Abstract Fusion with self-assembling amphipathic peptides (SAPs) could improve enzyme stability and activity in addition to their effects on protein expression. In this study, we proposed a metabolic regulation strategy based on an iterative SAP fusion with key enzymes in pathway. First, an SAP, S1nv10 (ANANARARANANARAR), was separately fused to the N-terminus of metabolite enzymes. To achieve the diversity in the enzyme activities, a library of the fused SAPs varied in hydrophobicity, length and net charge were generated by a multi-primer PCR procedure and the DATEL-assembly method. After simultaneous modification of three enzymes (CrtE, CrtY, and CrtI) using the SAP library, the Escherichia coli strain with 3.91-fold increase in β-carotene production was isolated. As indicated by western-blot and catalytic property analysis, SAP fusion induced the increases in both enzyme expression (CrtY and CrtE) and enzymatic activities (CrtY). The effectiveness of the strategy was further confirmed by the metabolic regulation of the eriodictyol and (2S)-naringenin, the yields of which were increased by 111.3% and 186.3% in E. coli, respectively. These results indicated that fusion with SAPs could effectively regulate metabolic pathway through optimization of the protein expression and catalytic properties of the key enzymes.

中文翻译：

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通过与自组装两亲肽融合优化代谢途径

摘要 与自组装两亲性肽 (SAPs) 融合除了对蛋白质表达有影响外，还可以提高酶的稳定性和活性。在这项研究中，我们提出了一种基于与途径中关键酶的迭代 SAP 融合的代谢调节策略​​。首先，将 SAP S1nv10 (ANANARARANANARAR) 分别融合到代谢酶的 N 端。为了实现酶活性的多样性，通过多引物 PCR 程序和 DATAL 组装方法生成了疏水性、长度和净电荷不同的融合 SAP 文库。使用 SAP 库同时修饰三种酶（CrtE、CrtY 和 CrtI）后，分离出 β-胡萝卜素产量增加 3.91 倍的大肠杆菌菌株。蛋白质印迹和催化性能分析表明，SAP融合诱导酶表达（CrtY和CrtE）和酶活性（CrtY）的增加。圣草酚和(2S)-柚皮素的代谢调节进一步证实了该策略的有效性，其在大肠杆菌中的产量分别增加了111.3%和186.3%。这些结果表明，与SAPs融合可以通过优化关键酶的蛋白质表达和催化特性来有效调节代谢途径。