Conditional Expression of the Small GTPase ArfA Impacts Secretion, Morphology, Growth, and Actin Ring Position in Aspergillus niger

Abstract

In filamentous fungi, growth and protein secretion occurs predominantly at the tip of long, thread like cells termed hyphae. This requires coordinated regulation of multiple processes, including vesicle trafficking, exocytosis, and endocytosis, which are facilitated by a complex cytoskeletal apparatus. In this study, functional analyses of the small GTPase ArfA from Aspergillus niger demonstrate that this protein functionally complements the Saccharomyces cerevisiae ARF1/2, and that this protein is essential for A. niger. Loss-of-function and gain-of-function analyses demonstrate that titration of arfA expression impacts hyphal growth rate, hyphal tip morphology, and protein secretion. Moreover, localization of the endocytic machinery, visualized via fluorescent tagging of the actin ring, was found to be abnormal in ArfA under- and overexpressed conditions. Finally, we provide evidence that the major secreted protein GlaA localizes at septal junctions, indicating that secretion in A. niger may occur at these loci, and that this process is likely impacted by arfA expression levels. Taken together, our results demonstrate that ArfA fulfills multiple functions in the secretory pathway of A. niger.

Disruption or reduced expression of the orotidine-5′-decarboxylase gene pyrG increases citric acid production: a new discovery during recyclable genome editing in Aspergillus niger

“…3). Consistent with the previous studies [31][32][33][34], Tet-on system is a useful method to functionally analyze essential genes, enabling analyses of null, loss-of-function, and over-expression in a single strain by addition of the inducer Dox. After co-transformation, ten randomly selected primary transformants were subcultured on MM plates, MM supplemented with 20 µg/mL Dox and MM supplemented with uridine.…”

Construction of an improved Aspergillus niger platform for enhanced glucoamylase secretion

“…Both observations indicate alternative routes of secretion at non-apical hyphal regions. We most recently obtained supporting indications for GlaA accumulation at septal regions in A. niger [ 44 ]. It will be therefore interesting to study in the future, whether there is any higher GlaA secretion via septa in the ∆ racA strain due to Tet-on driven overexpression of the glaA gene or not.…”

Moulding the mould: understanding and reprogramming filamentous fungal growth and morphogenesis for next generation cell factories

“…Do these models indeed form the conceptual basis for wet-lab data interpretation? In one recent example, conditional expression of the small GTPase encoding gene arfA in A. niger resulted in smaller pellet diameter in shake flask culture which occurred concomitantly with increased protein production [50]. These macromorphological effects were likely caused by decreased hyphal growth rate, reduced ability to maintain hyphal tip polarity, and a defective actin ring position at the hyphal tip due to altered arfA expression [50].…”