Location: Bioproducts Research
Project Number: 2030-21410-022-02-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2019
End Date: Mar 31, 2021
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), and 3) small rubber particle protein (SRPP). Our hypothesis is that these three proteins form the minimum required complex for natural rubber biosynthesis. We propose a collaborative effort to test this hypothesis. 1) Rubber transferase has CPT activity, catalyzing a condensation reaction of IPP units onto an FPP initiator in the cis conformation to make long chain rubber molecules. Various CPT genes have been studied in rubber-producing plants; overexpression of Hevea and Tk dandelion CPT in guayule (single gene) did not increase rubber production in guayule (McMahan et al., unpublished), suggesting it may be necessary but is not sufficient. 2) The cis-prenyl transferase binding protein (CBP) associated with rubber particles, only recently identified, was shown to associate with CPT and impact rubber biosynthesis in lettuce (Qu et al. 2015) and dandelion (Epping et al., 2015). 3) The small rubber particle protein (SRPP) is present in Hevea, guayule, and dandelion rubber particles. Functional genomics studies at UNR revealed a dramatic effect on natural rubber content and quality (Collins et al., 2012) in dandelion. We will develop protein expression systems for the three target proteins, and evaluate the impact of recombinant protein on rubber transferase activity in vitro. Our in vivo approach will be to prepare a stacked gene expression construct incorporating all 3 target genes and transform tobacco and/or Arabidopsis with the three proteins. The plants will be analyzed for evidence of natural rubber biosynthesis using extraction methods, NMR, and microscopy.