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PUFA polyketide synthase systems and uses thereof

A compound and polyketide technology, applied in the field of preparation and use of the non-bacterial PUFAPKS system disclosed in this paper, identification of bacteria and non-bacterial microorganisms containing the PUFAPKS system, can solve problems such as difficult plant expression

Inactive Publication Date: 2004-10-06
MARTEK BIOSCIENCES CORP (N D GES D STAATES DELAWARE) COLUMBIA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the marine bacterial PUFA PKS system is not expected to be easily adapted to plant expression under normal growth conditions

Method used

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  • PUFA polyketide synthase systems and uses thereof
  • PUFA polyketide synthase systems and uses thereof
  • PUFA polyketide synthase systems and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0241] The following example describes further analysis of PKS related sequences from Schizochytrium.

[0242] The inventors sequenced the full-length genome containing all three open reading frames (Orfs) in the Schizochytrium PUFA PKS system using the general methods outlined in Examples 8 and 9 of PCT Publication No. WO 0042195 and US Application Serial No. 09 / 231,899 DNA. The biologically active domain in the Schizochytrium PKS protein is in figure 1 is described graphically. The domain structure of the Schizochytrium PUFA PKS system is described in more detail below.

[0243] Open reading frame A (OrfA) :

[0244] The complete nucleotide sequence of OrfA is represented herein as SEQ ID NO:1. OrfA is a sequence of 8730 nucleotides (not including the stop codon), which encodes a sequence of 2910 amino acids, represented herein as SEQ ID NO:2. There are 12 domains within OrfA:

[0245] (a) a β-ketoacyl-ACP synthase (KS) domain;

[0246] (b) a malonyl-CoA:ACP acyltr...

Embodiment 2

[0289] The following examples describe the identification of three other non-bacterial organisms containing the PUFAPKS system according to the invention using the screening method of the invention.

[0290] Thraustochytrium 23B (ATCC 20892) was cultured following the screening method described in US Provisional Application Serial No. 60 / 298,796 and described in detail herein.

[0291] A biologically plausible screening system (using shake flask cultures) for the detection of microorganisms containing PUFA-producing PKS systems was performed as follows:

[0292] Place a 2 mL culture of the microorganism of the strain to be tested in a 250 mL baffled shake flask containing 50 mL of medium (aerobic treatment), and place another 2 mL culture of the same strain in a 250 mL unbaffled flask containing 200 mL of medium plate in shake flasks (anoxic treatment). Both shake flasks were placed on a shaker at 200 rpm. After 48-72 hours of incubation, the cultures were harvested by centr...

Embodiment 3

[0328] The following examples demonstrate that DHA and DPA synthesis in Schizochytrium does not involve membrane-bound desaturases or fatty acid elongases, as has been described for other eukaryotes (Parker-Bames et al., 2000, supra; Shanklin et al., 1998, cited above).

[0329] Schizochytrium accumulates large amounts of triacylglycerol rich in DHA and meningoleic acid (DPA; 22:5ω6); for example, 30% of dry weight DHA+DPA. In eukaryotes that synthesize 20- and 22-carbon PUFAs via elongation / desaturation pathways, the molecular repertoire of 18-, 20- and 22-carbon intermediates is quite large, so using [ 14 In vivo labeling experiments with C]-acetate revealed clear precursor-product kinetics of the predicted intermediates. Additionally, exogenous radiolabeled intermediates provided to the organism are converted to final PUFA products.

[0330] 2-day-old cultures were initially supplemented with a single pulse of [1- 14 C] Acetate. Cell samples are then harvested by centri...

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Abstract

The invention generally relates to polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systems isolated from or derived from non-bacterial organisms, to homologues thereof, to isolated nucleic acid molecules and recombinant nucleic acid molecules encoding biologically active domains of such a PUFA PKS system, to methods of making and using such system for the production of bioactive molecules of interest, and to novel methods for identifying new bacterial and non-bacterial microorganisms having such a PUFA PKS system.

Description

technical field [0001] The present invention relates to polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systems from microorganisms, including eukaryotes such as Thraustochytrid microorganisms. More specifically, the present invention relates to nucleic acids encoding non-bacterial PUFAPKS systems, to non-bacterial PUFAPKS systems, to genetically modified organisms containing non-bacterial PUFAPKS systems, and to methods of making and using the non-bacterial PUFAPKS systems disclosed herein. The present invention also relates to methods of identifying bacterial and non-bacterial microorganisms that contain the PUFAPKS system. Background technique [0002] The polyketide synthase (PKS) system is an enzyme complex derived from the fatty acid synthase (FAS) system generally known in the art, but it is often highly modified to produce specific products that generally bear little resemblance to fatty acids. The researchers tried to develop polyketide synthase (PKS) s...

Claims

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Application Information

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IPC IPC(8): A01H5/00A61K48/00A61P1/04A61P1/16A61P3/06A61P9/10A61P11/00A61P15/06A61P19/02A61P19/10A61P25/08A61P25/24A61P25/28A61P29/00A61P31/04A61P35/00A61P37/02A61P43/00C12N1/15C12N1/19C12N1/21C12N5/10C12N15/09C12N15/52C12N15/82C12P1/02C12P7/6427C12P7/6434C12P7/6472C12Q1/02C12Q1/68C12R1/645
CPCC12N9/93C12P7/6427C12P7/62C12P7/6472C12N15/8247C12N15/8273C12P7/625C12N15/52A61P1/00A61P1/04A61P1/16A61P11/00A61P15/06A61P19/02A61P19/10A61P25/00A61P25/08A61P25/18A61P25/24A61P25/28A61P27/02A61P29/00A61P31/00A61P31/04A61P35/00A61P3/06A61P37/02A61P37/06A61P43/00A61P7/00A61P9/00A61P9/10C12P7/6434
Inventor 詹姆斯·G·梅茨威廉·R·巴克利詹姆斯·H·弗拉特杰里·M·库纳
Owner MARTEK BIOSCIENCES CORP (N D GES D STAATES DELAWARE) COLUMBIA
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