(23g) Elucidating the Metabolism of Rhodococcus Opacus PD630 Via Parallel 13C-Metabolite Fingerprinting

Authors: 
Hollinshead, W. D., Washington University in St. Louis
Henson, W. R., Washington University in St. Louis
Abernathy, M., Washington University in St. Louis
Tang, Y., Washington University in St. Louis
Moon, T. S., Washington University in St. Louis

Rhodococcus opacus PD630 is a promising microbial host for lignocellulosic biofuel production, due to its high tolerance to aromatic compounds and accumulation of triacylglycerol, a biodiesel precursor. To quickly elucidate the metabolism of this non-model organism, several parallel 13C-fingerprinting experiments were designed to probe multiple pathways in Rhodococcus opacus PD630. Our 13C-fingerprinting approach resulted in several findings: 1) PD630 strongly prefers to utilize the Entner-Doudoroff (ED) pathway over Emden-Meyerhof (EMP) and the pentose phosphate pathway, 2) highly active malic enzyme and anaplerotic pathways, 3) uptake & utilization of exogenous amino acids, and 4) no carbon catabolite repression detected on growth with glucose and phenol. In addition, our results indicate that PD630 uses the ED pathway and gluconeogenesis concurrently for co-utilization of glucose and phenol. Gluconeogenesis is typically inhibited when glucose is catabolized through the EMP pathway, thus PD630’s preference for the ED pathway could be a key strategy to avoid this inhibition under growth with multiple carbon sources. These findings have revealed several interesting features of PD630 metabolism as well as demonstrated the potential of parallel 13C-metabolite fingerprinting.  This method is a qualitative alternative to 13C-Metabolic Flux Analysis that significantly reduces the time and expense for analyzing cell metabolisms. For labs that need quick and inexpensive characterization of non-model species, 13C-fingerprinting can be the most ideal approach. To promote this technique, we have developed a prototype 13C-fingerprinting toolkit that consist of 13C-tracers, an 8-well block that can be used as to cultivate small volumes (~4mL) of culture, a gassing manifold and instructions to enable other microbiology labs to easily adapt this tool into their characterization process of cell metabolisms.