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Compositions and methods for the increased production of isoprene and other products with 6 - phosphogluconolactonase (PGL)

A technology of isoprene and nucleic acid, applied in the field of improved compositions for increasing the production of isoprene in Escherichia coli, which can solve the problems of high cost, increased consumption of isoprene, insufficient isoprene, etc.

Inactive Publication Date: 2013-01-30
DANISCO US INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is a relatively expensive and energy-intensive process
Worldwide demand for petrochemical based products continues to increase, at the same time that consumption of isoprene is expected to rise to much higher levels in the long term and its availability is in any case limited
There are concerns that future supply of isoprene from petrochemical based sources will not be sufficient to meet planned demand and its price will rise to unprecedented levels

Method used

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  • Compositions and methods for the increased production of isoprene and other products with 6 - phosphogluconolactonase (PGL)
  • Compositions and methods for the increased production of isoprene and other products with 6 - phosphogluconolactonase (PGL)
  • Compositions and methods for the increased production of isoprene and other products with 6 - phosphogluconolactonase (PGL)

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0252] Example 1: Construction and expression of Saccharomyces cerevisiae gi1.2KKDyI operon, Populus alba isoprene synthesis Enzymes, Methanococcus mazei mevalonate kinase, pCL upstream MVA (Enterococcus faecalis mvaE and mvaS) and ybhE (pgl) E. coli strains

[0253] (i) Construction of bacterial strain EWL201 (BL21, Cm-GI1.2-KKDyI)

[0254] Escherichia coli BL21 (Novagen brand, from EMD Biosciences, Inc.) was the recipient strain, using MCM331P1 lysate (according to Ausubel et al. in Current Protocols in Molecular Biology. John Wiley and Sons, Inc. ( Lysate prepared as described in "Molecular Biology Laboratory Guide", John Wiley & Sons Publishing Company) was transduced. MCM331 cells contain the chromosomal construct gi1.2KKDyI (i.e., gi1.2 from S. cerevisiae) encoding S. - KKDyI operon; its construction is described in Example 10 of International Patent Publication No. WO 2009 / 076676A2 and US Patent Application No. 12 / 335,071 (US Patent Publication No. 2009 / 0203102...

example 2

[0293] Example 2: Improvement of heterogeneity by constitutive expression of ybhE(pgl) from a plasmid in E. coli Pentadiene production

[0294] This example shows isoprene production in a strain constitutively expressing E. coli ybhE(pgl) compared to a control strain expressing ybhE at wild-type levels (ie, EWL256). The gene ybhE(pgl) encodes an Escherichia coli 6-phosphogluconolactonase that inhibits post-translational glucoacylation of heterologously expressed proteins and improves product solubility and yield while also increasing biomass through the pentose phosphate pathway Yield and flux (Aon et al., Applied and Environmental Microbiology 74(4): 950-958, 2008 ).

[0295] i) Small scale analysis

[0296] Medium formula (per liter of fermentation medium): K 2 HPO 4 13.6g, KH 2 PO 4 13.6g, MgSO 4 *7H 2 O 2g, citric acid monohydrate 2g, ferric ammonium citrate 0.3g, (NH 4 ) 2 SO 4 3.2g, 1g of yeast extract, 1ml of 1000× trace metal solution. All components ...

example 3

[0310] Example 3: Recovery of Isoprene from Renewable Resources

[0311] Isoprene recovery from a set of four 14L scale fermentors in a two-step operation involving removal of isoprene from fermentation off-gas streams by adsorption to activated carbon followed by off-line steam desorption and condensed to obtain liquid isoprene ( Figure 16A and 16B ). The total amount of isoprene produced by the four fermentors was 1150 g (16.9 mol), of which 953 g (14 mol, 83%) were adsorbed by the carbon filter. After the vapor desorption / condensation step, the amount of liquid isoprene recovered was 810 g, corresponding to an overall recovery of 70%. The recovered isoprene was analyzed for the presence of impurities.

[0312] Analysis of isoprene liquid and its impurity distribution

[0313] The recovered isoprene liquid was analyzed by GC / MS and gas chromatography / flame ionization detection (GC / FID) to determine the nature and content of impurities. The product was found to be ...

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Abstract

Provided herein are improved compositions and methods for the increased production of isoprene. Also provided herein are improved compositions and methods for the increased production of heterologous polypeptides capable of biological activity.

Description

[0001] Cross-references to related patent applications [0002] This patent application claims priority to US Provisional Patent Application No. 61 / 289,959, filed on December 23, 2009, the disclosure of which is hereby incorporated by reference in its entirety. technical field [0003] The present disclosure relates to improved compositions and methods for increasing production of biochemicals in E. coli, and improved compositions and methods for increasing isoprene production in E. coli. Background technique [0004] Isoprene (2-methyl-1,3-butadiene) is an important organic compound that is widely used. For example, isoprene is used as an intermediate or feedstock in the synthesis of various chemical compositions and polymers. Isoprene is also an important biological material naturally synthesized by many plants and animals. [0005] When the stereoregular polymerization of isoprene entered the market in the early 1960s, isoprene became an important monomer for the synthe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P5/00C12P7/00C12N15/90
CPCC12P5/007C12N15/635C12P7/42C12N15/70C12N15/52
Inventor Z・Q・贝克M・A・塞尔温A・T・尼尔森C・M・派瑞斯
Owner DANISCO US INC
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