细胞色素P450酶在香精香料绿色生物合成中的应用

doi:10.13560/j.cnki.biotech.bull.1985.2022-1248

摘要/Abstract

摘要：

细胞色素P450酶是一类含亚铁血红素的蛋白超家族，来源广泛，几乎存在于所有的生命形式中。P450酶被誉为“黄金催化剂”，主要参与生物体内源物质合成、异源物质代谢及天然产物的生物合成，具有底物谱广、催化反应类型多样、催化立体专一性强等优点，使得它们在药物/毒物代谢、工程化生物合成等方面受到越来越多的关注。其中，P450酶可以实现常温、常压、中性条件下特定惰性C-H键选择性羟化，在高附加值精细化学品的生物合成中具有重要应用价值。本文介绍了P450选择性羟化的催化机制及几种常见的P450电子传递系统，并结合蛋白质工程与代谢工程，以柠檬烯含氧衍生物、诺卡酮、檀香醇、4-羟基异佛尔酮、大环内酯麝香等高附加值化合物的生物合成为例，综述了单酶催化的生物转化或全细胞从头合成的方式在天然香精香料生物合成中的探索与应用，探讨其面临的挑战并展望了其应用前景。

关键词: 细胞色素P450酶, 香精香料, 生物合成, 蛋白质工程, 代谢工程

Abstract:

Cytochromes P450 belong to a large superfamily of hemeproteins, which are widespread in almost all forms of life. P450s are known as “golden catalysts”, and they are mainly involved in the synthesis of endogenous substances, metabolism of xenobiotics, and biosynthesis of natural products in living organisms. P450- catalyzed reactions demonstrate various advantages including wide substrate spectrum, diverse types of catalytic reactions, and high catalytic regio-and stereospecificity, which make them increasingly attractive in drug/toxin metabolism, and engineered product biosynthesis. Among the P450 catalyzed reactions, the introduction of a hydroxyl group at a specific inert C-H bonds under mild conditions makes them highly useful in high value-added fine chemicals biosynthesis. Hereby the mechanism of selective hydroxylation of P450s and related several common types of electron transfer chain system are introduced. The biosynthesis of several natural flavors and fragrances such as limonene oxygenated derivatives, nootkatone, santalol, 4-hydroxyisophorone, and macrocyclic musk, which are catalyzed by single enzyme-catalyzed biotransformation or whole-cell de novo biosynthesis involving protein and metabolic engineering, are reviewed. Finally, the challenges and prospects of their practical applications are discussed.

Key words: cytochromes P450, flavor and fragrance, biosynthesis, protein engineering, metabolic engineering

郁慧丽, 李爱涛. 细胞色素P450酶在香精香料绿色生物合成中的应用[J]. 生物技术通报, 2023, 39(4): 24-37.

YU Hui-li, LI Ai-tao. Application of Cytochrome P450 in the Biosynthesis of Flavors and Fragrances[J]. Biotechnology Bulletin, 2023, 39(4): 24-37.

图/表 9

图1 P450BM3的晶体结构（PDB ID：2IJ2）

Fig. 1 Crystal structure of P450BM3（PDB ID: 2IJ2）

图2 细胞色素P450酶催化底物羟基化的反应机制

Fig. 2 Mechanism of substrate hydroxylation catalyzed by cytochrome P450

图3 P450酶氧化还原系统的类型

Fig. 3 Classes of P450 redox systems

图4 P450催化柠檬烯不同位点的羟化反应

Fig. 4 P450 catalyzes the hydroxylation of limonene at different sites

图5 P450参与的生物-化学两步转化（A）及生物法从头合成（-）-薄荷醇（B） L5H：柠檬烯-5-羟化酶；L3H：柠檬烯-3-羟化酶；IPDH：（-）-反式-异薄荷烯醇脱氢酶；IPR：（-）-异薄荷烯酮还原酶；IPGI：（+）-顺式-异胡薄荷酮异构酶；PGR：（+）-胡薄荷酮还原酶；MR：（-）-薄荷酮还原酶；PpCamFL：来源于恶臭假单胞菌的P450cam_Y96F/V247L 突变体；PaIPDHFV：来源于绿脓假单胞菌的IPDH_E95F/Y199V 突变体；KSI variant：来源于恶臭假单胞菌的Δ5-3-甾酮异构酶KSI_V88I/L99V/V101A/D103S 突变体

Fig. 5 P450 involved in the biotechnological-chemical processes（A）versus the de novo biosynthetic pathway for（-）-menthol production L5H: Limonene-5-hydroxylase; L3H: limonene-3-hydroxylase; IPDH:（-）-trans-isopiperitenol dehydrogenase; IPR:（-）-isopiperitenone reductase; IPGI:（+）-cis-isopulegone isomerase; PGR:（+）-pulegone reductase; MR:（-）-menthone reductase; PpCamFL: P450cam_Y96F/V247L variant from Pseudomonas putida; PaIPDHFV: IPDH_E95F/Y199V variant from Pseudomonas aeruginosa; KSI variant: Δ5-3-ketosteroid isomerase KSI_V88I/L99V/V101A/D103S variant from P. putida

图6 P450参与的生物法从头合成S-（-）-紫苏醇 AACT：乙酰CoA酰基转移酶；HMGS：HMG-CoA合酶；HMGR：HMG-CoA还原酶；MVK：甲羟戊酸激酶；PMVK：磷酸甲羟戊酸激酶；MVD：焦磷酸甲羟戊酸脱羧酶；IDI：IPP异构酶；GPPS：GPP合酶；LS：柠檬烯合酶

Fig. 6 P450 involved in the de novo biosynthesis of S-（-）-perillyl alcohol AACT: Acetyl-CoA C-acetyltransferase; HMGS: hydroxymethylglutaryl-CoA synthase; HMGR: hydroxymethylglutaryl-CoA reductase; MVK: mevalonate kinase; PMVK: phosphomevalonate kinase; MVD: diphosphomevalonate decarboxylase; IDI: isopentenyl-diphosphate delta-isomerase; GPPS: GPP synthase; LS: limonene synthase

图7 P450参与的单酶催化法（A）及全细胞从头合成（+）-诺卡酮（B） FPPS：FPP合酶；CnVS：来自阿拉斯加黄柏的瓦伦烯合酶；ADH：醇脱氢酶

Fig. 7 P450 involved in the biotransformation of（+）-valencene（A）and de novo biosynthesis of（+）-nootkatone（B） FPPS: FPP synthase; CnVS: valencene synthase from Chamaecyparis nootkatensis; ADH: alcohol dehydrogenase

图8 P450参与的全细胞从头合成檀香醇 SaSS：来自檀香树的檀香烯合酶

Fig. 8 P450 involved in de novo biosynthesis of（+）-nootkatone SaSS: Santalene synthase from Santalum album

图9 P450参与的“一锅法”催化α-异佛尔酮生成4KIP

Fig. 9 P450 involved in “one-pot” biocatalytic system from α-isophorone to 4KIP

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