Search

The economic viability of bioprocesses is constrained by the limited range of operating conditions that can be tolerated by the cell factory. Engineering of the microbial cell membrane is one strategy that can increase robustness and thus alter this range. In this work, we targeted cellular components that contribute to maintenance of appropriate membrane function, such as: flotillin-like proteins, membrane structural proteins, and membrane lipids. Specifically, we exploited the promiscuity of squalene hopene cyclase (SHC) to produce polycyclic terpenoids with properties analogous to cholesterol. Strains producing these cholesterol-like molecules were visualized by AFM and height features were observed. Production of these cholesterol-like molecules was associated with increased tolerance towards a diversity of chemicals, particularly alcohols, and membrane trafficking processes such as lipid droplet accumulation and production of extracellular vesicles. This engineering approach improved the production titers for wax-esters and ethanol by 80- and 10-fold, respectively. Expression of SHC resulted in the production of steroids. Strains engineered to also express truncated squalene synthase (tERG9) produced diplopterol and generally did not perform as well. Increased expression of several membrane-associated proteins, such as YqiK, was observed to impact vesicle trafficking and further improve tolerance relative to SHC alone, but did not improve bio-production. Deletion of YbbJ increased lipid droplet accumulation as well as production of intracellular wax esters. This work serves as a proof of concept for engineering strategies targeting membrane physiology and trafficking to expand the production capacity of microbial cell factories., Competing Interests: Declaration of competing interest LRJ and MCS declare no competing interests., (Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The cell membrane plays a central role in the fitness and performance of microbial cell factories and therefore it is an attractive engineering target. The goal of this work is to develop a systematic framework for identifying membrane features for use as engineering targets. The metrics that describe the composition of the membrane can be visualized as "knobs" that modulate various "outcomes", such as physical properties of the membrane and metabolic activity in the form of growth and productivity, with these relationships varying depending on the condition. We generated a set of strains with altered membrane lipid composition via expression of des, fabA and fabB and performed a rigorous characterization of these knobs and outcomes across several individual inhibitory conditions. Here, the knobs are the relative abundance of unsaturated lipids and lipids containing cyclic rings; the average lipid length, and the ratio of linear and non-linear lipids (L/nL ratio). The outcomes are membrane permeability, hydrophobicity, fluidity, and specific growth rate. This characterization identified significant correlations between knobs and outcomes that were specific to individual inhibitors, but also were significant across all tested conditions. For example, across all conditions, the L/nL ratio is positively correlated with the cell surface hydrophobicity, and the average lipid length is positively correlated with specific growth rate. A subsequent analysis of the data with the individual inhibitors identified pairs of lipid metrics and membrane properties that were predicted to impact cell growth in seven modeled scenarios with two or more inhibitors. The L/nL ratio and the membrane hydrophobicity were predicted to impact cell growth with the highest frequency. We experimentally validated this prediction in the combined condition of 42 °C, 2.5 mM furfural and 2% v/v ethanol in minimal media. Membrane hydrophobicity was confirmed to be a significant predictor of ethanol production. This work demonstrates that membrane physical properties can be used to predict the performance of biocatalysts in single and multiple inhibitory conditions, and possibly as an engineering target. In this manner, membrane properties can possibly be used as screening or selection metrics for library- or evolution-based strain engineering., (Copyright © 2021 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)