A recombinant bacterium with a high PHA yield and its construction method provide an engineered microorganism that can be used for the production of polyhydroxyalkanoate (PHA). A recombinant Ralstonia eutropha with a high PHA yield includes a promoter for upregulating a phaJ gene and further includes a mutant phaC gene. The recombinant Ralstonia eutropha can be used for producing 3-hydroxybutyrate-co-3-hydroxyhexanoate (PHBHHx) in PHA.

The present disclosure provides a microorganism and expression cassette useful for biologically producing PHA ho-mopolymers and/or PHA copolymers, including PHB-co-MCL copolymers of controllable or predetermined composition. In embodiments, the present disclosure provides a nucleic acid construct suitable for use in a microorganism and/or expression cassette including a nucleic acid construct including: one or more genes comprising a phaJ4 gene, a phaA gene, a phaB gene, a phaC1 gene, or combinations thereof; a cDNA that encodes one or more proteins comprising an enoyl-CoA hydratase 2, a -ketothiolase, an acetoacetyl-CoA reductase, a type II poly hydroxyalkanoate synthase, or combinations thereof; or one or more nucleic acid sequences that encode one or more proteins including an enoyl-CoA hydratase 2, a -ketothiolase, an acetoacetyl-CoA reductase, a type II poly hydroxyalkanoate synthase, or combinations thereof.

A method of manufacturing 1,4-butanediol through acetyl-CoA, acetoacetyl-CoA, 3-hydroxybutyryl-CoA, crotonyl-CoA, and 4-hydroxybutyryl-CoA by using a microbe and/or a culture thereof, wherein the microbe in the manufacturing method for 1,4-butanediol includes any one of genes among (a) a gene that has a base sequence of sequence number 1, (b) a gene that has a base sequence such that one or more bases are deleted, substituted, or added in a base sequence of sequence number 1, wherein the gene has a base sequence with an identity greater than or equal to 90% with respect to the base sequence of sequence number 1, and (c) a gene that hybridizes with a gene that has a base sequence complementary with a gene that has a base sequence described in sequence number 1 on a stringent condition, and includes any one or more genes among (d) genes that have base sequences of sequence numbers 2 to 9, (e) genes that have base sequences such that one or more bases are deleted, substituted, or added in base sequences of sequence numbers 2 to 9, wherein the genes have base sequences with an identity greater than or equal to 90% with respect to original base sequences thereof, and (f) genes that hybridize with genes that have base sequences complementary with genes that have base sequences of sequence numbers 2 to 9 on a stringent condition.

The present invention relates to the technical field of microorganisms, and specifically to engineered microorganisms expressing acetoacetyl-CoA reductase variants and methods for increasing the proportion of 3-hydroxyhexanoic acid in PHA. The acetoacetyl-CoA reductase variants and their coding genes provided by the present invention can significantly increase the proportion of 3-hydroxyhexanoic acid in PHA produced by strains; the proportion of 3-hydroxyhexanoic acid in PHA produced by the engineered Ralstonia eutropha constructed utilizing the acetoacetyl-CoA reductase variants and their coding genes provided by the present invention is significantly increased, which provides new genes and strain resources for strains producing poly(3-hydroxybutyrate-co-3-10 the development of engineered hydroxyhexanoate) with high proportion of 3-hydroxyhexanoic acid.

Recombinant microbes comprising a novel pathway for producing fatty acid derivatives, such as fatty acid methyl esters, fatty acid ethyl esters, fatty alcohols, and fatty alcohol acetates, while generating low levels of 3-hydroxy fatty acid byproducts are provided herein. The addition of a heterologous R-3-hydroxy-acyl-CoA dehydratase or a heterologous R-specific enoyl-CoA hydratase and optionally a heterologous trans-2-enoyl-CoA reductase to a recombinant microbe reduces the amount of 3-hydroxy fatty acid byproducts produced by the acyl-ACP dependent fatty acid biosynthesis pathway of the recombinant microbe. Methods of producing the fatty acid derivatives are also provided, in addition to cell cultures and fatty acid derivative compositions produced by the recombinant microbes. The fatty acid derivatives produced by the recombinant microbes may be used to produce fragrances, pheromones, pharmaceutical agents, nutraceuticals or precursors thereof.

A transformed microorganism having an ability to produce a polyhydroxyalkanoate copolymer containing 3-hydroxyalkanoate monomer units having 8 or more carbon atoms includes: an exogenous gene encoding a polyhydroxyalkanoate synthase having an amino acid sequence of any one of SEQ ID NOS: 1 to 4; or an exogenous gene encoding a protein that has an amino acid sequence having a sequence identity of at least 90% with the amino acid sequence of any one of SEQ ID NOS: 1 to 4 and that has polyhydroxyalkanoate synthase activity.

Microorganisms are genetically engineered to continuously produce fatty acids, fatty alcohols, cultured protein, or any combination thereof by microbial fermentation, particularly by microbial fermentation of a gaseous substrate. The microorganisms are C1-fixing. The production of fatty acids, fatty alcohols, and cultured proteins can be improved. This can be improved through the expression of 3-hydroxyacyl-ACP:CoA transacylases.