Cells cultured in a nutrient-limited environment can undergo adaptation, which confers improved fitness under long-term energy limitation. We have previously shown how a recombinant S. cerevisiae strain, producing a heterologous insulin product, under glucose-limited conditions adapts over time at the average population level. In this paper, we investigated this adaptation at the single-cell level by application of FACS and showed that three apparent phenotypes underlie the adaptive response observed at the bulk level: (1) cells that drastically reduced insulin production (23 %), (2) cells with reduced enzymatic capacity in central carbon metabolism (46 %), (3) cells that exhibited pseudohyphal growth (31 %). We speculate that the phenotypic heterogeneity is a result of different mechanisms to increase fitness. Cells with reduced insulin productivity have increased fitness by reducing the burden of the heterologous insulin production and the populations with reduced enzymatic capacity of the central carbon metabolism and pseudohyphal growth have increased fitness towards the glucose-limited conditions. The results highlight the importance of considering population heterogeneity when studying adaptation and evolution.

Growth decoupling can be used to optimize production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference (CRISPRi) to increase production of a single domain antibody (sdAb) by inhibiting growth during production. First, we screened 21 sgRNA targets in the purine and pyrimidine biosynthesis pathways, and found that repression of 11 pathway genes led to increased GFP production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve production of two versions of an expression-optimized sdAb. Proteomics analysis of the sdAb-producing pyrF, pyrG, and cmk growth decoupling strains showed significantly decreased RpoS levels and an increase of ribosome-associated proteins, indicating that the growth decoupling strains do not enter stationary phase and maintain their capacity for protein synthesis upon growth inhibition. Finally, sdAb production was scaled up to shake-flask fermentation where the product yield was improved 2.6-fold compared to the control strain with no sgRNA target sequence. An sdAb content of 14.6% was reached in the best-performing pyrG growth decoupling strain.