Location: Bioproducts Research
Project Number: 2030-21410-021-000-D
Project Type: In-House Appropriated
Start Date: Apr 13, 2015
End Date: Apr 12, 2020
Objective:
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.
Approach:
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.
