\u3ci\u3eIn silico\u3c/i\u3e Driven Metabolic Engineering Towards Enhancing Biofuel and Biochemical Production

The development of a secure and sustainable energy economy is likely to require the production of fuels and commodity chemicals in a renewable manner. There has been renewed interest in biological commodity chemical production recently, in particular focusing on non-edible feedstocks. The fields of metabolic engineering and synthetic biology have arisen in the past 20 years to address the challenge of chemical production from biological feedstocks. Metabolic modeling is a powerful tool for studying the metabolism of an organism and predicting the effects of metabolic engineering strategies. Various techniques have been developed for modeling cellular metabolism, with the underlying principle of mass balance driving the analysis. In this dissertation, two industrially relevant organisms were examined for their potential to produce biofuels. First, Saccharomyces cerevisiae was used to create biodiesel in the form of fatty acid ethyl esters (FAEEs) through expression of a heterologous acyl-transferase enzyme. Several genetic manipulations of lipid metabolic and / or degradation pathways were rationally chosen to enhance FAEE production, and then culture conditions were modified to enhance FAEE production further. The results were used to identify the rate-limiting step in FAEE production, and provide insight to further optimization of FAEE production. Next, Clostridium thermocellum, a cellulolytic thermophile with great potential for consolidated bioprocessing but a weakly understood metabolism, was investigated for enhanced ethanol production. To accomplish the analysis, two models were created for C. thermocellum metabolism. The core metabolic model was used with extensive fermentation data to elucidate kinetic bottlenecks hindering ethanol production. The genome scale metabolic model was constructed and tuned using extensive fermentation data as well, and the refined model was used to investigate complex cellular phenotypes with Flux Balance Analysis. The work presented within provide a platform for continued study of S. cerevisiae and C. thermocellum for the production of valuable biofuels and biochemicals

Metabolic engineering of microorganisms for the overproduction of fatty acids

Fatty acids naturally synthesized in many organisms are promising starting points for the catalytic production of industrial chemicals and diesel-like biofuels. However, bio-production of fatty acids in microbial hosts relies heavily on manipulating tightly regulated fatty acid biosynthetic pathways, thus complicating the engineering for higher yields. With the advent of systems metabolic engineering, we demonstrated an iterative metabolic engineering effort that integrates computationally driven predictions and metabolic flux analysis (MFA) was demonstrated to meet this challenge. With wild type E. coli fluxomic data, the OptForce procedure was employed to suggest genetic manipulations for fatty acid overproduction. In accordance with the OptForce prioritization of interventions, fabZ and acyl-ACP thioesterase were upregulated and fadD was deleted to arrive at a strain that produces 1.70 g/L and 0.14 g fatty acid/g glucose of C14-16 fatty acid in minimal medium. However, OptForce does not infer gene regulation, enzyme inhibition and metabolic toxicity. Along with transcriptomics and metabolomics analysis, we re-deployed OptForce simulation using the redefined flux distribution as constraints to generate predictions for the second generation fatty acid-overproducing strain. MFA identified the up-regulation of the TCA cycle and down-regulation of pentose phosphate pathway under fatty acid overproduction to replenish the need of energy and reducing molecules. The elevation of intracellular metabolite levels in the TCA cycle complemented the flux findings. With re-defined flux boundary of the first generation strain, OptForce suggested the interruption of TCA cycle such as removal of succinate dehydrogenase as the most prioritized genetic intervention to further improve fatty acid production. Meanwhilem, the whole genome transcriptional analysis revealed acid stress response, membrane disruption, colanic acid and biofilm formation during fatty acid production, thus pinpointing the targets for future metabolic engineering effort. These results highlight the benefit of using computational strain design and system metabolic engineering tools in systematically guiding the strain design to produce free fatty acids. Nonetheless, Saccharomyces cerevisiae is another attractive host organism for the production of biochemicals and biofuels. However, S. cerevisiae is very susceptible to octanoic acid toxicity. Transcriptomics analysis revealed membrane stress and intracellular acidification during octanoic acid stress. MFA illustrated the increase of flux in the TCA cycle possibly to facilitate the ATP-binding-cassette transporter activities. Further efforts can focus on improving membrane integrity or explore oleaginious yeasts to enhance the tolerance against fatty acids

Microbial Secondary Metabolites and Biotechnology

Many research teams are working to demonstrate that microorganisms can be our daily partners, due to the great diversity of biochemical transformations and molecules they are able to produce. This Special Issue highlights several facets of the production of microbial metabolites of interest. From the discovery of new strains or new bioactive molecules issued from novel environments, to the increase in their synthesis by traditional or innovative methods, different levels of biotechnological processes are addressed. Combining the new dimensions of "Omics" sciences, such as genomics, transcriptomics or metabolomics, microbial biotechnologies are opening up incredible opportunities for discovering and improving microorganisms and their production