US 2016/0355850 A1 · PROCESSES TO PREPARE ELONGATED 2-KETOACIDS AND C6-C10 COMPOUNDS THEREFROM VIA GENETIC MODIFICATIONS TO MICROBIAL METABOLIC PATHWAYS

Abstract

Modification of metabolic pathways includes genetically engineering at least one enzyme involved in elongating 2-ketoacids during leucine biosynthesis, and preferably at least isopropylmalate dehydrogenase or synthase (LeuB or LeuA in E. coli), to include at least such non-native enzyme, enzyme complex, or combination thereof to convert 2-ketobutyrate or 2-ketoisovalerate to a C7-C11 2-ketoacid, wherein the production of such is at a higher efficiency than if a purely native pathway is followed. The C7-C11 2-ketoacid may then be converted, via a native or genetically engineered thiamin dependent decarboxylase, to form a C6-C10 aldehyde having one less carbon than the C7-C11 2-ketoacid being converted. In some embodiments the C6-C10 aldehyde may then be converted via additional native or genetically engineered enzymes to form other C6-C10 products, including alcohols, carboxylic acids, and alkanes. This genetic engineering offers the opportunity for commercial scale of in vivo biosynthetic processes that may be more cost-efficient than non-biobased approaches to produce the same products.

Claims10

Dependent claims

A process for preparing a C7-C11 2-ketoacid comprising contacting

a substrate, selected from 2-ketobutyrate and 2-ketoisovalerate, and
(1) a native or genetically modified LeuA enzyme;
(2) a genetically modified LeuB′ enzyme, wherein the enzyme is
(a) obtained from Escherichia coli and has an amino acid sequence corresponding to Sequence Listing, SEQ ID 1;
the enzyme having been modified in that alanine, glycine, valine or leucine is independently substituted for Leu-96, Val-198, or a combination thereof;
or
(b) the enzyme has an amino acid sequence that is at least 60 percent homologous to the amino acid sequence of Sequence Listing, SEQ ID 1;
the enzyme having been modified as in (a;
and
(3) a native or genetically modified LeuCD′ enzyme complex;
under conditions such that the 2-ketobutyrate or 2-ketoisovalerate is converted, via one or more steps, to a C7-C11 2-ketoacid at a higher catalytic efficiency than would be exhibited by an otherwise identical process wherein a native LeuB enzyme is substituted for the genetically modified LeuB′ enzyme.

8 more elements

A process to prepare a C6-C10 aldehyde comprising

(1) contacting a carbon-containing substrate and one or more native or genetically modified enzymes under conditions to form 2-ketobutyrate or 2-ketoisovalerate;
(2) contacting the 2-ketobutyrate or 2-ketoisovalerate and
(a) a native or genetically modified LeuA enzyme;
(b) a genetically modified LeuB′ enzyme, wherein the LeuB′ enzyme is
(i) obtained from Escherichia coli and has an amino acid sequence corresponding to Sequence Listing, SEQ ID 1;
the enzyme having been modified in that alanine, glycine, valine or leucine is independently substituted for Leu-96, Val-198, or a combination thereof;
or
(ii) the enzyme has an amino acid sequence that is at least 60 percent homologous to the amino acid sequence of Sequence Listing, SEQ ID 1;
the enzyme having been modified as in (a);
and
(c) a native or genetically modified LeuCD′ enzyme complex;
under conditions such that the 2-ketobutyrate or 2-ketoisovalerate is converted, via one or more steps, to a C7-C11 2-ketoacid at a higher catalytic efficiency than would be exhibited by an otherwise identical process wherein a native LeuB enzyme is substituted for the genetically modified LeuB′ enzyme;
and
(3) contacting the C7-C11 2-ketoacid and a native or genetically modified thiamin dependent decarboxylase under conditions such that the C7-C11 2-ketoacid is converted to a C6-C10 aldehyde having one less carbon atom than the C7-C11 2-ketoacid being converted;
each of (1), (2) and (3) occurring in one or more steps;
wherein (1), (2) and (3) independently occur within or outside of a genetically modified microbial organism.

13 more elements

Dependencies are inferred from the claim text.

Processes to prepare elongated 2-ketoacids and c6-c10 compounds therefrom via genetic modifications to microbial metabolic pathways

Life of this patent

Processes to prepare elongated 2-ketoacids and c6-c10 compounds therefrom via genetic modifications to microbial metabolic pathways

Life of this patent

Abstract

Claims10