Genetic analysis of Comamonas acidovorans polyhydroxyalkanoate synthase and factors affecting the incorporation of 4-hydroxybutyrate monomer

Abstract

The polyhydroxyalkanoate (PHA) synthase gene of Comamonas acidovorans DS-17 (phaCCa) was cloned by using the synthase gene of Alcaligenes eutrophus as a heterologous hybridization probe. Complete sequencing of a 4.0-kbp SmaI-HindIII (SH40) subfragment revealed the presence of a 1,893-bp PHA synthase coding region which was followed by a 1,182-bp beta-ketothiolase gene (phaACa). Both the translated products of these genes showed significant identity, 51.1 and 74.2%, respectively, to the primary structures of the products of the corresponding genes in A. eutrophus. The arrangement of PHA biosynthesis genes in C. acidovorans was also similar to that in A. eutrophus except that the third gene, phaB, coding for acetoacetyl-coenzyme A reductase, was not found in the region downstream of phaACa. The cloned fragment complemented a PHA-negative mutant of A. eutrophus, PHB-4, resulting in poly-3-hydroxybutyrate accumulation of up to 73% of the dry cell weight when fructose was the carbon source. The heterologous expression enabled the incorporation of 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate monomers. The PHA synthase of C. acidovorans does not appear to show any preference for 4-hydroxybutyryl-coenzyme A as a substrate. This leads to the suggestion that in C. acidovorans, it is the metabolic pathway, and not the specificity of the organism's PHA synthase, that drives the incorporation of 4HB monomers, resulting in the efficient accumulation of PHA with a high 4HB content.

FIG. 1

Organization of the PHA biosynthetic genes of C. acidovorans in EV80 subfragment. (a) Subfragments relevant for nucleotide sequence analysis; (b) restriction map; (c) strategy for sequencing of the SH40 subfragment.

FIG. 2

Nucleotide sequence of a 3,973-bp region containing the phaC_Ca and phaA_Ca genes with the amino acid sequences. Stop codons are indicated by asterisks. SD, Shine-Dalgarno site (RBS).

FIG. 3

Alignment and identities of the deduced sequence of PHA synthase from C. acidovorans with those from Alcaligenes (Alc.) sp. strain SH-69 (GenBank accession no. U78047), A. eutrophus (29, 36), Aeromonas caviae (7), Rhodococcus ruber (30), and Chromatium vinosum (22), which have the ability to incorporate short-chain HA into PHA. Amino acids identical in at least four sequences are boxed in black.