Moore Simon J, Tosi Tommaso, Bell David, Hleba Yonek B, Polizzi Karen M, Freemont Paul S
Centre for Synthetic Biology and Innovation, Imperial College London, South Kensington Campus, London, UK.
Department of Medicine, Imperial College London, South Kensington Campus, London, UK.
Synth Biol (Oxf). 2021 Aug 20;6(1):ysab021. doi: 10.1093/synbio/ysab021. eCollection 2021.
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 of fruit. Due to toxicity, microbial production provides only low yields of up to 5-100 mg L. 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), 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.
无细胞提取物和基于纯化酶的系统为研究一系列化学品的生物合成策略提供了一个有吸引力的解决方案。4-(4-羟基苯基)-丁-2-酮，也称为树莓酮，是树莓果实的主要香气成分，被用作食品和体育行业的天然添加剂。目前对天然形式的树莓酮进行工业加工涉及从每千克果实约1-4毫克的产量中进行化学提取。由于毒性，微生物生产的产量仅低至5-100毫克/升。在此，我们报告了一种高效的无细胞策略，以探究一种合成酶途径，该途径可将L-酪氨酸或前体4-(4-羟基苯基)-丁-2-烯-1-酮转化为树莓酮，转化率高达100%。作为该策略的一部分，回收廉价的辅因子至关重要。具体而言，该途径中的最后一步酶促反应由树莓酮/姜辣素合酶（RZS1）催化，这是一种依赖NADPH的双键还原酶。为了放宽对无细胞生物合成中首选辅因子NADH的辅因子特异性，我们鉴定了一种对NADH具有强活性的变体（G191D）。我们在“一锅法”无细胞反应中实施RZS1 G191D变体，以高产率（61毫克/升）生产树莓酮，这为传统微生物生产提供了一条替代途径。总之，我们的无细胞策略补充了人们对工程合成酶级联反应以生产工业相关增值化学品日益增长的兴趣。
