Redundancy in the biosynthesis of triacylglycerol by Rhodococcus

Many mycolic acid-containing actinobacteria are oleaginous, accumulating high amounts of triacylglycerols (TAGs) under conditions of nutrient stress. These bacteria contain multiple copies of the genes involved in TAG biosynthesis: glycerol-phosphate acyltransferase (GPAT), acylglycerol-phosphate acyltransferase (AGPAT), phosphatidic acid phosphatase (PAP) and diglyceride acyltransferases (WS/DGAT), encoded by plsB, plsC, pap, and atf, respectively. Analysis of Rhodococcus jostii RHA1’s genome revealed that it carries 1 plsB, 8 plsC, 7 pap, and 16 atf. Quantitative, time-dependent data of six of these atf genes, selected based on previous transcriptomics data, revealed distinct expression patterns under nitrogen-limiting (N-) and carbon-limiting (C-) conditions. For example, the levels of atf10, atf3 and atf8 transcripts dropped ~10-fold upon growth substrate depletion, while the levels of atf4, atf6 and atf9 transcripts rose. Under N- conditions, RHA1 cells continued to accumulate TAGs for five days after ammonia depletion, during which time atf10 and atf8 transcripts remained abundant. Targeted deletion of any one of atf3, atf4, atf6, atf9 and atf10 did not significantly affect TAG accumulation under N- conditions, consistent with the redundancy of putative acyltransferases in the RHA1 genome. However, deletion of both atf8 and atf10 resulted in a 50% decrease in TAG accumulation. Furthermore, the fatty acid profile of the ∆atf8∆atf10 mutant was significantly perturbed, and was restored by complementation with either atf8 or atf10. RT-qPCR data analysis also revealed that the expression patterns of plsC (RS27555) and plsB were the same as that of atf9, consistent with their occurrence in an operon. Unexpectedly, deletion of plsB did not affect TAG accumulation, suggesting an alternative pathway for TAG and phospholipids biosynthesis. Finally, I identified three genes encoding HAD-type hydrolases as being putatively involved in TAG biosynthesis, including one that occurs as a fusion with plsC. The available data suggest that they act as PAPs. Overall, the results establish that there is a certain degree of functional redundancy in TAG biosynthesis, and that Atf8 and Atf10 play a major role in TAG accumulation. At the same time, the results also highlight important gaps in our knowledge of TAG biosynthesis in mycolic acid-containing oleaginous actinobacteria.Science, Faculty ofGraduat