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Methods, reagents and cells for biosynthesizing compound

A carboxyl-functionalized and reactive technology, applied in the field of carbon chain aliphatic backbone

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 the C7 building block into the extracellular milieu

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0319] Enzymatic activity of thioesterases that use pimeloyl-CoA as a substrate and form pimelic acid

[0320] The sequence encoding the N-terminal His-tag was added to the tesB gene (SEQ ID NO:21, see Figure 9 ), allowing the generation of N-terminally HIS-tagged ω-transaminases. The modified tesB gene was cloned into the pET15b expression vector under the control of the T7 promoter. The expression vector was transformed into a BL21[DE3] E. coli host. The resulting recombinant E. coli strains were grown in 500 mL shake flask cultures containing 50 mL of Luria Broth (LB) medium and antibiotic selection pressure at 37°C with shaking at 230 rpm. Each culture was induced overnight at 17°C with 0.5 mM IPTG.

[0321] The pellet from each induced shake flask culture was harvested by centrifugation. Resuspend the pellet and in Y-per TM solution (ThermoScientific, Rockford, IL). Cell debris was separated from the supernatant by centrifugation. Thioesterase was purified from th...

Embodiment 2

[0324] Enzymatic activity of omega-transaminase using pimelate semialdehyde as substrate and forming 7-aminoheptanoic acid

[0325] Sequences encoding N-terminal His-tags were added to omega-transaminases from Chromobacterium violaceum, Pseudomonas syringae, Rhodobacter sphaericus, and Vibrio fluvialus encoding the ω-transaminases of SEQ ID NOs: 13, 15, 16, and 18, respectively. gene (see Figure 9 ), 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.

[0326] The pellet from each induced shake flask culture was harvested by centrif...

Embodiment 3

[0332] Enzymatic activity of carboxylic acid reductases that use pimelic acid as a substrate and form pimelic semialdehyde

[0333] A sequence encoding a His-tag was added to the genes from Segniliparus rugosus and Segniliparus rotundus encoding the carboxylic acid reductases of SEQ ID NO: 9 and 12, respectively (see Figure 9), allowing the generation of an N-terminally HIS-tagged carboxylic acid reductase. Each modified gene was cloned into the pETDuet expression vector together with the sfp gene encoding the His-tagged phosphopantetheinyl transferase from Bacillus subtilis, both under the T7 promoter. Each expression vector was transformed into a BL21[DE3] E. coli host, and each resulting recombinant large intestine 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 Bacillus strains. Each culture was induced overnight at 37°C using autoinduction medium.

[0334] The pellet from each induc...

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Abstract

This document describes biochemical pathways for producing 2(E)-heptenedioyl-CoA methyl ester from precursors such as 2-oxo-glutarate, acetyl-CoA, or succinyl-CoA using one or more of a fatty acid O-methyltransferase, a thioesterase, a CoA-transferase, a CoA ligase, as well as recombinant hosts expressing one or more of such enzymes. 2(E)-heptenedioyl-CoA methyl ester can be enzymatically converted to pimeloyl-CoA using a trans-2-enoyl-CoA reductase, and a methylesterase. Pimeloyl-CoA 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 priority to US Application Nos. 62 / 012,674 and 62 / 012,735, both filed June 16, 2014, the disclosures of which are incorporated herein by reference in their entirety. [0003] field of invention [0004] The present invention relates to a method for masking a carbon chain aliphatic backbone functionalized with a terminal carboxyl group using a polypeptide having fatty acid O-methyltransferase activity in a recombinant host. The present invention also relates to methods of biosynthesizing heptopenoyl-CoA methyl ester in a host using one or more of: (i) a polypeptide having fatty acid O-methyltransferase activity (ii) a polypeptide having thioesterase activity or Polypeptides with CoA-transferase activity, or (iii) polypeptides having CoA ligase activity, and recombinant host cells involving the expression of one or more such enzymes. In addition, the present invention relates to the conversion of he...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/18C12P7/44C12P7/24C12P7/62C12P13/00C12P17/10C12N1/21C12R1/19
CPCC12P7/62C07C69/533C12N9/0008C12N9/1029C12N9/1096C12N9/16C12N9/18C12P7/18C12P7/24C12P7/40C12P7/42C12P7/44C12P11/00C12P13/001C12P13/005C12P17/10C12Y102/99006C12Y203/01016C12Y206/01018C12Y206/01019C12Y206/01029C12Y206/01048C12Y206/01082C12Y301/01085C12Y301/02
Inventor A.L.博特斯A.V.E.康拉迪
Owner INVISTA TEXTILES (U K) LTD
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