Harnessing nature’s ingenuity with microorganisms for industrial production is an attractive solution to today’s climate concerns. Nature’s innate diversity allows the production of many value-added chemicals and can be expanded on through genetic engineering. Although the use of microbial cell factories (MCFs) has been extremely successful at lab scale, the numbers of successful bioprocesses remain limited. High cell densities and long cultivation times lead to reductions in productivity over the course of the cultivation through the effects of genetic and expression instability of the strain. This instability leads to population diversification. In this review, we explore the roots of genetic instability in microorganisms, focusing on prokaryotic bioprocesses, and how organisms cope with this instability. We spotlight single-cell detection methods capable of monitoring populations within the bioprocess both in- and on-line. We also examine different approaches to minimizing population diversification, both through strain development and bioprocess engineering. With this review, we highlight the fact that population-averaged metrics overlook the single-cell stresses driving genetic and functional instability, leading to an overestimation of microbial bioprocess robustness. High-throughput single-cell monitoring in industry-like conditions remains essential to identify and select truly stable microbial cell factories and bioprocesses.

The hyperthermoacidophilic archaeon Sulfolobus acidocaldarius is a promising production host for industrial biotechnology applications due to its ability to thrive in extreme conditions. However, the lack of well-characterised genetic parts and tools, particularly promoters, limits its potential for metabolic engineering. In this study, we developed the first promoter library for S. acidocaldarius by randomising specific regions of the core promoter sequence of P _{Saci 2137} , a promoter known to function in both S. acidocaldarius and Escherichia coli . The library was initially screened in E. coli using mKate2, a red fluorescent reporter protein, and seven promoters were selected for characterisation in S. acidocaldarius using the thermostable β-galactosidase reporter LacS. The resulting promoter collection exhibited a 5-fold range of expression levels in S. acidocaldarius , spanning from low to high constitutive expression when compared to S. acidocaldarius promoters P _sac7d and P _malE . This study demonstrates a successful workflow for generating and characterising S. acidocaldarius promoters, providing a valuable toolkit for fine-tuning gene expression and optimising metabolic pathways in this extremophilic archaeon. The design principles established here can be extended to other archaeal systems with similar promoter architectures.