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

Abstract

Background: The 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.

Results: In 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.

Conclusions: Using 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.

Keywords: Mycelial morphology; Neurospora crassa; Pellet; Protein secretion; Viscosity.

Fig. 1

Screening of 90 morphological mutants in Neurospora crassa. Conidia from the wild type (WT) and morphological mutants were separately inoculated into Avicel medium and batch cultured for 7 days. The viscosities of culture broths altered by more than 50% compared with the WT are indicated as follows: blue dots, high-viscosity mutants; red dots, low-viscosity mutants

Fig. 2

Mycelial morphologies of wild type and Δgul-1 mutant during submerged cultivation. Conidia from the wild type (WT) and gul-1 mutant (Δgul-1) were separately inoculated into Avicel medium and batch cultured for 7 days, and the viscosities of the broths were measured at 24 h intervals. The Δgul-1 mutant grew in pellet form, whereas the wild type exhibited a clump type morphology. Blue line indicates WT; Red line indicates the Δgul-1 mutant. Scale bar is 200 μm. Values represent the means of at least three replicates, error bars show standard deviation

Fig. 3

Comparison of the viscosity of culture broths from WT, Δgul-1, Pc-gul-1 and Pn-gul-1 strains grown on Avicel medium for 7 days. The following strains were grown in 2% (w/v) Avicel media: the WT, the gul-1 gene knockout mutant (Δgul-1) and the complemented strain under either the control of the ccg-1 promoter (Pc-gul-1) or the native promoter (Pn-gul-1). The viscosity was measured and displayed after normalization to the WT control according to percentage. Values represent the means of at least three biological replicates, error bars show standard deviation. Statistical significance was performed using a two-tailed Student’s t test (**P < 0.01; ***P < 0.001; n.s., not significant)

Fig. 4

The subcellular localization of GUL-1 in Neurospora crassa. Locations of GUL-1 proteins were monitored by recording enhanced green fluorescent protein signal. Microscopic observation was performed with an OLYMPUS BX51 microscope. Scale bar is 10 μm

Fig. 5

Effect of different concentrations of chemicals on hyphal growth in WT and Δgul-1. Aliquots of 5 μL 1 × 10⁷ mL⁻¹ spore suspensions of WT and Δgul-1 were incubated at 28 °C for 18 h on MM plates incorporating H₂O₂ (10, 20 mM), diamide (6, 9 μg/mL), methyl-viologen (4.5, 9 μg/mL), NaCl (0.5, 1.0 M), Congo Red (1, 2 mg/mL) or Calcofluor White (200, 400 μg/mL), and then the diameter of each colony was measured. Relative diameter reduction means the reduction of growth rate on MM containing chemicals compared with the growth on MM only. Values represent the means of at least three biological replicates, error bars show standard deviation. Statistical significance was performed using a two-tailed Student’s t test (***P < 0.001)

Fig. 6

Phenotype of WT and Δgul-1 strains when grown on Avicel medium. Conidia from the wild type (WT) and the gul-1 knockout mutant (Δgul-1) were separately inoculated into Avicel medium and batch cultured. After 7 days, total extracellular protein concentration, endoglucanase activity and β-glucosidase activity were measured. Data were normalized to the WT control according to percentage. Values represent the means of at least three biological replicates, error bars show standard deviation. Statistical significance was performed using a two-tailed Student’s t test (**P < 0.01)

Fig. 7

Transcriptome analysis of the Δgul-1 strain on Avicel medium. a Expression levels of genes encoding cell wall proteins in Δgul-1 mutant relative to wild-type (WT) strain on Avicel. b Expression levels of major cellulase genes in Δgul-1 mutant relative to the wild-type (WT) strain on Avicel. c Expression levels from RNA-seq data of genes encoding major secreted proteins from WT and Δgul-1 when grown on Avicel medium. After Δgul-1 and WT conidia were grown on Avicel for 3 days, the transcriptional abundance of major cellulase genes and cell wall protein genes was evaluated by RNA-seq and quantitative real-time PCR. Values represent the means of at least three biological replicates, error bars show standard deviation. Statistical significance was performed using a two-tailed Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 8

Phenotype of ∆ncw-1∆Ncap3m∆gul-1 triple mutant when grown on Avicel medium. Conidia of the wild type (WT), the double deletion strain (∆ncw-1∆Ncap3m) and the triple deletion strain (∆ncw-1∆Ncap3m∆gul-1) were separately inoculated into Avicel medium, and then cultured for 7 days. a Total extracellular protein concentration, endoglucanase activity, β-glucosidase activity and the viscosity of culture broth were measured and normalized to the WT according to percentage. Values represent the means of at least three biological replicates, error bars show standard deviation. Statistical significance was performed using a two-tailed Student’s t test (***P < 0.001; n.s., not significant). b Mycelial morphologies of the triple deletion strain (∆ncw-1∆Ncap3m∆gul-1) and its parental strain (∆ncw-1∆Ncap3m) after 7 days cultivation. The images were acquired by an Olympus SZX-7 stereomicroscopy with a digital camera attached. Scale bar is 300 μm