College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, Jiangsu, China.
School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, 211816, Jiangsu, China.
Appl Microbiol Biotechnol. 2018 Sep;102(17):7455-7464. doi: 10.1007/s00253-018-9163-6. Epub 2018 Jul 2.
High level expression of penicillin G acylase (PGA) in Escherichia coli is generally constricted by a complex maturation process and multiple limiting steps. In this study, three PGAs isolated from Providencia rettgeri (PrPGA), Alcaligenes faecalis (AfPGA), and Achromobacter xylosoxidans (AxPGA) were efficiently expressed in E. coli by replacing with applicable signal peptide. Different bottlenecks of the expression process were analyzed for PrPGA, AfPGA, and AxPGA. Subsequently, five efficient signal peptides, including OmpA, pelB, Lpp, PhoA, and MalE, were used to replace the original signal peptides of the PGAs. With respect to AfPGA and AxPGA, translocation was the primary limitation, and the use of pelB signal peptide effectively overcame this barrier. For PrPGA, which was almost not expressed in wild type, the translation initiation efficiency was optimized by replacing with MalE signal peptide. In addition, low temperature (20 °C) slowed down the transcription and translation, thereby facilitating the posttranslational process and preventing the formation of inclusion bodies. Furthermore, combined induction with IPTG and arabinose not only enhanced the cell density but also remarkably improved the expression of PGAs. Final specific activities of the three PGAs reached 2100 (PrPGA), 9200 (AfPGA), and 1400 (AxPGA) U/L/OD, respectively. This simple and robust strategy by fitting replacement of signal peptide might dramatically improve the expression of PGAs from various bacteria, which was significant in the production of many valuable β-lactam antibiotics.
青霉素 G 酰化酶（PGA）在大肠杆菌中的高水平表达通常受到复杂的成熟过程和多个限制步骤的限制。在这项研究中，通过替换适用的信号肽，从普通变形杆菌（PrPGA）、粪产碱杆菌（AfPGA）和木糖氧化无色杆菌（AxPGA）中分离出的三种 PGA 在大肠杆菌中得到了有效表达。分析了 PrPGA、AfPGA 和 AxPGA 表达过程中的不同瓶颈。随后，使用五个有效的信号肽，包括 OmpA、pelB、Lpp、PhoA 和 MalE，替换了 PGAs 的原始信号肽。对于 AfPGA 和 AxPGA，易位是主要的限制因素，使用 pelB 信号肽有效地克服了这一障碍。对于在野生型中几乎不表达的 PrPGA，通过替换 MalE 信号肽优化了翻译起始效率。此外，低温（20°C）减缓了转录和翻译，从而有利于翻译后过程并防止包涵体的形成。此外，IPTG 和阿拉伯糖的联合诱导不仅提高了细胞密度，而且显著提高了 PGAs 的表达。三种 PGA 的最终比活分别达到 2100（PrPGA）、9200（AfPGA）和 1400（AxPGA）U/L/OD。这种通过替换信号肽来适应的简单而强大的策略可能会极大地提高来自各种细菌的 PGA 的表达水平，这对于生产许多有价值的β-内酰胺抗生素具有重要意义。
