Identification of genes coding for putative wax ester synthase/diacylglycerol acyltransferase enzymes in terrestrial and marine environments

Abstract

Synthesis of neutral lipids such as triacylglycerols (TAG) and wax esters (WE) is catalyzed in bacteria by wax ester synthase/diacylglycerol acyltransferase enzymes (WS/DGAT). We investigated the diversity of genes encoding this enzyme in contrasting natural environments from Patagonia (Argentina). The content of petroleum hydrocarbons in samples collected from oil-producing areas was measured. PCR-based analysis covered WS/DGAT occurrence in marine sediments and soil. No product was obtained in seawater samples. All clones retrieved from marine sediments affiliated with gammaproteobacterial sequences and within them, most phylotypes formed a unique cluster related to putative WS/DGAT belonging to marine OM60 clade. In contrast, soils samples contained phylotypes only related to actinomycetes. Among them, phylotypes affiliated with representatives largely or recently reported as oleaginous bacteria, as well as with others considered as possible lipid-accumulating bacteria based on the analysis of their annotated genomes. Our study shows for the first time that the environment could contain a higher variety of ws/dgat than that reported from bacterial isolates. The results of this study highlight the relevance of the environment in a natural process such as the synthesis and accumulation of neutral lipids. Particularly, both marine sediments and soil may serve as a useful source for novel WS/DGAT with biotechnological interest.

Fig. 1

Neighbour-joining dendogram showing the relationship of translated ws/dgat sequences (272 amino acids positions) obtained from Patagonian soils with reference sequences. The tree was calculated with a representative of each sequence type defined at 0.01 distance cut-off. For environmental sequence types, numbers in parenthesis indicate the accession numbers given in GenBank. Total number of sequences belonging to the same sequence type in the corresponding library from which they were retrieved is indicated in < and > symbols. AtfA from Acinetobacter baylyi ADP1 was used as outgroup. Relevant bootstrap values (1,000 replicates) higher than 50% are shown.

Fig. 2

Neighbour-joining dendogram showing the relationship of translated ws/dgat sequences (278 amino acids positions) obtained from Patagonian marine sediments with reference sequences. The tree was calculated with a representative of each sequence type defined at 0.01 distance cut-off. For environmental sequence types, numbers in < and > symbols indicate the total number of sequences in the corresponding library from which they were retrieved. The accession numbers of reference sequences and environmental clones obtained in this study are indicated. Acyltransferase from Thermobispora bispora was used as outgroup. Relevant bootstrap values (1,000 replicates) higher than 50% are shown.

Fig. 3

Multiple sequence alignments of Patagonian environmental clones and characterized WS/DGAT. Conserved regions and domains required for acyltransferase activity are marked in boxes. a LHIMP and LH = Las Heras soil more or less impacted, respectively. Atf-like acyltransferases from Rhodococcus opacus PD630, Rhodococcus jostii RHA1 and Thermomonospora curvata are included. b CR and BB = Comodoro Rivadavia port and Belvedere beach sediments, respectively. Atf from Acinetobacter baylyi ADP1 and Atf-like from Psychrobacter articus 273-4 are included.