NADP-dependent malic enzymes as a tool for improving oil production in Rhodococcus bacteria

Abstract

Rhodococcus bacteria are able to produce triacylglycerols (TAG) from diverse carbon sources, including industrial wastes. Oleaginous bacteria may serve as a source of lipids with potential application in the industry, such as in the production of biofuels, biolubricants, additives for cosmetics or feed, oleochemicals and other manufactured products. Thus, rhodococci could serve as lipid production platforms that could be rationally optimized via genetic engineering. Based on the well-known malic enzyme (ME) participation on lipid metabolism in several eukaryotic microorganisms, herein we investigated the occurrence and natural contribution of these enzymes in different Rhodococcus strains. Enzymatic activity measurements and the use of a metabolic inhibitor (sesamol) indicated that NADP+-dependent ME?s are contributing to the TAG biosynthesis as well as cell biomass production in the oleaginous R. jostii RHA1 and R. opacus PD630 strains. In addition, we functionally characterized a putative ME encoded by the RHA1_RS44255 gene in R. jostii RHA1. Heterologous expression of RHA1_RS44255 in E. coli BL21 (DE3) resulted in a two-fold increase in NADP+-ME activity. Over-expression of the gene in RHA1 and PD630 strains grown with glucose, promoted an increase up to 10% by cellular dry weight in total fatty acid production without sacrificing cellular biomass. Expression of this gene in R. fascians F7 resulted in an increase of 1.3-1.4-fold in lipids during cultivation on glycerol. Results confirmed the contribution of NADP+-ME on TAG accumulation in oleaginous rhodococci and the utility of these enzymes as an alternative approach to increase bacterial oil production from different carbon sources.