1. Introduction

A growing number of pharmaceuticals, food ingredients and other valuable chemicals are today produced commercially by recombinant microbial cell factories. Expression of heterologous proteins confers a burden to the host strain which can result in an adverse selection towards low-producing cells during production (Rugbjerg & Olsson, 2020).
The chemostat mode of cultivation is an efficient strategy for industrial production processes, especially in continuous manufacturing, as the cells in the bioreactor can be kept in a constant growth environment for long periods with a constant output (Wright, Rønnest, & Sonnenschein, 2020). In this steady-state growth environment, the cells are exposed to nutrient limitation, e.g. glucose, either as a kinetic limitation of the cellular transport of nutrients or as limitation of the metabolic enzymes converting the nutrients (Schreiber & Ackermann, 2020). The nutrient-limited conditions in the chemostat impose a constant selective pressure on the production organism. Isogenic cells cultured in glucose-limited environments often undergo adaptation increasing fitness benefits under long-term energy limitation.
We have previously shown how a recombinant S. cerevisiae strain producing a heterologous protein adapts over time with respect to the population average protein levels (Wright et al., 2020). Over approximately 30 generations of glucose-limited growth, we observed a drastic decrease in recombinant protein production to almost half of the maximum value together with significant changes in the intracellular proteome. The underlying mechanisms of this adaptation are currently unknown but could not be associated to genetic mutations (unpublished results).
In nature, microorganisms live in complex communities consisting of many different species, subspecies and genetic variants. Phenotypic heterogeneity can occur in populations of genetically identical cells with respect to different traits including metabolism and morphology (Davis & Isberg, 2016). Phenotypic population heterogeneity with respect to metabolic activity has been shown in chemostat cultivations of microbes (Kundu, Weber, Griebler, & Elsner, 2020; Maharjan, Seeto, & Ferenci, 2007).
In this study, we have investigated the adaptation of a recombinantS. cerevisiae strain in glucose-limited cultures at the single-cell level. The results show that the bulk adaptive outcomes observed at the culture level, i.e., protein levels, physiology changes, and loss of productivity, are a mixed response composed of at least three apparent phenotypes or subpopulations. The results highlight the importance of considering population heterogeneity when studying adaptation.