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Methods, reagents and cells for biosynthesizing compounds

An active, hydroxyalkyl technology, applied in the field of recombinant host cells and recombinant hosts

Inactive Publication Date: 2017-05-31
INVISTA TEXTILES (U K) LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] However, no wild-type prokaryote or eukaryote naturally overproduces or secretes such C5 building blocks into the extracellular environment

Method used

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  • Methods, reagents and cells for biosynthesizing compounds
  • Methods, reagents and cells for biosynthesizing compounds
  • Methods, reagents and cells for biosynthesizing compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0368] Enzymatic activity of omega-transaminase using glutaric semialdehyde as a substrate and forming 5-aminovaleric acid

[0369] Nucleotide sequences encoding N-terminal His-tags were added to the genes from Chromobacterium violaceum and Rhodococcus sphaeroides encoding ω-transaminases of SEQ ID NOs: 8 and 10, respectively (see Figure 10 ), allowing the production of N-terminal HIS-tagged omega-aminotransferases. Each resulting modified gene was cloned into the pET21a expression vector under the control of the T7 promoter, and each expression vector was transformed into a BL21[DE3] E. coli host. The resulting recombinant E. coli strains were grown in 250 mL shake flask cultures containing 50 mL LB medium and antibiotic selection pressure at 37°C with shaking at 230 rpm. Each culture was induced overnight at 16°C with 1 mM IPTG.

[0370] Pellets from each induced shake flask culture were harvested by centrifugation. Each pellet was resuspended and lysed by sonication. C...

Embodiment 2

[0376] Enzymatic activity of carboxylic acid reductase using 5-hydroxyvaleric acid as a substrate and forming 5-hydroxyvaleraldehyde

[0377] Nucleotide sequences encoding His-tags were added to M. marinum, M. smegmatis, M. smegmatis, Segniliparus from M. marinum, M. smegmatis, M. smegmatis, Segniliparus encoding carboxylic acid reductases of SEQ ID NOs: 2-4, 6 and 7, respectively Genes of rugosus, M. marseii, and Segniliparus rotundus (GenBank accession numbers ACC40567.1, ABK71854.1, EFV11917.1, EIV11143.1, and ADG98140.1, respectively) (see Figure 10 ), allowing the production of N-terminal HIS-tagged carboxylate reductases. Each modified gene was cloned into the pET Duet expression vector, both under the control of the T7 promoter, along with the sfp gene encoding the His-tagged phosphopantethelin transferase from Bacillus subtilis. Each expression vector was transformed into a BL21[DE3] E. coli host. Each resulting recombinant E. coli strain was grown in a 250 mL shake...

Embodiment 3

[0382] Omega-transaminase activity for 5-aminopentanol to form 5-oxopentanol

[0383] Nucleotide sequences encoding N-terminal His-tags were added to Chromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae, Rhodobacter sphaeroides, Escherichia coli encoding ω-transaminases of SEQ ID NOs: 8-13, respectively Bacillus and Vibrio fluvius genes (see Figure 10 ), allowing the production of N-terminal HIS-tagged omega-aminotransferases. The modified gene was cloned into the pET21a expression vector under the T7 promoter. Each expression vector was transformed into a BL21[DE3] E. coli host. Each resulting recombinant E. coli strain was grown in a 250 mL shake flask containing 50 mL of LB medium and antibiotic selection pressure at 37°C with shaking at 230 rpm. Each culture was induced overnight at 16°C with 1 mM IPTG.

[0384] Pellets from each induced shake flask culture were harvested by centrifugation. Each pellet was resuspended and lysed by sonication. Cell...

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Abstract

This document describes biochemical pathways for producing 5-hydroxypentanoate methyl ester and pentanoic acid pentyl ester using one or more of a fatty acid O-methyltransferase, an alcohol O-acetyltransferase, and a monooxygenase, as well as recombinant hosts expressing one or more of such exogenous enzymes. 5-hydroxypentanoate methyl esters and pentanoic acid pentyl esters can be enzymatically converted to glutaric acid, 5-aminopentanoate, 5-hydroxypentanoate, cadaverine, or 1,5-pentanediol.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims U.S. Provisional Application Serial No. 62 / 012,659, filed June 16, 2014, U.S. Provisional Application Serial No. 62 / 012,666, filed June 16, 2014, and U.S. Provisional Application Serial No. , filed June 16, 2014 No. 62 / 012,604, the disclosure of which is hereby incorporated by reference in its entirety. [0003] Field of Invention [0004] The present invention relates to methods for the biosynthesis of methyl 5-hydroxyvalerate and amyl valerate using one or more isolated enzymes, such as fatty acid O-methyltransferase, alcohol O-acetyltransferase and monooxygenase , and to recombinant host cells expressing one or more of these enzymes. The present invention also relates to the enzymatic conversion of methyl 5-hydroxyvalerate and amyl valerate to 5-hydroxyvalerate and 1,5-Pentanediol methods, and recombinant host cells expressing one or more of these enzymes. In addition, the present invention re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12P7/44C12P7/62C12P7/6436
CPCC12P7/44C12N9/1007C12P7/62C12N9/1096C12N9/0008C12N9/0077C12Y102/99006C12Y114/15001C12Y114/15003C12Y206/01018C12Y206/01019C12Y206/01029C12Y206/01048C12Y206/01082C12P7/42C12P7/6436C12P13/001C12P7/6409C12P7/24C07C59/01C08G63/06C12P7/40C07C47/02C07C47/12C07C59/325C07C223/02
Inventor A.L.博特斯A.V.E.康拉迪R.哈杜什
Owner INVISTA TEXTILES (U K) LTD
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