Insight in to the phylogeny of polyhydroxyalkanoate biosynthesis: Horizontal gene transfer

Abstract

Polyhydroxyalkanoates (PHAs) are gaining more and more importance the world over due to their structural diversity and close analogy to plastics. Their biodegradability makes them extremely desirable substitutes for synthetic plastics. PHAs are produced in organisms under certain stress conditions. Here, we investigated 253 sequenced (completely and unfinished) genomes for the diversity and phylogenetics of the PHA biosynthesis. Discrepancies in the phylogenetic trees for phaA, phaB and phaC genes of the PHA biosynthesis have led to the suggestion that horizontal gene transfer (HGT) may be a major contributor for its evolution. Twenty four organisms belonging to diverse taxa were found to be involved in HGT. Among these, Bacillus cereus ATCC 14579 and Xanthomonas axonopodis pv. citri str. 306 seem to have acquired all the three genes through HGT events and have not been characterized so far as PHA producers. This study also revealed certain potential organisms such as Streptomyces coelicolor A3(2), Staphylococcus epidermidis ATCC 12228, Brucella suis 1330, Burkholderia sp., DSMZ 9242 and Leptospira interrogans serovar lai str. 56601, which can be transformed into novel PHA producers through recombinant DNA technology.

Introduction

Man has relied extensively on biopolymers such as wool, leather, silk and cellulose. Plastics are the most widely used synthetic polymers as substitutes to natural ones. However, the growing reliance on synthetic polymers has raised a number of environmental and human health concerns (Kalia et al., 2003). Biodegradable plastics like polyhydroxyalkanoates (PHAs) can provide some relief from this recurrent problem. PHAs are produced in organisms under certain stress conditions. Microorganisms use different biological pathways for the synthesis of polyhydroxybutyrate (PHB), which are a type of PHA. PHB synthesis starts from acetyl-CoA and proceeds via acetoacetyl CoA and 3-hydroxybutyryl-CoA. Initially condensation of two acetyl-CoA molecules takes place to form acetoacetyl-CoA. The reaction is catalyzed by β-ketothiolase (phaA). Reduction of acetoacetyl-CoA is carried out by an NADPH-dependent acetoacetyl-CoA dehydrogenase (phaB). Lastly, the (R)-3-hydroxybutyryl-CoA monomers are polymerized into P(3HB) by P(3HB) polymerase (phaC).

PHB can also be synthesized via a five-step metabolic pathway. Here, NADH dependent acetoacetyl CoA reductase catalyzes the formation of S(+)-3-hydroxybutyryl-CoA. The S form is subsequently converted to R(−)-3-hydroxybutyryl-CoA by two stereospecific 2-enoyl-CoA-hydratases prior to polymerization (Lee and Choi, 1999). In addition to the classical three-step P(3HB) pathway (Madison and Huisman, 1999), some PHA producers use secondary pathways (Steinbüchel and Lutke-Eversloh, 2003, Aldor and Keasling, 2003). In methylmalonyl–CoA pathway, conversion of succinyl-CoA to (2R)-methylmalonyl-CoA proceeds through the action of coenzyme B12-dependent methylmalonyl-CoA mutase (Aldor et al., 2002). Methyl malonyl-CoA epimerase transforms R to S form. To convert (2S)-methylmalonyl-CoA to propionyl-CoA, two alternative enzymes are involved in Propionibacterium shermanii (Aldor et al., 2002) and Propionigenium modestum (Dimroth and Schink, 1998). Propionyl CoA then proceeds to the formation of polyhydroxyvalerate. In pseudomonads of rRNA homology group I, two additional pathways are found. These involve either β-oxidation or fatty acid biosynthesis intermediates for PHA production (Lee and Choi, 1999). The main route during growth on simple carbon compounds is the de novo fatty acid synthetic pathways, which provide the precursors for PHA biosynthesis (Yu, 2001). A pathway, which offers conversion of succinyl-CoA to propionyl-CoA for recombinant P(3HB-co-HV) production was discovered recently (Aldor et al., 2002).

Metabolic engineering is being extensively explored to enhance PHA synthesis by introducing new metabolic pathways to broaden the range of substrates. Screening of genome and metabolic databases revealed novel organisms with potential for PHA production (Kalia et al., 2003). On the other hand, recombinant DNA technology for transferring PHA producing ability of certain organisms into others, including non-PHA producers (Reddy et al., 2003) has met with limited success. It is perhaps necessary to assess the extent to which natural selection might have been effective in moulding genomes, the pattern of codon usage, the level of expression of a gene (Codon Adaptation Index, CAI) and its adaptation to their hosts (Sharp and Li, 1987). Felmlee et al. (1985) have reported that low CAI values of the four hemolysin genes in Escherichia coli are atypical of that species, which suggested that these genes are not well adapted and have been recently acquired. CAI as a measure of dominating codon bias has been used to predict gene expression levels in a large number of bacteria and certain eukaryotes (Carbone et al., 2003). Here we present the result of a phylogenetic analysis of the enzymes of PHA biosynthesis pathway. The genetic variability resulting from horizontal gene transfer (HGT) events during evolution can facilitate transformation of presently “non”-PHA producer to a producer.