Microbial metabolism involves complex, system-level processes implemented via the orchestration of metabolic reactions, gene regulation, and environmental cues. One canonical example of such processes is acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum, during which cells convert carbon sources to organic acids that are later reassimilated to produce solvents as a strategy for cellular survival. The complexity and systems nature of the process have been largely underappreciated, rendering challenges in understanding and optimizing solvent production. Here, we present a system-level computational framework for ABE fermentation that combines metabolic reactions, gene regulation, and environmental cues. We developed the framework by decomposing the entire system into three modules, building each module separately, and then assembling them back into an integrated system. During the model construction, a bottom-up approach was used to link molecular events at the singlecell level into the events at the population level. The integrated model was able to successfully reproduce ABE fermentations of the WT C. acetobutylicum (ATCC 824), as well as its mutants, using data obtained from our own experiments and from literature. Furthermore, the model confers successful predictions of the fermentations with various network perturbations across metabolic, genetic, and environmental aspects. From foundation to applications, the framework advances our understanding of complex clostridial metabolism and physiology and also facilitates the development of systems engineering strategies for the production of advanced biofuels.integrated modeling | ABE fermentation | clostridial physiology | systems biology | metabolic engineering M icrobial metabolism is a means by which a microbe uses nutrients and generates energy to live and reproduce. As one of the most fundamental cellular characteristics, it typically involves complex biochemical processes implemented through the orchestration of metabolic reactions and gene regulation, as well as their interactions with environmental cues (1-3). One representative example of such complex processes is solvent production by Clostridium acetobutylicum, a Gram-positive, anaerobic bacterium that is considered to be one of the most prominent species for industrial biofuel production (4).Solvent [acetone-butanol-ethanol (ABE)] fermentation of the species involves two physiological phases (5-8): During the first phase, the bacterium grows exponentially, and organic acids (acetic acid and butyric acid) are produced with the release of energy-the acidogenic phase. This process causes a dramatic drop in extracellular pH. In response to the substantial decrease of the pH, cells enter the stationary phase, and the organic acids formed are reassimilated to produce solvents including acetone, butanol, and ethanol-the solventogenic phase-thereby helping the bacterium to relieve the stress as a strategy for survival. Solventogenesis is subsequently accompanied by the onset of sporulati...

Synthetic biology is a newly emerged research discipline that focuses on the engineering of novel cellular behaviors and functionalities through the creation of artificial gene circuits. One important class of synthetic circuits currently under active development concerns the programming of bacterial cellular communication and collective population-scale behaviors. Because of the ubiquity of cell-cell interactions within bacterial communities, having an ability of engineering these circuits is vital to programming robust cellular behaviors. Here, we highlight recent advances in communication-based synthetic gene circuits by first discussing natural communication systems and then surveying various functional engineered circuits, including those for population density control, temporal synchronization, spatial organization, and ecosystem formation. We conclude by summarizing recent advances, outlining existing challenges, and discussing potential applications and future opportunities.

BackgroundContact-dependent inhibition (CDI) has been recently revealed as an intriguing but ubiquitous mechanism for bacterial competition in which a species injects toxins into its competitors through direct physical contact for growth suppression. Although the molecular and genetic aspects of CDI systems are being increasingly explored, a quantitative and systematic picture of how CDI systems benefit population competition and hence alter corresponding competition outcomes is not well elucidated.ResultsBy constructing a mathematical model for a population consisting of CDI+ and CDI- species, we have systematically investigated the dynamics and possible outcomes of population competition. In the well-mixed case, we found that the two species are mutually exclusive: Competition always results in extinction for one of the two species, with the winner determined by the tradeoff between the competitive benefit of the CDI+ species and its growth disadvantage from increased metabolic burden. Initial conditions in certain circumstances can also alter the outcome of competition. In the spatial case, in addition to exclusive extinction, coexistence and localized patterns may emerge from population competition. For spatial coexistence, population diffusion is also important in influencing the outcome. Using a set of illustrative examples, we further showed that our results hold true when the competition of the population is extended from one to two dimensional space.ConclusionsWe have revealed that the competition of a population with CDI can produce diverse patterns, including extinction, coexistence, and localized aggregation. The emergence, relative abundance, and characteristic features of these patterns are collectively determined by the competitive benefit of CDI and its growth disadvantage for a given rate of population diffusion. Thus, this study provides a systematic and statistical view of CDI-based bacterial population competition, expanding the spectrum of our knowledge about CDI systems and possibly facilitating new experimental tests for a deeper understanding of bacterial interactions.