A comprehensive review on integration of cellular metabolic engineering and cell-free systems for microbial platforms

In response to the demand for environment friendly synthesis of chemical feedstocks, two disciplines have emerged: cellular metabolic engineering (CME) and cell-free metabolic engineering (CFME). Cell free systems largely replicate cellular pathways in vitro to bypass the need of live cells to achieve greater control and process simplicity. However, not all cellular biochemical aspects can be replicated in vitro, as some are not hardwired but crucial modulators of biochemical processes. These include metabolic states defined by ensemble of small molecules that influence proteostasis, catalytic activity of enzymes, and redox power, influencing cellular anabolic and catabolic decisions. Despite the advancement of molecular biology techniques engineering such control systems remains largely a challenge for cellular metabolic engineering. This review thoroughly examines these limitations in both fields and explores the potential for implementing non-hardwired control systems in cell-free metabolic engineering, either independently or in combination with cellular metabolic engineering. Further, the integration of chemistry and machine learning models is considered, with a focus on how their combined strengths can be leveraged to develop novel synthetic schemes.