Natural rubber biosynthesis is catalyzed by a unilamella membrane-bound heterologous complex with multiple different subunits (rubber transferase, RTase). Two substrates and divalent metal cation activators are required, and their concentrations affect biosynthetic rate and polymer molecular weight. Rate, molecular weight, and complex stability are highly sensitive to Mg2+ and Mn2+ concentration, but studies are challenging because methods to control ion concentration may dislodge the elongating rubber polymers from the RTase complexes, halting synthesis and producing low-molecular-weight polymer. Here, programmable chemical actuators (PCAs) are used to electrochemically control rubber biosynthetic rate and subsequent molecular weight in enzymatically active rubber particles purified from Ficus elastica (Indian rubber tree). RTase activity was assayed using 3H-FPP (initiator) and 14C-IPP (monomer). Since only one FPP molecular is needed to initiate a new rubber polymer, the ratio of incorporated 3H-FPP to 14C-IPP was used to calculate the mean molecular weight of newly synthesized polymers. PCAs exchange ions in solution through REDOX reactions which we show control cation concentration without dislodging the elongating rubber polymers from the RTase. PCAs demonstrated highly tunable control over monomer incorporation and molecular weight in both Mg2+ and Mn2+ cations. REDOX cycling PCAs did not irreversibly inhibit the rubber transferase complex, and no indication of enzymatic damage was observed. Precise PCA control of RTase activity may pave the way for rubber eventually to be produced in bioreactors.
Evans et al. (Wed,) studied this question.