Filamentous fungi are harnessed as cell factories for the production of a diverse range of organic acids, proteins, and secondary metabolites. Growth and morphology have critical implications for product titres in both submerged and solid-state fermentations. Recent advances in systems-level understanding of the filamentous lifestyle and development of sophisticated synthetic biological tools for controlled manipulation of fungal genomes now allow rational strain development programs based on data-driven decision making. In this review, we focus on Aspergillus spp. and other industrially utilised fungi to summarise recent insights into the multifaceted and dynamic relationship between filamentous growth and product titres from genetic, metabolic, modelling, subcellular, macromorphological and process engineering perspectives. Current progress and knowledge gaps with regard to mechanistic understanding of product secretion and export from the fungal cell are discussed. We highlight possible strategies for unlocking lead genes for rational strain optimizations based on omics data, and discuss how targeted genetic manipulation of these candidates can be used to optimise fungal morphology for improved performance. Additionally, fungal signalling cascades are introduced as critical processes that can be genetically targeted to control growth and morphology during biotechnological applications. Finally, we review progress in the field of synthetic biology towards chassis cells and minimal genomes, which will eventually enable highly programmable filamentous growth and diversified production capabilities. Ultimately, these advances will not only expand the fungal biotechnology portfolio but will also significantly contribute to a sustainable bio-economy.

Corynebacterium glutamicum is an important industrial workhorse and advanced genetic engineering tools are urgently demanded. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) have revolutionized the field of genome engineering. The CRISPR/Cas9 system that utilizes NGG as protospacer adjacent motif (PAM) and has good targeting specificity can be developed into a powerful tool for efficient and precise genome editing of C. glutamicum. Herein, we developed a versatile CRISPR/Cas9 genome editing toolbox for C. glutamicum. Cas9 and gRNA expression cassettes were reconstituted to combat Cas9 toxicity and facilitate effective termination of gRNA transcription. Co-transformation of Cas9 and gRNA expression plasmids was exploited to overcome high-frequency mutation of cas9, allowing not only highly efficient gene deletion and insertion with plasmid-borne editing templates (efficiencies up to 60.0 and 62.5%, respectively) but also simple and time-saving operation. Furthermore, CRISPR/Cas9-mediated ssDNA recombineering was developed to precisely introduce small modifications and single-nucleotide changes into the genome of C. glutamicum with efficiencies over 80.0%. Notably, double-locus editing was also achieved in C. glutamicum. This toolbox works well in several C. glutamicum strains including the widely-used strains ATCC 13032 and ATCC 13869. In this study, we developed a CRISPR/Cas9 toolbox that could facilitate markerless gene deletion, gene insertion, precise base editing, and double-locus editing in C. glutamicum. The CRISPR/Cas9 toolbox holds promise for accelerating the engineering of C. glutamicum and advancing its application in the production of biochemicals and biofuels.