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Published on: April 22, 2016
Efficient multi-enzyme system for icaritin production from naringenin with synchronous prenol utilization in
Anyi Wu1, Junping Zhou2, Yinan Xue3
1The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, Zhejiang, 310014, PR China; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang, 310014, PR China; State Key Laboratory of Green Chemical Synthesis and Conversion, Zhejiang University of Technology, Hangzhou, Zhejiang, 310014, PR China.
Abstract:
Icaritin (ICT) shows various pharmacological activities and is used as an anti-hepatocellular carcinoma drug. While most of the studies focused on constructing yeast chassis cells for ICT production to replace the ICT extraction of Epimedium species, an efficient multi-enzyme system was first constructed here in the prokaryote Escherichia coli, for the successful biotransformation from naringenin (NAR) and prenol to ICT. The plant-derived flavonoid 3-hydroxylase (F3H) and flavonol synthase (FLS) were screened, and then fused together with protein linkers to efficiently catalyze the conversion of NAR to kaempferol (KAE). The isopentenol utilization (IU) pathway was subsequently introduced to increase dimethylallyl diphosphate (DMAPP) supply, along with catalytic activity improvement of prenyltransferase (PT) using N-terminal truncations and active site pocket engineering. Furthermore, by screening O-methytransferases (OMT) from different sources, EpOMT4 from Epimedium pseudowushanense was selected for the ICT production. With the combination of these strategies, 80.8 ± 3.6 mg/L ICT could be produced from NAR and prenol in the recombinant E. coli, from the original 0 % degree of conversion. The systematic engineering of multi-enzyme system for ICT biosynthesis has laid the foundation for the large-scale ICT production using the industrially important E. coli chassis cell.
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