Poster M20 - Engineering Filamentous Fungi for the Valorization of Agricultural Wastes

Introduction: Exposure to particulate matter less than 2.5µm in diameter (PM2.5) from biomass burning is the sixth-highest cause of mortality worldwide [Nunta, 2023]. Of all agricultural residues burned, rice straw waste is the single most common–accounting for 40% of this waste [Kumar, 2023]. As most agricultural waste streams–including rice straw–are voluminous and have minimal nutritional or commercial value, cost-effectiveness is essential for devising waste management solutions for smallholder farmers to reduce field burning rates. While attempts to disincentivize field burning by valorizing such wastes have been made, these efforts, mainly centered around processing waste into combustion fuels, still only consume 20% of global rice straw produced due to the high infrastructure costs [Singh, 2024]. We have selected the filamentous fungus Aspergillus Oryzae to develop a system for producing high-value commercial enzymes from rice straw as a model waste. While understudied compared to model organisms like S. Cerevisiae and E. coli, A. oryzae has proven to be far more robust and capable of lower cost fermentation in solid-state compared to more common submerged fermenters, though until recently the former organisms have dominated due to the ease with which they can be engineered. Recently, powerful CRISPR-based genetic engineering tools have been developed which we are deploying and further expanding to demonstrate the use of agricultural waste in solid-state fungal-based fermentors to produce valuable enzymes and curb field burning in a cost-effective and scalable manner.

Materials and

Methods: Our goal in this work was to produce and cultivate a variant of A. oryzae capable of inducibly expressing exogenous DNA and demonstrate growth of this modified strain on rice straw. Using a novel CRISPR-Cas9 method for genetic engineering of A. oryzae described in “Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit”, we set about transforming a uridine-uracil auxotroph of A. oryzae RIB40 with an expression vector that firstly restored prototrophy and secondly inserted a starch-inducible promoter and enhanced green fluorescent protein cassette (EGFP) gene at a locus of interest. In the case of this study, this locus was in the middle of the endogenous pigmentation gene, such that successful transformation would result in disruption of its expression. RIB40 protoplasts were generated by yatalase digestion and transformation was performed using the construct detailed previously. Selection against auxotrophs was conducted by recovering transformed protoplasts in uridine-uracil deficient medium and transformations were verified by treatment with dextrin and fluorescence microscopy to test for EGFP expression and orthogonally by observation of the pigmentation phenotype–as successful transformation interferes with wild-type (WT) pigment production.
In parallel, surplus rice straw was sourced and treated with calcium hydroxide and sterilized by autoclaving. Both WT and engineered RIB40 were cultivated on these substrates in the presence of varying amounts of supplemental yeast-peptone-dextrose (YPD) broth to accelerate growth.

Results, Conclusions, and Discussions: The results shown in Figure 1 indicate successful transformation with our dextrin-inducible EGFP expression system. The transformation was efficient for A. oryzae, with 5 cfu/µg DNA and minimal off-target mutagenesis (as indicated by the preponderance of non-pigmented colonies). EGFP expression was observed via fluorescence microscopy performed on liquid culture samples, observing consistent expression throughout (Figure 1). Expression was strongest in larger multicellular tissues rather than individual fungal spores, suggesting fundamental differences in expression patterns between these cell types. Substantial expression of EGFP in fungi cultivated on rice straw was also observed (Figure 2).
We confirmed A. oryzae’s viability as a model organism for genetic modifications and inducible expression of exogenous proteins. Engineered A. oryzae can survive on cellulosic wastes such as rice straw while maintaining expression of these exogenous proteins, critically demonstrating the feasibility of our approach. These results suggest that, with limited organismal modification, we can reliably grow A. oryzae in large single-batch solid-state cultures and engineer it to produce high-value enzymes (other than EGFP).
Our proof-of-concept study has demonstrated both the ability to engineer A. oryzae and successfully cultivate it on waste rice straw, and we are confident that further exploration to actualize the potential of our application is warranted and promising. In the immediate term, we will generate a construct that expresses a commercially-relevant enzyme, xylanase, which is widely used in industrial applications. When we generate this engineered strain, we will purify and quantify the yield and enzymatic activity of xylanase across different growth conditions to elucidate the effects of solid-state rice straw growth on protein expression and functionality.
Tools for engineering filamentous fungi on a cellular and organismal level are underdeveloped and artisanal compared to common model organisms. We propose investigation of alternative promoters with a broad array of inducers and further exploration of the fungal genome for creating a minimal-genome chassis capable of greater engineering modularity while preserving the inherent advantages of fungi over bacterial models. While current methods and expertise allow for significant engineering and improvement of these organisms, additional investigation is required to develop these tools and realize these organisms’ full potential.

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