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unknown but could not be associated to genetic mutations (unpublished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.