A Multiple Global Transcriptional Regulators Engineering Approach for Improved Free Fatty Acid and PHB production

Abstract

Due to the depletion of petroleum resources, the development of alternative fuels has been actively pursued over the past several decades. Microbial free fatty acids (FFAs) are among the most promising candidates due to their renewable and sustainable productivity. In our previous study, we achieved the highest FFA production (33.64 g/L) through a combinatorial metabolic engineering strategy. However, scaling up the process from flask to bioreactor revealed improper carbon flux change, such as a smaller increase in FFA production compared to biomass and the accumulation of high concentration of acetate. Engineering global transcriptional regulator (gTR), which regulate numerous genes, is generally used strategy for rewiring carbon flux through modulation of metabolic networks. Unlike conventional gTR engineering, which targets a single gTR, we aimed to develop a multiple gTRs engineering strategy targeting seven gTRs to maixmize the diversity of the mutant library with altered metabolic networks. The mutant library was constructed by simultaneously modulating the expression of the seven gTRs through RBS strength control using Multiplex Automated Genome Engineering (MAGE). To screen for high FFAs-producing variants, we first applied a negative selection strategy to eliminate low-producing strains, utilizing tetracycline resistance gene(tetA) encoded FFA biosensor, which expresses tetA in response to FFAs production. Upon cultivation with tetracycline, low-producing variants were selectively removed due to their insufficient tetA expression. Subsequently, positive selection was performed using nile red, which emits red fluorescence upon interaction with non-polar compounds such as FFAs and polyhydroxybutyrate (PHB). Nile red stained the cells depending on FFA production levels, the high red fluorescent emitting variants were selected by fluorescence activated cell sorting(FACS). Following both negative and positive selections, 113 variants were isolated. The selected variants indicated from 0.27% to 66% higher production than the control strain. To investigate the applicability of multiple gTRs engineering to other biochemical products, we applied the same strategy without negative selection to PHB production. Following positive selection, a high PHB-producing variant was selected, which exhibited a 29% increase in PHB contents compared to the control. This study demonstrates that multiple gTRs engineering is a promising strategy for enhancing the production of various biochemicals