(4em) Engineering Tailor-Made Microbial Cell Factories With Metabolic Engineering and Synthetic Biology

Global energy demand, rising petroleum prices and environmental concerns have stimulated increased efforts to develop more sustainable and cost-effective chemicals that are derived from renewable resources. Metabolic engineering entails targeted manipulation of biosynthetic pathways to maximize yields of desired products. As an emerging discipline, synthetic biology is becoming increasingly important to design, construct and optimize metabolic pathways leading to desired overproduction phenotypes. Exploitation of the diverse microbial pathways based on metabolic engineering and synthetic biology frameworks provides a promising solution to synthesis of fuel and pharmaceutical molecules that are previously produced from the petroleum-based resources. 

Harnessing cell factories for producing biofuel and pharmaceutical molecules has stimulated efforts to develop novel synthetic biology tools customized for modular pathway engineering and optimization. Here we present a versatile gene assembly platform ePathBricks supporting the modular assembly of multigene metabolic pathways and combinatorial generation of pathway diversities. Based on this gene assembly platform, we have combinatorially optimized the transcriptional levels of three genetic modules in the fatty acid pathway and identified conditions that balance the supply of acetyl-CoA and consumption of malonyl-CoA/ACP. Refining protein translation efficiency by customizing ribosome binding sites for both the upstream acetyl coenzyme A formation and the downstream fatty acid synthase module has enabled further production improvement. Fed-batch cultivation of the engineered strain led to a final fatty acid production of 8.6 g/L. The modular engineering strategies demonstrate a generalized approach to engineering cell factories for valuable metabolites production.

As heterologous pathways become larger and more complicated, it becomes increasingly difficult to optimize them with static regulatory control. An ideal approach would be to use dynamic regulatory networks to control gene expression so that the host cells can adjust their metabolic functions when the environmental condition changes. Here we report engineering both the positive and negative feedback controls for dynamic tuning of metabolic flux in E. coli. We have identified a dual transcriptional regulator which can act either as an activator or a repressor for two different promoters. The level of activation or repression is dependent on the level of intracellular malonyl-CoA. As a proof of concept, we demonstrated that the expression of two reporter proteins can be exclusively switched between the on and off state. By engineering this synthetic malonyl-CoA controller, we envision that both the malonyl-CoA source pathway and the malonyl-CoA sink pathway can be dynamically modulated so that carbon flux can be efficiently redirected to synthesize our target compounds. Implementation of this dynamic control will maintain the intracellular malonyl-CoA at the optimal level and improve both the productivity and yield of value-added metabolites in E. coli.