Method to control cellular (3R)-hydroxyacyl-CoA esters, precursor molecules for polyhydroxyalkanoate synthesis in genetically modified organisms
OwnerKalervo Hiltunen, Tuomo Glumoffinventors
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Abstract
This invention relates to a method for controlling the production of cellular (3R)-hydroxyacyl CoA esters of predetermined length in a chosen host cell or organism. In particular, the invention relates to a modified gene encoding a multifunctional 2-enoyl-CoA hydratase 2/(3R)-hydroxyacyl CoA dehydrogenase enzyme type 2 protein and DNA constructs and host comprising the gene. The modification of the gene results in the enrichment of cellular (3R)-hydroxyacyl CoA esters of predetermined length, when the gene is introduced and expressed in a host cell oxidizing exogenous or endogenous β-fatty acids.
Claims32
A method for controlling the production of cellular (3R)-hydroxyacyl CoA esters of predetermined length in a host cell or organism, characterized in that the method comprises the following steps:
- introducing a gene encoding a multifunctional 2-enoyl-CoA hydratase 2/(3R)-hydroxyacyl CoA dehydrogenase enzyme type 2 protein (MFE-2) comprising at least one gene region encoding a hydratase domain and at least one gene region encoding a dehydrogenase domain, and wherein at least one genetic change has been made to the gene region encoding dehydrogenase domain, resulting in the enrichment of cellular (3R)-hydroxyacyl CoA esters of predetermined length, when the gene is introduced and expressed in a host cell oxidizing exogenous or endogenous fatty acids, or a DNA construct or a vector comprising the gene, into a host cell or organism;
- and
- growing the host cell or organism under suitable growth conditions in the presence of a carbon source comprising fatty acids from an endogenous source or originating from exogenous additions, resulting in the enrichment of cellular (3R)-hydroxyacyl CoA esters of predetermined length.
A method for preparing a host cell or organism capable of producing PHAs, said host cell or organism expressing an endogenous or foreign gene or genes encoding (3R)-hydroxyacyl-CoA ester polymerizing enzyme or enzymes, characterized in that the method comprises the following steps:
- introducing a gene encoding a multifunctional 2-enoyl-CoA hydratase 2/(3R)-hydroxyacyl CoA dehydrogenase enzyme type 2 protein (MFE-2), which comprises at least one gene region encoding a hydratase domain and at least one gene region encoding a dehydrogenase domain, wherein at least one genetic change has been made to the gene region encoding dehydrogenase domain, resulting in the enrichment of cellular (3R)-hydroxyacyl CoA esters of predetermined length, when the gene is introduced and expressed in a host cell oxidizing exogenous or endogenous fatty acids, or a DNA construct or a vector comprising the gene, into the host cell or organism;
- and
- growing the host cell or organism under suitable growth conditions in the presence of a carbon source comprising fatty acids from an endogenous source or originating from exogenous additions.
A method for producing PHAs in a host cell or organism expressing endogenous or foreign gene or genes encoding (3R)-hydroxyacyl-CoA ester polymerizing enzyme or enzymes, characterized in that the method comprises the following steps:
- introducing a gene encoding a multifunctional 2-enoyl-CoA hydratase 2/(3R)-hydroxyacyl CoA dehydrogenase enzyme type 2 protein (MFE-2) comprising at least one gene region encoding a hydratase domain and at least one gene region encoding a dehydrogenase domain, wherein at least one genetic change has been made to the gene region encoding dehydrogenase domain, resulting in the enrichment of cellular (3R)-hydroxyacyl CoA esters of predetermined length, when the gene is introduced and expressed in a host cell oxidizing exogenous or endogenous fatty acids or a DNA construct or a vector comprising the gene, into a host cell or organism;
- growing the host cell or organism under suitable growth conditions in the presence of a carbon source comprising fatty acids from an endogenous source or originating from exogenous additions;
- and
- recovering the PHAs or their hydrolysis products.
A gene encoding a multifunctional 2-enoyl-CoA hydratase 2/(3R)-hydroxyacyl CoA dehydrogenase enzyme type 2 protein (MFE-2) comprising at least one gene region encoding a hydratase domain and at least one gene region encoding a dehydrogenase domain, characterized in that at least one genetic change has been made to the gene region encoding dehydrogenase domain, resulting in the enrichment of cellular (3R)-hydroxyacyl CoA esters of predetermined length, when the gene is introduced and expressed in a host cell oxidizing exogenous or endogenous fatty acids.
A yeast gene encoding a multifunctional 2-enoyl-CoA hydratase 2/(3R)-hydroxyacyl CoA dehydrogenase enzyme type 2 protein comprising one gene region encoding hydratase domain and two gene regions encoding dehydrogenase domains, characterized in that both gene regions encoding dehydrogenase domains are inactivated resulting in the enrichment of cellular (3R)-hydroxyacyl CoA esters of about C4-C20 length, when the gene is introduced and expressed in a host cell capable of β-oxidation of fatty acids.
Dependencies are inferred from the claim text.