Guest Seminar - Harnessing the reconstituted cell-free system to create a sustainable bioeconomy
Reconstituted cell-free protein synthesis systems (e.g., the PURE system) allow the expression of toxic proteins, hetero-oligomeric protein subunits, and polyketides with non-canonical amino acids with high levels of homogeneity. In these systems, an artificial ATP/GTP regeneration system is required to drive protein synthesis. Here, we demonstrate the use of one bi-functional polyphosphate kinase that phosphorylates nucleosides in an exchange reaction from polyphosphate. The optimized single-kinase system produced a final sfGFP concentration (~530 μg/mL), beyond that of the three-kinase system (~400 μg/mL), with a five-fold mRNA translation rate in the first 90 minutes. To further expand the repertoire of PURE systems, a renovated PURE system was developed to express Fe-S proteins that are involved in electron transfer in respiratory and metabolic networks. We established a facile and efficient protocol to express mature [4Fe-4S] proteins in the PURE system. Furthermore, to overcome the O2 lability of [4Fe-4S] Fe-S clusters, an O2-scavenging enzyme cascade was incorporated. These amendments enabled a one-pot synthesis of mature [4Fe-4S] proteins under aerobic conditions, yielding holo-aconitase with a maximum concentration of ~0.15 mg/mL. This renovated system greatly expands the potential of the PURE system, paving the way for the future reconstruction of redox-active synthetic cells and enhanced cell-free biocatalysis. Based on a concept of sustainable bioeconomy, algal-derived polyphosphate is selected as the energy source for ATP regeneration since it can be readily obtained from microalgae biomass generated in the P-rich wastewater. Moreover, we also established a facile biochemical method to decompose soybean pulp, a ubiquitous food waste rich in insoluble proteins, for amino acid harvesting, serving as the building blocks of proteins/peptides in the reconstituted cell-free systems. Altogether, the research efforts abovementioned enable the upcycling of P-rich microalgae sludge and soybean pulp to fuel reconstituted cell-free protein synthesis, creating a new sustainable bioeconomy platform.
Dr. Po-Hsiang Wang received his MS degree from the Graduate Institute of Natural Products, Chung Gung University, Taiwan. Before his Ph.D. studies, his research was focused on the biodegradation of androgens and estrogens under the supervision of Dr. Yin-Ru Chiang at the Biodiversity Research Center, Academia Sinica, Taiwan. Steroid sex hormones including androgens and estrogens are endocrine-disrupting, carcinogenic pollutants often found in the surface water of industrialized countries. He assisted Dr. Chiang to characterize the aerobic and anaerobic estrogen biodegradation pathways. The results of these studies have been written into 10 research articles and have been published in renowned peer-reviewed journals including Proceedings of the National Academy of Sciences USA. Later, he obtained his Ph.D. degree from the Department of Chemical Engineering & Applied Chemistry at University of Toronto, Canada, under the supervision of Prof. Elizabeth A. Edwards. His Ph.D. study was focused on (A) engineering anaerobic microbial consortia for removing chlorinated solvents and aromatic pollutants from groundwater and (B) enzymatic conversion of lignin derivatives into industrial materials and pharmaceuticals. The results of these studies have been published in renowned peer-reviewed journals including Cell Chemical Biology and the ISME Journal. During his Ph.D. study, he witnessed the power and potential of microbial enzymes in sustainable bioeconomy; however, their applications are often hampered by difficulties in enzyme production/purification. Therefore, he later joined Earth-Life Science Institute (ELSI), Tokyo Institute of Technology as a research scientist to study robust enzyme synthesis without cells and stable isotope fractionation enzymology. In 2020, he joined the Graduate Institute of Environmental Engineering, National Central University as an assistant professor, working on upcycling organic solid waste for a sustainable bioeconomy, while he continued his collaboration with ELSI on developing the reconstituted cell-free systems and synthetic cells. Since 2020, he has published 15 peer-reviewed articles and secured ~20M NTD of research funding.