This invention relates to a microorganism that produces a polyhydroxyalkanoate (PHA) copolymer with a regulated monomer composition ratio and comprises a (R)-specific enoyl-CoA hydratase gene in the genome DNA, wherein a nucleotide sequence upstream of the (R)-specific enoyl-CoA hydratase gene comprises a modification consisting of a substitution(s), a deletion(s), an insertion(s), and/or an addition(s) of one or a plurality of nucleotides so that the expression of the (R)-specific enoyl-CoA hydratase gene is regulated, and to a method for producing a PHA copolymer using the microorganism.

Cells and methods for producing polyhydroxyalkanoates. The cells comprise one or more recombinant genes selected from an R-specific enoyl-CoA hydratase gene, a PHA polymerase gene, a thioesterase gene, and an acyl-CoA-synthetase gene. The cells further have one or more genes functionally deleted. The functionally deleted genes include such genes as an enoyl-CoA hydratase gene, a 3-hydroxyacyl-CoA dehydrogenase, and a 3-ketoacyl-CoA thiolase gene. The recombinant cells are capable of using producing polyhydroxyalkanoates with a high proportion of monomers having the same carbon length from non-lipid substrates, such as carbohydrates.

Methods and systems for producing prescribed unit size poly(3-hydroxyalkanoate) (PHA) polymers and copolymers are provided. The methods and systems can employ recombinant bacteria that are not native producers of PHA or lack enzymes to degrade PHA once synthesized, metabolize short to long chain fatty acids without induction, and express an (R)-specific enoyl-CoA hydratase and a PHA synthase, the (R)-specific enoyl-CoA hydratase and PHA synthase having wide substrate specificities. The recombinant bacteria are fed at least one fatty acid substrate that is equal in carbon length to the prescribed or desired unit size of the PHA polymer to be produced. The prescribed unit size PHA that is produced is then isolated and/or purified.

The present disclosure describes a genetically engineered a KASIII-independent fatty acid biosynthetic pathway that makes use of the promiscuous nature of the rest of the FAS enzymes (3-ketoacyl-ACP synthetase, 3-ketoacyl-ACP reductase, 3-hydroxyacyl ACP dehydrase, enoyl-ACP reductase) to bypass the KASIII step by providing a Co-A precursor of two or higher than two carbons (such as the four carbon butyryl-CoA) as the starting molecule. Since many CoA-based starter molecules can be supplied for the fatty acid synthesis, much more diversified products can be obtained with various carbon-chain lengths. As such, this disclosure will serve as a powerful and efficient platform to produce low to medium chain length products carrying many different functional groups.

The use of microorganisms to make alpha-functionalized chemicals and fuels, (e.g. alpha-functionalized carboxylic acids, alcohols, hydrocarbons, amines, and their beta-, and omega-functionalized derivatives), by utilizing an iterative carbon chain elongation pathway that uses functionalized extender units. The core enzymes in the pathway include thiolase, dehydrogenase, dehydratase and reductase. Native or engineered thiolases catalyze the condensation of either unsubstituted or functionalized acyl-CoA primers with an alpha-functionalized acetyl-CoA as the extender unit to generate alpha-functionalized -keto acyl-CoA. Dehydrogenase converts alpha-functionalized -keto acyl-CoA to alpha-functionalized -hydroxy acyl-CoA. Dehydratase converts alpha-functionalized -hydroxy acyl-CoA to alpha-functionalized enoyl-CoA. Reductase converts alpha-functionalized enoyl-CoA to alpha-functionalized acyl-CoA. The platform can be operated in an iterative manner (i.e. multiple turns) by using the resulting alpha-functionalized acyl-CoA as primer and the aforementioned alpha-functionalized extender unit in subsequent turns of the cycle. Termination pathways acting on any of the four alpha-functionalized CoA thioester intermediates terminate the platform and generate various alpha-functionalized carboxylic acids, alcohols and amines with different -reduction degree.

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.

This document describes biochemical pathways for producing a difunctional product having an odd number of carbon atoms in vitro or in a recombinant host, or salts or derivatives thereof, by forming two terminal functional groups selected from carboxyl, amine, formyl, and hydroxyl groups in an aliphatic carbon chain backbone having an odd number of carbon atoms synthesized from (i) acetyl-CoA and propanedioyl-CoA via one or more cycles of methyl ester shielded carbon chain elongation or (ii) propanedioyl-[acp] via one or more cycles of methyl ester shielded carbon chain elongation. The biochemical pathways and metabolic engineering and cultivation strategies described herein rely on enzymes or homologs accepting methyl ester shielded aliphatic carbon chain backbones and maintaining the methyl ester shield for at least one further enzymatic step following one or more cycles of methyl ester shielded carbon chain elongation.

Recombinant cells and methods for improved yield of fatty alcohols. The recombinant cells harbor a recombinant thioesterase gene, a recombinant acyl-CoA synthetase gene, and a recombinant acyl-CoA reductase gene. In addition, a gene product from one or more of an acyl-CoA dehydrogenase gene, an enoyl-CoA hydratase gene, a 3-hydroxyacyl-CoA dehydrogenase gene, and a 3-ketoacyl-CoA thiolase gene in the recombinant cells is functionally deleted. Culturing the recombinant cells produces fatty alcohols at high yields.

A PHA copolymer which is slowly crystallized is improved in crystallization speed to improve the melt workability of the PHA copolymer in working such as injection molding, film molding, blow molding, fiber spinning, extrusion foaming or bead foaming, thereby improving the resultant articles in productivity. A method for the improvement is a method for producing a PHA mixture, including the step of culturing a microorganism having both of a gene encoding a PHA synthase that synthesizes a copolymer PHA (A) and that is derived from the genus Aeromonas, and a gene encoding a PHA synthase that synthesizes a PHA (B) different in melting point from the copolymer PHA (A) by 10 C. or more to produce, in a cell of the microorganism, two or more PHAs different in melting point from one another by 10 C. or more simultaneously.

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.