Title:

Characterizing and alleviating substrate limitations for improved in vitro ribosome construction

Authors:

Yi Liu, Brian R. Fritz, Mark J. Anderson, Jennifer A. Schoborg, Michael C. Jewett

Abstract:

We recently reported the development of an integrated synthesis, assembly, and translation (iSAT) method for in vitro construction of Escherichia coli ribosomes. iSAT allows simultaneous ribosomal RNA synthesis, ribosome assembly, and reporter protein expression as a measure of ribosome activity. Here we explore causes of iSAT reaction termination to improve efficiency and yields. We discovered that phosphoenolpyruvate (PEP), the secondary energy substrate, and nucleoside triphosphates (NTPs) were rapidly degraded during iSAT reactions. In turn, we observed a significant drop in the energy charge and termination of protein synthesis. Further, we identified that the accumulation of inorganic phosphate is inhibitory to iSAT. Fed-batch replenishment of PEP and magnesium glutamate (to offset the inhibitory effects of accumulating phosphate by repeated additions of PEP) prior to energy depletion prolonged the reaction duration 2-fold and increased yields 75%. By adopting a semi-continuous method, where passive diffusion enables substrate replenishment and byproduct removal, we prolonged iSAT reaction duration 5-fold and increased superfolder green
fluorescent protein yield 7-fold to 7.5 ± 0.7 µmol L-1. This protein yield is the highest ever
reported for iSAT reactions. Our results underscore the critical role energy substrates play in iSAT and highlight the importance of understanding metabolic processes that influence substrate depletion for cell-free synthetic biology.