Abstract:
In archaea the cell membrane consists of ether linked isoprenoid units, in contrast to bacteria and eukaryotes, where the cell membrane is made up of ester linked fatty acid and glycerol. So, it was expected that functional beta oxidation pathway should not be present in Archaea. The genome sequence of a hyperthermophilic archaeon Pyrobaculum calidifontis contains two individual sets of genes that are expected to involve in betaoxidation of fatty acids. Set one makes an operon “1” consists of 7 genes annotated as Pcal_1306, Pcal_1307, Pcal_1308, Pcal_1309, Pcal_1310, Pcal_1311 and Pcal_1312. Genes of set two are distributed in two neighboring operons. Operon 2A contains Pcal_0836, Pcal_0837, Pcal_0838 and operon 2B consists of Pcal_0839, Pcal_0840 and Pcal_0841. Another significant importance of these genes is their role in the biobutanol synthesis, a second generation biofuel In this study genes from operon 1 were cloned, expressed in Escherichia coli. All of the proteins except Pcal_1309 were purified by heat treatment, anion exchange chromatography and gel filtration chromatography. After purification these proteins were partially characterized through standard assay for each enzyme. The products of each enzyme assay was analysed by HPLC to confirm the nature of product. After characterization an attempt was made for in vitro reconstruction of fatty acid oxidation and bio-butanol pathway. The overexpression resulted in soluble expression of all genes except Pcal_1309. Pcal_1309 was expressed as insoluble fraction. Characterization showed that Pcal_1306 exhibited enoyl-CoA dehydratase, recombinant Pcal_1307 exhibited esterase activity, recombinant Pcal_1308 showed acyl-CoA dehydrogenase activity, recombinant Pcal_1311 was responsible for aldehyde reductase activity, and recombinant Pcal_1312 was found responsible for NADH dependent keto acyl CoA reductase activity. While function of recombinant Pcal_1310 could not be clearly identified. Although these proteins are known for their role in betoxidation but in reverse these may also be involved in fatty acid synthesis. Further studies are required to completely establish the role of these genes in archaea to find out the nature of the last universal common ancestor. A partial characterization of enzymes involved in beta oxidation has been made, so we expect there might be a function fatty acid oxidation system in Archaea.
Page(s):
59-59
DOI:
DOI not available
Published:
Journal: Abstract Book on Global Science Technology and Management Conference, Volume: 0, Issue: 0, Year: 2023