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Methods for producing dicarboxylic acids

A dicarboxylic acid and product technology, applied in the field of dicarboxylic acid production, can solve the problems of reducing activity and catalytic conversion

Inactive Publication Date: 2016-06-15
SYNTHETIC GENOMICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another disadvantage of this enzyme is the requirement of two additional enzymes, ferredoxin and ferredoxin reductase, which reduce the activity and catalytic turnover of this enzyme (Lawson, R.J et al., Biochemistry 2004, 43, 12390; Cryle, MJ , Schlichting I. PNAS, 2008, 105, 15696)

Method used

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  • Methods for producing dicarboxylic acids
  • Methods for producing dicarboxylic acids
  • Methods for producing dicarboxylic acids

Examples

Experimental program
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Effect test

Embodiment 1

[0073] Example 1 - Enzyme Engineering

[0074] This example shows the engineering of wild-type (wt) BM3 and wild-type (wt) YetO to generate a set of mutant enzymes with the desired activities of peroxidizing fatty acid subterminals and cleaving C-C bonds. Cloning of BM3 (or CYP102A1) from Bacillus megaterium and a proprietary homolog from a Bacillus strain (YetO from Bacillus 15_F03). There is common media homology between BM3 and YetO (60% identity, 75% similarity). Native gene sequences were used in all plasmids. The YetO gene was cloned from gDNAPCR, while for BM3, the natural DNA sequence was used to make the synthetic gene. Various site-specific mutants from these sequences were then created, as shown in Table 1.

[0075] Table 1: Original BM3 and cloned homologues

[0076]

[0077] This article shows the sequence. For F87 (in BM3) and F89 (in YetO), the phenylalanine site was mutated to alanine in BM3, and to alanine, isoleucine, serine, or valine to produce Mutan...

Embodiment 2

[0083] Example 2 - Oxidation of palmitic acid by BM3-YetO

[0084] Strain BL21DE3 / pSGI-040 (pET24a-BM3-yetO-WT) was grown in 150 mL of M9TV / TB (3 / 1 v / v) + glucose (2 g / L) + FeCl3 (0.05 mM) on. Cells were grown at 30°C for 3-4h until reaching OD600-0.5. At this point, 0.5 mM δ-aminolevulinic acid and 0.25 mM IPTG were added, and the flask was moved to a shaker at room temperature (~23°C), where it was allowed to shake at 120 rpm for an additional 16h.

[0085] Cells were harvested by centrifugation and frozen at -80°C for at least 1 h. Frozen cells were resuspended in 15 mL lysis buffer (100 mM potassium phosphate, 10% glycerol (v / v), 1 mM DTT and 1 mg / mL lysozyme). After thawing the pellet, it was incubated on ice for 15 min before the cells were sonicated twice. Lysed cells were centrifuged at 12,000 rpm to pellet insoluble cell debris. The clarified lysate was used in the enzymatic reaction without any further purification. A P450 of approximately 8-10 uM (or nanomoles...

Embodiment 3

[0088] Example 3 - Oxidation of Cyclohydrocarbons

[0089] This example demonstrates that a mutant CYP102 enzyme (BM3-F87A) oxidizes cyclic hydrocarbons, including cyclooctane and cyclodecane, and also produces cyclodecane C-C bond cleavage. It was found that sebacic acid (sebacic acid) was produced after peroxidation when the enzyme was placed in the presence of cyclodecane.

[0090] By performing the reaction in the presence of deuterated lauric acid, we were able to trace the carbon of the sebacic acid produced to cyclodecane, rather than lauric acid. Therefore using cyclodecane and fully deuterated lauric acid (C 12 D. 23 CO 2 H) to carry out the reaction.

[0091] BL21(DE3) / pSGI-004(pET24a-BM3-F87A) cells were grown and lysed as described above. P450 concentrations of -14-20 [mu]M were obtained under these conditions. Prepare the reaction by mixing 1.5 mL of reaction buffer and 0.5 mL of enzyme lysate. By adding 4 mM deuterated lauric acid (C 12 D. 23 CO 2 H), 1...

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Abstract

The present invention provides methods of producing dicarboxylic acids. The methods involve incubating a fatty acid or hydrocarbon substrate with an enzyme to produce a dicarboxylic acid product. The enzyme acts on the substrate to produce a product that has been both over-oxidized and has undergone cleavage of a C-C bond. In some embodiments the enzymes having these useful characteristics are mutants of a cytochrome P450 enzyme, for example an enzyme of the class CYP102 or a mutant thereof. The invention provides enzymes where these desirable characteristics can be found in a single enzyme, and thus in some embodiments the methods can be performed through the action of a single enzyme.

Description

[0001] This application claims the benefit of US Provisional Application Serial No. 61 / 906,819, filed November 20, 2013, which is hereby incorporated by reference in its entirety, including all tables, drawings, and claims. field of invention [0002] The present invention relates to the enzymatic production of dicarboxylic acids from hydrocarbons or fatty acids. [0003] Sequence Listing Incorporation [0004] The material in the accompanying Sequence Listing is hereby incorporated by reference into this application. The attached sequence listing text file, named SGI1760_1WO_Sequence_Listing_ST25, was created on November 18, 2014, and is 53KB. This file can be evaluated using Microsoft Word on a computer using Windows OS. Background of the invention [0005] Long-chain dicarboxylic acids are versatile chemical intermediates used in the synthesis of fragrances, polymers, adhesives and lubricants, and by the pharmaceutical industry in the synthesis of antibiotics. Diacids ...

Claims

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Application Information

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IPC IPC(8): C12P7/44C12P7/64C12N9/00
CPCC12P7/6409C12N9/001C12N9/0071C12Y114/14001
Inventor B·M·格里芬S·坎博乌拉卡斯
Owner SYNTHETIC GENOMICS INC
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