Aspergillus nidulans cell wall integrity kinase, MpkA, impacts cellular phenotypes that alter mycelial-material mechanical properties

Abstract

Mycelial materials are an emerging, natural material made from filamentous fungi that have the potential to replace unsustainable materials used in numerous commercial applications (e.g., packaging, textiles, construction). Efforts to change the mechanical properties of mycelial-materials have typically involved altering growth medium, processing approaches, or fungal species. Although these efforts have shown varying levels of success, all approaches have shown there is a strong correlation between phenotype (of both fungal mycelia and mycelial material's assembly) and resultant mechanical properties. We hypothesize that genetic means can be used to generate specific fungal phenotypes, leading to mycelial materials with specific mechanical properties. To begin to test this hypothesis, we used a mutant of the model filamentous fungus, Aspergillus nidulans, with a deletion in the gene encoding the last kinase in the cell wall integrity (CWI) signaling pathway, mpkA. We generated one set of mycelial materials from the ΔmpkA deletion mutant (A1404), and another from its isogenic parent (A1405; control). When subjected to tensile testing, and compared to material generated from the control, ΔmpkA material has similar elastic modulus, but significantly increased ultimate tensile strength, and strain at failure. When subjected to a fragmentation assay (i.e., resistance to shear-stress), the ΔmpkA material also had higher relative mechanical strength. To determine possible causes for this behavior, we carried out a comprehensive set of phenotype assessments focused on: three-dimensional structure, hyphal morphology, hyphal growth behaviors, and conidial development. We found, compared to the control, material generated from the ΔmpkA mutant manifests significantly less development, a modified cell wall composition, larger diameter hyphae, more total biomass, higher water capacity and more densely packed material, which all appear to impact the altered mechanical properties.

Fig. 1

Mycelial material generation. Mycelial discs were grown from the (A) control (A1405) and (B) ΔmpkA (A1404) fungal strains. (C) Average dry material biomass (g) from two disks for the control (●) and ΔmpkA (○) increased exponentially, with average specific growth rates (straight lines) of 0.041 h^− 1 and 0.043 h^− 1 and final biomass values of 0.20 g and 0.30 g respectively. No significant difference in growth rates (P = 0.84); significant difference in final weights (P = 0.001)

Fig. 2

Tensile testing of mycelial material. Material discs were dried and then (A) cut into strips, which were (B) mounted in a paper frame which was then (C) mounted on load-frame for testing. Details in Methods. Typical stress-strain curves for mycelial material generated from the (D) control (A1405) and (E) ΔmpkA (A1404) fungal mutant. In both cases, five test strips were cut from the center of a single disc of mycelial material. Numbers on each graph indicate the relative position of where coupons (panel A) were cut from the mycelial material. Positions 1–5 were immediately adjacent to each other, with position 3 in the middle of the material disk. For both fungal genotypes, the material shows linear elasticity before failure. Mechanical properties determined from stress-strain testing (n = 15) of material generated from control (A1405) and ΔmpkA (A1404) mutant. (F) Average Young’s modulus, (G) ultimate tensile strength and (H) strain at failure

Fig. 3

Representative SEM images of mycelial material three-dimensional structure and morphological features. (A) Control (A1405) sample surface showing an abundance of developmental structures (e.g., conidia, Hülle cells), loose packing of hyphae, and voids in the material. (B) Fracture point of the same control sample. (C) The ΔmpkA (A1404) sample surface shows fewer developmental structures, denser hyphal packing, and an absence of voids. (D) Fracture point of the same ΔmpkA sample

Fig. 4

Representative TEM images showing the hyphal morphology of (A) control (A1405) and (B) ΔmpkA (A1404) mutant. Size bars = 1 μm. Measurements of hyphal morphology show that the (C) average hyphal diameter (nm) is significantly wider in the mutant and (D) cell wall thickness (nm) is similar

Fig. 5

Relative material strength measured in a high-shear mixer. (A) Average size (n = 4) of fungal elements versus time in the high-shear mixer for material from the control (●) and ΔmpkA (○) mutant. Lines indicate exponential fit used to determine specific fragmentation rate. (B) Average size of fungal elements for the control and ΔmpkA mycelial material after 5s of high-shear mixing are significantly different (P = 0.02). (C) Fragmentation rate, determined from exponential fits in A, are not significantly different (P = 0.96)

Fig. 7

Composition of mobile carbohydrates. (A) Overlay of 1D spectra for the control (A1405) and ΔmpkA mutant (A1404), shown in orange and black, representing the highly mobile region, semi-dynamic region, and cumulative region using INEPT, 2s DP, and 35s DP experiments. The glucan types, their monomers, and carbon numbers are labeled and color-coded as follows: galactofuranose (Galf, pink), α-1,2-mannose (Mn^1,2, light brown), α-1,6-mannose (Mn^1,6, brown), galactopyranose (Galp, yellow), and N-acetyl galactosamine (GalNAc, magenta). (B) The through-bond correlations 2D 1 ¹³C-¹³C -DP J INADEQUATE correlation spectrum, shown in orange and grey for positive control and mutant. (C) The molar composition of these mobile components determined by analyzing the peak volumes in the 2D DP-INADEQUATE spectra. The GM molecules are color-coded in pink shades, while the GAG components are shaded in orange in the donut graph. The legend corresponds to the abbreviations, linking the monomers to their assignments in the INADEQUATE spectra

Fig. 6

Composition of rigid polysaccharide probed by high-resolution ssNMR. (A) Overlay CP spectra of positive control and ∆mpkA mutant, with the rigid polysaccharide shown in orange and black spectra. The glucan types and its carbon numbers are abbreviated, and color coded as: α-1,3glucan (A, green), (β-1,3-glucan (B, blue), chitin (Ch, orange), chitosan (Cs, pink). (B) The 2D ¹³C-¹³C CORD, correlation explores the spatial arrangement of cross-linkages among α-1,3-glucan, β-1,3-glucan, chitin, and chitosan. (C) The molar composition of these rigid components is determined by analyzing the peak volumes in the 2D ¹³C-¹³C CORD spectra

Fig. 8

Cell wall melanin content. Compared to the control, the ΔmpkA mutant produces significantly more (~ 80%) melanin (P = 0.01)

Fig. 9

Conidia collected, show that compared to the control, the ΔmpkA produces significantly fewer (40%) conidia (P = 0.02)

Fig. 10

FTIR traces for material grown from control (- - -) and ΔmpkA (––) mycelial materials. Annotated graph showing the difference in water between the control and the ΔmpkA mutant with a close up image highlighting additional traces. This graph also suggests that there might be additional chemical differences found between the control and the ∆mpkA mutant