Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Production of isoprene, isoprenoid precursors, and isoprenoids using acetoacetyl-coa synthase

A technology of acetoacetyl coenzyme and acetyl coenzyme, which is applied in the directions of acyltransferase, biochemical equipment and methods, and introduction of foreign genetic material using vectors, can solve the problem of expensive purification, uncommercially attractive and time-consuming isoprene yield. And other issues

Inactive Publication Date: 2014-09-10
DANISCO US INC +1
View PDF33 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, isoprene yields from naturally occurring organisms are not commercially attractive
Isoprene can also be obtained by fractionating petroleum, but purification of this material is expensive and time-consuming

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Production of isoprene, isoprenoid precursors, and isoprenoids using acetoacetyl-coa synthase
  • Production of isoprene, isoprenoid precursors, and isoprenoids using acetoacetyl-coa synthase
  • Production of isoprene, isoprenoid precursors, and isoprenoids using acetoacetyl-coa synthase

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0259] Example 1: Encoding MVA for the production of MVA by acetoacetyl-CoA synthase (NphT7) Construction of Upstream Pathway Plasmid

[0260] An expression plasmid was generated to encode the nphT7 gene, mvaS gene, and mvaR gene for expressing acetoacetyl-CoA synthase, HMG-CoA synthase, and HMG-CoA reductase, respectively. Simply put, forward and reverse primers are synthesized to amplify mvaS genes (MCM489 and MCM490), mvaR genes (MCM491 and MCM492) and nphT7 genes (MCM495 and MCM496) from synthetic genes encoding Streptomyces proteins (Table 1) . The MCM485 forward primer and MCM486 reverse primer were used to amplify the expression vector. The DNA template used for vector amplification is pMCM1225 (Table 2). The DNA template used to amplify mvaS and mvaR from Streptomyces is StrepCL190 (DNA2.0), which contains synthetic operons encoding mvaS and mvaR, and also encodes acetyl-CoA acetyltransferase (atoB). The pMCM1187 template including the synthetic gene encoding histidi...

example 2

[0273] Example 2: Construction of a plasmid encoding isoprene synthase and MVK for isoprene production

[0274] An expression plasmid for isoprene synthase and mevalonate kinase (MVK) with the bla gene encoding β-lactamase was generated. In short, primers MCM694 and MCM695 (Table 5) were used to amplify the bla gene from pUC19DNA (Invitrogen). The primers MCM696 and MCM697 were used to amplify the expression plasmid pDu65, which did not contain the cmR marker gene. use Seamless Cloning and Assembly Kit ( Seamless cloning and assembly kit) (#A13288) The amplicons are fused according to the manufacturer's protocol, and the product is then transformed into chemically competent MD09-314 cells. The fusion plasmid was selected on the LB / carb50 plate at 37°C overnight. A single colony was selected, cultured in 5mL LB / carb50 at 37°C and stored at -80°C.

[0275] Table 5. Primers used for pMCM1623 construction

[0276]

example 3

[0277] Example 3: Construction of thiolase-deficient Escherichia coli strain CMP861

[0278] Produce acetyl-CoA acetyltransferase (atoB) deficient strains. In short, the strain JW2218 (Baba et al. 2006. Mol. Syst. Biol. ("Molecular Systems Biology") 2:2006.0008) from the Keio collection (Keio collection) was used as a template, and the primer atoBrecF (5' -GCAATTCCCCTTCTACGCTGGG-3' (SEQ ID NO: 15)) and atoBrecR (5'-CTCGACCTTCACGTTGTTACGCC-3' (SEQ ID NO: 16)), by PCR amplifying a DNA fragment containing the atoB gene disrupted by the kanamycin marker . The polymerase Herculase II Fusion (Agilent, Santa Clara, CA) was used according to the manufacturer's instructions. The obtained PCR product was used in a recombineering reaction (Recombineering Reaction) to integrate the PCR product at the atoB locus of the strain CMP451 according to the recommendation of the manufacturer (Gene Bridges, Heidelberg, Germany). CMP451 is CMP258 with two modifications (see US Patent Application No...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

This invention relates to a recombinant microorganism capable of producing isoprene and isoprene production with the use of such recombinant microorganism with good efficiency. In this invention, the acetoacetyl-CoA synthase gene encoding an enzyme capable of synthesizing acetoacetyl-CoA from malonyl-CoA and acetyl-CoA and one or more genes involved in isoprene biosynthesis that enables synthesis of isoprene from acetoacetyl-CoA are introduced into a host microorganism.

Description

[0001] Cross reference to related patent applications [0002] This application claims the benefits of U.S. Provisional Application No. 61 / 515,300 filed on August 4, 2011, and the disclosure of the provisional application is incorporated herein by reference in its entirety. Technical field [0003] The present invention generally relates to methods for producing isoprene, isoprenoid precursors, and / or isoprenoids from cultured cells and compositions containing these cultured cells. Background of the invention [0004] The products of the mevalonate-dependent pathway are isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP). IPP and DMAPP are precursors of isoprene and isoprenoids. [0005] Isoprene (2-methyl-1,3-butadiene) is a key raw material for many synthetic polymers (most notably synthetic rubber). Isoprene is naturally produced by a variety of microorganisms, plants and animal species. Specifically, two pathways for isoprene biosynthesis have been identified:...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C08F36/08C12P5/00C12N9/10C12N9/00
CPCC12P5/007C12N15/81C12Y402/03027C12N9/1085C08F36/08C12N9/88C12Y203/01194C12N15/70C12N9/1029Y02P20/582Y02E50/30
Inventor I·S·阿尔多Z·Q·贝克M·C·米勒C·M·派瑞斯
Owner DANISCO US INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com