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

A technology for synthesizing enzymes and free fatty acids, applied in biochemical equipment and methods, and microbial measurement/inspection, which can solve problems such as inaccurate mechanism definition

Inactive Publication Date: 2014-04-30
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
Comparison scheme
Effect test

Embodiment 1

[0434] This example describes the generation of a FAS knockout strain of Schizochytrium for biochemical studies.

[0435] 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. 20050191679 A1). Using the method described in US Pat. No. 7,001,772, a Schizochytrium FAS knockout (FAS-KO) construct was obtained. The EcoRV fragment of about 10.0 kB in genomic DNA containing most of the FAS Orf (from about 728 bp downstream of the putative ATG start codon to about 680 bp downstream of the stop codon) was cloned into Stratagene Bluescript In the vector (pBSK), the vector is located in the EcoRV site of the polycloning 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 above US patent ...

Embodiment 2

[0437] The following examples describe general protocols for the preparation of cell-free extracts of the following strains: Schizochytrium Ac66, PUFA synthase KO strain derived from Schizochytrium Ac66, and Schizochytrium Ac66-derived FAS-KO strain.

[0438] 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 of >about 2.5 and <about 5. Cells in 50 mL of medium were collected by centrifugation in 50 mL plastic tubes (benchtop centrifuge, approximately 1200 rpm x 4 min). The supernatant was decanted and the cells were resuspended in 5 mL of Buffer A (100 mM phosphate (pH 7.2), 10% (w / v) glycerol, 1 mM EDTA and 2 mM DTT), then as before. Centrifuge as described. Discard the supernatant and resuspe...

Embodiment 3

[0440] This example describes general conditions for in vitro assays of FAS and PUFA synthetase activity.

[0441] Examples of protocols for in vitro activity assays for FAS and PUFA synthase activity are as follows. Mix the enzyme preparation and Buffer A (both components in a volume of 90 μL) and the following components (added as a cocktail (10 μL)) to make a final volume of 100 μL to give the indicated in parentheses Final concentration: Malonyl-CoA (50 μM - non-radioactive malonyl-CoA and malonyl-2- 14 A mixture of C-CoA with a final concentration of radiolabeled at 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 performed in glass tubes in a room temperature (about 21°C) water bath. The time of incubation depends on the needs of the experiment. The reaction was stopped using one of two work-up protocol based methods. For the use of ...

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Abstract

The present application discloses novel acyl-CoA synthases and novel acyltransferases, nucleic acid molecules encoding the acyl-CoA synthases and acyltransferases, recombinant nucleic acid molecules and recombinant host cells containing the nucleic acid molecules, and recombinant host cells containing the nucleic acid molecules. Genetically modified organisms (microorganisms and plants) of the recombinant nucleic acid molecules and recombinant host cells and methods of obtaining and using the organisms. The present application also discloses genetically modified organisms (eg, plants or microorganisms) that have been genetically modified to express a PKS-like system (PUFA PKS system or PUFA synthase) to produce PUFA, wherein the organism has been genetically modified to express an acyl-CoA synthetase, has been genetically modified to express an acyltransferase, has been genetically modified to remove or inactivate a fatty acid synthase (FAS) expressed by said organism to convert malonyl in said organism CoA reduces competition with PUFA synthase or increases malonyl-CoA levels, and in one aspect, has been genetically modified to inhibit KASII or KASIII. In addition to PUFAs and oils derived from the above-mentioned organisms, the present application also discloses other modifications and methods of obtaining and using the above-mentioned organisms and various products including the above-mentioned PUFAs and oils.

Description

technical field [0001] The present invention generally relates to accessory proteins and targets for improving polyunsaturated fatty acids (PUFAs), particularly long chain PUFAs (LCPUFAs), in host organisms Use in which the host organism has been genetically modified with a PKS-like system (ie, the PUFA PKS system or PUFA synthase) that produces the PUFAs described above. The present invention also relates to organisms that have been genetically modified to express the accessory proteins described above or modified with respect to the targets described above, and methods of making and using such 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 commercial needs. Vegetable oils from oil seed crops are relatively inexpensive and do not have the contaminati...

Claims

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Application Information

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Patent Type & Authority Patents(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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