ReviewTriacylglycerol and wax ester-accumulating machinery in prokaryotes
Graphical abstract
Triacylglycerol inclusion bodies in the oleaginous Rhodococcus opacus PD630.
Introduction
Most bacteria are able to survive and thrive in environments with fluctuating nutritional conditions. Moreover, bacterial cells also interact with multiple stress factors that simultaneously occur in natural environments. The production of neutral lipids, such as wax esters (WE) and triacylglycerols (TAG), may be part of the complex strategic survival mechanisms evolved by some prokaryotes, which allow them to colonize and thrive in natural environments. These lipids are convenient storage compounds for carbon and energy, which can be utilized for cell survival in energy-poor environments. Since the carbon atoms of acyl moieties of TAG and WE are in their most reductive form; the degradation of these biomolecules produces a maximum yield of energy in comparison to other storage compounds produced by bacteria, such as glycogen and polyhydroxyalkanoates [1]. The energy obtained by the slow mobilization of stored lipids may provide cells of energetic autonomy and a temporal independence from the environment and contribute to cell survival when they do not have access to energy resources in the environment. Lipid stored by bacteria may be important not only for their energy potential but also as a reservoir of metabolic water under desiccation conditions, since fatty acid oxidation releases large amounts of metabolic water [2]. In addition, storage lipids possess other important functions in cells, such as the regulation of the fatty acid composition of membrane lipids, as a sink for reducing equivalents and physiological active and potentially toxic metabolic intermediates for balancing the metabolism under environmental fluctuating conditions, as precursor source for biosynthesis of essential lipids, among other possible functions [3].
The biosynthesis and accumulation of TAG and/or WE are stimulated when an excess of a carbon source is available and the nitrogen source is limiting [4], [5]. These special conditions are frequently found in soil and marine environments. The ability to accumulate storage lipids demands the presence of a genetic and enzymatic endowment in the microorganism and the capability for maintaining the balance of precursors and reducing equivalents since the lipid accumulation is an energy-expensive process, which compete with cellular growth. The process of neutral lipid accumulation and their involved components have been well studied in eukaryotic organisms, such as plants and yeasts [6], [7]. The pioneer studies on WE and TAG accumulation in prokaryotes were mainly performed in members of Acinetobacter [4], Mycobacterium [8], Streptomyces [9] and Rhodococcus [10] genera. The important role of TAG in the pathogenesis of Mycobacterium tuberculosis, and the relationship of TAG metabolism with antibiotic biosynthesis by Streptomyces coelicolor have stimulated the basic research on such lipids in those microorganisms. On the other hand, members of Acinetobacter and Rhodococcus genera, such as Acinetobacter baylyi ADP1 and Rhodococcus opacus PD630 have been used as models for deciphering different aspects on WE/TAG biosynthesis and accumulation. More recently, other bacteria with the ability to produce WE and/or TAG have emerged as model organisms for different studies in this field, including Marinobacter hydrocarbonoclasticus [11], Alcanivorax borkumensis [12] and Rhodococcus jostii [13]. The potential application of such neutral lipid-producing microorganisms as a source of single cell oil useful for the production of biofuels or other derived industrial products, promoted further studies which contributed with our understanding of the process. Single cell oils are lipids extracted from microorganisms, which could serve as alternative oil sources for the production of biofuels with similar efficiency as petroleum diesel. The use of microorganisms for lipid production provides some advantage over agricultural sources with regards to the enormous variability of fatty acid composition depending on the carbon source used for cultivation of cells, and the better accessibility of microorganisms to genetic and metabolic engineering. Current research efforts are being focused on the biochemistry and genetics of oil-accumulating bacteria for designing a scalable and commercially viable oil-producing system from inexpensive feedstocks. In this context, the application of omic approaches as well as the functional identification and characterization of key genes/proteins from model bacteria, enabled significant advances in the fundamental knowledge on WE/TAG metabolism. This review article provides a comprehensive view on the composition of the WE/TAG-accumulating machinery necessary for supporting biosynthesis and accumulation of such lipids in prokaryotes.
Section snippets
Synthesis and accumulation of WE/TAG by bacteria
TAG as well as WE are synthesized by a diversity of bacteria. However, there are some qualitative and quantitative differences in their accumulation profiles. The synthesis and accumulation of TAG and WE have been reported for Gram negative hydrocarbon-degrading bacteria belonging to Acinetobacter, Marinobacter, Thalassolituus and Alcanivorax genera [4], [14], [15]. These microorganisms are able to produce TAG and WE during cultivation of cells on acetate, pyruvate or hexadecane as sole carbon
Key acyltransferase enzymes for TAG and WE synthesis in bacteria
The synthesis of TAG and WE in prokaryotes depends on the presence of a CoA-dependent acyltransferase enzyme known as wax ester synthase/diacylglycerol acyltransferase (WS/DGAT). This enzyme can exhibit simultaneously both, acyl-CoA:fatty alcohol acyltransferase (wax ester synthase, WS) and diacylglycerol acyltransferase (DGAT) activities (Fig. 1). The first prokaryotic WS/DGAT was reported for A. baylyi ADP1 by Kalscheuer and Steinbüchel [16]. Later, several WS/DGATs were identified, cloned
Routes that feed precursors for neutral lipid biosynthesis
The key metabolic intermediates feeding lipid biosynthesis are pyruvate, acetyl-CoA and glycerol-3-phosphate. Pyruvate is the end product of glycolysis, which is one of the switch points for carbon flux distribution within the central metabolism. This metabolic intermediate can serve as precursor for sugar phosphate synthesis through the gluconeogenesis or can be used to replenish TCA cycle intermediates that are bled off for anabolic processes (anaplerotic reactions) [30]. In addition,
Triacylglycerol- and wax ester-biosynthetic machinery in Gram negative bacteria
Gram negative bacteria belonging to Acinetobacter, Marinobacter, and Alcanivorax are the most studied microorganisms regarding neutral lipid accumulation, principally WE. Among them, A. baylyi ADP1 is the main model representative in the field. Biosynthesis of WE by strain ADP1 involves three enzymatic steps; firstly, an acyl-CoA is reduced to a corresponding long-chain aldehyde by a NADPH dependent fatty acyl-CoA reductase (called Acr1) [35], the resulting fatty aldehyde is further reduced to
Triacylglycerol- and wax ester-accumulating machinery in Gram positive actinobacteria
Actinobacteria usually accumulate TAG during cultivation of cells on diverse carbon sources under nitrogen limiting conditions, and in some cases when cells are grow on n-alkanes o n-alcohols, they also accumulate WE [8], [9], [10]. Despite recent advances in our understanding of neutral lipid metabolism occurred in the last years, the understanding of this process in actinobacteria is still fragmentary. Members of Streptomyces, Mycobacterium and Rhodococcus genera are the most investigated
Concluding remarks
It is now clear that the difference between a WE/TAG-accumulating bacterial strain; from other that is not able to produce such storage lipids, is not simply the presence/absence of key enzymes involved in their synthesis. WS/DGAT enzymes are certainly indispensable for storage lipid biosynthesis, but they must work within an integrated metabolic and regulatory network in lipid-accumulating bacteria. This metabolic network, which is dynamic and changing, can enable a cell to efficiently respond
Acknowledgment
The author would like to thank all collaborators and colleagues, who contributed to the advances in the bacterial WE/TAG research field. Our studies on this topic are being currently financed by the SCyT of the University of Patagonia San Juan Bosco, the Agencia Comodoro Conocimiento (MCR), Oil m&s SA Company, Project PIP-CONICET Nro. 0764, Project PFIP CHU-25 (COFECyT) and Project PICT2012 Nro. 2031 (ANPCyT), Argentina. Alvarez H.M is a career investigator of the Consejo Nacional de
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