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Methods, reagents and cells for biosynthesizing compounds

A reactive, hydroxyalkyl-based technology for biosynthesis of compounds, reagents and cells

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 C7 building blocks into the extracellular milieu

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

[0373] Enzymatic activity of omega-transaminase using pimelate semialdehyde as substrate and forming 7-aminoheptanoic acid

[0374] The nucleotide sequence encoding the N-terminal His-tag was added to the genes from Chromobacter violaceum, Pseudomonas syringae, Rhodobacter sphaericus and Vibrio fluvialus encoding the ω-transaminases of SEQ ID NO: 8, 11 and 13, respectively (see Figure 10), allowing the generation of N-terminally HIS-tagged ω-transaminases. Each of the resulting modified genes 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.

[0375] The pellet from each induced shake flask culture was harvested by centrifugation. E...

Embodiment 2

[0381] Carboxylate reductase activity using pimelic acid as a substrate and forming pimelic semialdehyde

[0382] The nucleotide sequence encoding the His-tag was added to the genes from Segniliparus rugosus and Segniliparus rotundus encoding the carboxylic acid reductases of SEQ ID NOs: 4 (EFV11917.1) and 7 (ADG98140.1), respectively (see Figure 10 ), allowing the generation of an N-terminally HIS-tagged carboxylic acid reductase. Each modified gene was cloned into the pET Duet expression vector together with the sfp gene encoding the His-tagged phosphopantetheinyltransferase from Bacillus subtilis, both under the T7 promoter. Each expression vector was transformed into a BL21[DE3] E. coli host, and each resulting recombinant E. coli was grown in a 250 mL shake flask culture containing 50 mL LB medium and antibiotic selection pressure at 37°C with shaking at 230 rpm strain. Each culture was induced overnight at 37°C using autoinduction medium.

[0383] The pellet from eac...

Embodiment 3

[0387] Enzyme activity of carboxylic acid reductase using 7-hydroxyheptanoic acid as substrate and forming 7-hydroxyheptanal

[0388] The nucleotide sequence encoding the His-tag was added to the carboxylic acid reductases from Mycobacterium marinum, Mycobacterium smegmatis, Segniliparus rugosus, Mycobacterium smegmatis, Marseilles respectively encoding the carboxylic acid reductase of SEQ ID NO:2-7. Mycobacterium, and Segniliparus rotundus genes (GenBank accession numbers ACC40567.1, ABK71854.1, EFV11917.1, ABK75684.1, EIV11143.1, and ADG98140.1, respectively) (see Figure 10 ), allowing the generation of an N-terminally HIS-tagged carboxylic acid reductase. Each modified gene was cloned into the pET Duet expression vector together with the sfp gene encoding the His-tagged phosphopantetheinyl transferase from Bacillus subtilis, all under the control of the T7 promoter. Together with the expression vector from Example 3, each expression vector was transformed into a BL21[DE3]...

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Abstract

This document describes biochemical pathways for producing 7-hydroxyheptanoate methyl ester and heptanoic acid heptyl 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. 7-hydroxyheptanoate methyl esters and heptanoic acid heptyl esters can be enzymatically converted to pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, or 1,7-heptanediol.

Description

[0001] Cross References 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 7-hydroxyheptanoate and heptyl heptanoate 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 7-hydroxyheptanoate and heptyl heptanoate to 7-hydroxyheptanoate and 1,7-Heptanediol methods, and recombinant host cells expressing one or more of these enzymes. In addition, the prese...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12P7/44C12P7/62C12N1/21C12R1/19C12P7/6436
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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