No abstract

The CRISPR/Cas9 system is a revolutionary genome editing tool. However, in eukaryotes, search and optimization of a suitable promoter for guide RNA expression is a significant technical challenge. Here we used the industrially important fungus, Aspergillus niger, to demonstrate that the 5S rRNA gene, which is both highly conserved and efficiently expressed in eukaryotes, can be used as a guide RNA promoter. The gene editing system was established with 100% rates of precision gene modifications among dozens of transformants using short (40-bp) homologous donor DNA. This system was also applicable for generation of designer chromosomes, as evidenced by deletion of a 48 kb gene cluster required for biosynthesis of the mycotoxin fumonisin B1. Moreover, this system also facilitated simultaneous mutagenesis of multiple genes in A. niger. We anticipate that the use of the 5S rRNA gene as guide RNA promoter can broadly be applied for engineering highly efficient eukaryotic CRISPR/Cas9 toolkits. Additionally, the system reported here will enable development of designer chromosomes in model and industrially important fungi.

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.