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

Abstract

Members of the genus Rhodococcus are specialists in the biosynthesis and accumulation of triacylglycerols (TAGs). As no transport protein related to TAG metabolism has yet been characterized in these bacteria, we used the available genomic information of Rhodococcus jostii RHA1 to perform a broad survey of genes coding for putative lipid transporter proteins in this oleaginous micro-organism. Among the seven genes encoding putative lipid transporters, ro05645 (now called ltp1: lipid transporter protein) coding for an ATP-binding cassette protein was found clustered with others genes encoding enzymes catalysing the three putative acylation reactions of the Kennedy pathway for TAG synthesis. Overexpression of ltp1 in the RHA1 strain led to an increase of approximately sixfold and threefold in biomass and TAG production, respectively, when cells were cultivated on palmitic acid and oleic acid. Moreover, overexpression of ltp1 also promoted a significant increase in the uptake of a fluorescently labelled long-chain fatty acid (LCFA), as compared with the WT strain RHA1, and its further incorporation into the TAG fraction. Gluconate-grown cells showed increasing amounts of intracellular free fatty acids, but not of TAG, after overexpressing ltp1. Thus, for the first time to our knowledge, a transporter functionally related to TAG metabolism was identified in oleaginous rhodococci. Our results suggested that Ltp1 is an importer of LCFAs that plays a functional role in lipid homeostasis of R. jostii RHA1.

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

“…749 and 750), that implies that the diffusion of xenobiotics through phospholipid bilayers in intact cells is normally negligible. It is now clear that transporters enhance (and are probably required for) the transmembrane transport even of hydrophobic molecules such as alkanes, 1316–1321 terpenoids, 1319,1322,1323 long-chain, 1324–1328 and short-chain 1329–1332 fatty acids, and even CO 2 . 1333,1334 This may imply a significantly enhanced role for transporter engineering in whole cell biocatalysis.…”

How drugs get into cells: tested and testable predictions to help discriminate between transporter-mediated uptake and lipoidal bilayer diffusion

“…While other mechanisms are also possible (Dikicioglu et al, 2014), the ABC transporter (Sá-Correia et al, 2009) Pdr18 (Teixeira et al, 2012) and the glyceroaquaporin Fps1 (Teixeira et al, 2009) have properties consistent with such a role as ethanol transporters in yeast, a fact of considerable biotechnological relevance (Dunlop et al, 2011). In the context of biofuels production (and ethanol is a biofuel), and based on similar strategies of toxicity resistance to the one that we exploited earlier (Lanthaler et al, 2011), we now also know them for a variety of other rather lipophilic substances such as alkanes (Tsukagoshi and Aono, 2000; Fernandes et al, 2003; Ankarloo et al, 2010; Chen et al, 2013; Doshi et al, 2013; Foo and Leong, 2013; Ling et al, 2013; Nishida et al, 2013), arenes (Kieboom et al, 1998b), terpenoids (Jasiński et al, 2001; Yazaki, 2006; Foo and Leong, 2013), long-chain fatty acids (Wu et al, 2006a,b; Khnykin et al, 2011; Lin and Khnykin, 2014; Villalba and Alvarez, 2014), short-chain fatty acids (Gimenez et al, 2003; Islam et al, 2008; Moschen et al, 2012; Sá-Pessoa et al, 2013), etc. These are all substances for which bilayer lipoidal diffusion was “once widely assumed” (and presumably still is in some quarters).…”

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

“…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.…”