Strategies to increase tolerance and robustness of industrial microorganisms

Abstract

The development of a cost-competitive bioprocess requires that the cell factory converts the feedstock into the product of interest at high rates and yields. However, microbial cell factories are exposed to a variety of different stresses during the fermentation process. These stresses can be derived from feedstocks, metabolism, or industrial production processes, limiting production capacity and diminishing competitiveness. Improving stress tolerance and robustness allows for more efficient production and ultimately makes a process more economically viable. This review summarises general trends and updates the most recent developments in technologies to improve the stress tolerance of microorganisms. We first look at evolutionary, systems biology and computational methods as examples of non-rational approaches. Then we review the (semi-)rational approaches of membrane and transcription factor engineering for improving tolerance phenotypes. We further discuss challenges and perspectives associated with these different approaches.

Keywords: Genome-scale model; Membrane engineering; Novel synthetic biology tools; Systems biology; Toxicity; Transcription factor engineering.

Fig. 1

Different causes of cellular stress (left), origin (middle) and examples (right). Lignocellulosic feedstock is composed of hemicellulose, cellulose, and lignin. During the pre-treatment, inhibitors such as furans, weak acids, or phenolic compounds can be formed. The natural and engineered metabolism can generate intermediates, by-products, or products that can generate cell stress, e.g. alcohols, organic acids, short chain fatty acids or aromatic compounds. Parameters such as pH, osmotic pressure, and temperature can further stress cells and often change during the up-scaling of a production process. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Overview of approaches and methods to increase tolerance and robustness in microorganisms discussed in this review. Evolutionary, systems biology, and computational approaches are non-rational approaches that can identify genetic targets to increase stress tolerance in the host organism. While those approaches target the whole cell, more rational approaches can target specific parts of the cell, e.g., the membrane or transcription factors (TF). Circled terms represent exemplary technologies discussed in this review. ALE – Adapted Laboratory Evolution, GEM – Genome-scale Model, TF – transcription factor. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Overview of different membrane engineering strategies to increase stress tolerance and resistance. A) Engineering of the fatty acid composition of the membrane by altering the degree of saturation, the average chain length, or integrating cyclopropane-fatty acids. B) Engineering lipid composition by altering the sphingolipid or sterol content or changing the phospholipid headgroup (PG Phosphatidylglycerol, PI Phosphatidylinositol, PS Phosphatidylserine, PE Phosphatidylethanolamine, PC Phosphatidylcholine). C) Integrating transporter proteins into the membrane, which can either be passive channel proteins or active energy-consuming efflux pumps. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)