Green method of synthesizing <sc>l</sc>-malate from <sc>d</sc>-glucose via CO2 fixation using an ATP-free in vitro synthetic enzymatic biosystem

In conjunction with the pressing global issue of climate change and the associated concern over global warming, increasing interest has emerged in the exploration of carbon dioxide (CO2) as a resource for the generation of a diverse array of products intended to serve societal needs. This study presents the development of an ATP-free and NAD-balanced in vitro synthetic enzymatic biosystem (ivSEB), which comprises only five cascade thermophilic enzymes, designed for the synthesis of l-malate through CO2 fixation powered by the utilization of d-glucose as a substrate. This designed ivSEB yields two moles of l-malate from one mole of d-glucose and two moles of CO2. Through meticulous refinement of reaction conditions and enzyme loading amounts, this ivSEB has demonstrated its capability to produce 6.85 mM of l-malate via CO2 fixation from an initial 5 mM of d-glucose with a molar product yield of 68.5%, and 2.45 mM of l-lactate as a byproduct. In the pursuit of assessing the industrial feasibility of this ivSEB, the study further subjected the system to the utilization of a high concentration (45.70 mM) of d-glucose. Although this endeavor necessitates additional optimization for enhanced efficiency, the present findings herald the emergence of an alternative avenue for the sustainable production of l-malate through CO2 fixation, thus bearing substantial promise for addressing ecological and industrial imperatives.