Metabolic flux analysis and metabolomics of Methylotrophs

Abstract

Methylotrophs are a group of microbes that use C1 compounds as the sole carbon and energy source, which are of interest to be used as platforms for bioconversion of C1 substrates to valuable chemicals and fuels. Methylobacterium extorquens AM1 is the best studied model organism for methylotrophs, and it uses the serine cycle for assimilation of C1 units. Methylomicrobium buryatense 5GB1 is a Type I methanotroph that uses the ribulose monophosphate (RuMP) pathway for assimilation, and it is also an industrially promising candidate for bio-conversion of methane into valuable chemicals. Metabolic flux analysis and metabolomics are two important approaches to investigate cell behavior at the level of metabolites and fluxes, which are integrative consequences of gene expression. These approaches will provide critical information for a rational strain design process in metabolic engineering iterations. In the first part of the thesis, the metabolic flux analysis approach was successfully applied to M. exorquens AM1 to investigate the metabolic network response to the trade-off between growth rate and biomass yield. Then the method was tailored to apply to M. buryatense 5GB1 for flux elucidation in core metabolism under methane growth to provide baseline information for better prediction of performance. At the end, the combined approaches were used to investigate the core metabolism of M. buryatense 5GB1 response to two substrates, methane and methanol. This work has resulted in major new insights into methylotrophic metabolism. For the serine cycle methylotroph, it was demonstrated that the C3/C4 interconversion reactions, previously thought to be side reactions of little relevance to core metabolism, are critical to the ultimate values of growth rate and yield. This new insight now provides targets for strain engineering towards either maximum growth rate, or maximum yield. For the RuMP cycle methanotroph, it was determined first, that contrary to decades of assumptions in the literature, the Type I methanotroph studied here operates a complete, oxidative TCA cycle. That finding changes the entire metabolic network balance, especially with regards to NADH generation, and this knowledge is essential for generating accurate predictive metabolic models. Second, it was determined that the partial serine cycle present in Type I methanotrophs does not play a significant role during growth on methane. However, it was discovered that it does play a role during growth on methanol. Growth on methanol was discovered to involve a substantially rearranged metabolic network compared to methane, in complete contrast to literature expectations. In this case, the TCA cycle is broken, NADH is generated mainly from formate oxidation, and flux shifts from the Embden-Meyer-Parnas pathway to the Entner-Douderoff pathway. In addition, substantial carbon is redirected to synthesis of glycogen and excretion of formate. We have concluded that both metabolic flux analysis and metabolomics are extremely valuable tools in understanding the metabolic network of methylotrophs, and their use has corrected metabolic misunderstanding that has been in the literature for decades. These methods must be tailored for the C1 assimilation system, and the results presented in this thesis demonstrate an approach for the two main types of methylotrophs. These new techniques are now ready to use for investigating other methylotrophs in the future, and to serve as important tools for future metabolic engineering of methylotrophs for biotechnology applications.