The glyoxylate shunt is essential for CO2-requiring oligotrophic growth of Rhodococcus erythropolis N9T-4

Abstract

Rhodococcus erythropolis N9T-4 shows extremely oligotrophic growth requiring atmospheric CO2 and forms its colonies on an inorganic basal medium (BM) without any additional carbon source. Screening of a random mutation library constructed by a unique genome deletion method that we established indicated that the aceA, aceB, and pckG genes encoding isocitrate lyase, malate synthase, and phosphoenolpyruvate carboxykinase, respectively, were requisite for survival on BM plates. The aceA- and aceB deletion mutants and the pckG deletion mutant grew well on BM plates containing L-malate and D-glucose, respectively, suggesting that the glyoxylate (GO) shunt and gluconeogenesis are essential for the oligotrophic growth of N9T-4. Interestingly, most of the enzyme activities in the TCA cycle were observed in the cell-free extract of N9T-4, with perhaps the most important exception being α-ketoglutarate dehydrogenase (KGDH) activity. Instead of the KGDH activity, we detected a remarkable level of α-ketoglutarate decarboxylase (KGD) activity, which is the activity exhibited by the E1 component of the KGDH complex in Mycobacterium tuberculosis. The recombinant KGD of N9T-4 catalyzed the decarboxylation of α-ketoglutarate to form succinic semialdehyde (SSA) in a time-dependent manner. Since N9T-4 also showed a detectable SSA dehydrogenase activity, we concluded that N9T-4 possesses a variant TCA cycle, which uses SSA rather than succinyl-CoA. These results suggest that oligotrophic N9T-4 cells utilize the GO shunt to avoid the loss of carbons as CO2 and to conserve CoA units in the TCA cycle.

Expression of an alcohol dehydrogenase gene in a heterotrophic bacterium induces carbon dioxide-dependent high-yield growth under oligotrophic conditions

“…At the very least, we can say that the formaldehyde metabolism seems not to be involved in the adhX-dependent HYGO phenotype, since we could not detect formaldehyde dismutase activity of AdhX (data not shown). Recently, using a random mutagenesis strategy for N9T-4 cells, it was revealed that (i) the glyoxylate shunt and gluconeogenesis are essential for the oligotrophic growth of N9T-4 cells and (ii) N9T-4 has a functional bypass route on the TCA cycle connecting α-ketoglutarate with succinate via succinic semialdehyde [11]. It was suggested that such carbon metabolic pathways are involved in the carbon and energy conservation mechanisms needed for oligotrophy, although the fundamental CO 2 -fixation mechanism in N9T-4 -including the roles of MDH and nFADH -is still unknown.…”

“…This bacterium requires CO 2 for its oligotrophic growth but shows none of the known activities and none of the genes for the key enzymes in microbial CO 2 fixation pathways identified to date, suggesting that it has a unique carbon metabolism and CO 2 fixation system [10]. Using a random mutagenesis strategy for N9T-4 cells, Yano et al [11] revealed that (i) the glyoxylate shunt and gluconeogenesis are essential for the oligotrophic growth of N9T-4 and (ii) N9T-4 has a functional bypass route on the tricarboxylic acid (TCA) cycle connecting α-ketoglutarate with succinate via succinic semialdehyde, suggesting that such carbon metabolic pathways are involved in the carbon and energy conservation mechanisms needed for oligotrophy. However, the core CO 2 -fixation mechanism of this strain under oligotrophic conditions is still unknown.…”

Intracellular accumulation of trehalose and glycogen in an extreme oligotroph, Rhodococcus erythropolis N9T-4

“…Using a random mutagenesis strategy for N9T-4 cells, we revealed that the glyoxylate shunt and gluconeogenesis are essential for its oligotrophic growth. 7) Furthermore, we found that N9T-4 has a functional bypass route on the TCA cycle connecting α-ketoglutarate with succinate via succinic semialdehyde. 7) We suggest that the carbon metabolic pathways described above are involved in the carbon and energy conservation mechanisms needed for oligotrophy.…”

“…7) Furthermore, we found that N9T-4 has a functional bypass route on the TCA cycle connecting α-ketoglutarate with succinate via succinic semialdehyde. 7) We suggest that the carbon metabolic pathways described above are involved in the carbon and energy conservation mechanisms needed for oligotrophy. From the perspective of application, we believe that this oligotrophic feature of N9T-4 would provide good cost performance for the production of useful compounds such as biofuels, since the cells can be cultivated under the very low nutrient conditions described above.…”

“…We previously revealed that phosphoenolpyruvate carboxykinase is essential for oligotrophic growth in N9T-4, suggesting that gluconeogenesis plays an important role in oligotrophic carbon metabolism. 7) The glucose 1-phosphate produced by gluconeogenesis is the branch point of both trehalose and glycogen metabolism. In the OtsA/B pathway, trehalose 6-phosphate is produced from UDPglucose and glucose 1-phosphate by OtsA and then dephosphorylated by OtsB.…”