Ma Kexuan, Zhang Panpan, Zhao Jian, Qin Yuqi
State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China.
National Glycoengineering Research Center, Shandong University, Qingdao, China.
Biotechnol Biofuels Bioprod. 2025 Feb 22;18(1):20. doi: 10.1186/s13068-025-02624-7.
The production of cellulases by filamentous fungi is a crucial aspect of sustainable bioproduction from renewable lignocellulosic biomass. Following the transcription of cellulase genes in the nucleus, a complex pathway involving translation, folding, and secretion is required to produce extracellular cellulases. Most studies about cellulase production have focused on examining transcriptional regulatory mechanisms and enhancement of enzyme gene levels; comparatively, little is known about protein translation and secretion for cellulase production.
A translation-machinery-associated (TMA) protein PoTma15 was identified in cellulosic Penicillium oxalicum. The PoTma15 is conserved in various filamentous fungi, but not in yeast, plants, or animals. All homologous proteins of PoTma15 have previously been uncharacterized. PoTma15 was initially thought to be one of the putative interactors of transcription factor PoXlnR, as it was preyed by tandem affinity purification (TAP) coupled with the mass spectrometry (TAP-MS) technique using PoXlnR as the bait. Subsequent research revealed that PoTma15 is associated with the translation machinery. The top three proteins associated with PoTma15 are orthologs of Saccharomyces cerevisiae translation-machinery-associated protein (Tma19), translation elongation factor eIF5A, and ribosomal protein S28, respectively. PoTma15 is widely distributed in fungal hyphae and positively correlates with the production of cellulases and extracellular proteins. Deleting the Potma15 gene (Δtma15) decreased cellulase production, while overexpressing the Potma15 gene (OEtma15) increased cellulase production. However, the Δtma15 mutant was not observed to have downregulated transcript levels of major (hemi)cellulase and amylase genes, compared to the P. oxalicum wild type (WT). The production of extracellular cellulases and extracellular proteins of the Δtma15 mutant was less affected by cycloheximide, an inhibitor of eukaryotic translation elongation, compared to the WT strain and OEtma15 mutant, suggesting a stronger resistance to the translation-inhibiting effects of cycloheximide in the Δtma15 mutant. The results demonstrate that PoTma15 is a translation-machinery-associated protein that affects translation elongation and, consequently, the production of enzyme proteins.
PoTma15 is the first TMA protein characterized in cellulosic filamentous fungi and the first TMA protein used in fungi to increase cellulase production. PoTma15's role in the production of cellulases and total extracellular proteins suggests that not only can it be used to widen the cellulase production pathway, but can even be engineered as a target to improve the production of other heterologous protein or bioproducts using filamentous fungi as cell factories in the future.
丝状真菌产生纤维素酶是可再生木质纤维素生物质可持续生物生产的关键环节。纤维素酶基因在细胞核中转录后，需要一个涉及翻译、折叠和分泌的复杂途径来产生细胞外纤维素酶。大多数关于纤维素酶生产的研究都集中在研究转录调控机制和提高酶基因水平上；相比之下，对于纤维素酶生产中的蛋白质翻译和分泌了解较少。
在产纤维素的草酸青霉中鉴定出一种与翻译机器相关（TMA）的蛋白质PoTma15。PoTma15在各种丝状真菌中保守，但在酵母、植物或动物中不保守。PoTma15的所有同源蛋白此前均未被表征。PoTma15最初被认为是转录因子PoXlnR的假定相互作用蛋白之一，因为在以PoXlnR为诱饵的串联亲和纯化（TAP）结合质谱（TAP-MS）技术中它是猎物。随后的研究表明PoTma15与翻译机器相关。与PoTma15相关的前三种蛋白质分别是酿酒酵母翻译机器相关蛋白（Tma19）、翻译延伸因子eIF5A和核糖体蛋白S28的直系同源物。PoTma15广泛分布于真菌菌丝中，与纤维素酶和细胞外蛋白的产生呈正相关。删除Potma15基因（Δtma15）会降低纤维素酶的产生，而过表达Potma15基因（OEtma15）会增加纤维素酶的产生。然而，与草酸青霉野生型（WT）相比，未观察到Δtma15突变体中主要（半）纤维素酶和淀粉酶基因的转录水平下调。与WT菌株和OEtma15突变体相比，Δtma15突变体的细胞外纤维素酶和细胞外蛋白的产生受真核翻译延伸抑制剂环己酰亚胺的影响较小，这表明Δtma15突变体对环己酰亚胺的翻译抑制作用具有更强的抗性。结果表明PoTma15是一种与翻译机器相关的蛋白质，它影响翻译延伸，从而影响酶蛋白的产生。
PoTma15是在产纤维素丝状真菌中鉴定出的首个TMA蛋白，也是真菌中首个用于提高纤维素酶产量的TMA蛋白。PoTma15在纤维素酶和总细胞外蛋白产生中的作用表明，它不仅可用于拓宽纤维素酶生产途径，甚至未来还可被设计为一个靶点，以利用丝状真菌作为细胞工厂来提高其他异源蛋白或生物产品的产量。
