Polyunsaturated fatty acid production in heterologous organisms using pufa polyketide synthase systems

A technology of organisms and enzyme synthesis, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as inaccurate mechanism definition

Inactive Publication Date: 2009-11-04
DSM IP ASSETS BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, to the best of the present inventors' knowledge, the precise mechanism for transferring PUFAs from the enzyme to those target sites had not been defined prior to the present invention

Method used

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  • Polyunsaturated fatty acid production in heterologous organisms using pufa polyketide synthase systems
  • Polyunsaturated fatty acid production in heterologous organisms using pufa polyketide synthase systems
  • Polyunsaturated fatty acid production in heterologous organisms using pufa polyketide synthase systems

Examples

Experimental program
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Embodiment 1

[0436] This example describes the generation of Schizochytrium FAS knockout strains for biochemical studies.

[0437] Schizochytrium contains a large gene encoding the FAS enzyme responsible for the production of short-chain saturated fatty acids (described in US Patent Application Publication No. 20050191679A1 ). Schizochytrium FAS knockout (FAS-KO) constructs were obtained using the methods described in US Patent No. 7,001,772. The ~10.0 kB EcoRV fragment in genomic DNA containing the majority of the FAS Orf (from ~728 bp downstream of the putative ATG start codon to ~680 bp downstream of the stop codon) was cloned into Stratagene Bluescript In the vector (pBSK), the vector is located at the EcoRV site of the multiple cloning region. The approximately 3.5 kB internal BglII fragment was removed from the cloned Schizochytrium DNA and replaced with the approximately 1.1 kB BamHI fragment from pTubZeo11-2 (which contains a Zeocin resistance cassette) (see the aforementioned US ...

Embodiment 2

[0439] The following examples describe general protocols for the preparation of cell-free extracts of the following strains: Schizochytrium Ac66, the PUFA synthase KO strain derived from Schizochytrium Ac66, and the PUFA synthase derived from Schizochytrium Ac66 FAS-KO strain.

[0440] An example of a protocol for preparing a cell-free homogenate (CFH) from a cell wall deficient strain of Schizochytrium is as follows. Cells were grown in A50-3 medium and then diluted into M2B medium. The medium used to grow the KO strains was supplemented with appropriate fatty acids. Cells were grown in M2B medium to an OD600nm > about 2.5 and < about 5. Cells in 50 mL of medium were collected by centrifugation in a 50 mL plastic tube (tabletop centrifuge, about 1200 rpm x 4 minutes). Decant the supernatant, then resuspend the cells in 5 mL of Buffer A (BufferA) (100 mM phosphate (pH 7.2), 10% (w / v) glycerol, 1 mM EDTA, and 2 mM DTT), and then Centrifuge as described above. Discard the s...

Embodiment 3

[0442] This example describes general conditions for in vitro FAS and PUFA synthetase activity assays.

[0443] Examples of protocols for in vitro activity assays for FAS and PUFA synthase activity are as follows. The enzyme preparation and buffer A (both components in a volume of 90 μL) and the following components (added as a cocktail (10 μL)) were mixed to a final volume of 100 μL to obtain the Final Concentration: Malonyl-CoA (50 μM - non-radioactive malonyl-CoA and malonyl-2- 14 A mixture of C-CoA with radiolabeled at a final concentration of 0.65 μCi / mL), NADH (1 mM), NADPH (1 mM) and acetyl-CoA (10 μM). These and additional components can be adjusted based on the needs of a particular experiment. The assay reactions were carried out in glass tubes in a water bath at room temperature (about 21°C). The time of incubation depends on the needs of the experiment. The reaction was stopped using one of two methods based on a work-up protocol. For the conversion of fatty a...

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Abstract

Disclosed are novel acyl-CoA synthetases and novel acyltransferases, nucleic acid molecules encoding the same, recombinant nucleic acid molecules and recombinant host cells comprising such nucleic acid molecules, genetically modified organisms (microorganisms and plants) comprising the same, and methods of making and using the same. Also disclosed are genetically modified organisms (e.g., plants, microorganisms) that have been genetically modified to express a PKS-like system for the production of PUFAs (a PUFA PKS system or PUFA synthase), wherein the organisms have been modified to express an acyl-CoA synthetase, to express an acyl transferase, to delete or inactivate a fatty acid synthase (FAS) expressed by the organism, to reduce competition for malonyl CoA with the PUFA synthase or toincrease the level of malonyl CoA in the organism, and in one aspect, to inhibit KASII or KASIII. Additional modifications, and methods to make and use such organisms, in addition to PUFAs and oils o btained from such organisms, are disclosed, alone with various products including such PUFAs and oils.

Description

technical field [0001] The present invention generally relates to the improvement of polyunsaturated fatty acids (polyunsaturated fatty acid, PUFA) in host organisms (host organism) by accessory protein (accessory protein) and target (target), especially long-chain PUFA (long-chain PUFA, LCPUFA) Use in the production of said host organisms which have been genetically modified with a PKS-like system (ie PUFA PKS system or PUFA synthetase) for the production of the above-mentioned PUFAs. The invention also relates to organisms that have been genetically modified to express the above-mentioned accessory proteins or modified for the above-mentioned targets and methods of making and using the above-mentioned organisms. Background technique [0002] Polyunsaturated fatty acids (PUFAs) are believed to be useful for nutritional, pharmaceutical, industrial, and other purposes. However, the current supply of PUFAs from natural sources and chemical synthesis is insufficient to meet co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 詹姆斯·G·梅茨杰里·M·库纳詹姆斯·C·利普梅尔
Owner DSM IP ASSETS BV
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