Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae

Abstract

A 5.0-kbp EcoRV-EcoRI restriction fragment was cloned and analyzed from genomic DNA of Aeromonas caviae, a bacterium producing a copolyester of (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyhexanoate (3HHx) [P(3HB-co-3HHx)] from alkanoic acids or oils. The nucleotide sequence of this region showed a 1,782-bp poly (3-hydroxyalkanoate) (PHA) synthase gene (phaC Ac [i.e., the phaC gene from A. caviae]) together with four open reading frames (ORF1, -3, -4, and -5) and one putative promoter region. The cloned fragments could not only complement PHA-negative mutants of Alcaligenes eutrophus and Pseudomonas putida, but also confer the ability to synthesize P(3HB-co-3HHx) from octanoate or hexanoate on the mutants' hosts. Furthermore, coexpression of ORF1 and ORF3 genes with phaC Ac in the A. eutrophus mutant resulted in a decrease in the polyester content of the cells. Escherichia coli expressing ORF3 showed (R)-enoyl-coenzyme A (CoA) hydratase activity, suggesting that (R)-3-hydroxyacyl-CoA monomer units are supplied via the (R)-specific hydration of enoyl-CoA in A. caviae. The transconjugant of the A. eutrophus mutant expressing only phaC Ac effectively accumulated P(3HB-co-3HHx) up to 96 wt% of the cellular dry weight from octanoate in one-step cultivation.The utilization of biological systems for production of biodegradable materials is becoming important as a solution of the problems concerning plastic waste and the global environment. Poly(3-hydroxyalkanoates) (PHA) are produced by a wide variety of bacteria as intracellular carbon-and energystorage materials from renewable carbon resources, such as sugars or plant oils (2,6,18,25). Since these bacterial PHA are biodegradable thermoplastics, they have attracted industrial attention as possible candidates for large-scale biotechnological products. At present, more than 90 different monomeric units have been found as constituents of PHA (37).Bacterial PHA can be divided into two groups, depending on the number of carbon atoms in the monomeric units (35). One group of bacteria, including Alcaligenes eutrophus, produces short-chain-length PHA with C 3 -to-C 5 monomer units, while the other group, including Pseudomonas oleovorans, synthesizes medium-chain-length PHA with C 6 -to-C 14 monomer units. Only a few reports are available for bacteria which can synthesize PHA consisting of both short-and medium-chainlength monomer units. For example, Rhodospirillum rubrum (3), Rhodocyclus gelatinosus (19), and Rhodococcus ruber (12) produce terpolymers consisting of C 4 , C 5 , and C 6 3-hydroxyalkanoate (3HA) units from hexanoate, and some pseudomonad strains accumulate PHA consisting of C 4 -to-C 12 3HA units (16,38). Our laboratory has found that a random copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx), P(3HB-co-3HHx), is produced by Aeromonas caviae FA440 isolated from soil (7,32). This bacterium synthesizes the copolyester from alkanoic acids of even carbon numbers or from plant oils up to approximately 30 wt% of the cellular dry weight, with a 3HHx fra...

Cloning and expression of the PHA synthase gene from a locally isolated Chromobacterium sp. USM2

“…Previously, Kolibachuk and co-workers noticed that 3HHx monomer was accumulated by recombinant C. necator PHBˉ4 harboring the PHA synthase gene of C. violaceum when fed with even carbon numbered fatty acids (Kolibachuk et al, 1999). Similar studies were also done to generate 3HHx monomer by expressing PHA synthase gene of A. caviae in mutant C. necator PHB -4 (Fukui and Doi, 1997). A. caviae is known to synthesize both SCL and MCL PHA containing four to six carbon atoms (Shimamura et al, 1994).…”

Mesaconyl-Coenzyme A Hydratase, a New Enzyme of Two Central Carbon Metabolic Pathways in Bacteria

“…Similar genes were found in other bacteria that have in common with R. sphaeroides the absence of a functional glyoxylate cycle for acetate assimilation. However, the overall sequence identity of these proteins to the characterized member of this so-called MaoC or FkbR2 family, an R-specific 3-hydroxyacyl-CoA dehydratase from Aeromonas caviae (18), is only in the range of 20%. Furthermore, the expected molecular mass of the encoded enzyme is about 38 kDa, which is larger than the masses of normal enoyl-CoA hydratases (14 to 28 kDa).…”

PhaC and PhaR Are Required for Polyhydroxyalkanoic Acid Synthase Activity in Bacillus megaterium

“…One is PHA synthase of Thiocapsa pfennigii, which has two subunits, PhaC and PhaE, with approximately 85% similarity, respectively, to those of A. vinosum (class III), but unlike A. vinosum it can incorporate medium-as well as short-chain-length HACoAs into the polymer (11). The other exception is PHA synthase of Aeromonas caviae, which has approximately 45% similarity to that of R. eutropha (class I) and can incorporate 3-hydroxyhexanoyl thioester monomers into the polymer (5).…”