Research Progress in Area 4 方向 ( 四 ) 課題進展 187 Abstract Marine microorganisms are a rich source of medicinal natural products, of which lasso peptides are a family of under-explored natural peptide products with a unique structural topology and interesting biological activities. Prof. Guo’s team will develop both in vivo and in vitro systems to reconstitute the synthesis of new lasso peptide natural products. In addition, they will carry out biochemical and structural studies to fully elucidate the molecular mechanism of the formation of the lariat knot structure through biochemical studies combined with protein X-ray crystallography. Using the insights gained in the mechanism investigation, the in vitro reconstitution system will be used to engineer new lasso peptides for screen of novel biological activities. These efforts aim to fully harness the potential of marine lasso peptides as new drugs. Research Activities and Progress • Overexpressed the Microcin J25 biosynthetic precursor McjA. • Overexpressed the Microcin J25 biosynthetic enzymes McjB and McjC. • Co-expressed McjB and McjC,McjA and McjB in the pET-Duet vector • Developed a method to purify the soluble McjB and McjC. • Constructed a plasmid to successfully express the Microcin J25 biosynthetic cluster. • Developed a method to isolate the lasso peptide Microcin J25. Key Findings • BothN-andC-terminal hexahistidine taggedMcjB and McjC were not to bind the Ni2+-NTA column, indicating that the N- and C-terminal tags may be hidden inside the proteins and inaccessible for affinity binding. • McjB and McjC were not co-purified by the Ni2+- NTA column chromatography. • No activity was found for the purified proteins, no matter whether the hexahisitine tag was attached at the N- or C-terminus. • Addition of a C-terminal tag to McjC disabled the synthesis of the lasso peptide but other modifications had no effect on its production. The activities of the proteins were retained by including dithiothreitol in the buffers. Research Output Publication 1 Trained personnel 6 Marine Lasso Peptides: Reconstitution and Mechanism of Their Biosynthesis Prof. Zhihong Guo The Hong Kong University of Science and Technology Fig 1. Structure and biosynthe tic enzymes of Microcin J25.
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