High-yield 'one-pot' biosynthesis of raspberry ketone, a high-value fine chemical

DOI: 10.1093/synbio/ysab021

Authors: Simon J Moore (Imperial College London) , Tommaso Tosi (Imperial College London) , David Bell (Imperial College London) , Yonek B Hleba (Imperial College London) , Karen M. Polizzi (Imperial College London) , Paul S. Freemont (Imperial College London)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Synthetic Biology

State: Published (Approved)
Published: August 2021
Diamond Proposal Number(s): 12579

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.

Journal Keywords: Synthetic biology; fine chemical; raspberry ketone; polyketides cell-free

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I04-Macromolecular Crystallography

Added On: 11/08/2021 09:02

Documents:
ysab021.pdf

Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Organic Chemistry Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)