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

Novel Systems Approach for Rational Engineering of Robust Microbial Metabolic Pathways


Laura Jarboe1* (, Ethan Bush1, Yannick Bomble2, Robert L. Jernigan1, Peter C. St. John2, Kejue Jia1, Pranav M. Khade1, Ambuj Kumar1, Jeff Law2, Onyeka Onyenemezu1, Jetendra K. Roy1, Chao Wu2


1Iowa State University; 2National Renewable Energy Laboratory


The goal of this project is to develop and implement a process for improving bioproduction under conditions that are appealing for industrial processes, such as high temperature and low pH. The approach addresses the failure of metabolic reactions due to inhibition, denaturation, misfolding or disorder of enzymes. Researchers have developed and implemented a framework for identifying these enzymes and selection of robust replacement enzymes, using high temperature and low pH as model stressors in Escherichia coli. The engineering strategy of replacing enzymes to improve bio-production is well-established, but rarely applied to system-wide stressors. This approach is complementary to improvement of microbial robustness by engineering the cell membrane and has advantages relative to evolutionary-based organism improvement by prioritizing bioproduction rather than growth.

Temperature sensitivity: There is a wealth of data available regarding enzyme structural integrity at high temperatures. Researchers are using an in vitro metabolomics approach for proteome-wide analysis of enzyme activity at high temperatures. Candidate bottleneck enzymes have been identified and investigated, including homoserine O-succinyltransferase (MetA), biotin synthase (BioB), ketol-acid reductoisomerase (IlvC), and 3-oxoacyl-ACP synthase 1 (FabB). For these candidate enzymes, sequences from thousands of various microorganisms with differing temperature ranges of growth have been collected and aligned. Experimental and predicted structures of these enzymes are used to query the enzyme dynamics, with the goal of identifying which sequence differences account for the ability of these critical enzymes to function at elevated temperatures.

Acid sensitivity: The pH tolerance efforts have prioritized modeling the effect of pH on the allocation of cellular resources. The metabolic model accounts for the effect of intracellular acidification on cellular energetics, the thermodynamic characteristics of metabolic reactions and on enzyme activity. Each of these three model adjustments contribute to the predicted flux distribution. A sensitivity analysis is in progress to identify the most critical enzymes for replacement. Borrowing from the abundant proteomic data of enzyme stability in the presence of increasing temperatures, researchers have developed a proteomic approach for enzymes with structural sensitivity to decreasing pH. This approach has identified several enzymes critical for central metabolism with poor acid tolerance. Researchers are also using models of enzyme temperature sensitivity as inspiration in the development of predictive sequence- and structure-based models of enzyme pH sensitivity.

Funding Information

This research was supported by the DOE Office of Science, BER Program, grant no. DE-SC0022090. This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. DOE under Contract No. DE-AC36-08GO28308. Funding provided U.S. DOE, Office of Science, through the GSP, BER Program under FWP ERW3526. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government.