The future of self-selecting and stable fermentations
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FERMENTATION, CELL CULTURE AND BIOENGINEERING - MINI REVIEW
The future of self‑selecting and stable fermentations Peter Rugbjerg1,2 · Lisbeth Olsson2 Received: 30 June 2020 / Accepted: 17 October 2020 © The Author(s) 2020
Abstract Unfavorable cell heterogeneity is a frequent risk during bioprocess scale-up and characterized by rising frequencies of lowproducing cells. Low-producing cells emerge by both non-genetic and genetic variation and will enrich due to their higher specific growth rate during the extended number of cell divisions of large-scale bioproduction. Here, we discuss recent strategies for synthetic stabilization of fermentation populations and argue for their application to make cell factory designs that better suit industrial needs. Genotype-directed strategies leverage DNA-sequencing data to inform strain design. Selfselecting phenotype-directed strategies couple high production with cell proliferation, either by redirected metabolic pathways or synthetic product biosensing to enrich for high-performing cell variants. Evaluating production stability early in new cell factory projects will guide heterogeneity-reducing design choices. As good initial metrics, we propose production half-life from standardized serial-passage stability screens and production load, quantified as production-associated percent-wise growth rate reduction. Incorporating more stable genetic designs will greatly increase scalability of future cell factories through sustaining a high-production phenotype and enabling stable long-term production. Keywords Production load · Metabolic burden · Evolutionary stability · Production robustness · Production stability · Genetic heterogeneity · Phenotypic heterogeneity
Introduction To accelerate commercialization of the growing number of advanced bioproducts, new solutions are needed to minimize the development costs of scaling bioprocesses to large volumes [43, 63]. Often, large-scale biomanufacturing is restricted by the long-term stability of the production phenotype. Metabolic burdens and toxicities confer a production load (defined as percent-wise reduction in specific growth rate associated with production). As cultures are scaled to industrial numbers of cell generations (e.g. > 40 generations), this production load selects against high production and enriches for spontaneously emerging low-producing subpopulations [50, 66]. The economic consequences of a poorly scalable process can be significant with estimates of full bioprocess scale-up costs ranging between 100 million * Peter Rugbjerg [email protected] 1
Enduro Genetics ApS, Copenhagen, Denmark
Department of Biology and Biological Engineering, Industrial Biotechnology, Chalmers University of Technology, Gothenburg, Sweden
2
and 1 billion USD including costs of pilot and manufacturing plants [12]. In large bioproduction plants, physiochemical heterogeneity is another, well-described phenomenon that occurs as a consequence of suboptimal mixing gradients causing variation in substrate, oxygen and pH regulato
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