Electronic activation and inhibition of natural rubber biosynthesis catalyzed by a complex heterologous membrane-bound complex

Authors: EVANS, J. PARKER - The Ohio State University; SUNDARENSAN, VISHNU - The Ohio State University; Cornish, Katrina

Submitted to: Processes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/19/2026
Publication Date: 1/20/2026
Citation: Evans, J.P., Sundarensan, V.B., Cornish, K. 2026. Electronic activation and inhibition of natural rubber biosynthesis catalyzed by a complex heterologous membrane-bound complex. Processes. 14(2):374. https://doi.org/10.3390/pr14020374.
DOI: https://doi.org/10.3390/pr14020374

Interpretive Summary: A programmable chemical actuator (PCA) has been developed and proven that it can be used to regulate the catalytic activity of the enzyme complex, rubber transferase, from all the way on to all the way off, without causing the release of the elongating rubber polymer from the active site. The PCA regulates the concentration of divalent cation activators without changing the volume of reaction solution. The actuation of metal ions did not influence the initiation of new rubber molecules in Mg2+-activated or Mn2+-activated reactions. PCAs demonstrated highly tunable control over monomer incorporation and molecular weight in both cations. It is anticipated that PCAs will facilitate additional insight into the functioning of metal cations in rubber biosynthesis and other metal-activated enzymes or complexes.

Technical Abstract: High molecular weight natural rubber (cis-1,4-polyisoprene) is a nonfungible commodity possessing unique physical properties. Rubber biosynthesis is catalyzed by a unilamella membrane-bound heterologous complex with multiple different subunits (rubber transferase, RTase). Two substrates (an allylic pyrophosphate initiator and the nonallylic isopentenyl pyrophosphate monomer) and divalent metal cations (C2+) are required for rubber biosynthesis, and their concentrations affect biosynthetic rate and polymer molecular weight. Rate, molecular weight and complex stability are highly sensitive to C2+ concentration, but studies are challenging because methods to control ion concentration may dislodge the elongating rubber particles from the RTase complexes, halting synthesis and producing low molecular weight polymer. This study shows the use of programmable chemical actuators to electrochemically control rubber biosynthetic rate and subsequent molecular weight in enzymatically active rubber particles purified form Ficus elastica (Indian rubber tree). PCAs exchange ions in solution through REDOX reactions which showed control cation concentration without dislodging the elongating rubber polymers from the RTase. The actuation of metal ions did not influence the initiation of new rubber molecules in Mg2+-activated or Mn2+-activated reactions. PCAs demonstrated highly tunable control over monomer incorporation and molecular weight in both cations. In general, Mg2+-activated reactions incorporated more IPP than Mn2+-activated reactions, but when reduction pulses were started 3 hours into the reaction, IPP incorporation did not differ significantly between Mg2+-activated reactions (1.3 ' 0.9 µmol*gdw-1*4hr-1) and Mn2+-activated reactions (1.3 ' 0.4 µmol*gdw-1*4hr-1). Mg2+-activated reactions produced a maximum molecular weight of 37.7 ' 3.1 Mg*mol-1 compared to 13.8 ' 1.1 Mg*mol-1 for Mn2+-activated reactions, both much higher molecular weights than synthesized in vivo. REDOX cycling PCAs did not irreversibly inhibit the rubber transferase complex, and no indication of enzymatic damage was observed. The time at [C2+]MAX correlated to molecular weight. Results demonstrate that PCAs can change ion concentration and regulate the biosynthetic rate and the molecular weight of natural rubber produced by a membrane-bound protein complex in vitro. It is anticipated that PCAs will facilitate additional insight into the functioning of metal cations in rubber biosynthesis and other metal-activated enzymes or complexes.