Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Knowledge-Guided Interrogation of the Plastid Fatty Acid Biofactory

Authors:

Gabriel Lemes Jorge1* (glj2d5@missouri.edu), Chaofeng Wang2, Rui Guan2, Eleana Kang2, Abdul Ghani1, Yen On Chan1, Chunhui Xu1, Trey Shaw1, Sai Akhil Choppararu1, Yongfang Qin1, Maneesh Lingwan3, Doug K. Allen3,4, Samuel Andrzejewski5, Annette Thompson5, Jerome M. Fox5, Dong Xu1, Trupti Joshi1, Jay J. Thelen1, Edgar B. Cahoon2* (ecahoon2@unl.edu)

Institutions:

1University of Missouri; 2University of Nebraska–Lincoln; 3Donald Danforth Plant Science Center; 4USDA-ARS; 5University of Colorado–Boulder

URLs:

Goals

  • Develop comparative systems datasets for typical (pennycress) vs. extreme (Cuphea) seed fatty acid synthase (FAS).
  • Develop kinetic model for pennycress vs. Cuphea FAS.
  • Reconstitute and ratiometric-optimize FAS for increased fatty acid and C10 fatty acid production and kinetic model refinement.
  • Generate synthetic biology–based FAS and acetyl-CoA carboxylase (ACCase) pennycress and camelina biodesigns for increased oil and C10 fatty acid production.

Abstract

B5: Bigger Better Brassicaceae Biofuels and Bioproducts aims to dissect the biochemical underpinnings of fatty acid biosynthesis in plant plastids. This work will enable the predictable design of the nonfood Brassicaceae oilseeds Camelina and pennycress for increased seed oil and tailored fatty acid chain lengths to support U.S. bioenergy and oleochemical sectors. In oilseeds, energy-dense fatty acids are produced in plastids by type II FASs, which consist of discrete enzymes that function in concert to carry out the iterative elongation of fatty acid chains, two carbons at a time. FAS is fueled by malonyl-CoA generated by acetyl-CoA carboxylase (ACCase), the rate-limiting enzyme in fatty acid synthesis. Studies explore the hypothesis that the changes in the stoichiometry of both FAS enzymes and multimeric complexes of ACCase and associated regulatory proteins can enable fine-tuning of total fatty acid production and fatty acid chain lengths. The project’s investigation of FAS composition and stoichiometry is informed by comparative analyses of seeds with a “typical” FAS (pennycress) that produces C16 and C18 fatty acids vs. seeds with an “extreme” FAS (Cuphea viscosissima) that produces ~75% 10:0 (Figure 1). As a first step, the team generated PacBio transcriptomes of developing pennycress and C. viscosissima seeds representing ~14,000 and ~21,000 gene families, respectively, which will be used for all integrative multiomics studies on data generated in this project. Through mining of these transcriptomes, researchers have generated a comprehensive set of cDNAs for FAS enzymes, ACCase enzymes, and regulatory proteins from seeds of both species. In pennycress seeds, researchers identified 37 genes, orthologous to Arabidopsis, involved in de novo fatty acid synthesis in the plastid. The team confirmed 31 key FAS and ACCase genes through global proteomic analysis of developing pennycress seeds. For absolute quantitation, 25 new AQUA peptides were synthesized alongside nine AQUA peptides shared with Arabidopsis. Researchers also expressed and purified recombinant pennycress FAS polypeptides, which will be used as standards in absolute quantitative proteomic studies and as constituents of in vitro FASs to support mathematical modeling of fatty acid synthesis. Early modeling efforts have focused on the integration of regulatory interactions associated with ACCase and FAS enzymes. To support the analysis of FAS compositions in developing seeds, the team also generated polyclonal antibodies against several recombinant FAS polypeptides that have yielded high-resolution western blots. Guided by prior research of the type II FAS of Escherichia coli, researchers are exploring the impact of changes in the expression of β-ketoacyl-acyl carrier protein synthase I (KASI), which acts as the initial condensing enzyme in fatty acid elongation, on yields of decanoic acid (C10) in pennycress and Camelina seeds engineered for the overproduction of this product. Overall, these collaborative efforts are generating fundamental knowledge that will be combined with B5 synthetic biology tool development for predictive design of optimized pennycress and Camelina feedstocks for biofuels and bioproducts.

Image

Figure 1. Simplified model of acetyl-CoA carboxylase (ACCase) and typical vs. extreme fatty acid synthase (FAS).

Funding Information

This research was supported by the DOE Office of Science, BER program, grant no. DE-SC0023142.