Chemo-optogenetics-based Isobutanol production via Metabolic Engineering in Zymomonas mobilis

Objective

Competition for metabolic/cellular resources between biomass formation (“growth”) and product synthesis (“production”) is a major bottleneck in microbial bioproduction, leading to reduced yields. Synthetic genetic circuits have aimed to decouple growth and production phases to improve yields, but current approaches based on chemical inducers or optogenetics remain inadequate for achieving efficient phase separation. CHIME-Z introduces chemo-optogenetics, a technology that combines small molecules and light to control gene expression, offering precise control, modularity, rapid reversibility, and orthogonality, thereby overcoming the limitations of existing tools. By applying chemo-optogenetics to engineer Zymomonas mobilis for isobutanol (a biofuel) production, CHIME-Z will deliver proof-of-concept for chemo-optogenetics-driven circuit design and metabolic engineering, which will find application in bioproduction. The project pursues three objectives. RO1: To develop chemo-optogenetic expression systems in E. coli and Z. mobilis, by screening libraries of DNA-binding domains and activation domains to identify combinations with minimal leakiness and high transcriptional activation. RO2: To model and construct a chemo-optogenetic circuit in Zymomonas mobilis to achieve separation of growth and isobutanol production phases, using Python-based computational modeling to predict system behavior, and genome engineering to implement a NIMPLY gate-based circuit that separates growth and isobutanol production phases. RO3: To optimize fermentation and scale-up parameters for isobutanol production from glucose and pretreated lignocellulosic biomass using the chemo-optogenetically engineered Zymomonas mobilis, through lab-scale process optimization, metabolomics-based validation, and subsequent bioreactor-based scale-up. Notably, CHIME-Z supports European Green Deal and the UNSDGs by converting lignocellulosic biomass into isobutanol through sustainable bioproduction.