Cloning and characterization of a gene involved in triacylglycerol biosynthesis and identification of additional homologous genes in the oleaginous bacterium Rhodococcus opacus PD630

Abstract

The oleaginous bacterium Rhodococus opacus strain PD630 serves as a model organism to investigate the metabolism of storage triacylglycerols (TAGs) in bacteria. The key enzyme catalysing the last step of TAG biosynthesis in bacteria is a promiscuous acyltransferase (Atf), exhibiting acyl-CoA acyltransferase activity to both diacylglycerols (DGAT activity) and fatty alcohols (wax ester synthase, WS activity). An 800 bp PCR product was obtained from chromosomal DNA of strain PD630 by using degenerate primers designed from conserved stretches of Atf proteins of Acinetobacter baylyi strain ADP1 and Mycobacterium smegmatis mc 2 155. The atf fragment was used as a probe on a strain PD630 gene library, resulting in the identification of a 3948 bp chromosomal DNA fragment containing the complete atf1 gene. An atf1 disruption mutant of strain PD630 exhibited a TAG-leaky phenotype and accumulated up to 50 % less fatty acids than the wild-type, with significantly reduced oleic acid content when cultivated in the presence of gluconate or oleic acid. Whereas DGAT activity was drastically reduced in comparison to the wild-type, WS activity remained almost unchanged in the mutant.RT-PCR analysis of gluconate-grown cells of strain PD630 showed that there is expression of atf1 under conditions of TAG synthesis. To identify additional Atfs in strain PD630, PCR employing non-degenerate primers deduced from Rhodococcus jostii RHA1 sequence data was used. This yielded nine additional atf-homologous genes exhibiting 88-99 % sequence identity to the corresponding strain RHA1 enzymes. Besides Atf1 only Atf2 exhibited high DGAT and/or WS activity when heterologously expressed in Escherichia coli.

Functional expression and characterization of five wax ester synthases in Saccharomyces cerevisiae and their utility for biodiesel production

“…However, no preference for specific acyl-CoA or alcohol substrates was reported in that study. In E. coli , the enzyme activity was measured using [1- 14 C] palmitoyl-CoA and 1-hexadecanol [20] and found to be 4.65 ± 0.04 pmol/mg protein/min, which is clearly lower than the activity found here in S. cerevisiae , which was 28.9 ± 3.29 pmol/mg protein/min (Table 1). …”

“…This enzyme has also been expressed in E. coli and has shown low DGAT activity [20]. However, no preference for specific acyl-CoA or alcohol substrates was reported in that study.…”

“…WS/DGAT from Acinetobacter baylyi ADP1 has been adopted in E. coli for ester production [4,8,16-18]. WS homologs are frequently found in the genomes of actinomycetes such as Rhodococcus [19,20] or in the genome databases of several marine bacteria like Marinobacter [19,21] and Psychrobacter [19]. Few reports are available about WS from mammals.…”

Improving microbial oil production with standard and native oleaginous yeasts by using Taguchi design

“…The bands were observed after staining the TLC plate by iodine vapor. So, the qualitative and semiquantitative analyses of intracellular lipid were carried out by thin-layer chromatography (Alvarez et al 2008). …”

Identification of a novel ATP-binding cassette transporter involved in long-chain fatty acid import and its role in triacylglycerol accumulation in Rhodococcus jostii RHA1

“…More recently, advances in the molecular biology and genetics of TAG metabolism have occurred as a result of the increased availability of genomic information and the development of new genetic tools for rhodococci (Holder et al, 2011;Chen et al, 2013;Villalba et al, 2013). These studies have been focused principally on some enzymes of TAG biosynthesis, such as the wax esters/diacylglycerol acyltransferases (WS/DGAT) (Alvarez et al, 2008;Hernández et al, 2013), NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase (MacEachran & Sinskey, 2013), and phosphatidic acid phosphatase (M. A. Hernández, A. Arabolaza, S. Comba, H. Gramajo & H. M. Alvarez, unpublished). Moreover, some genes coding for TAG granule-associated proteins have been identified and characterized in Rhodococcus opacus and Rhodococcus jostii (MacEachran et al, 2010; Ding et al, 2012;Chen et al, 2013).…”