Efficient microbial production of stylopine using a Pichia pastoris expression system.

Kentaro Hori,Shunsuke Okano,Fumihiko Sato

doi:10.1038/srep22201

Kentaro Hori, Shunsuke Okano + Show 1 more

Open Access

https://doi.org/10.1038/srep22201

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Journal: Scientific Reports	Publication Date: Feb 29, 2016
Citations: 19	License type: CC BY 4.0

Affiliation: Kyoto University

Abstract

Stylopine is a protoberberine-type alkaloid that has potential biological activities. Based on the successful microbial production of (S)-reticuline, we attempted to produce stylopine from (S)-reticuline by the reaction of berberine bridge enzyme, cheilanthifoline synthase (CYP719A5), and stylopine synthase (CYP719A2). Biosynthetic enzyme expression was examined in a methanol-utilizing yeast (Pichia pastoris), and both a “consolidated” system with all genes expressed in one cell and a “co-culture” system with three cell lines that each express a single gene were examined. Although both systems efficiently converted reticuline to stylopine, the consolidated system was more rapid and efficient than the co-culture system. However, substrate-feeding experiments revealed a decrease in the conversion efficiency in the consolidated system during successive cultures, whereas the conversion efficiency in the co-culture system remained constant. Thus, the final amount of stylopine produced from reticuline after successive feedings in the co-culture system was more than 150 nmoles from 750 nmoles of (R, S)-reticuline (375 nmoles of (S)-reticuline). The advantages and drawbacks of the “consolidated” system and the “co-culture” system are discussed.

Highlights

Stylopine is a protoberberine-type alkaloid that has potential biological activities
The RNA silencing of the gene for an enzyme that is downstream of stylopine in sanguinarine biosynthesis, i.e., tetrahydroprotoberberine N-methyltransferase, is feasible, as shown for the successful accumulation of reticuline in transgenic Eschscholzia californica cells with an RNA interference (RNAi) vector for berberine bridge enzyme (BBE) to convert reticuline to scoulerine[6]
Stylopine is synthesized from (S)-reticuline in a reaction consisting of three steps, i.e., berberine bridge enzyme (BBE)[8], cheilanthifoline synthase (e.g., CYP719A5 from E. californica, CHS)[9], and stylopine synthase (e.g., CYP719A2/A3 from E. californica, STS)[10]; a previous report indicated that the BBE step was inefficient for expression in a microbial system, especially in yeast[11]

Summary

Introduction

Stylopine is a protoberberine-type alkaloid that has potential biological activities. Based on the successful microbial production of (S)-reticuline, we attempted to produce stylopine from (S)-reticuline by the reaction of berberine bridge enzyme, cheilanthifoline synthase (CYP719A5), and stylopine synthase (CYP719A2). Biosynthetic enzyme expression was examined in a methanol-utilizing yeast (Pichia pastoris), and both a “consolidated” system with all genes expressed in one cell and a “coculture” system with three cell lines that each express a single gene were examined. Both systems efficiently converted reticuline to stylopine, the consolidated system was more rapid and efficient than the co-culture system. Reconstruction of the entire pathway in single cells is preferred due to the simplicity of handling and management, we first examined the stepwise optimization of the pathway to overcome the inefficiency of BBE

Methods

Results

Conclusion