Abstract
Recombinant protein production is mostly realized with large-scale cultivations and monitored at the level of the entire population. Detailed knowledge of cell-to-cell variations with respect to cellular growth and product formation is limited, even though phenotypic heterogeneity may distinctly hamper overall production yields, especially for toxic or difficult-to-express proteins. Unraveling phenotypic heterogeneity is thus a key aspect in understanding and optimizing recombinant protein production in biotechnology and synthetic biology. Here, microfluidic single-cell analysis serves as the method of choice to investigate and unmask population heterogeneities in a dynamic and spatiotemporal fashion. In this study, we report on comparative microfluidic single-cell analyses of commonly used E. coli expression systems to uncover system-inherent specifications in the synthetic M9CA growth medium. To this end, the PT7lac/LacI, the PBAD/AraC and the Pm/XylS system were systematically analyzed in order to gain detailed insights into variations of growth behavior and expression phenotypes and thus to uncover individual strengths and deficiencies at the single-cell level. Specifically, we evaluated the impact of different system-specific inducers, inducer concentrations as well as genetic modifications that affect inducer-uptake and regulation of target gene expression on responsiveness and phenotypic heterogeneity. Interestingly, the most frequently applied expression system based on E. coli strain BL21(DE3) clearly fell behind with respect to expression homogeneity and robustness of growth. Moreover, both the choice of inducer and the presence of inducer uptake systems proved crucial for phenotypic heterogeneity. Conclusively, microfluidic evaluation of different inducible E. coli expression systems and setups identified the modified lacY-deficient PT7lac/LacI as well as the Pm/XylS system with conventional m-toluic acid induction as key players for precise and robust triggering of bacterial gene expression in E. coli in a homogeneous fashion.
Highlights
While in natural environments, cell-to-cell variations in gene expression and growth may prove beneficial and are considered as bet-hedging or division of labor strategies to enhance environmental adaptability within an isogenic bacterial population [1,2], such phenotypic heterogeneity is unfavorable in biotechnology and synthetic biology
In synthetic biology and biotechnology, expression processes are mainly observed on averagebased population scale, ignoring phenotypic heterogeneity especially in case of adequate overall yields and functionality
Unraveling phenotypic heterogeneity is a key aspect for the optimization of biotechnological and synthetic biology applications; well-defined conditions have to be applied to avoid the influence of environmental heterogeneity on microbial expression setups
Summary
Cell-to-cell variations in gene expression and growth may prove beneficial and are considered as bet-hedging or division of labor strategies to enhance environmental adaptability within an isogenic bacterial population [1,2], such phenotypic heterogeneity is unfavorable in biotechnology and synthetic biology. Phenotypic homogeneity is needed to reliably predict and control target gene expression [3,4]. Catabolic regulatory networks such as those for lactose, arabinose or benzoate utilization were employed as useful tools for heterologous gene expression [5,6,7]. These expression systems commonly consist of native or mutagenized promoters and a corresponding transcriptional regulator that represses, derepresses or activates target gene expression in the presence of a specific inducer that can enter the cell via an appropriate transport system or by passive diffusion
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
