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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Bioproducts Research » Research » Research Project #428635

Research Project: Domestic Production of Natural Rubber and Industrial Seed Oils

Location: Bioproducts Research

2016 Annual Report

Objective 1: Develop varieties and commercially-viable post-harvest practices that maximize the market value of U.S.-produced guayule and Kazak dandelion. Sub-objective 1A: Genetically modify guayule for improved rubber yields. Sub-objective 1B: Identify biochemical regulation of enzymes in the isoprenoid pathways that will lead to increased yield of rubber. Sub-objective 1C: Develop an effective protocol for highly efficient genetic transformation of Kazak dandelion. Objective 2: Enable new commercially-viable processes for expanding the manufacture of industrial products based on guayule and Kazak dandelion. Subobjective 2A: Modify the protein components of guayule rubber to increase its market value. Subobjective 2B: Elucidate the roles of lipid in the biosynthesis of rubber and on the mechanical properties of dry rubber. Subobjective 2C: Develop novel processes to fractionate crude guayule resin into value-added components. Objective 3: Enable the commercial production of hydroxy fatty acids from oilseed crops already grown in the U.S. Sub-objective 3A. Develop knowledge of HFA synthesis in lesquerella to accelerate development of HFA-producing domestic oilseed crops. Sub-objective 3B. Develop HFA-producing camelina.

Subobjective 1A: Genetically modify guayule for improved rubber yields- we will engineer guayule for over-expression of isoprenoid genes and/or down-regulation of carbon-competing pathways to increase rubber content. Independently transformed lines and controls will be analyzed: gene expression, rubber/resin content, rubber transferase activity, inulin, squalene, lipids and TAGs. We will apply the knowledge with that developed in 1) regulation of biochemical pathways 2) storage of hydrocarbons in plants 3) guayule genomics tools. Sub-objective 1B: Identify biochemical regulation of enzymes- we will use yeast, S. cerevisiae, a single-celled eukaryote that responds to IPP by producing ergosterol, as a model to study HMGR, IDI, and FPP synthase impact on ergosterol. Results will be translated to tobacco to evaluate post-translational modifications in a model plant. Sub-objective 1C. Develop genetic transformation of Kazak dandelion- a robust transformation system will be developed by 1) screening diploid seedlings to identify highly regenerating lines 2) optimizing culture conditions 3) evaluating explant sources (hypocotyl, stem, leaf petiole), and 4) assessing seed production. Self-compatible lines will facilitate genetic studies on relationships among transgene dosage, gene expression level, and rubber content. Subobjective 2A- We will attempt to elucidate the roles of naturally-occurring proteins in Hevea rubber particles. This knowledge will inform modification of the chemical, physical, and/or biological properties of guayule and Kazak dandelion rubbers to meet industrial requirements. We will study interactions of proteins, amino acids, and lipids with rubber, then employ biobased post-harvest treatments. If unsuccessful, we will apply chemical treatments. Subobjective 2B: To elucidate the roles of lipid in rubber biosynthesis- the molecular species of various lipid classes in rubber particles of guayule, Kazak dandelion and Hevea will be quantified using HPLC and MS. Lipid profiles from the native guayule and Kazak dandelion will be compared to those from genetically modified plants. Subobjective 2C: Develop novel processes to fractionate crude guayule - we will evaluate a series of processes including 1) re-precipitation, 2) microfiltration, 3) liquid-liquid extraction, and 4) microfiltration/ultrafiltration to de-rubberize and fractionate guayule resin into major components. Sub-objective 3A. Develop knowledge of hydroxy fatty acid (HFA) synthesis in lesquerella- we will engineer key genes to increase HFA levels in lesquerella seed oil. We will apply Agrobacterium-mediated transformation, and identify stable transgenic lines by germinating T1 seeds in selection medium. Plants/seeds will be characterized (transgene copy number for T1 using qPCR, fatty acid and TAG composition in using T2 seeds). If the total HFA content does not reach the target 70% in transgenic lesquerella, alternative promoters will be studied. Sub-objective 3B. Develop HFA-producing camelina- knowledge gained from engineering lesquerella for increased HFA content will inform strategies to raise HFA-production in the domestic oilseed crops camelina.

Progress Report
This report covers the project, Domestic Production of Natural Rubber and Industrial Seed Oils, which began in April of 2015, and is part of National Program 306 Quality and Utilization of Agricultural Products (Component 2: Non-Food). Progress was made advancing all objectives during FY16. In domestic natural rubber research, under sub-objective 1A: Genetically modify guayule for improved rubber yields, previously modified lines were characterized to better understand the effect of the genetic modification on the plants’ properties, especially natural rubber yield. Modification of guayule to reduce the amount of stored carbohydrate might divert carbon to natural rubber production. In laboratory studies, guayule plants modified to downregulate the first step to carbohydrate synthesis, the sucrose-1-fructosyltransferase gene 1-SST, increased rubber and resin yield for laboratory grown (in vitro) plants, while fructan carbohydrate production was lowered, suggesting deviated carbon flux from carbohydrate to natural rubber production. However, results for greenhouse plants showed more impact in root tissue, where more of the fructan is stored. In other studies, increased rubber content was found for modified plants overexpressing two key natural rubber biosynthesis pathway genes, HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) and FPPs (farnesyl pyrophosphate synthase), especially when using a cold-inducible promoter. A patent has been filed. Under sub-objective 1B: Identify biochemical regulation of enzymes in the isoprenoid pathways that will lead to increased yield of rubber, an understanding of how plants control natural rubber accumulation will aid strategies to higher yield. Studies have been initiated to understand biochemical regulation of two key pathway genes, HMGR and IPPi (the isopentenyl pyrophosphate-dimethylallyl pyrophosphate isomerase). The HMGR protein has been previously expressed in the yeast, S. cerevisiae. Antibody development for both enzymes is underway. Further natural rubber research took place under sub-objective 1C: Develop an effective protocol for highly efficient genetic transformation of Kazak dandelion. Plants that grow well, and produce new plants by shoot regeneration, under laboratory conditions are needed for an effective protocol. A total number of 76 individual Kazak dandelion seedlings were screened for their shoot regeneration efficiency from root explants. Individuals with high efficiency of shoot regeneration from root explants have been identified. Another important aspect is the ability to distinguish modified plants from controls. The use of an antibiotic, Kanamycin (at concentration at 25 mg/l) has been demonstrated to be effective to eliminate non-transgenic shoots. In subobjective 2A: Modify the protein components of guayule rubber to increase its market value, a series of commercial proteins (gelatin, soy, albumin, casein, zein, gliadin and gluten) were added to guayule natural rubber as a latex blend. In general, protein addition reduced bulk viscosity and improved thermo-oxidative stability. Gel and green strength of the polymer-protein blends were increased, with the exception of gliadin, but not to levels observed for Hevea. Effects on vulcanization and mechanical properties in compounds were surprisingly influenced by the antioxidants used. Our results demonstrate the potential of proteins as bio-based rubber compounding additives. Results have been submitted for publication. In subobjective 2B: Elucidate the roles of lipid in the biosynthesis of rubber and on the mechanical properties of dry rubber we also seek to enhance the properties, and thereby the market value, of domestic latex and rubber. The outstanding features of the incumbent Hevea natural rubber have been attributed to the presence of non-rubber constituents, mainly proteins and lipids, which contribute to strain-induced crystallization. Guayule latex particles have similar lipid content, but quite different lipid composition compared to Hevea, which may be responsible for the disparity in properties. Hevea lipids were isolated from latex particles; the primary components were confirmed by LC-MS as furanoid type phospholipids, in agreement with literature reports. Blends of guayule latex with Hevea lipids and with a model furanoid lipid have been prepared. Use of guayule crops co-products could significantly impact its economic sustainability. In subobjective 2C: Develop novel processes to fractionate crude guayule resin into value-added components, chemical, solvent and enzymatic treatments to isolate components of guayule resin have been evaluated. In collaboration with an industrial partner, guayule resin in 3 forms, crude extract, purified, and leaf extract, have been evaluated. The industrial purification process reduced residual rubber to below 2%. An initial protocol for resin fractions’ extraction and GC-MS characterization has been developed. In addition, we have discussed collaboration with SRRC on application of some resin components in developing wood products resistant to termites. Oilseeds research progressed under sub-objective 3A: Develop knowledge of hydroxyl fatty acids (HFA) synthesis in lesquerella to accelerate development of HFA-producing domestic oilseed crops. Castor oil is composed of triacylglycerols (TAGs) containing about 90% of hydroxy fatty acids and has many industrial uses because of the structure. However, since castor contains toxic substances, it is desirable to produce TAGs with high hydroxy fatty acid (castor oil substitute) content in other oilseed plants. Lesquerella is an arid-land crop that could be used to produce hydroxyl fatty acids (HFA) similar to valuable castor oil. Genetic improvement of lesquerella could be an effective approach, and the effect of several genes on HFA content was studied. A key gene is HFA biosynthesis in plants is castor lysophosphatidic acid acyltransferase (RcLPAT2). The RcLPAT2 gene from castor plant was successfully transferred to lesquerella. The resulting lesquerella showed increased production of HFA at the sn-2 position, similar to castor. The increase was significant, from 2% to 17%, demonstrating that RcLPAT2 can be used to engineer a new HFA-producing crop, such as lesquerella, Camelina and canola. Another key enzymatic step (elongase) on the biosynthetic pathway of triacylglycerols in lesquerella has been identified and can be blocked to increase the content of hydroxy fatty acid of triacylglycerols in lesquerella. Genetic transformations of lesquerella with another castor LPAT gene, RcLPAT299 were also performed. However, RcLPA299 did not increase HFA content at the sn-2 position of seed TAGs. The results provide important information for re-directing future research. In addition, fatty acid composition was changed in 16 transgenic lesquerella lines with lowered expression for two other genes: 3-Ketoacyl-Coenzyme A Synthase (KCS3RNAi) and omega-3 fatty acid desaturase (FAD3RNAi). As expected, castor oil-like HFA was increased in positive lines. In summary, research identified key enzymatic steps in lesquerella can be used to produce castor oil for industrial uses. Camelina is already established as an industrial oilseed producing crop in the western United States. It is possible that it can also be used to produce valuable hydroxyl fatty acids (HFA)s. Under Sub-objective 3B: Develop HFA-producing camelina, a modification strategy was designed that required the use of the lipid biosynthesis gene PIFAH12 (oleate 12 hydroxylase). This gene was isolated and cloned from Physaria lindheimeri (Lindheimer’s bladderpod), a cruciferous flowering plant. A transformation vector carrying the PlFAH12 gene has been designed and constructed. The transformation vector has been sent to a Montana State University collaborator, for testing the effect of PlFAH12 in Camelina. Separately, data mining of a lesquerella seed transcriptome and expression profiling of lesquerella diacylglycerol acyltranstransferase (PfDGAT), Phospholipid:DAG acyltransferase (PfPDAT), and PC:DAG phosphocholine transferase (PfPDCT) gene family members have been performed. The results have been published.

1. Field evaluation of improved guayule lines. Increasing yield of natural rubber in guayule plants is the main goal of current genetic improvement studies. ARS scientists at Albany, California, discovered a single gene modification that can increase rubber content by up to four fold in the laboratory. During 2016, greenhouse evaluations confirmed the increase in rubber content, also discovered the same gene results in larger, greener plants. This remarkable combination could increase grower yields through both a higher percent of rubber and biomass. In collaboration with an industrial partner, a field trial was initiated. More than 1,500 tissue cultured plants were transported from ARS in Albany, California, to the field location, transitioned to soil and planted out in the Arizona field.

2. Identification of a specific gene to enhance production of castor oil in lesquerella. Castor seed oil is a conventional source of valuable hydroxy fatty acid (HFA) which has numerous industrial applications. Lesquerella seed oil also contains HFA, but unlike like castor seed which contain toxin ricin, lesquerella seeds represent a safe source of HFA. If lesquerella oil can be engineered to resemble castor oil, it would provide an alternative source of castor oil that is safe, cost-competitive, and readily adaptable by existing industrial technologies. ARS scientists discovered a key castor gene as a target for genetic engineering a castor oil-producing lesquerella crop. This gene, lysophosphatidic acid acyltransferase 2 (RcLPAT2), was used to create transgenic lesquerella plants in collaboration with Washington State University at Pullman and Rothamsted Research at United Kingdom. The result is the first demonstration that RcLPAT2 can be used to increase HFA, a valuable property for the engineering of a new castor oil-producing crop, such as lesquerella, Camelina, and canola.

Introduction of guayule as a commercial American agricultural crop provides a potential source of income for rural Native Americans. Guayule is indigenous to U.S. southwest desert land, now part of Native American reservations. Production farms include locations on reservation land in Arizona. U.S. producers of guayule latex and rubber operate agricultural and green manufacturing operations at or near the Gila River Indian community.

Review Publications
Chen, G.Q., Van Erp, H., Martin-Moreno, J., Johnson, K., Morales, J.S., Browse, J., Eastmond, P.J., Lin, J.T. 2016. Expression of castor LPAT2 enhances ricinoleic acid content at the sn-2 position of triacylglycerols in lesquerella seed. International Journal of Molecular Sciences. 17(4):507. doi: 10.3390/ijms17040507.
McKeon, T.A., Brandon, D.L., He, X. 2015. Improved method for extraction of castor seed for toxin determination. Biocatalysis and Agricultural Biotechnology. 5:56-57. doi: 10.1016/j.bcab.2015.12.007.
Hou, C.T., Lin, J.T., Ray, K. 2015. Identification of molecular species of polyol oils produced from soybean oil by Pseudomonas aeruginosa E03-12 NRRL B-59991. Biocatalysis and Agricultural Biotechnology. 4(4):500-505. doi: 10.1016/j.bcab.2015.08.017.
Orts, W.J., McMahan, C.M. 2016. Biorefinery developments for advanced biofuels from a widening array of biomass feedstocks. BioEnergy Research. 9(2):430-446. doi: 10.1007/s12155-016-9732-4.
Torres, L., McMahan, C.M., Ramadan, L.E., Holtman, K.M., Tonoli, G.H., Flynn, A., Orts, W.J. 2015. Effect of multi-branched PDLA additives on the mechanical and thermomechanical properties of blends with PLLA. Journal of Applied Polymer Science. doi: 10.1002/app.42858.