Engineered living materials grown from programmable Aspergillus niger mycelial pellets

Abstract

The development of engineered living materials (ELMs) has recently attracted significant attention from researchers across multiple disciplines. Fungi-derived ELMs represent a new type of macroscale, cost-effective, environmentally sustainable materials. However, current fungi-based ELMs either have to undergo a final process to heat-kill the living cells or rely on the co-culture with a model organism for functional modification, which hinders the engineerability and versatility of these materials. In this study, we report a new type of ELMs - grown from programmable Aspergillus niger mycelial pellets - by a simple filtration step under ambient conditions. We demonstrate that A. Niger pellets can provide sufficient cohesion to maintain large-area self-supporting structures even under low pH conditions. Subsequently, by tuning the inducible expression of genes involved in melanin biosynthesis, we verified the fabrication of self-supporting living membrane materials with tunable colors in response to xylose concentration in the surroundings, which can be further explored as a potential biosensor for detecting xylose level in industrial wastewater. Notably, the living materials remain alive, self-regenerative, and functional even after 3-month storage. Thus, beyond reporting a new engineerable fungi chassis for constructing ELMs, our study provides new opportunities for developing bulk living materials for real-world applications such as the production of fabrics, packaging materials, and biosensors.

Keywords: Aspergillus niger; Engineered living material; Filamentous fungus; Genetic circuit; Melanin.

Graphical abstract

Fig. 1

Schematic showing the design and production of a living membrane material grown from A. niger. A, Schematic showing the typical steps to produce mycelial pellets from A. niger in shaking cultures; B, Schematic showing the major components of the hypha; C, Schematic showing the melanin biosynthesis pathway and genomic organization of the genes involved in melanin biosynthesis; D, The typical protocol to produce a living membrane material grown from the engineered A. niger.

Fig. 2

Fabrication of living membrane materials from A. niger. A, Reprentative images of mycelial pellets produced by A. nigar under different pH conditions; B, SEM images of mycelial pellets produced by A. nigar under different pH conditions; C, Distribution of hyphal diameters of the living material grown from the Spxyn-pptA strain under different pH conditions; D, Typical images of the as-prepared membrane, membrane after drying, and self-dyed membrane; E, TGA thermograms of dried membrane and self-dyed membrane; F, representative stress/strain curves of dried membrane and self-dyed membrane.

Fig. 3

Design and characterization of xylose-sensing living membrane materials based on the two A. nigar strains. A, Schematic showing the construction of the SΔpptA and Spxyn-pptA strains for producing living membrane materials. 5′f and 3′f represent the upstream and downstream flanking sequences of pptA gene. RB and LB represent the right and left border sequences, respectively. B, The typical morphologies of living membrane materials grown from the wild type A. niger and the mutants (SΔpptA, and Spxyn-pptA), respectively. The indicated stains were cultured on PDA supplemented with different concentrations of 0.01, 0.03, 0.05, 0.07, and 0.1 ​wt% xylose for 3 days. C, UV–Vis spectra for the strain Spxyn-pptA 0.01, 0.03, 0.05, 0.07, and 0.1 ​wt% xylose. D, ATR−FTIR spectra for induced with 0.01, 0.03, 0.05, 0.07, and.

Fig. 4

Fabrication, physical, and structural characteristics of two different kinds of living membrane materials. A-D, Digital camera images (A–B) and SEM images (C–D) showing the surface morphology of living membrane materials grown from engineered A.nigar SΔpptA and engineered A.nigar Spxyn-pptA strains, respectively; E, representative stress/strain curves of living materials produced from engineered A. nigar SΔpptA and Spxyn-pptA strains in the presence 0.1 ​wt% xylose added in culture media; F, Bending curve profile for living membrane materials grown from engineered A. nigar SΔpptA and Spxyn-pptA strains in the presence of 0.1 ​wt % xylose added to culture media. G-H, schematic and digital images showing the tunable colors of selfdyed living membrane materials grown from engineered A. niger Spxyn-pptA strain, which secretes a controllable amount of melanin under different concentration of xylose. Note that the degree of color in turn reflects the amount of xylose concentration, implying a potential biosensor for detecting xylose in industrial waste. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Self-regeneration of living membrane materials. (A–D) Optical images of selfregeneration of living membrane material on agar plates for two continuous generations. From left to right: the xylose-induced first generation of living membrane material (GenI), partial fragments (∼3 ​mg) of the induced (GenI) cultured in media, the second generation of living membrane material (GenII), the xylose-induced GenII. (E) The regeneration of a piece of living membrane material stored for 3 months at 4 ​°C in a fresh agar plate.