| Summary: | Lipase B from Pseudozyma antarctica (PalB), had been expressed in several recombinant protein hosts and showed very good transesterification activity for biodiesel production. However, the functional expression could not be demonstrated until recently in the most popular recombinant protein expression system, e.g. Escherichia coli, and the expression performance stands improvement. The probable reason is that PalB is a lipase with more hydrophobic surface and three disulfide bonds and thus may not be easily expressed in E. coli functionally. This thesis focuses on the identification of factors limiting heterologous expression of PalB in E. coli through a systematic study by using several strategies, including the different expression compartments, fusion tags, folding factors, and host strains. Functional expression of PalB in the cytoplasm of E. coli was explored using BL21(DE3) and its mutant derivative Origami B(DE3) as the host. Bioactive PalB was obtained in the reduced cytoplasm of BL21(DE3), implying that the formation of disulfide bond was not strictly required for functional expression. However, the expression was ineffective and was primarily limited by formation of PalB inclusion bodies and growth arrest, both of which were associated with PalB misfolding and deteriorated physiology. The culture performance in terms of cell growth and PalB expression level could be significantly improved by simultaneous coexpression of multiple chaperones of trigger factor and GroEL/ES, but not individual coexpression of either one of them. It was proposed that the two chaperones mediate the early stage and late stage of cytoplasmic PalB folding and would be required simultaneously for boosting both the overall PalB synthesis rate and the cytoplasmic folding efficiency. On the other hand, a much higher bioactive PalB was produced in Origami B(DE3) harboring the same PalB expression vector. Furthermore, the significant high bioactive PalB was produced by coexpression of periplasmic folding factor without a signal ...
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