A fast and simple method to estimate relative, hyphal tensile‐strength of filamentous fungi used to assess the effect of autophagy

Abstract

Fungal hyphal strength is an important phenotype which can have a profound impact on bioprocess behavior. Until now, there is not an efficient method which allows its characterization. Currently available methods are very time consuming, thus, compromising their applicability in strain selection and process development. To overcome this issue, a method for fast and easy, statistically verified quantification of relative hyphal tensile strength was developed. It involves off-line fragmentation in a high shear mixer followed by quantification of fragment size using laser diffraction. Particle size distribution (PSD) is determined, with analysis time on the order of minutes. Plots of PSD 90th percentile versus time allow estimation of the specific fragmentation rate. This novel method is demonstrated by estimating relative hyphal strength during growth in control conditions and rapamycin-induced autophagy for Aspergillus nidulans (parental strain) and a mutant strain (ΔAnatg8) lacking an important autophagy gene. Both strains were grown in shake flasks and relative hyphal tensile strength was compared. The mutant strain grown in control conditions appears to be weaker than the parental strain, suggesting that Anatg8 may play a role in other processes involving cell wall biosynthesis. Furthermore, rapamycin-induced autophagy resulted in apparently weaker cells even for the mutant strain. These findings confirm the utility of the developed method in strain selection and process development.

Dynamic Transcriptomic and Phosphoproteomic Analysis During Cell Wall Stress in Aspergillus nidulans

“…Changes in gene expression take a significantly longer period as shown by previously published qPCR data (11) involving micafungin induced expression changes. Based on these previous findings, we drew samples for transcriptomic analysis at 0, 5,10,15,20,25,30,40,50,60,75,90, and 120 min (13 samples total).…”

“…From this group, we tested each non-essential kinase for its involvement in cell wall maintenance and repair by growing kinase deletion mutants in shake-flask culture and measuring mycelial size-distribution using laser particle-size analysis. We developed this approach previously ( 50 ) and have shown (that for similar growth rates) smaller mycelia can result from either (1) compact ( i.e. highly branched) morphology, or (2) hyphal fragmentation during shaking.…”

“…Apparent Cell Wall Strength-To measure apparent cell wall strength in shake flask culture mycelia were removed from shake flasks (ϳ5 ml) during exponential growth phase and subjected to particle size analysis using a Mastersizer 3000 instrument with a Hydro SM manual sample dispersion unit following published methods (29,50). The average mycelial size (S 90 ) for each biological replicate was calculated from at least 3 timepoints (between 17 and 24 h growth) using duplicate technical replicates (p value set at 0.05, nՆ3).…”

Understanding gradients in industrial bioreactors

“…Moreover, it is very complicated to reach industrially relevant tip speed values in small reactors due to equipment constraints. Tip speed affects the shear rate, causing changes in the rheology and morphology of filamentous fungi (Quintanilla et al, 2018). Other functional aspects to consider are the increased risk of vortex formation due to increased agitation speeds in J o u r n a l P r e -p r o o f scale down systems, the differences in volume dynamics (e.g.…”