Directed evolution of Gammaproteobacteria engineered with the BHAC

Microbial carbon metabolism often offers multiple routes to generate or assimilate certain compounds. Some of these pathways have evolved to be more efficient in terms of carbon or energy balance than others. Ethylene glycol is a promising next generation feedstock for bioprocesses, since it can be derived from degradation of the ubiquitous plastic PET or by electrochemical conversion of syngas. However, biotechnologically relevant bacteria mainly use a wasteful ethylene glycol assimilation route, the glycerate pathway. In contrast, the recently characterized beta-hydroxyaspartate cycle (BHAC) provides a more efficient way of ethylene glycol utilization. It has so far been unclear whether the growth performance of microorganisms on ethylene glycol can be improved by metabolic engineering with the BHAC. Using growth-coupled selection with increasing demands for pathway flux, we show here that Escherichia coli can assimilate glycolate and glycine via a three-enzyme BHA shunt, and that this engineered pathway is able to sustain complete biomass production from these compounds in evolved strains. Subsequently, we demonstrate the use of the four-enzyme BHAC as a heterologous ethylene glycol assimilation pathway in Pseudomonas putida. Directed evolution on ethylene glycol resulted in an improved strain efficiently growing on ethylene glycol. Taken together, this study establishes that the BHAC can be utilized as plug-and-play module for the metabolic engineering of two important microbial platform organisms. This BioProject contains the NGS data of E. coli and P. putida strains that were engineered with the BHA shunt or BHAC and subsequently subjected to directed evolution on ethylene glycol.