AN INTEGRATED APPROACH TO STUDYING AND ENGINEERING BACTERIAL TYPE II POLYKETIDE CORE STRUCTURE BIOSYNTHESIS

Abstract

Natural products including alkaloids, phenylpropanoids, polyketides, terpenoids and antibiotics provide abundant resources for drugs and drug leads. Type II polyketides (PK-IIs) are a large array of aromatic polyketides that have exhibited various bioactivities including anti-tumor, anti-cancer, anti-fungal and anti-bacteria. Based on the lengths of carbon chains and the cyclization patterns in their core structures, most bacterial PK-IIs have been classified into 6 typical classes: benzoisochromanequinone (BIQ), anthracycline (ANT), angucycline (ANG), tetracycline (TET), tetracenomycin (TCM) and pentangular (PEN). Bioengineering for diversification of PK-II core structures will generate libraries of bioactivities, which can serve the drug discovery in this family of compounds. However, although some typical PK-II core structures have been expressed, the general approach to producing different PK-II core structures had not been proposed. We validated the functional sequence of PactI promoter, tested different expression plasmid designs, and compared the production of compound in different culture media. These knowledge allowed us to develop a general approach to producing different PK-II core structures, that expressing non-redundant PK-II core structure biosynthetic pathways in a general way in the same host strain grown in the same culture medium. This approach not only improved our understanding to PK-II core structures and their biosynthetic pathways, but also provided a convenient way for generating the diverse-oriented libraries of PK-II molecules. We attempted the BIQ halogenation. On the other hand, the approach provided a bottom-up method to study functions of proteins involved in PK-II biosynthesis. We studied ActVI-ORFA, ActVI-ORF3, ActVA-ORF3 and Aln5 by expressing derived BIQ biosynthetic pathways. Crystallization and structure solutions were carried out for ActVI-ORFA and its two homologs. Our studies have provided clues to deducing their functions. Tests of a Aln5 homolog has revealed that the C3 reduction in BIQ biosynthesis can happen medium dependently in the absence of the C3 ketoreductase