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

Improving Bioprocess Robustness by Cellular Noise Engineering

Authors:

Georgios Daletos1* (daletosg@mit.edu), Andreas Vasdekis2, Gregory Stephanopoulos1

Institutions:

1Massachusetts Institute of Technology; 2University of Idaho–Moscow

Goals

The overall goal of this project is to enhance the robustness of biofuel-producing microbes in adverse and fluctuating environments, such as media containing toxic hydrolysates or elevated temperatures, by introducing cellular noise in gene expression. The project’s approach involves the identification of factors in the transcription process that increase cellular noise and the deployment of such factors to generate cells exhibiting increased cellular noise. The project uses modeling and single-cell analysis workflow to engineer Yarrowia lipolytica variants that can tolerate, grow, and efficiently synthesize biofuel precursors under steady state, albeit dynamically stressful, conditions. Overall, the team anticipates that strains with optimal levels of cellular noise will also exhibit robustness that maintains production under time-varying stresses.

Abstract

Robustness represents a system-level trait that allows cell populations to maintain function under adverse and fluctuating environments. When observed at the cellular or subcellular level, an isogenic cell population exhibits increased cell-to-cell variability, or noise, even under steady-state conditions. In this context, isogenic cells can undergo division of labor, with some expressing the pathways that enable them to continue functioning in a new environment. This concept guides the project in developing workflows for introducing and manipulating cellular noise to enhance cellular tolerance to environmental stressors. Focus has been placed on the construction of Y. lipolytica strains with the double phenotype of tolerance and high lipid productivity. In its first steps on cellular noise engineering, the project refined gene editing toolboxes that can deterministically vary the level of cellular noise in protein expression levels. Accordingly, the team introduced two to eight tandem upstream activating sequences to the pTEF promoter. The synthetic hybrid promoters were placed upstream of a green fluorescent protein, fused into plasmids, and stably integrated into the genome of Y. lipolytica. Each transformant was screened separately by flow cytometry to categorize them into expression and noise levels. The impact of activators or repressors on the promoters was likewise investigated. As a next step, researchers introduced key genes that play a significant role in viability at varying inhibitor levels. To this end, the team applied rational design to develop a cellulosic oil Y. lipolytica strain that is tolerant to the primary lignocellulosic inhibitor furfural. To enable tolerance to furfural, researchers constructed Y. lipolytica overexpressing an endogenous aldehyde dehydrogenase that converts furfural to the less toxic furoic acid. The project finally evaluated front‐runner Y. lipolytica strains under both stressful and non‐stressful conditions to quantify the effects of noise and expression levels on furfural tolerance. The team has also identified the mechanisms and related gene targets that could enable Y. lipolytica to withstand elevated temperatures, which form the next cellular noise engineering goal in this project.

Funding Information

This research was supported by the DOE Office of Science, BER program, grant no. DE‐SC0022016.