Optimality of extracellular enzyme production and activity in dynamic flux balance modeling

In microbial communities, many vital metabolic functions, including the degradation of cellulose, proteins and other complex macromolecules, are carried out by costly, extracellularly secreted enzymes. While significant effort has been dedicated to analyzing genome-scale metabolic networks for individual microbes and communities, little is known about the interplay between global allocation of metabolic resources in the cell and extracellular enzyme secretion and activity. Here we introduce a method for modeling the secretion and catalytic functions of extracellular enzymes using dynamic flux balance analysis. This new addition, implemented within COMETS (Computation Of Microbial Ecosystems in Time and Space), simulates the costly production and secretion of enzymes and their diffusion and activity throughout the environment, independent of the producing organism. After tuning our model based on data for a Saccharomyces cerevisiae strain engineered to produce exogenous cellulases, we explored the dynamics of the system at different cellulose concentrations and enzyme production rates. We found that there are distinct rates of constitutive enzyme secretion which maximize either growth rate or biomass yield. These optimal rates are strongly dependent on enzyme kinetic properties and environmental conditions, including the amount of cellulose substrate available. Our framework will facilitate the development of more realistic simulations of microbial community dynamics within environments rich in complex macromolecules, with applications in the study of soil and plant-associated ecosystems, and other natural and engineered microbiomes.https://www.biorxiv.org/content/10.1101/2021.11.01.466736v1First author draf

Metabolic Engineering of Serratia marcescens

The potential value of the chitin biomass (e.g. food waste) is recently considered being ignored by landfill. Chitin can be a potential cheap carbon source for converting into value-added chemicals by microorganisms. Serratia marcescens is a chitinolytic bacterium that harbors endogenous chitinase systems. With goals of characterzing S. marcescens chitinolytic capabilities and applying S. marcescens to chemical production from chitin, my dissertation main content includes five chapters: 1) Chapter 1 highlights background information of chitin source, S. marcescens and potential metabolic engineering targets using chitin as a substrate; 2) Chapter 2 demonstrates that ChiR is a key regulator in regulating 9 chitinase-related genes in S. marcescens Db11 and manipulation of chiR can be a useful and efficient genetic target to enhance chitin utilization; 3) Chapter 3 reports the production of N-acetylneuraminic acid (Neu5Ac) from chitin by a bottom-up approach of engineering the nonconventional chitinolytic bacterium, Serratia marcescens, including native constitutive promoter characterization and transcriptional and translational pathway balancing; 4) Chapter 4 describes improvement of S. marcescens chitinolytic capability by an adaptive evolution approach; 5) Chapter 5 elucidates S. marcescens intracellular metabolite profile using a constraint-based genome-scale metabolic model (iSR929) based on genomic annotation of S. marcescens Db11. Overall, the dissertation work is the first report of demonstrating the concept of chitin-based CBP using S. marcescens and the computational model and genetic molecular tools developed in this dissertation are valuable but not limited to design-build-test of S. marcescens for contributing to the field of biological science and metabolic engineering applications