Characterization of key triacylglycerol biosynthesis processes in rhodococci

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.

The pleiotropic transcriptional regulator NlpR contributes to the modulation of nitrogen metabolism, lipogenesis and triacylglycerol accumulation in oleaginous rhodococci

“…() demonstrated by transcriptomic analysis that the complete cluster where nlpR gene is located was highly expressed during incubation of R. jostii RHA1 cells in sterile soil containing both ammonium and nitrate at very low levels. Other transcriptomic data reported in different actinobacteria including M. smegmatis (Williams et al ., ), M. tuberculosis (Williams et al ., ), R. opacus PD630 (Chen et al , ) and more recently, R. jostii RHA1 (Amara et al ., ), also revealed the upregulation of nlpR orthologs under ammonium‐limiting conditions. All these results confirmed that nitrogen deprivation is the key condition to induce the expression of NlpR in rhodococci and other actinobacteria, probably through a mechanism mediated by the global regulator GlnR, as has been reported for S. coelicolor (Amin et al ., ).…”

“…For these reasons, we decided to explore the role of some transcriptional regulatory proteins that are significantly upregulated during TAG accumulation in oleaginous rhodococci. After analyzing different ‘omics’ studies in R. jostii RHA1 and R. opacus PD630, we observed a reproducible induction of a putative transcriptional regulator coded by the RHA1_RS31140 (formerly RHA1_ ro06368 ) gene under nitrogen‐limiting conditions (Chen et al ., ; Dávila Costa et al ., ; Amara et al ., ). This protein was always negligible under nitrogen‐rich conditions, which promote cell growth but not TAG accumulation.…”

“…In the last years, several genes/proteins and their contributions on TAG metabolism have been well described in R. opacus PD630 or R. jostii RHA1 used as models. Examples of these studies include proteins such as diacylglycerol acyltransferase enzymes (DGAT) (Alvarez et al , ; Hernández et al ., ; Amara et al ), a NADPH‐dependent glyceraldehyde‐3‐phosphate dehydrogenase (TadD) (MacEachran and Sinskey, ), a phosphatidic acid phosphatase type 2 enzyme (PAP2) (Hernández et al ., ), an ATP‐binding cassette transporter (Lpt1) (Villalba and Alvarez, ) and structural proteins involved in lipid body ontogeny (TadA) (MacEachran et al , ; Ding et al , ). Besides enzymes, transporters and structural proteins, the TAG‐accumulating machinery of rhodococci may include an unknown regulatory network probably integrated by global and specific regulatory proteins, which could coordinate the transition to a TAG‐producing phenotype.…”

A Fatty Acyl Coenzyme A Reductase Promotes Wax Ester Accumulation in Rhodococcus jostii RHA1

“…For example, the soil bacterium Rhodococcus jostii RHA1 (referred to as RHA1 here) accumulates neutral lipids up to 70% of cellular dry weight (CDW) in response to environmental stresses, such as nitrogen limitation (1)(2)(3). These neutral lipids are primarily triacylglycerides (TAGs) and are stored in lipid bodies within the cytoplasm (4).…”

A Review on The Bioconversion of Lignin to Microbial Lipid with Oleaginous Rhodococcus opacus

“…Transcriptomic and proteomic studies have also identified TAG biosynthetic genes and proteins [48][49][50][51][52][53]. In previous years, the Gram-positive, oleaginous, soil bacterium Rhodococcus opacus has been studied for its ability to accumulate intracellular lipids >20%, based on cell dry weight (CDW) [54][55][56][57][58][59][60].…”