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- Title
High-yield 'one-pot' biosynthesis of raspberry ketone, a high-value fine chemical.
- Authors
Moore, Simon J; Tosi, Tommaso; Bell, David; Hleba, Yonek B; Polizzi, Karen M; Freemont, Paul S
- 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 from 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 into a synthetic enzyme pathway that converts either L-tyrosine or the precursor, 4-(4-hydroxyphenyl)-buten-2-one, 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 catalyzed 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.
- Subjects
KETONES; RASPBERRIES; PLANT product synthesis; ENZYME biotechnology; SYNTHETIC enzymes
- Publication
Synthetic Biology (23977000), 2021, Vol 6, Issue 1, p1
- ISSN
2397-7000
- Publication type
Article
- DOI
10.1093/synbio/ysab021