Fruit residues as substrates for single-cell oil production by Rhodococcus species: physiology and genomics of carbohydrate catabolism.

Strains belonging to R. opacus, R. jostii, R. fascians, R. erythropolis and R. equi exhibited differential ability to grow and produce lipids from fruit residues (grape marc and apple pomace), as well as single carbohydrates, such as glucose, gluconate, fructose and sucrose. The oleaginous species, R. opacus (strains PD630 and MR22) and R. jostii RHA1, produced higher yields of biomass (5.1-5.6 g L-1) and lipids (38-44% of CDW) from apple juice wastes, in comparison to R. erythropolis DSM43060, R. fascians F7 and R. equi ATCC6939 (4.1-4.3 g L-1 and less than 10% CDW of lipids). The production of cellular biomass and lipids were also higher in R. opacus and R. jostii (6.8-7.2 g L-1 and 33.9-36.5% of CDW of lipids) compared to R. erythropolis, R. fascians, and R. equi (3.0-3.6 g L-1 and less than 10% CDW of lipids), during cultivation of cells on wine grape waste. A genome-wide bioinformatic analysis of rhodococci indicated that oleaginous species possess a complete set of genes/proteins necessary for the efficient utilization of carbohydrates, whereas genomes from non-oleaginous rhodococcal strains lack relevant genes coding for transporters and/or enzymes for the uptake, catabolism and assimilation of carbohydrates, such as gntP, glcP, edd, eda, among others. Results of this study highlight the potential use of the oleaginous rhodococcal species to convert sugar-rich agro-industrial wastes, such as apple pomace and grape marc, into single-cell oils.

Insights into the Metabolism of Oleaginous Rhodococcus spp.

Some species belonging to the Rhodococcus genus, such as Rhodococcus opacus, R. jostii, and R. wratislaviensis, are known to be oleaginous microorganisms, since they are able to accumulate triacylglycerols (TAG) at more than 20% of their weight (dry weight). Oleaginous rhodococci are promising microbial cell factories for the production of lipids to be used as fuels and chemicals. Cells could be engineered to create strains capable of producing high quantities of oils from industrial wastes and a variety of high-value lipids. The comprehensive understanding of carbon metabolism and its regulation will contribute to the design of a reliable process for bacterial oil production. Bacterial oleagenicity requires an integral configuration of metabolism and regulatory processes rather than the sole existence of an efficient lipid biosynthesis pathway. In recent years, several studies have been focused on basic aspects of TAG biosynthesis and accumulation using R. opacus PD630 and R. jostii RHA1 strains as models of oleaginous bacteria. The combination of results obtained in these studies allows us to propose a metabolic landscape for oleaginous rhodococci. In this context, this article provides a comprehensive and integrative view of different metabolic and regulatory attributes and innovations that explain the extraordinary ability of these bacteria to synthesize and accumulate TAG. We hope that the accessibility to such information in an integrated way will help researchers to rationally select new targets for further studies in the field.

Rhodococcus bacteria as a promising source of oils from olive mill wastes.

The accumulation of triacylglycerols (TAG) is a common feature among actinobacteria belonging to Rhodococcus genus. Some rhodococcal species are able to produce significant amounts of those lipids from different single substrates, such as glucose, gluconate or hexadecane. In this study we analyzed the ability of different species to produce lipids from olive oil mill wastes (OMW), and the possibility to enhance lipid production by genetic engineering. OMW base medium prepared from alperujo, which exhibited high values of chemical oxygen demand (127,000 mg/l) and C/N ratio (508), supported good growth and TAG production by some rhodococci. R. opacus, R. wratislaviensis and R. jostii were more efficient at producing cell biomass (2.2-2.7 g/l) and lipids (77-83% of CDW, 1.8-2.2 g/l) from OMW than R. fascians, R. erythropolis and R. equi (1.1-1.6 g/l of cell biomass and 7.1-14.0% of CDW, 0.1-0.2 g/l of lipids). Overexpression of a gene coding for a fatty acid importer in R. jostii RHA1 promoted an increase of 2.2 fold of cellular biomass value with a concomitant increase in lipids production during cultivation of cells in OMW. This study demonstrates that the bioconversion of OMW to microbial lipids is feasible using more robust rhodococal strains. The efficiency of this bioconversion can be significantly enhanced by engineering strategies.

Formation of indigoidine derived-pigments contributes to the adaptation of Vogesella sp. strain EB to cold aquatic iron-oxidizing environments.

We investigated previously under explored cold aquatic environments of Andean Patagonia, Argentina. Oily sheens similar to an oil spill are frequently observed at the surface of water in creeks and small ponds in these places. Chemical analysis of a water sample revealed the occurrence of high concentrations of iron and the presence of a free insoluble indigoidine-derived pigment. A blue pigment-producing bacterium (strain EB) was isolated from the water sample and identified as Vogesella sp. by molecular analysis. The isolate was able to produce indigoidine and another derived-pigment (here called cryoindigoidine) with strong antifreeze properties. The production of the pigments depended on the cell growth at cold temperatures (below 15 °C), as well as on the attachment of cells to solid surfaces, and iron limitation in the media. The pigments produced by strain EB showed an inhibitory effect on the growth of diverse microorganisms such as Candida albicans, Escherichia coli and Staphylococcus aureus. In addition, pigmented cells were more tolerant to freezing than non-pigmented cells, suggesting a role of cryoindigoidine/indigoidine as a cold-protectant molecule. The possible roles of the pigments in strain EB physiology and its interactions with the iron-rich environment from which the isolate was obtained are discussed. Results of this study suggested an active role of strain EB in the investigated iron-oxidizing ecosystem.

Physiological and genetic differences amongst Rhodococcus species for using glycerol as a source for growth and triacylglycerol production.

We analysed the ability of five different rhodococcal species to grow and produce triacylglycerols (TAGs) from glycerol, the main byproduct of biodiesel production. Rhodococcus fascians and Rhodococcus erythropolis grew fast on glycerol, whereas Rhodococcus opacus and Rhodococcus jostii exhibited a prolonged lag phase of several days before growing. Rhodococcus equi only exhibited poor growth on glycerol. R. erythropolis DSMZ 43060 and R. fascians F7 produced 3.9-4.3 g cell biomass l(-1) and 28.4-44.6% cellular dry weight (CDW) of TAGs after 6 days of incubation; whereas R. opacus PD630 and R. jostii RHA1 produced 2.5-3.8 g cell biomass l(-1) and 28.3-38.4% CDW of TAGs after 17 days of growth on glycerol. Genomic analyses revealed two different sets of genes for glycerol uptake and degradation (here named clusters 1 and 2) amongst rhodococci. Those species that possessed cluster 1 (glpFK1D1) (R. fascians and R. erythropolis) exhibited fast growth and lipid accumulation, whereas those that possessed cluster 2 (glpK2D2) (R. opacus, R. jostii and R. equi) exhibited delayed growth and lipid accumulation during cultivation on glycerol. Three glycerol-negative strains were complemented for their ability to grow and produce TAGs by heterologous expression of glpK2 from R. opacus PD630. In addition, we significantly reduced the extension of the lag phase and improved glycerol assimilation and oil production of R. opacus PD630 when expressing glpK1D1 from R. fascians. The results demonstrated that rhodococci are a flexible and amenable biological system for further biotechnological applications based on the reutilization of glycerol.

Label-free and redox proteomic analyses of the triacylglycerol-accumulating Rhodococcus jostii RHA1.

The bacterium Rhodococcus jostii RHA1 synthesizes large amounts of triacylglycerols (TAGs) under conditions of nitrogen starvation. To better understand the molecular mechanisms behind this process, we performed proteomic studies in this oleaginous bacterium. Upon nitrogen starvation, we observed a re-routing of the carbon flux towards the formation of TAGs. Under these conditions, the cellular lipid content made up more than half of the cell's dry weight. On the proteome level, this coincided with a shift towards non-glycolytic carbohydrate-metabolizing pathways. These pathways (Entner-Doudoroff and pentose-phosphate shunt) contribute NADPH and precursors of glycerol 3-phosphate and acetyl-CoA to lipogenesis. The expression of proteins involved in the degradation of branched-chain amino acids and the methylmalonyl-CoA pathway probably provided propionyl-CoA for the biosynthesis of odd-numbered fatty acids, which make up almost 30 % of RHA1 fatty acid composition. Additionally, lipolytic and glycerol-degrading enzymes increased in abundance, suggesting a dynamic cycling of cellular lipids. Conversely, abundance of proteins involved in consuming intermediates of lipogenesis decreased. Furthermore, we identified another level of lipogenesis regulation through redox-mediated thiol modification in R. jostii. Enzymes affected included acetyl-CoA carboxylase and a ß-ketoacyl-[acyl-carrier protein] synthase II (FabF). An integrative metabolic model for the oleaginous RHA1 strain is proposed on the basis of our results.