Heterologous Expression of Fungal Enzymes in two Aspergillus oryzae strains: A Comparative Study for Sustainable Plastic Recycling

Abstract

This thesis explores the heterologous expression of six fungal enzymes with potential polyurethane (PUR) degrading activity in two strains of the filamentous fungus Aspergillus oryzae: the standard Ory6 and a knock-out variant with disrupted transcription factors to potentially reduce native protease activity. Each enzyme was expressed in three gene formats: a native version (including any introns), exon-only version (excluding any introns), and a codon-optimized synthetic version (excluding any introns). The aim was to evaluate how gene design and host background influence protein expression in A. oryzae as a model fungal cell factory.
The results showed that while codon-optimized constructs generally yielded higher transformation success, functional protein expression, detectable via fluorescence, was inconsistent. In several cases, gene formats with confirmed integration failed to express detectable protein, which underscores the influence of factors such as protein folding and post-transcriptional processing. Notably, the exon-only version of the gene coding for an amidase from Fusarium solani (Enzyme 4) was successfully expressed, which indicates that intron removal via fusion PCR can serve as a cost-effective alternative to full codon optimized gene synthesis. On the contrary, the introns-containing native version of an amidase from Penicillium griseofulvum (Enzyme 5) outperformed both the exon-only and synthetic versions suggesting that introns can be critical for proper expression.
Overall, this study highlights the complexity and unpredictability of heterologous protein expression in the filamentous fungus A. oryzae. It demonstrates that even with codon optimization and confirmed genomic integration, successful expression cannot be assumed. These findings contribute to a deeper understanding of gene design strategies in fungal expression systems and support future efforts in enzyme-based plastic recycling solutions.

Popular Abstract

Plastic-Eating Fungi: A Study of Gene Expression in Aspergillus oryzae
In our modern world, plastics are everywhere, from sneakers and mattresses to insulation and packaging. Their widespread use comes from their excellent material properties. But those same properties also make certain plastics extremely difficult to recycle. One of the most challenging types is the plastic type called polyurethane. Unlike many plastics, polyurethane can’t simply be melted down and be reused. This has led to serious environmental problems. Imagine a mattress sitting in a landfill for hundreds of years, without being broken down. But what if the solution to this recycling problem comes from fungi, nature’s natural decomposers?
In my thesis, I explored whether a fungal species called Aspergillus oryzae could be genetically engineered to produce enzymes from other fungi capable of breaking down polyurethane. To support this research, I used a newly developed and efficient CRISPR/Cas9-based method to introduce six different enzyme genes into Aspergillus oryzae. Each gene had potential polyurethane-degrading activity, and I tested whether the fungi could successfully produce the corresponding enzymes. Each gene was introduced in three formats: the original version (which includes non-coding segments of DNA known as introns), an “exon-only” version (containing only the coding regions), and a codon-optimized synthetic version of the gene which was genetically rewritten for optimal expression in the fungi. I also compared two strains of Aspergillus oryzae: a standard version, and a “knock-out” version that’s missing two genes involved in protease regulation. This was to test whether reducing the fungus natural protein-degrading activity would help protect these foreign enzymes from being broken down by the fungi itself. To track whether the enzymes were successfully expressed, I used a yellow fluorescent marker fused to the enzymes that were to be expressed. If the fungi glowed under the UV light, it meant that the protein was successfully expressed.
From this study, it became clear that simply inserting a gene isn’t enough, many enzymes weren’t expressed even when the gene was correctly integrated. Successful expression was more common when the gene was synthetically codon-optimized. However, one exon-only version (without codon optimization) also worked, suggesting optimization isn’t always necessary. Interestingly, another enzyme was only expressed when introns were included, indicating that these non-coding segments may play a crucial role in gene function and proper protein folding.
Identifying successful polyurethane degrading enzymes requires deeper bioinformatic knowledge to understand their function, but also a better understanding of the challenges and strategies involved in heterologous expression to utilize these enzymes for sustainable recycling. This thesis highlights how gene expression in fungi is more complex and unpredictable than one might assume. The biology of fungi doesn’t always follow the engineering logic we expect.

• author: Ewaldh, Axel
• supervisor: Johan Svensson Bonde; Uffe Hasbro Mortensen; Robert Hansen Jagrelius
• organization: Pure and Applied Biochemistry
• course: KBKM05 20251
• year: 2025
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• keywords: Heterologous expression, Aspergillus oryzae, CRISPR/Cas9, Rolling Circle Integration, DIVERSIFY-platform, Enzyme-based plastic recycling, Applied biochemistry
• language: English
• id: 9199322
• date added to LUP: 2025-06-16 13:47:22
• date last changed: 2025-06-16 13:47:22