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

Systems Biology to Enable Modular Metabolic Engineering of Fatty Acid Production in Cyanobacteria


Baltazar Zuniga1* (, Joshua P. Abraham6, Hawkins S. Shepard3, Jody C. May3, Maneesh Lingwan5, Juthamas Jaroensuk5, Somnath Koley5, Bo Wang2, John A. McLean3, Doug K. Allen4,5, Brian F. Pfleger6, Jamey D. Young1,2


1Molecular Physiology and Biophysics, Vanderbilt University; 2Chemical and Biomolecular Engineering, Vanderbilt University; 3Chemistry, Vanderbilt University; 4U.S. Department of Agriculture, Agricultural Research Service; 5Donald Danforth Plant Science Center; 6Chemical and Biological Engineering, University of Wisconsin–Madison


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 research team plans to test the central hypothesis and accomplish the overall objective of this project by pursuing the following specific aims:

(1) Identify upstream metabolic control points regulating acetyl-CoA precursor availability. The working hypothesis is that engineering glycolytic pathways in Synechococcus sp. strain PCC 7002 will reveal rate-controlling steps that can be manipulated to maximize acetyl-CoA availability.

(2) Assess flux bottlenecks in the downstream fatty acid biosynthesis pathway. The working hypothesis is that multi-omics analyses of thioesterase-expressing strains will elucidate regulatory nodes that control FFA production and overall lipid metabolism in strain PCC 7002.


Cyanobacteria, such as the halotolerant Synechococcus sp. strain PCC 7002, are attractive hosts for FFA biofuel precursor production because they produce renewable chemicals directly from photosynthesis, grow in nutrient-poor environments, and readily incorporate genetic modifications. Despite the advantages of cyanobacterial FFA synthesis, production rates are currently too low to support its adoption as a source of renewable fuel.

The overall objective of this project is to enhance cyanobacterial FFA production by identifying and eliminating metabolic bottlenecks upstream and downstream of the FFA building-block acetyl-CoA. Upstream of acetyl-CoA, the approach uses a lactate-producing pyruvate sink in an engineered PCC 7002 strain as a model to study the metabolic changes associated with increased flux toward the acetyl-CoA precursor, pyruvate. Applying 13C metabolic flux analysis has revealed that enhanced pyruvate flux in the L-lactate-producing strain is associated with increased flux through the malic enzyme shunt but no change in flux through pyruvate kinase. These data suggest that pyruvate kinase may constitute a metabolic bottleneck limiting overall pyruvate-generating flux.

Downstream of acetyl-CoA, work has centered around the ketosynthase FabH, a putative bottleneck in cyanobacterial FFA biosynthesis previously identified in vitro. Heterologous ketosynthase expression was found to enhance cyanobacterial FFA production, but this effect heavily depends upon the level and timing of ketosynthase induction.

This project leverages a suite of systems biology approaches and novel analytical methodologies (e.g., Fast-Pass DESI-MSI for high-throughput screening and AEXpurif for acyl carrier protein analysis) to identify and investigate distinctive FFA production phenotypes. Ultimately, this work will enable integrated strategies that simultaneously address both upstream and downstream metabolic bottlenecks to enhance FFA production in cyanobacteria.


Calvin Cycle

Calvin (CBB) Cycle

Funding Information

This research was supported by the DOE Office of Science, BER Program, grant no. DE-SC0022207.