Pathway engineering for the production of β-amyrin and cycloartenol in Escherichia coli—a method to biosynthesize plant-derived triterpene skeletons in E. coli

Pathway engineering for the production of β-amyrin and cycloartenol in Escherichia coli—a... Cycloartenol is biosynthetically the first sterol skeleton, which is metabolized to phytosterols such as β-sitosterol and stigmasterol. β-Amyrin is the most commonly occurring aglycone skeleton for oleanane-type saponins such as glycyrrhizin and saikosaponins. It has been regarded that these cyclic triterpenes are unable to be produced in Escherichia coli, while no reports are available on their production with E. coli. Here, we describe a method to synthesize triterpene skeletons from higher plants, including cycloartenol and β-amyrin. We introduced into E. coli the biosynthetic pathway genes from farnesyl diphosphate (FPP) to cycloartenol or β-amyrin, which contained Arabidopsis (Arabidopsis thaliana)-derived squalene synthase (AtSQS) and squalene epoxidase (AtSQE) genes in addition to the Arabidopsis cycloartenol synthase (AtCAS1) gene, or the β-amyrin synthase (EtAS) gene of the petroleum plant Euphorbia tirucalli, along with the isopentenyl diphosphate isomerase (HpIDI) gene from a green algae Haematococcus pluvialis. The order of genes, HpIDI, AtSQS, AtSQE, driven by transcriptional read-through from a tac promoter to an rrnB terminator, was crucial for their functional expression in E. coli to produce cycloartenol or β-amyrin. The co-expression of a bacterial NADPH-regenerating gene (zwf or gdh) as well as bacterial redox partner protein genes (camA and camB, or NsRED and NsFER) was found to increase the amounts of these triterpenes several fold. The present study could open up opportunities not only to carry out functional analysis of a higher-plant-derived oxidosqualene cyclase (OSC) gene in E. coli but also to produce functional triterpenes that originate from medicinal or herbal plants. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Microbiology and Biotechnology Springer Journals

Pathway engineering for the production of β-amyrin and cycloartenol in Escherichia coli—a method to biosynthesize plant-derived triterpene skeletons in E. coli

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Publisher
Springer Berlin Heidelberg
Copyright
Copyright © 2017 by Springer-Verlag GmbH Germany
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Biotechnology
ISSN
0175-7598
eISSN
1432-0614
D.O.I.
10.1007/s00253-017-8409-z
Publisher site
See Article on Publisher Site

Abstract

Cycloartenol is biosynthetically the first sterol skeleton, which is metabolized to phytosterols such as β-sitosterol and stigmasterol. β-Amyrin is the most commonly occurring aglycone skeleton for oleanane-type saponins such as glycyrrhizin and saikosaponins. It has been regarded that these cyclic triterpenes are unable to be produced in Escherichia coli, while no reports are available on their production with E. coli. Here, we describe a method to synthesize triterpene skeletons from higher plants, including cycloartenol and β-amyrin. We introduced into E. coli the biosynthetic pathway genes from farnesyl diphosphate (FPP) to cycloartenol or β-amyrin, which contained Arabidopsis (Arabidopsis thaliana)-derived squalene synthase (AtSQS) and squalene epoxidase (AtSQE) genes in addition to the Arabidopsis cycloartenol synthase (AtCAS1) gene, or the β-amyrin synthase (EtAS) gene of the petroleum plant Euphorbia tirucalli, along with the isopentenyl diphosphate isomerase (HpIDI) gene from a green algae Haematococcus pluvialis. The order of genes, HpIDI, AtSQS, AtSQE, driven by transcriptional read-through from a tac promoter to an rrnB terminator, was crucial for their functional expression in E. coli to produce cycloartenol or β-amyrin. The co-expression of a bacterial NADPH-regenerating gene (zwf or gdh) as well as bacterial redox partner protein genes (camA and camB, or NsRED and NsFER) was found to increase the amounts of these triterpenes several fold. The present study could open up opportunities not only to carry out functional analysis of a higher-plant-derived oxidosqualene cyclase (OSC) gene in E. coli but also to produce functional triterpenes that originate from medicinal or herbal plants.

Journal

Applied Microbiology and BiotechnologySpringer Journals

Published: Jul 15, 2017

References

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