Tanaka Kenya, Yukawa Takahiro, Bamba Takahiro, Wakiya Miho, Kumokita Ryota, Jin Yong-Su, Kondo Akihiko, Hasunuma Tomohisa
Engineering Biology Research Center, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
Graduate School of Science, Innovation and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
Appl Microbiol Biotechnol. 2025 Jan 28;109(1):30. doi: 10.1007/s00253-025-13417-1.
The fermentative production of valuable chemicals from lignocellulosic feedstocks has attracted considerable attention. Although Saccharomyces cerevisiae is a promising microbial host, it lacks the ability to efficiently metabolize xylose, a major component of lignocellulosic feedstocks. The xylose oxidative pathway offers advantages such as simplified metabolic regulation and fewer enzymatic steps. Specifically, the pathway involves the conversion of xylose into 2-keto-3-deoxy-xylonate, which can be channeled into two distinct pathways, the Dahms pathway and the Weimberg pathway. However, the growth of yeast on xylose as the sole carbon source through the xylose oxidative pathway has not been achieved, limiting its utilization. We successfully engineered S. cerevisiae to metabolize xylose as its sole carbon source via the xylose oxidative pathways, achieved by enhancing enzyme activities through iron metabolism engineering and rational enzyme selection. We found that increasing the supply of the iron-sulfur cluster to activate the bottleneck enzyme XylD by BOL2 disruption and tTYW1 overexpression facilitated the growth of xylose and the production of ethylene glycol at 1.5 g/L via the Dahms pathway. Furthermore, phylogenetic analysis of xylonate dehydratases led to the identification of a highly active homologous enzyme. A strain possessing the Dahms pathway with this highly active enzyme exhibited reduced xylonate accumulation. Furthermore, the introduction of enzymes based on phylogenetic tree analysis allowed for the utilization of xylose as the sole carbon source through the Weimberg pathway. This study highlights the potential of iron metabolism engineering and phylogenetic enzyme selection for the development of non-native metabolic pathways in yeast. KEY POINTS: • A 1.5 g/L ethylene glycol was produced via the Dahms pathway in S. cerevisiae. • Enzyme activation enabled growth on xylose via both the Dahms and Weimberg pathways. • Tested enzymes in this study may expand the application of xylose oxidative pathway.
利用木质纤维素原料发酵生产有价值的化学品已引起了广泛关注。尽管酿酒酵母是一种很有前景的微生物宿主，但它缺乏有效代谢木糖的能力，而木糖是木质纤维素原料的主要成分。木糖氧化途径具有代谢调控简化和酶促步骤较少等优势。具体而言，该途径涉及木糖转化为2-酮-3-脱氧木糖酸，其可进入两条不同的途径，即达姆斯途径和魏姆贝格途径。然而，通过木糖氧化途径使酵母以木糖作为唯一碳源生长尚未实现，这限制了其利用。我们通过铁代谢工程和合理的酶选择来增强酶活性，成功改造了酿酒酵母，使其能够通过木糖氧化途径将木糖作为唯一碳源进行代谢。我们发现，通过破坏BOL2和过表达tTYW1来增加铁硫簇的供应以激活瓶颈酶XylD，促进了木糖的生长，并通过达姆斯途径以1.5 g/L的产量生产了乙二醇。此外，对木糖酸脱水酶的系统发育分析导致鉴定出一种高活性的同源酶。具有该高活性酶的达姆斯途径菌株表现出木糖酸积累减少。此外，基于系统发育树分析引入酶使得能够通过魏姆贝格途径利用木糖作为唯一碳源。本研究突出了铁代谢工程和系统发育酶选择在酵母中非天然代谢途径开发方面的潜力。要点：• 酿酒酵母通过达姆斯途径生产了1.5 g/L的乙二醇。• 酶的激活使酵母能够通过达姆斯途径和魏姆贝格途径在木糖上生长。• 本研究中测试的酶可能会扩大木糖氧化途径的应用。
