Emergence of Phenotypically Distinct Subpopulations Is a Factor in Adaptation of Recombinant Saccharomyces cerevisiae under Glucose-Limited Conditions.

Cells cultured in a nutrient-limited environment can undergo adaptation, which confers improved fitness under long-term energy limitation. We have shown previously how a recombinant Saccharomyces cerevisiae strain, producing a heterologous insulin product, under glucose-limited conditions adapts over time at the average population level. Here, we investigated this adaptation at the single-cell level by application of fluorescence-activated cell sorting (FACS) and showed that the following three apparent phenotypes underlie the adaptive response observed at the bulk level: (i) cells that drastically reduced insulin production (23%), (ii) cells with reduced enzymatic capacity in central carbon metabolism (46%), and (iii) 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 toward the glucose-limited conditions. The results highlight the importance of considering population heterogeneity when studying adaptation and evolution. IMPORTANCE The yeast Saccharomyces cerevisiae is an attractive microbial host for industrial production and is used widely for manufacturing, e.g., pharmaceuticals. Chemostat cultivation mode is an efficient cultivation strategy for industrial production processes as it ensures a constant, well-controlled cultivation environment. Nevertheless, both the production of a heterologous product and the constant cultivation environment in the chemostat impose a selective pressure on the production organism, which may result in adaptation and loss of productivity. The exact mechanisms behind the observed adaptation and loss of performance are often unidentified. We used a recombinant S. cerevisiae strain producing heterologous insulin and investigated the adaptation occurring during chemostat growth at the single-cell level. We showed that three apparent phenotypes underlie the adaptive response observed at the bulk level in the chemostat. These findings highlight the importance of considering population heterogeneity when studying adaptation in industrial bioprocesses.

CRISPR interference of nucleotide biosynthesis improves production of a single-domain antibody in Escherichia coli.

Growth decoupling can be used to optimize the 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 to increase the 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 the repression of 11 pathway genes led to the increased green fluorescent protein production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve the 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.

Single-Cell Technologies to Understand the Mechanisms of Cellular Adaptation in Chemostats.

There is a growing interest in continuous manufacturing within the bioprocessing community. In this context, the chemostat process is an important unit operation. The current application of chemostat processes in industry is limited although many high yielding processes are reported in literature. In order to reach the full potential of the chemostat in continuous manufacture, the output should be constant. However, adaptation is often observed resulting in changed productivities over time. The observed adaptation can be coupled to the selective pressure of the nutrient-limited environment in the chemostat. We argue that population heterogeneity should be taken into account when studying adaptation in the chemostat. We propose to investigate adaptation at the single-cell level and discuss the potential of different single-cell technologies, which could be used to increase the understanding of the phenomena. Currently, none of the discussed single-cell technologies fulfill all our criteria but in combination they may reveal important information, which can be used to understand and potentially control the adaptation.

Scale-down of continuous protein producing Saccharomyces cerevisiae cultivations using a two-compartment system.

In the biotechnological industry, economic decisions in investment are typically based on laboratory-scale experiments. Scale-down as a tool is therefore of high industrial importance to transfer the processes into larger production scale without loss in performance. In this study, large-scale prolonged continuous cultivations with a heterologous protein producing Saccharomyces cerevisiae strain have been scaled-down to a two-compartment scale-down reactor system. The effects of glucose, pH, and oxygen concentration gradients have been investigated by comparison with corresponding 300 mL standard continuous cultivations. It was found that substrate gradients within a limited range result in increased productivity of the heterologous protein under regulation of glycolytic TPI promoter and delay the decrease of protein and trehalose production during continuous cultivation. Based on these results, it is argued that introduction of variations in substrate concentration can be beneficial for industrial continuous cultivations.