High-yield ‘one-pot’ biosynthesis of raspberry ketone, a high-value fine chemical
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
Cell-free extract and purified enzyme-based systems provide an attractive solution to study biosynthetic strategies towards a range of chemicals. 4-(4-hydroxyphenyl)-butan-2-one, also known as raspberry ketone, is the major fragrance component of raspberry fruit and is used as a natural additive in the food and sports industry. Current industrial processing of the natural form of raspberry ketone involves chemical extraction with a yield of ~1-4 mg kg-1 of fruit. Due to toxicity, microbial production provides only low yields of up to 5-100 mg L-1. Herein, we report an efficient cell-free strategy to probe a synthetic enzyme pathway that converts either L-tyrosine or the precursor, 4-(4-hydroxyphenyl)-buten-2-one (HBA), into raspberry ketone at up to 100% conversion. As part of this strategy, it is essential to recycle inexpensive cofactors. Specifically, the final enzyme step in the pathway is catalysed by raspberry ketone/zingerone synthase (RZS1), an NADPH-dependent double bond reductase. To relax cofactor specificity towards NADH, the preferred cofactor for cell-free biosynthesis, we identify a variant (G191D) with strong activity with NADH. We implement the RZS1 G191D variant within a ‘one-pot’ cell-free reaction to produce raspberry ketone at high-yield (61 mg L-1), which provides an alternative route to traditional microbial production. In conclusion, our cell-free strategy complements the growing interest in engineering synthetic enzyme cascades towards industrially relevant value-added chemicals.
Date Issued
2021-08-20
Date Acceptance
2021-08-03
Citation
Synthetic Biology, 2021, 6 (1), pp.1-8
ISSN
2397-7000
Publisher
Oxford University Press
Start Page
1
End Page
8
Journal / Book Title
Synthetic Biology
Volume
6
Issue
1
Copyright Statement
© The Author(s) 2021. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
License URL
Sponsor
Engineering & Physical Science Research Council (EPSRC)
Identifier
https://academic.oup.com/synbio/advance-article/doi/10.1093/synbio/ysab021/6338191?login=true
Grant Number
EP/K038648/1
Subjects
cell-free
fine chemical
polyketides
raspberry ketone
synthetic biology
Publication Status
Published
Date Publish Online
2021-08-20