Isolation and characterization of genes of alcaligenes lactus encoding enzymes capable of synthesizing polyhydroxyalkanoic acid

Abstract

To identify the polyhydroxyalcanoic acid(PHA) biosynthetic gene of Alcaligenes lactus, a genomic library was constructed and screened with two types of single stranded probes which were originated from both ends of open reading frames of phb CAB operon of A. eutrophus, a known bacterial strain producing PHA. In screening the PHA biosynthetic gene with plaque hybridization method, several positive clones were isolated with only probe 3`` which corresponds to 3`` end region of phb CAB operon(Reductase gene). These $\lambda$PHA clones were confirmed to contain 5`` region of PHA biosynthetic gene with chromosome walking and three $\lambda$PHA clones($\lambda$PHA1,3,4) were selected. In the Sall/EcoRI fragment(7Kb) of $\lambda$PHA 4 clone, there are four homology regions covering evenly among three genes(synthetase, thiolase, reductase) of phb CAB operon of A. eutrophus. As like phb CAB operon, three genes of PHA synthesis of A. lactus are clustered together and the nucleotide sequence homology between putative PHA operon of A. lactus and phb CAB operon of A. eutrophus is about 75\% and that of amino acid sequence is 80 $ \sim$ 85\% But the arrangement of genes in putative operon is different from phb CAB operon in that putative PHA operon has extra sequence between phb A(thiolase gene) and phb B(reductase gene) which of size is 1.3 kb. This fragment shows no homology with any regions of phb CAB operon. Due to this reason Sall/EcoRI fragment(7kb) of $\lambda$PHA 4 clone contains partial phb CAB operon lacks of 5`` end region(2KB) but the orientation of $\lambda$PHA 4 is antiparallel to $\lambda$PHA 3 clone which has some overlapping region with $\lambda$PHA 4 clone. So ultimately we got putative full operon of PHA biosynthetic gene encoding three enzymes of A. lactus.