Liu Jianming,Zhang Han,Xu Yingying,Meng Hao,Zeng An-Ping
The use of gaseous and air-captured CO 2 for technical biosynthesis is highly desired, but elusive so far due to several obstacles including high energy (ATP, NADPH) demand, low thermodynamic driving force and limited biosynthesis rate. Here, we present an ATP and NAD(P)H-free chemoenzymatic system for amino acid and pyruvate biosynthesis by coupling methanol with CO 2 . It relies on a re-engineered glycine cleavage system with the NAD(P)H-dependent L protein replaced by biocompatible chemical reduction of protein H with dithiothreitol. The latter provides a higher thermodynamic driving force, determines the reaction direction, and avoids protein polymerization of the rate-limiting enzyme carboxylase. Engineering of H protein to effectively release the lipoamide arm from a protected state further enhanced the system performance, achieving the synthesis of glycine, serine and pyruvate at g/L level from methanol and air-captured CO 2 . This work opens up the door for biosynthesis of amino acids and derived products from air.
Lin Fan,Shangshang Sun,Zhidan Zhang,Yanmei Qin,Peter Ruhdal Jensen,Chun You
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.
Wei Xinlei,Yang Xue,Hu Congcong,Li Qiangzi,Liu Qianqian,Wu Yue,Xie Leipeng,Ning Xiao,Li Fei,Cai Tao,Zhu Zhiguang,Zhang Yi-Heng P. Job,Zhang Yanfei,Chen Xuejun,You Chun
In vitro biotransformation (ivBT) facilitated by in vitro synthetic enzymatic biosystems (ivSEBs) has emerged as a highly promising biosynthetic platform. Several ivSEBs have been constructed to produce poly-3-hydroxybutyrate (PHB) via acetyl-coenzyme A (acetyl-CoA). However, some systems are hindered by their reliance on costly ATP, limiting their practicality. This study presents the design of an ATP-free ivSEB for one-pot PHB biosynthesis via acetyl-CoA utilizing starch-derived maltodextrin as the sole substrate. Stoichiometric analysis indicates this ivSEB can self-maintain NADP + /NADPH balance and achieve a theoretical molar yield of 133.3%. Leveraging simple one-pot reactions, our ivSEBs achieved a near-theoretical molar yield of 125.5%, the highest PHB titer (208.3 mM, approximately 17.9 g/L) and the fastest PHB production rate (9.4 mM/h, approximately 0.8 g/L/h) among all the reported ivSEBs to date, and demonstrated easy scalability. This study unveils the promising potential of ivBT for the industrial-scale production of PHB and other acetyl-CoA-derived chemicals from starch.
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