opacus PD630 were incubated in a nitrogen-free mineral medium (MSM0) containing gluconate as the sole carbon source and in the presence of an inhibitor of lipid metabolism. Cerulenin inhibits the de novo fatty acid biosynthesis pathway by binding to the active sites of ketoacyl synthases I and II (Funabashi et al., 1989). We used gluconate as a carbon source for cultivation experiments because this substrate supports higher amounts of triacylglycerol accumulation by cells of strain PD630 than other carbon sources (Alvarez et al., 1996). Thus, the inhibition of lipid biosynthesis may have a significant Nutlin-3a ic50 effect on glycogen

accumulation. Cerulenin (25 μg mL−1) inhibited fatty acid biosynthesis and subsequent accumulation of triacylglycerols as is shown in Fig. 2. The triacylglycerol content of cells cultivated in the presence of the inhibitor was likely to have been produced by cells during their preculture in NB as suggested by the results shown in Fig. 2b. In addition, the total amounts of glycogen and polyhydroxyalkanoates were also affected by cerulenin as shown by an approximately twofold and 30-fold increase in the polyhydroxyalkanoates and glycogen contents, respectively. Glycogen accumulation was also studied in two mutants of R. opacus PD630 defective in triacylglycerol accumulation (Table 4). Mutant PDM41 was obtained by chemical mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine, whereas an atf1ΩKm mutant was constructed by

a specific kanamicyn cassette disruption of the atf1 gene of strain PD630 (Alvarez et al., STA-9090 solubility dmso 2008). The atf1 gene is one of the genes involved in the biosynthesis of triacylglycerols in R. opacus PD630 as has been reported previously (Alvarez et al., 2008). Atf proteins (diacylglycerol acyltransferase enzyme) catalyze the condensation of acyl-CoA and diacylglycerol with the formation of triacylglycerols. Other isoenzymes besides Atf1 must contribute to triacylglycerol accumulation in strain PD630, because disruption of the atf1 gene resulted in a

very decrease in the cellular triacylglycerol content, but not in the absence of triacylglycerols in cells (Alvarez et al., 2008). Triacylglycerol synthesis of both mutants used in this study was reduced as compared with the wild type as shown in Table 4. The atf1ΩKm mutant showed an approximately twofold increase in the glycogen content in comparison with the wild type, whereas the polyhydroxyalkanoate content in both strains was similar (Table 4). Interestingly, mutant PDM41, which, from gluconate, accumulated 9.1% triacylglycerols compared with 63.8% in the wild type, exhibited an approximately twofold and sevenfold increase in the polyhydroxyalkanoates and glycogen content, respectively, in comparison with the wild type (Table 4). In this study, we analyzed the genetic potential and the physiological ability of diverse species of the genus Rhodococcus to synthesize glycogen from gluconate or glucose as the sole carbon sources.