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Engineered trans-enoyl COA reductases and methods of making and using same

A trans-enoyl, engineering technology, applied in the field of engineered trans-enoyl COA reductase and its preparation and use, can solve the problems of increasing the cost and complexity of biosynthetic compounds, reducing product efficiency or yield, etc.

Pending Publication Date: 2022-04-01
GENOMATICA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, engineered microorganisms can produce undesired by-products due to undesired enzymatic activity on pathway intermediates and final products
Thus, such by-products and impurities increase the cost and complexity of biosynthetic compounds and may reduce the efficiency or yield of desired products

Method used

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  • Engineered trans-enoyl COA reductases and methods of making and using same
  • Engineered trans-enoyl COA reductases and methods of making and using same
  • Engineered trans-enoyl COA reductases and methods of making and using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0194] Example 1. Identification of 5-carboxy-2-pentenoyl-CoA reductase.

[0195] Genes encoding 5-carboxy-2-pentenoyl-CoA reductase (Ter) were identified from the literature and bioinformatically from public databases using the Basic Local Alignment Search Tool (BLAST) (Table 3). Genes encoding each reductase were synthesized, expressed in E. coli, and 5-carboxy-2-pentenoyl-CoA (CPCoA) reductase activity was assessed using an enzyme coupled assay.

[0196] The genes encoding the TER enzyme candidates of Table 3 were cloned into low copy vectors under constitutive promoters and the constructs were transformed into E. coli using standard techniques. The transformants were cultured overnight at 35° C. in LB medium containing antibiotics, and then the cells were centrifuged at room temperature at a speed of 15000 rpm. To prepare lysates, remove supernatant and resuspend Ter gene-expressing E. coli cells in chemical lysis solution containing lysozyme, nuclease, and 10 mM DTT. Us...

Embodiment 2

[0207] Example 2. In vivo activity of 5-carboxy-2-pentenoyl-CoA reductase.

[0208] Two identified 5-carboxy-2-pentenoyl-CoA reductases were also tested in an in vivo assay in which they were modified to express the enzyme encoding 3-oxoadipyl-CoA thiolase (Thl), 3 Escherichia coli strains containing the genes for -oxoadipyl-CoA dehydrogenase (Hbd) and 3-oxoadipyl-CoA dehydratase ("crotonase" or Crt) were used to contain 5-carboxy-2-pentane Construct transformation of the enoyl-CoA reductase gene. The gene encoding the TER enzyme of Candida tropicalis (SEQ ID NO:1, referred to as candidate #1) and the gene encoding the TER of Drosophila melanogaster (SEQ ID NO:24, candidate #24) were respectively cloned into low copy in the plasmid vector under a constitutive promoter. The amino acid sequences of Candida and Drosophila TER are about 40% identical; image 3 shown. The vector expressing the Ter gene was transformed into a Thl / Hbd / Crt E. coli strain. The transformants were t...

Embodiment 3

[0214] Example 3. Targeted mutagenesis of Ter gene.

[0215] Using structure-guided design, and then by codons at Q52, V105, S148, V149, T153, V301, T302 and N307, and Q11, A39, S48, S59, G97, S103, H104, N106, F107, I147, L152 , L156, R200, D201, R202, E303, K306, N308, and L316 codons to mutate the gene encoding the Candida TER enzyme (SEQ ID NO: 1) to generate the Candida TER enzyme encoding (SEQ ID NO: 1) gene variants. Similarly, structure-guided design was used followed by mutating the gene encoding the Candida TER enzyme (SEQ ID NO: 1) at the codons for S96, L98, V129, M274, T275 to generate Variants of the gene for the enzyme (SEQ ID NO: 1). Amino acid position substitutions were performed using degenerate primer sequences and PCR, wherein the altered gene sequence mixture was transformed into E. coli.

[0216] Transformants were tested in lysate assays as described in Example 1 and re-tested in lysate assays to provide assays that exhibited higher activity than wil...

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Abstract

Trans-enoyl CoA reductase (TER) enzymes and nucleic acids encoding the same are disclosed. In some cases, the TER enzyme is a non-natural engineered trans-enoyl CoA reductase. Also disclosed are biosynthetic methods and engineered microorganisms that enhance or improve the biosynthesis of 6-aminocaproic acid, hexamethylenediamine, caproic acid, caprolactone, or caprolactam. The engineered microbial organisms include an exogenous TER, and in some cases also include an engineered TER.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. serial numbers 62 / 837,888 filed April 24, 2019, 62 / 860,123 filed June 11, 2019, and 62 / 860,160 filed June 11, 2019 The benefit of the Provisional Patent Application, the contents of which are hereby incorporated by reference in their entirety. [0003] Incorporated into sequence listing [0004] This application contains a sequence listing named "GNO0098WO Sequence Listing.txt", which was created on April 22, 2020 and is 151KB in size. The Sequence Listing is incorporated herein by reference. Background technique [0005] Nylon is a polyamide that can be synthesized by polycondensation of diamines and dicarboxylic acids or polycondensation of lactams. Nylon 6,6 is produced by the reaction of hexamethylenediamine (HMD) and adipic acid, while nylon 6 is produced by ring-opening polymerization of caprolactam. Therefore, adipic acid, hexamethylenediamine and caprolactam are important intermed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12P7/24C12P7/40C12P7/42C12P7/44C12P7/62C12P13/00C12P13/02C12P19/32
CPCC12P13/001C12P13/005C12P7/40C12P17/10C12P7/62C12P17/08C12P7/18C12N9/0008C12Y103/01038C12N9/001C12P7/44C12N15/70C12N9/78C12Y102/01003C12N1/20C12N15/52C12Y103/01044C12N9/1096C12Y206/01
Inventor 阿米特·M·沙阿哈里什·纳加拉让迈克尔·A·诺博
Owner GENOMATICA INC
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