Automated strain construction facilities (biofoundries) employing advanced molecular cloning strategies provide the possibility of constructing a large number of genetically diverse production strains economically. Common molecular cloning tasks include synthesizing operons of varying induction strength, new-to-nature pathways, or rewired (or synthetic) genetic circuits, all encoded episomally or functionally integrated into the host genome. A new concept to meet the increasing demand for large genetic construct libraries is the standardization and modularization of genetic elements utilizing the Modular Cloning (MoClo) - principle.

In this study, a MoClo toolbox for rapid plasmid construction for episomal gene expression and CRISPR/Cas12a-based genome modifications was developed for C. glutamicum. A library of basic genetic parts (promoters, ribosome binding sites, enzyme encoding DNA sequences, and terminators) for transcription units was established, which facilitated the construction of plasmids pEVCg(K)_AE and pJYS3_MoClo for episomal gene expression and CRISPR/Cas12a-genome modifications, respectively.

Promoter characterization experiments (expression of fluorescent reporter protein genes) confirmed the functionality of the individual parts and resulted in a set of twelve C. glutamicum and three E. coli promoters, which can be readily used for future library constructions. To address more complex cloning needs, the parts library was expanded to enable standardized construction of synthetic operons constituted of up to four pre-assembled transcription units. The MoClo principle was then adapted for genome editing using CRISPR/Cas12a, yielding plasmid pJYS3_MoClo. Since only a limited number of genomic integration sites is known and validated for applications using C. glutamicum, the standardization and modularity of pJYS3_MoClo will be subsequently used to generate a data set of suitable genomic integration loci in C. glutamicum ATCC 13032.

Taken together, this work presents a versatile molecular toolbox for strain engineering of C. glutamicum, paving the way for the automated metabolic engineering of this industrial microbial workhorse.