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Genetically engineered microorganisms for the production of poly-4-hydroxybutyrate

A technology of hydroxybutyrate and products, applied in the field of genetically engineered microorganisms for the production of poly-4-hydroxybutyrate, which can solve the problems of low cytotoxicity, low amount of desired product, low polymer, etc.

Inactive Publication Date: 2015-01-28
CJ CHEILJEDANG CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, these methods are disadvantageous in that the amount of polymer in the biomass is relatively low, which further leads to the subsequent low amount of desired product
Therefore, there is a need to generate genetically modified organisms with increased amounts of polymers (e.g., poly-4-hydroxybutyrate), which in turn are processed into green chemicals, thereby overcoming the low yields, Disadvantages of cytotoxicity and low purity

Method used

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  • Genetically engineered microorganisms for the production of poly-4-hydroxybutyrate
  • Genetically engineered microorganisms for the production of poly-4-hydroxybutyrate
  • Genetically engineered microorganisms for the production of poly-4-hydroxybutyrate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0199] Example 1: Improvement of P4HB production by using alpha-ketoglutarate decarboxylase from Pseudonocardia dioxanivorans

[0200] Several metabolic pathways have been proposed to produce succinic semialdehyde (SSA) from the tricarboxylic acid (TCA) cycle (reviewed by Steinbüchel and Lütke-Eversloh, Biochem. Engineering J. 16:81–96 (2003) and Efe et al., Biotechnology and Bioengineering 99:1392-1406 (2008)). One such pathway converts α-ketoglutarate to SSA by α-ketoglutarate decarboxylase encoded by kgdM (Tian et al., Proc. Natl. Acad. Sci. U.S.A. 102:10670-10675 (2005)). Previous attempts have utilized the kgdM gene from Mycobacterium tuberculosis (Tian et al., Proc. Natl. Acad. Sci. U.S.A. 102:10670-10675 (2005); figure 1 , Reaction No. 6) The production of P4HB was not successful, and only a very small amount of P4HB was produced (Van Walsem et al., patent application number WO 2011100601A1).

[0201] This example demonstrates that homologues of M. tuberculosis KgdM ...

Embodiment 2

[0210] Example 2: Development of a growth selection strategy to obtain genes with improved α-ketoglutarate decarboxylase activity

[0211] The P4HB titers of recombinant hosts expressing the kgd homologue from P.dioxanivorans (hereafter referred to as kgdP) were only positive for those expressing sucD from Clostridium krujiri. Ck Approximately two-thirds of the titers were obtained in strains with the * gene (see Tables 2 and 3). Therefore, a growth selection method was developed to obtain mutated kgdP genes with improved α-ketoglutarate decarboxylase activity. To this end, Escherichia coli MG1655ΔsucAB strain was constructed, which lacked α-ketoglutarate dehydrogenase activity ( figure 1 , Reaction 4). MG1655 containing the deletion of sucAB was constructed using the above-mentioned known biotechnology means and methods. Due to the lack of α-ketoglutarate dehydrogenase (ΔsucAB) and any native α-ketoglutarate decarboxylase activity in E. Grow in E2 minimal medium as the s...

Embodiment 3

[0215] Example 3: Improved P4HB production by expressing a mutated alpha-ketoglutarate decarboxylase kgdP-M38 from Pseudonocardia dioxanivorans

[0216] Improved P4HB production in strains expressing kgdP-M38

[0217] In this example, the production of P4HB was compared in strains expressing native kgdP and in strains expressing the mutated kgdP-M38 from Pseudonocardia dioxanivorans. Therefore, the following two strains were constructed using the well-known biotechnological tools and methods described above, the genomes of both strains contain chromosomal deletions of yneI, gabD, pykF and pykA, and overexpression of orfZ from Clostridium krusii Ck genes, ppc gene from Escherichia coli, PHA synthase phaC3 / C1* and ssaR from Arabidopsis At *Gene. In addition, strain 7 with P trc The promoter expresses the native kgdP gene, while strain 8 also expresses the P trc The promoter expressed mutated kgdP-M38 (Table 5).

[0218] Table 5: Microbial strains used in this part of Exampl...

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Abstract

Methods and genetically engineered hosts for the production of poly-4-hydroxybutrate and 4-carbon products are described herein.

Description

[0001] related application [0002] This application claims priority to US Provisional Application No. 61 / 613,388, filed March 20, 2012, which is hereby incorporated by reference in its entirety. [0003] This application incorporates by reference the Sequence Listing contained in the following ASCII files, filed concurrently with this application: [0004] a. File name: 46141007001SEQ.txt; generated on February 26, 2013, with a size of 76.3975KB. Background technique [0005] Bio-based biodegradable polymers (such as polyhydroxyalkanoate (PHA)) in a variety of biomass systems (such as plant biomass, microbial biomass (such as bacteria, including cyanobacteria, yeast, fungi) or produced from algae biomass). Genetically modified biomass systems for the production of a wide variety of biodegradable PHA polymers and copolymers have recently been developed (Lee (1996), Biotechnology & Bioengineering 49:1-14; Braunegg et al. (1998), J. Biotechnology 65:127-14. 161; Madison, L.L...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N9/02C12N9/04C12N9/12C12N9/88C12P7/62C12N1/21
CPCC12N9/1205C12Y207/01086C12Y604/01001C12N9/001C12Y401/01071C12N9/0008C12Y103/01006C12N9/93C12Y102/01075C12N9/88C12P7/625C12Y602/01005C12Y101/01077C12N15/52C12N9/0006C12Y101/01002C12Y602/01004
Inventor W·R·法默C·W·J·麦克查利彻T·M·拉姆塞尔Z·张D-E·常J·比克迈耶J·比利C·莫泽
Owner CJ CHEILJEDANG CORP