Systems Biology to Enable Modular Metabolic Engineering of Fatty Acid Production in Cyanobacteria
Baltazar Zuniga1* (email@example.com), Joshua P. Abraham1, Margarita Orlova2, Hawkins S. Shepard2, Jody C. May2, Yao Xu2, Maneesh Lingwan3, Juthamas Jaroensuk3, Somnath Koley3, Bo Wang1, Carl H. Johnson1, 2, John A. McLean2, Doug K. Allen3, 4, Brian F. Pfleger1, and Jamey D. Young1, 2
1University of Wisconsin–Madison; 2Vanderbilt University; 3Donald Danforth Plant Science Center; and 4USDA-ARS St. Louis, MO
The overall objective of this project is to use systems biology to identify metabolic control points and bottlenecks that regulate flux to free fatty acids (FFAs) in cyanobacteria. The central hypothesis is that cyanobacterial lipid metabolism can be modularized into pathways “upstream” and “downstream” of the nodal metabolite acetyl-CoA, which can be separately studied and optimized to enhance overall FFA production. The team plans to test its central hypothesis and accomplish the overall objective of this project by pursuing the following specific aims:
- Identify (upstream) metabolic control points regulating acetyl- CoA precursor availability. The working hypothesis is that engineering glycolytic pathways in PCC 7002 will reveal rate-controlling steps that can be manipulated to maximize acetyl-CoA availability.
- Assess flux bottlenecks in the (downstream) fatty acid biosynthesis pathway. The working hypothesis is that multiomics analyses of thioesterase-expressing strains will elucidate regulatory nodes that control FFA production and overall lipid metabolism in PCC 7002.
Cyanobacteria are attractive hosts for biomanufacturing because of their ability to rapidly fix CO2, grow in nutrient-poor environments, and produce renewable chemicals directly from photosynthesis. Unlike triacylglycerol production in green algae, producing free fatty acids (FFAs) using genetically engineered cyanobacteria results in the secretion of the product into the culture medium for efficient recovery. However, an incomplete understanding of the regulation of cyanobacterial lipid metabolism limits the ability to engineer high-titer FFA-producing strains rationally. The overall objective of this project is to use systems biology to identify metabolic control points and bottlenecks that regulate flux to FFAs in the fast-growing, halotolerant Synechococcus sp. strain PCC 7002 via the modular optimization of metabolic pathways that are “upstream” and “downstream” of the nodal metabolite acetyl-CoA in a “push-pull” metabolic engineering strategy. Recent work has centered around the previously identified bottleneck of cyanobacterial FFA synthesis, FabH. Unexpectedly, overexpressing the kinetically superior E.coli FabH inhibits FFA production in a C8-producing PCC 7002 strain. Team members are using a suite of systems biology approaches, including 13C flux analysis, metabolomics, lipidomics, and proteomics to investigate this and other distinctive FFA production phenotypes. These data will allow us to identify and correct metabolic bottlenecks limiting FFA biosynthesis, “i.e., pull,” and optimize the carbon flux directed towards FFA synthesis, “i.e., push.” This approach will provide a deeper understanding of how fatty acid flux is regulated upstream and downstream of acetyl-CoA, enabling integrated “push-pull” metabolic engineering strategies to produce lipid products directly from photosynthetic CO2 fixation in cyanobacteria.
This research was supported by the DOE Office of Science, Office of Biological and Environmental Research (BER), grant no. DE-SC0022207.