The fermentative metabolism of Escherichia coli has been extensively studied and represents the primary metabolic mode of interest for industrial applications. However, most of this knowledge originates from the study of metabolic components in isolation and is rather incomplete. In contrast to previous studies, we have used a system-level approach that provides an understanding of fermentative metabolism otherwise not achievable by the use of classical biochemical and genetic approaches. To this end, we used in silico and in vivo metabolic flux analysis (MFA) to investigate the fermentative utilization of glucuronate, glucose, and glycerol. These three substrates were chosen because of their differences in the oxidation state of carbon, which in turn largely dictates the distribution of metabolic fluxes in central metabolism and fermentative and biosynthetic pathways.
The fermentative metabolism of glucuronate and glucose was investigated with emphasis on the dissimilation of pyruvate via pyruvate formate-lyase (PFL) and pyruvate dehydrogenase (PDH). Prior to our studies the in vivo activity of PDH in anaerobic cultures was considered negligible and the role of this enzyme in the fermentative metabolism of E. coli remained unknown. However, we found that the growth of a PDH-deficient strain on either glucose or glucuronate was significantly impaired. Metabolite balancing, biosynthetic 13C labeling of proteinogenic amino acids, and isotopomer balancing all indicated a large increase in the flux of the oxidative branch of the pentose phosphate pathway (ox-PPP) in response to the PDH deficiency. Since both the ox-PPP and PDH generate CO2 and reducing equivalents, the aforementioned findings led the hypothesis that the role of PDH is to provide CO2 (metabolism of glucose) and biosynthetic power (metabolism of glucuronate) for cell growth. Biochemical and molecular genetic approaches confirmed the proposed roles of PDH. In vivo and in vitro MFA were also used to investigate the fermentative metabolism of the highly reduced substrate glycerol. This analysis confirmed a previously reported metabolic model in which the synthesis of both ethanol and 1,2-propanediol are required for the anaerobic fermentation of glycerol. The system level analysis predicted an important role for pathways not previously reported to be involved in the fermentative utilization of glycerol. These predictions were validated experimentally, thus further underscoring the importance of using a systems level approach to study microbial metabolism.
Murarka, A., Clomburg, J., and Gonzalez, R. (2010). Metabolic flux analysis of wild-type Escherichia coli and mutants deficient in pyruvate-dissimilating enzymes during fermentative metabolism of glucuronate. Microbiology-SGM 156 (6) (doi 10.1099/mic.0.036251-0).
Murarka, A., Clomburg, J. M., Moran, S., Shanks, J.V., and Gonzalez, R. (2010). Metabolic analysis of wild-type Escherichia coli and a pyruvate dehydrogenase (PDH)-deficient derivative reveals the role of PDH in the fermentative metabolism of glucose. (MS in Review)