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Scattered Branched-Chain Fatty Acids And Biological Production Thereof

a technology of branched-chain fatty acids and biological production, which is applied in the direction of enzymology, ligases, transferases, etc., can solve the problems of not producing medium-chain branched-chain fatty acids, fatty acids may not be available or easily isolated from a natural organism in meaningful quantities,

Inactive Publication Date: 2011-07-07
THE PROCTER & GAMBLE COMPANY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these organisms do not produce branched-chain fatty acids in amounts that are commercially useful.
Another limitation of these natural organisms is that they apparently do not produce medium-chain branched-chain fatty acids, such as those with 11 or 13 carbons.
In addition, if fatty acids having particular chain lengths, branches on particular carbons, or branches at positions other than the iso and anteiso positions are desired, these fatty acids may not be available or easily isolated from a natural organism in meaningful quantities.

Method used

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  • Scattered Branched-Chain Fatty Acids And Biological Production Thereof

Examples

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example 1

Construction of Methylmalonyl-CoA Mutase Expression Vector

There are numerous genes annotated to encode the two subunits of methylmalonyl-CoA mutase. Janibacter sp. HTCC2649 encodes two such genes. Synthetic versions of these genes were prepared, with the codon usage altered to match that used by many E. coli genes (i.e., the coding sequence was codon-optimized for expression in E. coli). By analogy to other methylmalonyl-CoA mutase genes, these synthetic genes were named mutA (SEQ ID NO: 1) and mutB (SEQ ID NO: 2), corresponding to the MutA (SEQ ID NO: 3) and MutB (SEQ ID NO: 4) protein subunits. In the synthetic DNA, an extra three base pairs were added (encoding an alanine residue immediately after the initiation methionine) in mutA to facilitate introduction of an NcoI site. An XhoI restriction site was also placed after the coding sequence of mutB for insertion into the pBAD vector (Invitrogen). The NcoI / XhoI fragment was cloned into pBAD.

example 2

Construction of Methylmalonyl-CoA Epimerase Expression Vector

There are numerous genes annotated to encode methylmalonyl-CoA mutase. One such gene is from Streptomyces sviceus. A synthetic gene can be constructed (SEQ ID NO: 5) using codon usage similar to E. coli genes and with EcoRI and Hind III sites flanking the coding region. An E. coli Shine-Dalgarno sequence can be added between the EcoRI site and the initiation codon for the epimerase gene. The predicted protein product is the same as the predicted protein product from the S. sviceus gene (SEQ ID NO: 6). The epimerase gene can be cloned into the pBAD-mutAB construct using the EcoRI and Hind III restriction sites (downstream of mutB) to form the pBAD-mutAB-epimerase gene plasmid. E. coli cultures can be grown at 27° C. after induction with arabinose and supplemented with hydroxycobalamin to achieve expression of functional methylmalonyl-CoA mutase and branched-chain fatty acid production.

example 3

Construction of Propionyl-CoA Carboxylase Expression Vector

Nucleotide sequences (SEQ ID NO: 7 and SEQ ID NO: 8) encoding the two propionyl-CoA carboxylase subunits AccA1 (GenBank Accession NO. AF113603.1; SEQ ID NO: 9) and PccB (GenBank Accession No. AF113605.1; SEQ ID NO: 10)), respectively, from the Streptomyces coelicolor A3(2) propionyl-CoA carboxylase (Rodriguez E., Gramajo H., Microbiology. 1999 November; 145:3109-19), were codon-optimized for E. coli expression. A gene construct for expressing propionyl-CoA carboxylase was constructed with the following elements sequentially 1) PLlac0-1 promoter and operator plus T7 gene10 ribosomal binding site (SEQ ID NO: 11); 2) optimized accA1 (SEQ ID NO: 12); 3) three restriction site sequences including BglII, NotI and XbaI and a T7 gene10 ribosome binding site (SEQ ID NO: 13); and 4) codon-optimized pccB (SEQ ID NO: 14). The synthesized DNA fragments were cloned into the XhoI and PstI sites of expression vector pZA31-MCS (Expressys, Ru...

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Abstract

Methods and cells for producing scattered branched-chain fatty acids are provided. For example, the invention provides a method for producing branched-chain fatty acid comprising a methyl on one or more even number carbons. The method comprises culturing a cell comprising an exogenous or overexpressed polynucleotide comprising a nucleic acid sequence encoding a polypeptide that catalyzes the conversion of propionyl-CoA to methylmalonyl-CoA and / or an exogenous or overexpressed polynucleotide comprising a nucleic acid sequence encoding a polypeptide that catalyzes the conversion of succinyl-CoA to methylmalonyl-CoA, under conditions allowing expression of the polynucleotide(s) and production of branched-chain fatty acid. The cell produces more branched-chain fatty acid comprising a methyl on one or more even number carbons than an otherwise similar cell that does not comprise the polynucleotide(s). A cell that produces branched-chain fatty acid and the branched-chain fatty acid also are provided.

Description

FIELD OF THE INVENTIONThe invention relates to cells and methods for producing fatty acids, and more particularly relates to cells and methods for producing scattered branched-chain fatty acids.BACKGROUND OF THE INVENTIONBranched-chain fatty acids are carboxylic acids with a methyl or ethyl branch on one or more carbons that can be either chemically synthesized or isolated from certain animals and bacteria. While certain bacteria, such as Escherichia coli, do not naturally produce branched-chain fatty acids, some bacteria, such as members of the genera Bacillus and Streptomyces, can naturally produce these fatty acids. For example, Streptomyces avermitilis and Bacillus subtilis both produce branched-chain fatty acids with from 14 to 17 total carbons, with the branches in the iso and anteiso positions (Cropp et al., Can. J. Microbiology 46: 506-14 (2000); De Mendoza et al., Biosynthesis and Function of Membrane Lipids, in Bacillus subtilis and Other Gram-Positive Bacteria, Sonenshein...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07C53/126C12P7/64C12N1/00C12N1/21
CPCC12N9/90C12N9/93C12Y604/01003C12Y504/99002C12P7/6409
Inventor SAUNDERS, CHARLES WINSTONXU, JUNLAUGHLIN, II, LEO TIMOTHYKHAMBATTA, ZUBIN SAROSHGREEN, PHILLIP RICHARD
Owner THE PROCTER & GAMBLE COMPANY
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