Supplementary Material for: Omics-based investigation on mechanisms controlling cellular internalization of keratin monomers during biodegradation by Stenotrophomonas maltophilia DHHJ

Introduction: The global poultry industry produces millions of tons of waste feathers every year, which can be degraded to make feed, fertilizer, and daily chemicals. However, feather degradation is a complex process that is not yet fully understood. This results in low degradation efficiency and difficulty in industrial applications. Omics-driven system biology research offers an effective solution to quickly and comprehensively understand the molecules and mechanisms involved in a metabolic pathway. Methods: In the early stage of this process, feathers are hydrolyzed into water-soluble keratin monomers. In this study, we used high-throughput RNA-seq technology to analyze the genes involved in the internalization and degradation of keratin monomers in S. maltophilia DHHJ strain cells. Moreover, we used Co-IP with LC-MS/MS technology to search for proteins that interact with recombinant keratin monomers. Results: We discovered TonB transports and molecular chaperones associating with the keratin monomer, which may play a crucial role in the transmembrane transport of keratin. Meanwhile, multiple proteases belonging to distinct families were identified as binding partners of keratin monomers, among which ATPases associated with diverse cellular activities (AAA+) family proteases are overrepresented. Four genes, including JJL50_15620, JJL50_17955 (TonB-dependent receptors), JJL50_03260 (ABC transporter ATP-binding protein), and JJL50_20035 (ABC transporter substrate-binding protein), were selected as representatives for determining their expressions under different culture conditions using qRT-PCR and they were found to be upregulated in response to keratin degradation consistent with the data from RNA-seq and Co-IP. Conclusion: This study highlights the complexity of keratin biodegradation in S. maltophilia DHHJ, in which multiple pathways are involved such as protein folding, protein transport, and several protease systems. Our findings provide new insights into the mechanism of feather degradation.

引言：全球家禽养殖业每年产生数百万吨废弃羽毛，这些羽毛可被降解用于制备饲料、肥料及日用化学品。然而，羽毛降解是一个尚未被完全阐明的复杂过程，导致降解效率低下且难以实现工业应用。以组学（Omics）为驱动的系统生物学研究，为快速且全面地解析代谢通路中涉及的分子与作用机制提供了有效解决方案。
方法：在该降解过程的早期阶段，羽毛被水解为水溶性角蛋白单体。本研究采用高通量RNA测序（RNA-seq）技术，分析嗜麦芽窄食单胞菌（S. maltophilia）DHHJ菌株细胞内参与角蛋白单体内化与降解的基因。此外，本研究结合免疫共沉淀（Co-IP）与液相色谱-串联质谱（LC-MS/MS）技术，筛选与重组角蛋白单体相互作用的蛋白质。
结果：本研究发现了与角蛋白单体结合的TonB转运蛋白及分子伴侣，它们可能在角蛋白的跨膜转运过程中发挥关键作用。同时，多个隶属于不同家族的蛋白酶被鉴定为角蛋白单体的结合伴侣，其中ATP酶关联多种细胞活动（AAA+）家族蛋白酶的占比显著偏高。本研究选取4个基因作为代表，包括JJL50_15620、JJL50_17955（TonB依赖型受体）、JJL50_03260（ABC转运体ATP结合蛋白）以及JJL50_20035（ABC转运体底物结合蛋白），通过实时定量荧光PCR（qRT-PCR）检测它们在不同培养条件下的表达水平，结果显示这些基因在角蛋白降解过程中均出现上调表达，与RNA-seq及Co-IP的实验数据一致。
结论：本研究揭示了嗜麦芽窄食单胞菌DHHJ菌株对角蛋白的生物降解过程具有复杂性，该过程涉及蛋白质折叠、蛋白质转运以及多种蛋白酶系统等多条通路。本研究结果为阐明羽毛降解的作用机制提供了全新的视角。