Efficient secretory production of large-size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution.,Biotechnology and Bioengineering

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Efficient secretory production of large-size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution.
Biotechnology and Bioengineering ( IF 3.5 ) Pub Date : 2020-06-26 , DOI: 10.1002/bit.27478
Shan Liu ₁ , Juan Wang ₁ , Zhiguang Zhu ₁ , Ting Shi ₁ , Yi-Heng P Job Zhang ₁

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Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large‐size heterologous enzymes with a special focus on 83‐kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5‐IA was improved by directed evolution with two novel double‐layer Petri‐dish‐based high‐throughput screening (HTS) methods. The high‐sensitivity HTS relied on the detection of high‐activity Cel5 on the carboxymethylcellulose/Congo‐red assay. The second modest‐sensitivity HTS focused on the detection of low‐activity IA on the amylodextrin‐I₂ assay. After six rounds of HTS, a secretory Cel5‐IA level was increased to 234 mg/L, 155 times the wild‐type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large‐size heterologous proteins in B. subtilis.

中文翻译：

枯草芽孢杆菌中大尺寸异源酶的高效分泌生产：分泌伙伴和定向进化。

重组蛋白的分泌生产为酶工业中目标蛋白的生产和纯化提供了一种简单的方法。我们从开发分泌生产的三个大尺寸的外源酶的联合战略，特别注重83 kDa的异淀粉酶（IA）的古硫化叶菌的细菌枯草芽孢杆菌。首先，枯草杆菌家族 5 糖苷水解酶内切葡聚糖酶 (Cel5)的分泌蛋白与 NprB 信号肽一起用作融合伙伴，以促进 IA 的分泌产生。这种分泌伙伴策略对于分泌另外两种大酶是有效的：来自Clostridium phytofermentas 的家族 9 糖苷水解酶和来自热纤梭菌的纤维糊精磷酸化酶。其次，Cel5-IA 的分泌通过使用两种新型双层培养皿高通量筛选 (HTS) 方法的定向进化得到改善。高灵敏度 HTS 依赖于在羧甲基纤维素/刚果红测定中检测高活性 Cel5。第二个中等灵敏度的 HTS 侧重于在淀粉糊精-I ₂测定中检测低活性 IA 。六轮 HTS 后，分泌性 Cel5-IA 水平增加到 234 mg/L，是仅使用 NprB 信号肽的野生型 IA 的 155 倍。这种组合策略可用于增强枯草芽孢杆菌中大尺寸异源蛋白质的分泌产生。

更新日期：2020-06-26

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