Abstract
Oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcripts were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δatf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production.
Highlights
Atf[6] in RHA1, resulted in a 30–50% decreased fatty acid (FA) content during stationary phase compared to the wild-type using gluconate as a growth substrate[13,15]
RHA1 in particular has been identified as a promising starting point for metabolic engineering based on its ability to grow on a lignin-enriched stream derived from corn stover
In this study of TAG accumulation in RHA1, we used benzoate as a model of lignin-depolymerization products degraded via the β -ketoadipate pathway to succinate and acetyl-CoA
Summary
Atf[6] in RHA1, resulted in a 30–50% decreased fatty acid (FA) content during stationary phase compared to the wild-type using gluconate as a growth substrate[13,15]. Proteomics and transcriptomics studies have identified a number of other genes involved in TAG biosynthesis and have begun to provide a more integrated view of this biosynthesis with respect to cellular metabolism. Proteomic studies have identified 228 LD-associated proteins in RHA1, the two most abundant of which were RHA1_RS10270 (formerly Ro02104) and PspA5 The former includes a predicted apolipoprotein domain and was annotated as “microorganism lipid droplet small” (MLDS) because its deletion yielded larger LDs. 177 LD-associated proteins were found to be differentially produced under lipid-accumulating conditions in PD630, including the MLDS homolog[16], previously identified as TadA18. 177 LD-associated proteins were found to be differentially produced under lipid-accumulating conditions in PD630, including the MLDS homolog[16], previously identified as TadA18 In these studies, the LD-associated proteins were predicted to be involved in a remarkably wide variety of cellular processes. The findings are discussed with respect to the engineering of rhodococcal strains for biotechnological applications
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