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

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.

A curcumin direct protein biosensor for cell‐free prototyping

“…We therefore created a synthetic enzyme pathway to supply these precursors and study the biosynthesis of curcumin. For this, we used the TAL, p ‐coumaroyl‐CoA ligase (PCL) and malonyl‐CoA synthetase (MatB), as described in our previous study [24], along with a curcumin synthase (CUS) from Oryza sativa [25]. To establish initial reaction conditions, 1 mM of L‐tyrosine, the cofactors (ATP, malonate, CoA, and Mg 2+ ) and 1 μM of purified enzymes (TAL, PCL, MatB and CUS) were incubated at 30°C to synthesise the analogue of curcumin, bisdemethoxycurcumin (BDMC).…”

“…Routine molecular biology was performed as described previously [23]. The TAL, PCL and MatB expression constructs were described in our previous work [24]. CUS [25] was synthesised by ThermoFisher Scientific and codon optimised for E. coli K12 expression with compatibility for EcoFlex cloning [23].…”

A curcumin direct protein (DiPro) biosensor for cell-free prototyping

“…During our study, the highly related VvCurA structure (PDB: 5ZXN) was released with NADP + bound 24 . We compared VvCurA to our own related reductase structure (PDB: 6EOW) with NADP+ and half-curcumin (p-hydroxybenzalacetone) bound 27 . Based on this, we speculate that curcumin fluorescence (in the absence of NADPH), is generated through stabilization of p-p* transitions upon excitation (450 nm), leading to an excited state intramolecular charge transfer species 21 .…”

“…We therefore created a synthetic enzyme pathway to supply these precursors and study the biosynthesis of curcumin. For this we used the tyrosine ammonia lyase (TAL), p-coumaroyl-CoA ligase and malonyl-CoA synthetase (MatB), described in our previous study 27 , along with a curcumin synthase (CUS) from Oryza sativa 29 . Using 1 mM L-tyrosine as a substrate, the cofactors (ATP, malonate, CoA, Mg 2+ ) and purified enzymes were included (see methods), to attempt to synthesize the analog of curcumin, bisdemethoxycurcumin (BDMC).…”

“…Routine molecular biology was performed as described previously 33 . The TAL, PCL and MatB expression constructs were described in our previous work 27 . CUS 29 was synthesised by ThermoFisher Scientific and codon optimised for E. coli K12 expression with compatibility for EcoFlex cloning 33 .…”