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

A C6-C10, genetic modification technology, applied in the field of engineering enzymes, can solve problems such as non-octanol and small n-octanol

Inactive Publication Date: 2016-07-20
DOW GLOBAL TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the amount of n-octanol produced by this invention is too small to be commercially viable

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Modified LeuA (ie, "LeuA'") enzymes are prepared.

[0036] This example represents one embodiment for making an engineered LeuA' enzyme. This is accomplished by starting with an E. coli organism transformed with a plasmid containing a modified LeuA gene to generate a 2-isopropylmalate synthase variant (LeuA') for capturing the 2-ketoacid of interest for catalysis, the 2-isopropylmalate synthase variant has a higher than average catalytic efficiency (K cat / K M ). For this purpose, the specific gene identified as suitable is LeuA; GenBank Accession No. NC_000913.3 Gene ID: 947465.

[0037] In formula K cat / K M Medium, K cat It is "conversion number", unit = sec -1 ;K M is the Michaelis-Menten constant; the conversion number is equal to VMax / [E], where V Max is the maximum speed, [E] is the enzyme concentration. This equation, applied to reactions obeying Michaelis-Menten kinetics, generally provides the amount (in moles) of substrate converted to product in 1 ...

Embodiment 2

[0041] I. Preparation of E. coli LeuB' (isopropylmalate dehydrogenase) variants with increased activity towards 3-hexylmalate (3-HM).

[0042] During the biosynthesis of 2-ketononanoic acid via the recursive activity of the LeuABCD pathway, 3-alkylmalates of various lengths are formed as substrates for LeuB. For efficient biosynthesis of 2-ketononanoic acid, LeuB is expected to efficiently capture 3-ethylmalate (intermediate III, n=2; figure 1 ), 3-propyl malic acid (intermediate III, n=3; figure 1 ), 3-butylmalic acid (3-BM; Intermediate III, n=4; figure 1 ), 3-amylmalic acid (intermediate III, n=5; figure 1 ) and 3-hexylmalic acid (3-HM; Intermediate III, n=6; figure 1 ) for catalysis. Native LeuB is relatively inefficient at capturing longer unnatural 3-alkylmalate substrates. In order to improve the efficiency of native LeuB in capturing 3-hexylmalate for catalysis, the active site of native LeuB was modified using protein engineering techniques described below. ...

Embodiment 3

[0084] Modified LeuCD (ie, "LeuCD'") enzymes are prepared.

[0085] In this embodiment of Example 3, a method similar to that used in Example 1 for the LeuA' enzyme is described. The method begins with an E. coli organism transformed with a plasmid containing the LeuC and LeuD genes, resulting in the production of a modified 2-isopropylmalate isomerase complex that complexes body in the 2-alkylmalic acid ( figure 1 Among them, n=1-5 in the intermediate II) isomerized to their corresponding 3-alkyl malic acid ( figure 1 In intermediate III, n=1-5) has higher catalytic efficiency (k cat / K M ). By changing some genetic codes of the LeuC and LeuD genes (LeuC: GenBank Accession No. NC000913.3 Gene ID: 945076; and LeuD: GenBank Accession No. NC000913.3, Gene ID: 945642) obtained from GenBank, that is, substituting the amino acid sequence One or more amino acids, synthesize the modified LeuC and LeuD genes. Each engineered variant was prepared by substituting one or more res...

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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.

Description

[0001] Cross references to related patent applications [0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 61 / 915,040, filed December 12, 2013, entitled "Process to Prepare Octanol From Syngas Via Genetic Modifications to Microbial Metabolic Pathways," by It is incorporated herein by reference in its entirety. technical field [0003] The present invention relates to the field of production of C6-C10 aldehydes and products prepared therefrom using biological enzymes. More specifically, it relates to the field of utilizing engineered enzymes that can be expressed by genetically modified microorganisms to convert suitable substrates to C6-C10 aldehydes through one or more metabolic pathways, or incubated in vitro to convert 2 - Conversion of ketobutyrate or 2-ketoisovalerate to extended 2-keto acids followed by conversion of 2-keto acids to C6-C10 aldehydes. Background technique [0004] Concerns about future scarcity, cost, and en...

Claims

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Application Information

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IPC IPC(8): C12P7/26C12P7/40C12N9/10C12N9/04C12P7/24
CPCC12P7/24C12P7/26C12P7/40C12N15/52C12N9/0006C12N9/1025C12N9/88C12Y101/01085C12Y203/03013C12Y402/01033Y02E50/10C12N1/14C12N1/20C12P7/6409
Inventor P·C·桑格哈尼C·C·斯托瓦斯B·A·罗德里格斯A·文卡特斯瓦兰
Owner DOW GLOBAL TECH LLC
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