Genetic analysis of acyl-CoA carboxylases involved in lipid accumulation in Rhodococcus jostii RHA1

Abstract

In actinomycetes, the acyl-CoA carboxylases, including the so-called acetyl-CoA carboxylases (ACCs), are biotin-dependent enzymes that exhibit broad substrate specificity and diverse domain and subunit arrangements. Bioinformatic analyses of the Rhodococcus jostii RHA1 genome found that this microorganism contains a vast arrange of putative acyl-CoA carboxylases domains and subunits. From the thirteen putative carboxyltransferase domains, only the carboxyltransferase subunit RO01202 and the carboxyltransferase domain present in the multidomain protein RO04222 are highly similar to well-known essential ACC subunits from other actinobacteria. Mutant strains in each of these genes showed that none of these enzymes is essential for R. jostii growth in rich or in minimal media with high nitrogen concentration, presumably because of their partial overlapping activities. A mutant strain in the ro04222 gene showed a decrease in triacylglycerol and mycolic acids accumulation in rich and minimal medium, highlighting the relevance of this multidomain ACC in the biosynthesis of these lipids. On the other hand, RO01202, a carboxyltransferase domain of a putative ACC complex, whose biotin carboxylase and biotin carboxyl carrier protein domain were not yet identified, was found to be essential for R. jostii growth only in minimal medium with low nitrogen concentration. The results of this study have identified a new component of the TAG-accumulating machinery in the oleaginous R. jostii RHA1. While non-essential for growth and TAG biosynthesis in RHA1, the activity of RO04222 significantly contributes to lipogenesis during single-cell oil production. Furthermore, this study highlights the high functional diversity of ACCs in actinobacteria, particularly regarding their essentiality under different environmental conditions.