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Methods, reagents and cells for biosynthesizing compounds

A functionalization and conversion technology, applied in biochemical equipment and methods, organic chemistry, oxidoreductase, etc., can solve problems such as no reports

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

However, no information on the use of these strategies was reported (Jang et al., Biotechnology & Bioengineering, 2012, 109(10), 2437–2459)

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

[0301] Enzyme activity of thioesterases using adipyl-CoA as substrate and forming adipate

[0302] The sequence encoding the N-terminal His tag was added to the gene from Escherichia coli encoding the thioesterase of SEQ ID NO:21 and SEQ ID NO:28 (see Figure 9 ), allowing the generation of N-terminal HIS-tagged thioesterases. Each resulting modified gene was cloned into the pET15b expression vector under the control of the T7 promoter. Each expression vector was transformed into a BL21[DE3] E. coli host. The resulting recombinant E. coli strain was cultured at 37° C. under shaking at 230 rpm in a 250 mL shake flask containing 50 mL of Luria Broth (LB) medium and antibiotic selection pressure. Cultures were induced overnight at 17°C with 0.5 mM IPTG.

[0303] Pellets from induced shake flask cultures were harvested via centrifugation. Resuspend each pellet, and in Y-per TM solution (ThermoScientific, Rockford, IL). Cell debris and supernatant were separated via centrifug...

Embodiment 2

[0306] Enzymatic activity of ω-transaminase using adipate semialdehyde as substrate and forming 6-aminocaproic acid

[0307] The nucleotide sequence encoding the His tag was added to the omega-transaminases encoding SEQ ID NOs: 13, 14, 15, 16, and 18 from Bacillus viridans, Pseudomonas aeruginosa, Pseudomonas syringae, spheroids Rhodobacter and Vibrio genes (see Figure 9 ), allowing the generation of N-terminally HIS-tagged ω-transaminases. Each resulting modified gene was cloned into a 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.

[0308] Pellets from each induced shake flask culture were harvested via centrifugation. Each pellet was resuspended ...

Embodiment 3

[0314] Enzymatic activity of carboxylic acid reductases that use adipate as a substrate and form adipate semialdehyde

[0315] The nucleotide sequence encoding the HIS-tag was added to the genes from Segniliparus rugosus and Segniliparus rotundusta 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 pET Duet 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 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, shaking at 230 rpm. Each culture was induced overnight at 37°C using autoinduction medium.

[0316] The pellet from each induced shake ...

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Abstract

This document describes biochemical pathways for producing 2,3-dehydroadipyl-CoA methyl ester from precursors such as 2-oxoglutarate using one or more of a fatty acid O-methyltransferase, a thioesterase, a CoA-transferase and a CoA ligase, as well as recombinant hosts expressing one or more of such enzymes. 2,3-dehydroadipyl-CoA methyl ester can be enzymatically converted to adipyl-CoA using a trans-2-enoyl-CoA reductase, and a methylesterase, which in turn can be enzymatically converted to adipic acid, 6-aminohexanoate, 6-hydroxyhexanoate, caprolactam, hexamethylenediamine, or 1,6-hexanediol.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of 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. technical field [0003] The present invention relates to a method for masking a carbon chain aliphatic backbone functionalized with a terminal carboxyl group in a recombinant host using a polypeptide having fatty acid O-methyltransferase activity. The present invention also relates to a method for biosynthesizing 2,3-dehydroadipyl CoA methyl ester in a host using one or more of: (i) a polypeptide having fatty acid O-methyltransferase activity, (ii) a polypeptide having Polypeptides with thioesterase activity or CoA transferase activity, or (iii) polypeptides having CoA ligase activity, and recombinant host cells expressing one or more such exogenous enzymes. The present invention also relates to the conversion of 2,3-dehydroadipy...

Claims

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Application Information

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IPC IPC(8): C12P7/18C12P7/44C12P7/62C12P13/00C12P17/10
CPCC12N9/0008C12N9/1029C12N9/1096C12N9/16C12N9/18C12P7/18C12P7/44C12P7/62C12P13/001C12P13/005C12P17/10C12Y102/99006C12Y203/01016C12Y206/01018C12Y206/01019C12Y206/01029C12Y206/01048C12Y206/01082C12Y301/01085C12Y301/02C07C69/533C12P7/24C12P7/40C12P7/42C12P11/00
Inventor A.L.博特斯A.V.E.康拉迪
Owner INVISTA TEXTILES (U K) LTD
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