provide isoprenoid compositions with fewer impurities and greater uniformity, isoprenoids, such as rubber, are typically produced synthetically. [0005] Isoprene (2-methyl-1,3-butadiene) is a volatile hydrocarbon that is insoluble in wate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/52
CPCC12N9/88C12N15/63C12N15/74C12N15/79C12P5/005C12Y402/03027
Inventor Z·Q·贝克D·A·埃斯特尔C·L·莱福D·H·韦尔斯J·V·米勒
Owner DANISCO US INC
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