Disruption of gul-1 decreased the culture viscosity and improved protein secretion in the filamentous fungus Neurospora crassa

Liangcai Lin,Zhiyong Sun,Chaoguang Tian,Qian Liu,Wenliang Sun,Jingen Li,Yong Chen

doi:10.1186/s12934-018-0944-5

Abstract

BackgroundThe cellulolytic fungus Neurospora crassa is considered a potential host for enzyme and bioethanol production. However, large scale applications are hindered by its filamentous growth. Although previous investigations have shown that mycelial morphology in submerged culture can be controlled by altering physical factors, there is little knowledge available about the potential for morphology control by genetic modification.ResultsIn this study, we screened morphological mutants in the filamentous fungus N. crassa. Of the 90 morphological mutants screened, 14 mutants exhibited considerably higher viscosity compared with that of the wild type strain, and only two mutants showed low-viscosity morphologies in submerged culture. We observed that disruption of gul-1 (NCU01197), which encodes an mRNA binding protein involved in cell wall remodeling, caused pellet formation as the fermentation progressed, and resulted in the most significant decrease in viscosity of culture broth. Moreover, over-expression of gul-1 caused dramatically increased viscosity, suggesting that the gul-1 had an important function in mycelial morphology during submerged cultivation. Transcriptional profiling showed that expression of genes encoding eight GPI-anchored cell wall proteins was lowered in Δgul-1 while expression of genes associated with two non-anchored cell wall proteins was elevated. Meanwhile, the expression levels of two hydrophobin genes were also significantly altered. These results suggested that GUL-1 affected the transcription of cell wall-related genes, thereby influencing cell wall structure and mycelial morphology. Additionally, the deletion of gul-1 caused increased protein secretion, probably due to a defect in cell wall integrity, suggesting this as an alternative strategy of strain improvement for enzyme production. To confirm practical applications, deletion of gul-1 in the hyper-cellulase producing strain (∆ncw-1∆Ncap3m) significantly reduced the viscosity of culture broth.ConclusionsUsing the model filamentous fungus N. crassa, genes that affect mycelial morphology in submerged culture were explored through systematic screening of morphological mutants. Disrupting several candidate genes altered viscosities in submerged culture. This work provides an example for controlling fungal morphology in submerged fermentation by genetic engineering, and will be beneficial for industrial fungal strain improvement.

Highlights

The cellulolytic fungus Neurospora crassa is considered a potential host for enzyme and bioethanol production
Loss of NCU01197, which was annotated as gul-1 previously [23], resulted in the pellet growth form and reduced viscosity by more than 80% compared with wild type (WT)
In Penicillium chrysogenum, down-regulation of chitin synthase gene resulted in pellet growth in submerged culture, and caused enhanced penicillin production [49]

Summary

Introduction

The cellulolytic fungus Neurospora crassa is considered a potential host for enzyme and bioethanol production. Large scale applications are hindered by its filamentous growth. Filamentous fungi are widely used for industrial scale production of antibiotics, proteins and many other useful chemicals [1,2,3]. Lin et al Microb Cell Fact (2018) 17:96 significant impact preliminary Erlenmeyer flask batch fermentations, when scaled up there are negative impacts on nutrient consumption and oxygen uptake, and a decrease in productivity. The simplest strategy to overcome this problem is to increase agitation speed. High impeller speed increases power consumption, and produces high shear stress that often damages fungal mycelia reducing product yield [5]. The pellet morphology is preferred for many industrial processes, and it would greatly facilitate cost-effective production if one could design a way of controlling mycelial morphology during the fermentation process

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