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Abstract
Engineering synthetic pathways in microbial hosts can afford the bioproduction of industrially-important fuels, chemicals, or medicines from renewable carbon sources. However, the intricate biochemical network and metabolic burden within microbial hosts consistently challenge the performance of these pathways. Unlike endogenous pathways, which benefit from precise regulation networks, introducing heterologous pathways into microbial hosts often leads to unbalanced enzyme expression and carbon flux distribution, hindering the construction of highly efficient microbial biosynthesis systems. Herein, we developed regulation tools as well as constructed autonomous regulation networks to dynamically control the heterologous pathways, engineered the endogenous metabolic network to enhance the efficiency of foreign pathways, establishing efficient microbial cell factories. First, we engineered the p-coumaric acid biosensor system in Escherichia coli (E. coli) through promoter engineering and protein rational design for versatile dynamic performance. Then, the engineered biosensor system was then leveraged to develop an Autonomous Cascaded Artificial Dynamic (AutoCAD) regulation system, mimicking natural regulation in cells to automatically fine-tune enzyme expression in naringenin pathway. The AutoCAD regulation system, consisting of intermediate-based feedforward and product-based feedback control genetic circuits, resulted in a 16.5-fold increase in naringenin titer compared with the static control. Next, to expand the usability of dynamic regulation tools, we established tunable genetic logic gates with versatile dynamic performance by varying regulatory parameters. Finally, using mevalonate-based isoprenol pathway as a proof-of-principle demonstration, the CRISPR/dCas9 interference (CRISPRi) screening was applied to identify endogenous gene targets intervening or promoting isoprenol biosynthesis in E. coli for improved isoprenol titer. Our work demonstrated the design of dynamic regulation tools to construct autonomous regulation systems, as well as the characterization of new isoprenol biosynthesis-related genes in E. coli using the CRISPRi strategy.