Location: Bioproducts Research
Project Number: 2030-21410-022-002-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2019
End Date: Jul 31, 2023
Natural rubber is synthesized on subcellular vesicles called rubber particles. Purified rubber particles alone contain all necessary factors for rubber production. The enzymes and intermediates comprising synthesis of the natural rubber initiator, farnesyl pyrophosphate (FPP), and monomer, isopentenyl pyrophosphate (IPP), are well documented. However, the specific machinery that constitutes the Rubber Transferase (RuT) biosynthetic complex is still unknown. The University of Nevada, Reno, the ARS Rubber Lab, and others have previously used genomic approaches to identify expressed genes associated with rubber-producing tissues and proteomics to identify proteins associated with rubber particles. These particle-associated, membrane-localized proteins form a complex to biosynthesize NR from soluble monomer then store the insoluble polymer within the rubber particles. Identification of the specific and necessary components comprising the RuT would identify targets for bioengineering in guayule, and further enable natural rubber production in alternative plants (i.e. tobacco), microbes (bacteria, yeast), or synthetic nanoscale enzymatic constructs (i.e. nanolipoprotein particles (NLPs)). We propose a collaborative effort to identify the components of the rubber transferase.
The rubber particle protein targets will be 1) cis-prenyl transferase (CPT), 2) cis-prenyl transferase binding protein (CBP), 3) small rubber particle protein (SRPP), and rubber elongation factor (REF). Protein expression systems for the three target proteins will be developed/improved, and the impact of recombinant proteins, on rubber transferase activity will be evaluated in vitro. Plants transformed in prior years of this agreement will be evaluated for quantitative gene expression and for phenotypes including isoprene production using extraction methods, NMR, and microscopy.