Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany.
Institute of Biotechnology, RWTH Aachen University, 52074, Aachen, Germany.
Microb Cell Fact. 2021 Feb 17;20(1):49. doi: 10.1186/s12934-021-01539-w.
In recent years, the production of inclusion bodies that retained substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) were formed by genetic fusion of an aggregation inducing tag to a gene of interest via short linker polypeptides and overproduction of the resulting gene fusion in Escherichia coli. The resulting CatIBs are known for their high stability, easy and cost efficient production, and recyclability and thus provide an interesting alternative to conventionally immobilized enzymes.
Here, we present the construction and characterization of a CatIB set of the lysine decarboxylase from Escherichia coli (EcLDCc), constructed via Golden Gate Assembly. A total of ten EcLDCc variants consisting of combinations of two linker and five aggregation inducing tag sequences were generated. A flexible Serine/Glycine (SG)- as well as a rigid Proline/Threonine (PT)-Linker were tested in combination with the artificial peptides (18AWT, L6KD and GFIL8) or the coiled-coil domains (TDoT and 3HAMP) as aggregation inducing tags. The linkers were fused to the C-terminus of the EcLDCc to form a linkage between the enzyme and the aggregation inducing tags. Comprehensive morphology and enzymatic activity analyses were performed for the ten EcLDCc-CatIB variants and a wild type EcLDCc control to identify the CatIB variant with the highest activity for the decarboxylation of L-lysine to 1,5-diaminopentane. Interestingly, all of the CatIB variants possessed at least some activity, whilst most of the combinations with the rigid PT-Linker showed the highest conversion rates. EcLDCc-PT-L6KD was identified as the best of all variants allowing a volumetric productivity of 457 g L d and a specific volumetric productivity of 256 g L d g. Noteworthy, wild type EcLDCc, without specific aggregation inducing tags, also partially formed CatIBs, which, however showed lower activity compared to most of the newly constructed CatIB variants (volumetric productivity: 219 g L d, specific volumetric activity: 106 g L d g). Furthermore, we demonstrate that microscopic analysis can serve as a tool to find CatIB producing strains and thus allow for prescreening at an early stage to save time and resources.
Our results clearly show that the choice of linker and aggregation inducing tag has a strong influence on the morphology and the enzymatic activity of the CatIBs. Strikingly, the linker had the most pronounced influence on these characteristics.
近年来，已证明可以生产保留大量催化活性的包涵体。这些催化活性包涵体（CatIB）是通过短连接肽将聚集诱导标签与目的基因进行基因融合，并在大肠杆菌中过量生产所得基因融合而形成的。由此产生的 CatIB 具有高稳定性、易于低成本生产、可回收利用等特点，因此为传统固定化酶提供了一种有趣的替代选择。
本文通过 Golden Gate 组装构建并表征了一组大肠杆菌赖氨酸脱羧酶（EcLDCc）的 CatIB，共构建了十种由两种连接肽和五种聚集诱导标签序列组合而成的 EcLDCc 变体。测试了柔性丝氨酸/甘氨酸（SG）和刚性脯氨酸/苏氨酸（PT）连接肽与人工肽（18AWT、L6KD 和 GFIL8）或卷曲螺旋结构域（TDoT 和 3HAMP）作为聚集诱导标签的组合。这些连接肽与 EcLDCc 的 C 末端融合形成酶与聚集诱导标签之间的连接。对十种 EcLDCc-CatIB 变体和野生型 EcLDCc 对照进行了全面的形态学和酶活性分析，以确定在 L-赖氨酸脱羧生成 1,5-二氨基戊烷方面具有最高活性的 CatIB 变体。有趣的是，所有的 CatIB 变体都具有一定的活性，而大多数与刚性 PT-连接肽的组合表现出最高的转化率。EcLDCc-PT-L6KD 被确定为所有变体中最好的，其体积产率为 457g L d，比活体积产率为 256g L d g。值得注意的是，没有特定聚集诱导标签的野生型 EcLDCc 也部分形成了 CatIB，但与大多数新构建的 CatIB 变体相比，其活性较低（体积产率：219g L d，比活体积产率：106g L d g）。此外，我们证明微观分析可以作为一种工具来寻找产生 CatIB 的菌株，从而可以在早期进行预筛选，以节省时间和资源。
我们的结果清楚地表明，连接肽和聚集诱导标签的选择对 CatIB 的形态和酶活性有很大的影响。引人注目的是，连接肽对这些特性的影响最为显著。
