Introduction

With the growing global emphasis on sustainable development and the urgent pursuit of carbon neutrality, the use of renewable resources to replace traditional fossil feedstocks for biomanufacturing has become a key research focus in the field of industrial biotechnology. Methanol, which can be synthesized from CO₂ via photocatalysis or electrochemical reduction, has emerged as a renewable chemical feedstock with broad availability, ease of transportation, and strong compatibility with existing fermentation infrastructures. It not only enables the recycling of carbon resources but also reduces dependence on fossil fuels, thereby contributing to the achievement of carbon neutrality goals. Consequently, methanol is regarded as a highly promising next-generation low-carbon substrate for biomanufacturing (Li H., et al., Science, 2020; Kattel S., et al., Science, 2017; Graciani J., et al., Science, 2014).

In addition, as a liquid carbon source, methanol is easier to store and transport compared to gaseous CO₂, while also seamlessly integrating with current industrial fermentation facilities, which grants it significant advantages for large-scale industrial application^{(Saha D., et al., Chemical Reviews, 2016, Olah G. A., et al., Angewandte Chemie International Edition, 2013)}. Therefore, the development of novel microbial chassis with both efficient methanol-utilization capacity and broad product-synthesis potential is of great importance for advancing the industrialization of the methanol-based bioeconomy.

Methylotrophs are microorganisms capable of growing on methanol as the sole carbon source, making them ideal hosts for methanol-based fermentation.

Among eukaryotic microorganisms, Pichia pastoris, an important non-conventional yeast, has attracted considerable attention in synthetic biology and metabolic engineering due to its high protein expression capacity and unique methanol assimilation pathways. In recent years, extensive studies have focused on P. pastoris fermentation using methanol as the carbon source, including metabolic pathway optimization, application of gene editing technologies, and integration of systems and synthetic biology tools.

However, although natural methylotrophic microorganisms such as P. pastoris exhibit excellent methanol utilization capacity, their relatively simple metabolic networks and limited product spectra constrain their application in diversified biosynthesis^{(Chen T. J., et al., International Journal of Biological Macromolecules, 2020, Zou X., et al., Critical Reviews in Biotechnology, 2019, Zhao S. F., et al., Antonie van Leeuwenhoek, 2019)}. In contrast, Aureobasidium spp., a yeast-like fungus isolated from mangrove environments and belonging to the subphylum Pezizomycotina of Ascomycota, has been widely applied in industry, particularly for the high-yield production of pullulan (with titers up to 100 g/L)^{(Xue S. J., et al., International Journal of Biological Macromolecules, 2019)}. Moreover, this organism is capable of synthesizing a variety of high value-added products, including polymalic acid, gluconic acid, β-glucans, glycine, and melanin, demonstrating remarkable biotechnological potential^{(Chen T. J., et al., International Journal of Biological Macromolecules, 2020, Zou X., et al., Critical Reviews in Biotechnology, 2019, Zhao S. F., et al., Antonie van Leeuwenhoek, 2019)}.

Currently, the fermentation of Aureobasidium spp. primarily relies on traditional carbon sources in microbial engineering, such as sucrose and glucose. These substrates are costly and may compete with food resources, thereby limiting economic feasibility and sustainability^{(Xue S. J., et al., International Journal of Biological Macromolecules, 2019, Xue S. J., et al., Food Chemistry, 2019, Jiang H., et al., Food Chemistry, 2018)}. Therefore, introducing methanol assimilation pathways into Aureobasidium spp. to construct a methanol-based cell factory would not only broaden the product spectrum of methanol bioconversion and reduce production costs, but also enable the recycling of methanol and CO₂ byproducts generated in industrial processes. Such an approach aligns with strategies for energy conservation, emission reduction, and carbon neutrality, and offers new opportunities for sustainable green biomanufacturing.

Methanol metabolism in eukaryotic microorganisms relies on the coordinated function of specific subcellular compartments, with peroxisomes serving as critical sites for methanol oxidation and detoxification of the toxic intermediate formaldehyde.

Subcellular compartments and organelles contain specific proteins that determine their structural and catalytic capabilities. In certain cases, the same protein can be found in multiple compartments, indicating dual or multiple targeting, a phenomenon referred to as “dual targeting.” In this process, a single protein is localized to different subcellular compartments (e.g., mitochondria, cytosol, nucleus, or endoplasmic reticulum) via distinct mechanisms, participating in different biochemical pathways and sometimes performing entirely different functions^{(Karniely S., et al., EMBO Reports, 2005)}. Dual targeting plays an important role in methanol metabolism.

Peroxisomes are key organelles for methanol metabolism in eukaryotic microorganisms (Jiang W., et al., Nature Chemical Biology, 2021). In fungi, ribosome readthrough of peroxisomal targeting signals (PTS1) represents an important post-translational regulatory mechanism. Studies have shown that ribosomal readthrough of PTS1 enables the dual localization of glycolytic enzymes to both the cytosol and peroxisomes (Gould S. J., et al., The Journal of Cell Biology, 1989). Specifically, when the stop codon (TGA) of the PGK1 gene is replaced by a serine codon (TCA), translation continues to produce a C-terminally extended pgk1 pex isoform that contains a functional PTS1 signal (-PKL*), thereby directing the protein to peroxisomes (Freitag J., et al., Nature, 2012).

This project aims to elucidate and optimize the methanol metabolism of Aureobasidium melanogenum P16, and to construct a cell factory capable of efficiently producing glucose using methanol as the sole carbon source. Although the native methanol tolerance and metabolic capacity of P16 are lower than those of Pichia pastoris, it possesses diverse biosynthetic pathways, offering significant potential for metabolic engineering.

To further investigate and engineer the methanol metabolic pathway in P16, our team established a CRISPR-Cas9 gene knockout system specifically designed for this strain. This system enables efficient and precise editing of target genes. The reliability and accuracy of the system were validated by knocking out the Ade2 gene, which encodes phosphoribosyl pyrophosphate amidotransferase. This breakthrough provides a powerful tool platform for functional genomics research in non-model fungi.

Preliminary laboratory studies revealed that P16 can naturally tolerate a certain level of methanol and is able to grow on media with methanol as the sole carbon source for a limited period. However, after 24 hours, growth enters the stationary phase, and cell viability begins to decline after 36 hours. The methanol tolerance and utilization capability of P16 are weaker than those of P. pastoris.

Through genomic analysis and subcellular fluorescence co-localization experiments, we identified key methanol metabolism genes in P16, including AOX, DAS, and DAK. While AOX contains a typical peroxisomal targeting signal (PTS), DAS and DAK lack this signal. Based on literature, we hypothesized that their peroxisomal localization may rely on a ribosomal readthrough mechanism.

To test this, we designed simulated readthrough experiments to verify whether DAS and DAK in P16 depend on ribosomal readthrough for peroxisomal targeting. The results indicated that DAK localization to the peroxisome is ribosomal readthrough-dependent, whereas DAS showed suboptimal localization.

To address this limitation, we heterologously overexpressed the DAS gene. Introduction of the DAS gene from Komagataella pastoris significantly improved P16 growth on methanol medium, confirming the effectiveness of this strategy.

Following gene localization and functional validation, we conducted methanol metabolic engineering and carbon flux optimization experiments in P16. Heterologous expression of yihX, the haloacid dehalogenase-like phosphatase gene from Escherichia coli, was implemented to enhance conversion of metabolic intermediates to glucose; however, glucose production was not significantly increased. Subsequently, using a CRISPR-Cas9 plasmid designed by our team, we successfully knocked out the pfk gene, encoding phosphofructokinase, to reduce carbon flux competition in the glycolytic pathway, creating favorable conditions for methanol utilization. Product analysis demonstrated that the engineered P16 strain exhibited significantly increased glucose production on methanol medium.

This project not only represents the first successful application of CRISPR-Cas9 technology in Aureobasidium melanogenum P16, but also systematically elucidates and optimizes its methanol metabolic potential through a comprehensive metabolic engineering strategy. The study provides a novel biotechnological route to support the “methanol economy” and carbon neutrality initiatives, while demonstrating the considerable potential of P16 as a green microbial cell factory capable of producing high value-added products such as polysaccharides, organic acids, and enzymes. This exploration not only expands the scope of iGEM research into non-model fungi but also highlights the broad applications of synthetic biology in promoting sustainable development.

① Breakthrough in gene editing tools: a CRISPR-Cas9 gene knockout system was successfully constructed and validated in A. melanogenum P16 for the first time, providing a novel technical platform for genetic manipulation and functional genomics research in non-model fungi.

② Elucidation of methanol metabolism mechanisms: the subcellular localization characteristics of methanol metabolism-related genes in P16 were systematically analyzed. Using simulated ribosomal readthrough and heterologous gene overexpression strategies, the peroxisomal targeting mechanisms of key genes were verified.

③ Metabolic engineering strategies: by knocking out the glycolytic key gene pfk and introducing the yihX gene from Escherichia coli, carbon flux competition was effectively reduced, leading to enhanced glucose synthesis efficiency.

Looking forward, this project aims to systematically analyze the methanol metabolic network of P16 on a broader scale, identify additional key genes and regulatory factors, and construct highly efficient and stable methanol-utilizing cell factories through multi-gene editing and metabolic pathway reconstruction. Using the P16 platform, high value-added products such as polysaccharides, organic acids, and enzymes can be synthesized, providing biotechnological support for the development of the “methanol economy” and carbon neutrality strategies.

1. [1] Li, H., Qiu, C., Ren, S., Dong, Q., Zhang, S., Zhou, F., Liang, X., Wang, J., Li, S., & Yu, M. (2020). Na+-gated water-conducting nanochannels for boosting CO2 conversion to liquid fuels. Science, 367(6478), 667–671.
2. [2] Kattel, S., Ramírez, P. J., Chen, J. G., Rodriguez, J. A., & Liu, P. (2017). Response to Comment on "Active sites for CO2 hydrogenation to methanol on Cu/ZnO catalysts". Science, 357(6354), eaan8210.
3. [3] Graciani, J., Mudiyanselage, K., Xu, F., Baber, A. E., Evans, J., Senanayake, S. D., Stacchiola, D. J., Liu, P., Hrbek, J., Fernández Sanz, J., & Rodriguez, J. A. (2014). Highly active copper-ceria and copper-ceria-titania catalysts for methanol synthesis from CO₂. Science, 345(6196), 546–550.
4. [4] Tedeeva, M. A., Kustov, A. L., Batkin, A. M., Garifullina, C., Zalyatdinov, A. A., Yang, D., Dai, Y., Yang, Y., & Kustov, L. M. (2024). Catalytic systems for hydrogenation of CO2 to methanol. Molecular Catalysis, 566, 114403.
5. [5] Saha, D., Grappe, H. A., Chakraborty, A., & Orkoulas, G. (2016). Postextraction Separation, On-Board Storage, and Catalytic Conversion of Methane in Natural Gas: A Review. Chemical Reviews, 116(19), 11436–11499.
6. [6] Olah, G. A. (2013). Towards oil independence through renewable methanol chemistry. Angewandte Chemie International Edition, 52(1), 104–107.
7. [7] Gao, J., Li, Y., Yu, W., & Zhou, Y. J. (2022). Rescuing yeast from cell death enables overproduction of fatty acids from sole methanol. Nature Metabolism, 4(7), 932–943.
8. [8] Jiang, W., Hernández Villamor, D., Peng, H., Chen, J., Liu, L., Haritos, V., & Ledesma-Amaro, R. (2021). Metabolic engineering strategies to enable microbial utilization of C1 feedstocks. Nature Chemical Biology, 17(8), 845–855.
9. [9] Chen, T. J., Liu, G. L., Chen, L., Yang, G., Hu, Z., Chi, Z. M., & Chi, Z. (2020). Alternative primers are required for pullulan biosynthesis in Aureobasidium melanogenum P16. International Journal of Biological Macromolecules, 147, 10–17.
10. [10] Zou, X., Cheng, C., Feng, J., Song, X., Lin, M., & Yang, S. T. (2019). Biosynthesis of polymalic acid in fermentation: advances and prospects for industrial application. Critical Reviews in Biotechnology, 39(3), 408–421.
11. [11] Zhao, S. F., Jiang, H., Chi, Z., Liu, G. L., Chi, Z. M., Chen, T. J., Yang, G., & Hu, Z. (2019). Genome sequencing of Aureobasidium pullulans P25 and overexpression of a glucose oxidase gene for hyper-production of Ca2+-gluconic acid. Antonie van Leeuwenhoek, 112(5), 669–678.
12. [12] Xue, S. J., Jiang, H., Chen, L., Ge, N., Liu, G. L., Hu, Z., Chi, Z. M., & Chi, Z. (2019). Over-expression of Vitreoscilla hemoglobin (VHb) and flavohemoglobin (FHb) genes greatly enhances pullulan production. International Journal of Biological Macromolecules, 132, 701–709.
13. [13] Wang, P., Jia, S.-L., Liu, G.-L., Chi, Z., & Chi, Z.-M. (2022). Aureobasidium spp. and their applications in biotechnology. Process Biochemistry, 116, 72–83.
14. [14] Xue, S. J., Chen, L., Jiang, H., Liu, G. L., Chi, Z. M., Hu, Z., & Chi, Z. (2019). High pullulan biosynthesis from high concentration of glucose by a hyperosmotic resistant, yeast-like fungal strain isolated from a natural comb-honey. Food Chemistry, 286, 123–128.
15. [15] Jiang, H., Xue, S. J., Li, Y. F., Liu, G. L., Chi, Z. M., Hu, Z., & Chi, Z. (2018). Efficient transformation of sucrose into high pullulan concentrations by Aureobasidium melanogenum TN1-2 isolated from a natural honey. Food Chemistry, 257, 29–35.
16. [16] Karniely, S., & Pines, O. (2005). Single translation--dual destination: mechanisms of dual protein targeting in eukaryotes. EMBO Reports, 6(5), 420–425.
17. [17] Gould, S. J., Keller, G. A., Hosken, N., Wilkinson, J., & Subramani, S. (1989). A conserved tripeptide sorts proteins to peroxisomes. The Journal of Cell Biology, 108(5), 1657–1664.
18. [18] Freitag, J., Ast, J., & Bölker, M. (2012). Cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi. Nature, 485(7399), 522–525.