Xu Yali, Hsieh Ming-Yi, Narayanan Niju, Anderson William A, Scharer Jeno M, Moo-Young Murray, Chou C Perry
Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
Biotechnol Prog. 2005 Sep-Oct;21(5):1357-65. doi: 10.1021/bp0501120.
Penicillin acylase (PAC) precursor, proPAC, was overproduced in a soluble or insoluble form in the cytoplasm of Escherichia coli through the expression of the leader-less pac gene (ll-pac) devoid of the coding region for the signal peptide of PAC. Also, a portion of the overexpressed proPAC was further processed to form mature PAC, indicating that the posttranslational processing steps for PAC maturation can occur in both the periplasm and the cytoplasm of E. coli. The cultivation performance for ll-pac expression was limited by several factors, including (1) misfolding of proPAC, resulting in the aggregation of insoluble proPAC as inclusion bodies, (2) intracellular proteolysis, leading to the degradation of the overexpressed gene products, and (3) inefficient PAC maturation, limiting the formation of active PAC. The effect of coexpression of various cytoplasmic chaperones, including trigger factor, GroEL/ES, DnaK/J-GrpE, and their combinations, on ll-pac expression was investigated. Intracellular proteolysis of the overexpressed gene products could be prevented by coexpression of GroEL/ES. On the other hand, coexpression of trigger factor appeared to be able to facilitate the folding of soluble proPAC and to improve PAC maturation. The roles of trigger factor and GroEL/ES could be coordinated to significantly improve ll-pac expression performance. DnaK/J-GrpE had an effect for solublization of proPAC and perhaps, similar to trigger factor, for improving PAC maturation. The ll-pac expression performance was also significantly improved through the simultaneous coexpression of DnaK/J-GrpE and GroEL/ES. The results of the study suggest that the folding and/or processing of proPAC could be a major issue limiting the overproduction of PAC in E. coli and the bottleneck could be eliminated through the coexpression of appropriate chaperone(s).
青霉素酰化酶（PAC）前体，即proPAC，通过在大肠杆菌细胞质中表达不含PAC信号肽编码区的无引导序列pac基因（ll-pac），以可溶或不可溶形式过量表达。此外，部分过表达的proPAC进一步加工形成成熟的PAC，这表明PAC成熟的翻译后加工步骤可在大肠杆菌的周质和细胞质中发生。ll-pac表达的培养性能受到几个因素的限制，包括：（1）proPAC的错误折叠，导致不溶性proPAC以包涵体形式聚集；（2）细胞内蛋白水解，导致过表达的基因产物降解；（3）PAC成熟效率低下，限制了活性PAC的形成。研究了包括触发因子、GroEL/ES、DnaK/J-GrpE及其组合在内的各种细胞质伴侣蛋白共表达对ll-pac表达的影响。GroEL/ES的共表达可防止过表达基因产物的细胞内蛋白水解。另一方面，触发因子的共表达似乎能够促进可溶性proPAC的折叠并改善PAC成熟。触发因子和GroEL/ES的作用可以协同，显著提高ll-pac的表达性能。DnaK/J-GrpE对proPAC的溶解有作用，并且可能与触发因子类似，有助于改善PAC成熟。通过同时共表达DnaK/J-GrpE和GroEL/ES，ll-pac的表达性能也得到了显著提高。研究结果表明，proPAC的折叠和/或加工可能是限制大肠杆菌中PAC过量生产的主要问题，通过共表达合适的伴侣蛋白可以消除这一瓶颈。
