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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Bioproducts Research » Research » Research Project #438153

Research Project: Domestic Production of Natural Rubber and Resins

Location: Bioproducts Research

2021 Annual Report

Objective 1: Genetically modify guayule for improved commercial rubber yields. Sub-objective 1A: Over-express enzymes and proteins involved in natural rubber synthesis and accumulation in guayule, including components of the rubber transferase complex. Sub-objective 1B: Increase natural rubber yield in guayule through controlled expression of transcription factors related to plant stress response. Sub-objective 1C. Apply CRISPR/Cas9 technology to improve rubber yield in guayule. Objective 2: Develop environmentally sustainable, commercially viable processes for fractionation and modification of guayule resin co-product into higher value products. Sub-objective 2A: Enrich the high-value terpene fraction of guayule resin via environmentally-friendly extraction and filtration processes. Sub-objective 2B: Chemically modify guayule resin components to enhance their market value. Objective 3: Enable marketable natural rubber composites incorporating food waste and byproducts. Sub-objective 3A: Evaluate the use of heat-treated agricultural residues as bio-based reinforcing fillers in natural rubber compounds. Sub-objective 3B: Assess the feasibility of using meat-processing byproducts and other agricultural residues as bio-based rubber compound additives. Sub-objective 3C: Develop bio-based antioxidants for stabilization of natural rubber and resin.

Sub-objective 1A: Over-express enzymes and proteins involved in natural rubber synthesis –Co-expression of genes associated with the Rubber Transferase and the MVA pathway will provide targets for preparation of a vector using GAANTRY technology that can insert multiple transgenes simultaneously into plants. We will generate at least 10 independent transformed guayule lines using Agrobacterium-mediated transformation. Transgene insertion will be confirmed, and genotype and phenotype analysis performed. Sub-objective 1B: Controlled expression of transcription factors – We will construct transformation vectors, overexpressing transcription factors. Guayule transformation, transgene confirmation, transgene expression, and other phenotypes including rubber content will be determined. Sub-objective 1C. Apply CRISPR/Cas9 technology to improve rubber yield. – Using plant codon optimized synthetic Cas9 nuclease, to reduce off-target effects, Agrobacterium-mediated transformation of guayule will be performed to target the reporter gene GUS (ß-glucuronidase). Once the Cas9 is proven to be functional in guayule, we will target the AOS1 for gene editing. Transgenic lines will be further evaluated by standard methods. Sub-objective 2A: Enrich the high-value terpene fraction of guayule resin –The utility of filtration technology for fractionation of guayule resins will be evaluated, with a focus on green solvents and low temperature processing. Sub-objective 2B: Chemically modify guayule resin components – We will determine if saponification and methanolysis of complex guayule resin mixtures can/should be applied as a fractionation strategy, as a means to valuable products. Sub-objective 3A: Evaluate the use of heat-treated agricultural residues – We will conduct torrefaction of the guayule bagasse and other crop residues. Natural rubber composites will be prepared and characterized using with the torrefied biobased fillers compared to conventionally used fillers. Sub-objective 3B: Assess the feasibility of using agricultural residues – We will focus on protein sources from agricultural operations, initially meat by-products. Materials will be characterized for chemical and physical properties, and protein stability. Model natural rubber compounds will be formulated in which commercial meat by-products will be added to, or used in place of, synthetic anti-degradants and vulcanization aids, and the impact on compound performance assessed. Sub-objective 3C: Develop bio-based antioxidants – We will determine the efficacy of in vivo stabilization of guayule rubber by tocopherols, and ex vivo use of biobased antioxidants for guayule extraction processing and compounding.

Progress Report
In support of Sub-objective 1A, natural rubber is synthesized by at least a cis-prenyl transferase (CPT) and a cis-prenyl transferase binding-protein (CBP). A (guayule homologue of) the small rubber particle (RP) protein (SRPP) may function in stabilization and coagulation of rubber molecules in RP. We constructed a conventional transformation vector carrying three guayule genes, CPT3, CBP and SRPP to evaluate their effect for engineering rubber production in guayule. Progress was also made in Sub-objective 1B. Rubber synthesis in guayule is highly upregulated by cold, drought, wounding, and other stresses in its natural environment or under controlled conditions. Transcription factors (TFs) are regulatory proteins induced by signals such as environmental stresses. A well-studied TF gene family, DREBs (dehydration responsive element binding proteins) regulate many stress-responsive genes, and a guayule DREB1D (PaDREB1D) was highly induced in cold-treated stem tissue where active rubber synthesis and accumulation occurred. ARS scientists in Albany, California, have successfully cloned and constructed PaDREB1D in a binary vector for studying the function of PaDREB1D. The success of this project allows the ability of transgenic guayule lines overexpressing the PaDREB1D to be engineered for increased rubber synthesis in the absence of a cold signal. In support of Sub-objective 1C, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology was applied to improve rubber yield in guayule, and progress was made toward designing constructs for gene editing of guayule. In collaboration with the Joint Genome Institute, a draft genomic DNA assembly was prepared, providing some of the critical plant DNA sequences needed for precise editing. In subordinate project 2030-21410-022-01R, Sustainable Bioeconomy for Arid Regions, guayule plants were transformed to downregulate genes that promote flowering. It is possible that plants with fewer flowers may produce more rubber, or have other desirable traits. Evaluation of the plants is continuing, Progress was also made on Sub-objective 3A. Natural rubber composites were prepared using torrefied biobased fillers as full or partial replacements for carbon black, the conventional petroleum-based filler. Moderate levels of biobased fillers could be used without compromising the material performance. To address Sub-objective 3B, a series of experimental compounds were prepared. The antioxidant performance of meat by-product extracts was evaluated.

1. Genomic resources for guayule. Modern breeding techniques for the natural rubber-producing crop, Parthenium argentatum (guayule), require background information characterizing the plant DNA. In collaboration with the Joint Genome Institute, a draft guayule DNA assembly was successfully developed from a vigorous hybrid line used in commercial crop development. In addition, ARS researchers in Albany, California, measured the differences in gene expression between high and low rubber-producing field plants by RNA sequencing. The resulting RNA assembly, or transcriptome, highlights which genes are expressed driving production of rubber, resins, and carbohydrates in guayule. The two new databases featuring the DNA and RNA assembled sequences are now available to researchers and commercial developers worldwide, linked and hosted by ARS GrainGenes servers.

Review Publications
Torres, L.F., McCaffrey, Z., Washington, W., Williams, T.G., Wood, D.F., Orts, W.J., McMahan, C.M. 2021. Torrefied agro-industrial residue as filler in natural rubber compounds. Journal of Applied Polymer Science. 138(28). Article e50684.
Cornish, K., Dacosta, B., McMahan, C.M. 2020. Temporal analysis of natural rubber transferases reveals intrinsic distinctions for in vitro synthesis in two rubber-producing species. Current Topics in Biochemical Research. 21:45-58.
Chen, G.Q., Kim, W., Johnson, K., Park, M., Lee, K., Kim, H. 2021. Transcriptome analysis and identification of lipid genes in Physaria lindheimeri, a genetic resource for hydroxy fatty acids in seed oil. International Journal of Molecular Sciences. 22(2). Article 514.
Chen, G.Q., Johnson, K., Nazarenus, T.J., Ponciano, G.P., Morales, E., Cahoon, E.B. 2021. Genetic engineering of lesquerella with increased ricinoleic acid content in seed oil. Plants. 10(6). Article 1093.