Nature provides unique insights into design strategies evolved by living organisms to construct robust materials with a combination of mechanical properties that are challenging to replicate synthetically. Hereby, inspired by the impact‐resistant dactyl club of the stomatopod, a mineralized biocomposite is rationally designed and produced in the complex shapes of dental implant crowns exhibiting high strength, stiffness, and fracture toughness. This material consists of an expanded helicoidal organization of cellulose nanocrystals (CNCs) mixed with genetically engineered proteins that regulate both binding to CNCs and in situ growth of reinforcing apatite crystals. Critically, the structural properties emerge from controlled self‐assembly across multiple length scales regulated by rational engineering and phase separation of the protein components. This work replicates multiscale biomanufacturing of a model biological material and also offers an innovative platform to synthesize multifunctional biocomposites whose properties can be finely regulated by colloidal self‐assembly and engineering of its constitutive protein building blocks.

Class I hydrophobins are functional amyloids secreted by fungi. They self-assemble into organized films at interfaces producing structures that include cellular adhesion points and hydrophobic coatings. Here, we present the first structure and solution properties of a unique Class I protein sequence of Basidiomycota origin: the Schizophyllum commune hydrophobin SC16 (hyd1). While the core β-barrel structure and disulphide bridging characteristic of the hydrophobin family are conserved, its surface properties and secondary structure elements are reminiscent of both Class I and II hydrophobins. Sequence analyses of hydrophobins from 215 fungal species suggest this structure is largely applicable to a high-identity Basidiomycota Class I subdivision (IB). To validate this prediction, structural analysis of a comparatively distinct Class IB sequence from a different fungal order, namely the Phanerochaete carnosa PcaHyd1, indicates secondary structure properties similar to that of SC16. Together, these results form an experimental basis for a high-identity Class I subdivision and contribute to our understanding of functional amyloid formation.

Hydrophobins are small fungal proteins that are amphiphilic and have a strong tendency to assemble at interfaces. By taking advantage of this property, hydrophobins have been used for a number of applications: as affinity tags in protein purification, for protein immobilization, such as in foam stabilizers, and as dispersion agents for insoluble drug molecules. Here, we used site-directed mutagenesis to gain an understanding of the molecular basis of their properties. We especially focused on the role of charged amino acids in the structure of hydrophobins. For this purpose, fusion proteins consisting of Trichoderma reesei hydrophobin I (HFBI) and the green fluorescent protein (GFP) that contained various combinations of substitutions of charged amino acids (D30, K32, D40, D43, R45, K50) in the HFBI structure were produced. The effects of the introduced mutations on binding, oligomerization, and partitioning were characterized in an aqueous two-phase system. It was found that some substitutions caused better surface binding and reduced oligomerization, while some showed the opposite effects. However, all mutations decreased partitioning in surfactant systems, indicating that the different functions are not directly correlated and that partitioning is dependent on finely tuned properties of hydrophobins. This work shows that not all functions in self-assembly are connected in a predictable way and that a simple surfactant model for hydrophobin function is insufficient.

The heterologous expression of proteins is often a crucial first step in not only investigating their function, but also in their industrial application. The functional assembly and aggregation of hydrophobins offers intriguing biotechnological applications from surface modification to drug delivery, yet make developing systems for their heterologous expression challenging. In this article, we describe the development of Pichia pastoris KM71H strains capable of solubly producing the full set of predicted Cordyceps militaris hydrophobins CMil1 (Class IA), CMil2 (Class II), and CMil3 (IM) at mg/L yields with the use of 6His-tags not only for purification but for their detection. This result further demonstrates the feasibility of using P. pastoris as a host organism for the production of hydrophobins from all Ascomycota Class I subdivisions (a classification our previous work defined) as well as Class II. We highlight the specific challenges related to the production of hydrophobins, notably the challenge in detecting the protein that will be described, in particular during the screening of transformants. Together with the literature, our results continue to show that P. pastoris is a suitable host for the soluble heterologous expression of hydrophobins with a wide range of properties.

The basidiomycete Phanerochaete carnosa is a white-rot species that has been mainly isolated from coniferous softwood. Given the particular recalcitrance of softwoods to bioconversion, we conducted a comparative transcriptomic analysis of P. carnosa following growth on wood powder from one softwood (spruce; Picea glauca) and one hardwood (aspen; Populus tremuloides). P. carnosa was grown on each substrate for over one month, and mycelia were harvested at five time points for total RNA sequencing. Residual wood powder was also analyzed for total sugar and lignin composition. Following a slightly longer lag phase of growth on spruce, radial expansion of the P. carnosa colony was similar on spruce and aspen. Consistent with this observation, the pattern of gene expression by P. carnosa on each substrate converged following the initial adaptation. On both substrates, highest transcript abundances were attributed to genes predicted to encode manganese peroxidases (MnP), along with auxiliary activities from carbohydrate-active enzyme (CAZy) families AA3 and AA5. In addition, a lytic polysaccharide monooxygenase from family AA9 was steadily expressed throughout growth on both substrates. P450 sequences from clans CPY52 and CYP64 accounted for 50% or more of the most highly expressed P450s, which were also the P450 clans that were expanded in the P. carnosa genome relative to other white-rot fungi. The inclusion of five growth points and two wood substrates was important to revealing differences in the expression profiles of specific sequences within large glycoside hydrolase families (e.g., GH5 and GH16), and permitted co-expression analyses that identified new targets for study, including non-catalytic proteins and proteins with unknown function.