Caldicellulosiruptor bescii is the most thermophilic, cellulolytic bacterium known and has the native ability to utilize unpretreated plant biomass. Cellulase A (CelA) is the most abundant enzyme in the exoproteome of C. bescii and is primarily responsible for its cellulolytic ability. CelA contains a family 9 glycoside hydrolase and a family 48 glycoside hydrolase connected by linker regions and three carbohydrate-binding domains. A truncated version of the enzyme (TM1) containing only the endoglucanase domain is thermostable and actively degrades crystalline cellulose. A catalytically active TM1 was successfully produced via the attachment of the PelB signal peptide (P-TM1), which mediates post-translational secretion via the SecB-dependent translocation pathway. We sought to enhance the extracellular secretion of TM1 using an alternative pathway, the signal recognition particle (SRP)-dependent translocation pathway. The co-translational extracellular secretion of TM1 via the SRP pathway (D-TM1) resulted in a specific activity that was 4.9 times higher than that associated with P-TM1 overexpression. In batch fermentations, the recombinant Escherichia coli overexpressing D-TM1 produced 1.86 ± 0.06 U/ml of TM1 in the culture medium, showing a specific activity of 1.25 ± 0.05 U/mg cell, 2.7- and 3.7-fold higher than the corresponding values of the strain overexpressing P-TM1. We suggest that the TM1 secretion system developed in this study can be applied to enhance the capacity of E. coli as a microbial cell factory for the extracellular secretion of this as well as a variety proteins important for commercial production.

中文翻译：

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肽聚糖网络的扰动和信号识别粒子依赖性途径的利用增强了大肠杆菌中 CelA 截短突变体的细胞外产生

Caldicellulosiruptor bescii 是已知的最嗜热的纤维素分解细菌，具有利用未经预处理的植物生物质的天然能力。纤维素酶 A (CelA) 是贝氏梭菌外蛋白质组中最丰富的酶，主要负责其纤维素分解能力。CelA 包含一个家族 9 糖苷水解酶和一个家族 48 糖苷水解酶，通过接头区域和三个碳水化合物结合结构域连接。仅包含内切葡聚糖酶结构域的酶 (TM1) 的截短版本是热稳定的，并且会主动降解结晶纤维素。通过附着 PelB 信号肽 (P-TM1) 成功产生了具有催化活性的 TM1，该信号肽通过 SecB 依赖性易位途径介导翻译后分泌。我们试图使用另一种途径，即信号识别粒子 (SRP) 依赖性易位途径来增强 TM1 的细胞外分泌。TM1 通过 SRP 途径 (D-TM1) 的共翻译细胞外分泌导致比与 P-TM1 过表达相关的活性高 4.9 倍。在分批发酵中，过表达 D-TM1 的重组大肠杆菌在培养基中产生 1.86 ± 0.06 U/ml 的 TM1，显示比活性为 1.25 ± 0.05 U/mg 细胞，比相应值高 2.7 倍和 3.7 倍过表达 P-TM1 的菌株。我们建议本研究开发的 TM1 分泌系统可用于增强大肠杆菌的能力。