The Rhodococcus opacus TadD protein mediates triacylglycerol metabolism by regulating intracellular NAD(P)H pools

Daniel P Maceachran,Anthony J Sinskey

doi:10.1186/1475-2859-12-104

Abstract

BackgroundThe Gram-positive actinomycete Rhodococcus opacus is widely studied for its innate ability to store large amounts of carbon in the form of triacylglycerol (TAG). Several groups have demonstrated that R. opacus PD630 is capable of storing anywhere from 50 to 76% of its cell dry weight as TAG. While numerous studies have focused on phenomenological aspects of this process, few have sought to identify the underlying molecular and biochemical mechanisms responsible for the biosynthesis and storage of this molecule.ResultsHerein we further our previous efforts to illuminate the black box that is lipid metabolism in actinomycetes using a genetic approach. Utilizing a simple, colorimetric genetic screen, we have identified a gene, referred to herein as tadD (triacylglycerol accumulation deficient), which is critical for TAG biosynthesis in R. opacus PD630. Furthermore, we demonstrate that the purified protein product of this gene is capable of oxidizing glyceraldehyde-3-phosphate, while simultaneously reducing NAD(P)+ to NAD(P)H. Supporting this biochemical data, we observed that the ratio of NAD(P)H to NAD(P)+ is elevated in wildtype cultures grown under lipid production conditions as compared to cultures grown under vegetative growth conditions, while the mutant strain demonstrated no change irrespective of growth conditions. Finally, we demonstrate that over-expressing a putative phosphorylative glyceraldehyde-3-phosphate dehydrogenase leads to decreased TAG production during growth on TAG accumulation conditions.ConclusionTaken together, the data support the identification of a key metabolic branch point separating vegetative growth and lipid accumulation lifestyles in Rhodococcus.

Highlights

The Gram-positive actinomycete Rhodococcus opacus is widely studied for its innate ability to store large amounts of carbon in the form of triacylglycerol (TAG)
The non-phosphorylative glyceraldehyde 3-phosphate dehydrogenase (NP-G3P) family of enzymes, a sub-family of the larger aldehyde dehydrogenase family, was originally associated with green eukaryotes, plants and algae primarily, wherein it catalyzes the irreversible oxidation of glyceradehyde-3-phosphate (G3P) to 3-phosphoglycerate (3PG) while concomitantly reducing NAD(P)+ to NAD(P) H [19]
This is in contrast to the canonical phosphorylative glyceraldehyde 3-phosphate dehydrogenase (GapA) which oxidizes G3P to 1,3-bisphosphoglycerate (1,3BPG) while reducing NAD+ to NADH. 1,3-BPG is subsequently dephosphorylated to 3-PG by the enzyme phosphoglycerate kinase yielding one molecule of adenosine triphosphate (ATP) (Figure 1) [20]

Summary

Introduction

The Gram-positive actinomycete Rhodococcus opacus is widely studied for its innate ability to store large amounts of carbon in the form of triacylglycerol (TAG). The non-phosphorylative glyceraldehyde 3-phosphate dehydrogenase (NP-G3P) family of enzymes, a sub-family of the larger aldehyde dehydrogenase family, was originally associated with green eukaryotes, plants and algae primarily, wherein it catalyzes the irreversible oxidation of glyceradehyde-3-phosphate (G3P) to 3-phosphoglycerate (3PG) while concomitantly reducing NAD(P)+ to NAD(P) H [19] This reaction is mediated by the GapN protein in most organisms. Instead of yielding one molecule of NADH and ATP, as would be the case in the phosphorylative branch of glycolysis, the non-phosphorylative branch yields a single molecule of NAD(P)H, an essential reducing equivalent in most biosynthetic pathways including fatty acid biosynthesis While this family of enzymes was initially associated strictly with green eukaryotes, sequence and functional homologs have been identified in a number of eubacteria and archaea [21,22,23]

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