A synthetic biology approach to study carotenoid production in Corynebacterium glutamicum: Read-out by a genetically encoded biosensor combined with perturbing native gene expression by CRISPRi.

Metabolic engineering for the development of microbial production strains, such as carotenoid overproducing bacteria, has a long history in industrial biotechnology. In contrast to classical strain development that mostly relies on the generation and screening of mutant libraries, rational strain development relies on the identification of a genetic target that has to be engineered in order to overcome metabolic bottlenecks facilitating the production of the desired valuable compounds. In this work, two synthetic biology approaches, namely, a CRISPRi-library and a genetically encoded biosensor, are demonstrated as tools for metabolic engineering purposes with a focus on carotenoid biosynthesis in C. glutamicum. The methods presented here gave insights into carotenoid biosynthesis and facilitated development of new metabolic engineering strategies. The use of a genetically encoded biosensor, the screening of a CRISPRi-library, and their combination can be transferred to study a wide range of organisms and target compounds.
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