药学学报

Name	Source	Substrate(s)	Reaction(s)	Ref
I. CYP450s in the natural biosynthetic pathway of bile acids
CYP7A1	Vertebrates	Cholesterol	C7α-Hydroxylation	[53]
CYP8B1	Vertebrates	7α-Hydroxycholest-4-en-3-one, 5β-cholestane-3α,7α-diol, 3α,7α-dihydroxy-5β-cholestanoic acid, chenodeoxycholic acid, 7α-hydroxycholesterol, 7α,26-dihydroxycholest-4-en-3-one, 7α-hydroxy-3-oxo-4-cholestenoic acid	C12α-Hydroxylation	[54-56]
CYP7B1	Vertebrates	27-Hydroxycholesterol, 25-hydroxycholesterol, pregnenolone, dehydroepiandrosterone	C7α-Hydroxylation	[58]
5α-Androstane-3β,17β-diol	C6α-, C7α-Hydroxylation
CYP4A21	Sus scrofa	Chenodeoxycholic acid, taurochenodeoxycholic acid	C6α-Hydroxylation	[47]
CYP3A4	Higher vertebrates	Lithocholic acid	C3-Dehydrogenation; C1β-, C6α-hydroxylation	[52]
Deoxycholic acid	C3-Dehydrogenation; C1β-hydroxylation
Chenodeoxycholic acid	C3-Dehydrogenation; C6α-hydroxylation
Ursodeoxycholic acid	C3-Dehydrogenation; C1β-, C22-hydroxylation
Cholic acid	C3-Dehydrogenation
CYP27A1	Vertebrates	5β-Cholestane-3α,7α-diol, 5β-cholestane-3α,7α,12α-triol, 3β-hydroxy-Δ⁵ sterols (cholesterol, 7α-hydroxycholesterol, 24-hydroxycholesterol, 25-hydroxycholesterol, cholesterol sulfate) 3-oxo-Δ⁴ sterols (4-cholesten-3-one, 7α-hydroxy-4-cholesten-3-one, 24-hydroxy-4-cholesten-3-one, 25-hydroxy-4-cholesten-3-one)	C27-Hydroxylation and further oxidation	[57]
II. CYP450s used for heterologous biosynthesis of bile acids
CYP107D1 (OleP)	Streptomyces antibioticus, heterologously expressed in Escherichia coli	Lithocholic acid, deoxycholic acid	C6β-Hydroxylation	[59]
CYPg3484	Fusarium sporotrichioides, heterologously expressed in Pichia pastoris	Deoxycholic acid	C7β-Hydroxylation	[60]
P450_FG	Fusarium graminearum, heterologously expressed in Pichia pastoris	Deoxycholic acid	C7β-Hydroxylation	[60]
P450_FE	Fusarium equiseti HG18, heterologously expressed in Pichia pastoris	Lithocholic acid	C7β-Hydroxylation	[61]
P450_FL	Fusarium longipes, heterologously expressed in Pichia pastoris	Lithocholic acid	C7β-Hydroxylation	[62]
P450_AA	Allokutzneria albata, heterologously expressed in Escherichia coli	Lithocholic acid	C7β-Hydroxylation	[63]

Name	Source	Substrate(s)	Reaction(s)	Ref
I. CYP450s in the natural biosynthetic pathway of bile acids
CYP7A1	Vertebrates	Cholesterol	C7α-Hydroxylation	[53]
CYP8B1	Vertebrates	7α-Hydroxycholest-4-en-3-one, 5β-cholestane-3α,7α-diol, 3α,7α-dihydroxy-5β-cholestanoic acid, chenodeoxycholic acid, 7α-hydroxycholesterol, 7α,26-dihydroxycholest-4-en-3-one, 7α-hydroxy-3-oxo-4-cholestenoic acid	C12α-Hydroxylation	[54-56]
CYP7B1	Vertebrates	27-Hydroxycholesterol, 25-hydroxycholesterol, pregnenolone, dehydroepiandrosterone	C7α-Hydroxylation	[58]
5α-Androstane-3β,17β-diol	C6α-, C7α-Hydroxylation
CYP4A21	Sus scrofa	Chenodeoxycholic acid, taurochenodeoxycholic acid	C6α-Hydroxylation	[47]
CYP3A4	Higher vertebrates	Lithocholic acid	C3-Dehydrogenation; C1β-, C6α-hydroxylation	[52]
Deoxycholic acid	C3-Dehydrogenation; C1β-hydroxylation
Chenodeoxycholic acid	C3-Dehydrogenation; C6α-hydroxylation
Ursodeoxycholic acid	C3-Dehydrogenation; C1β-, C22-hydroxylation
Cholic acid	C3-Dehydrogenation
CYP27A1	Vertebrates	5β-Cholestane-3α,7α-diol, 5β-cholestane-3α,7α,12α-triol, 3β-hydroxy-Δ⁵ sterols (cholesterol, 7α-hydroxycholesterol, 24-hydroxycholesterol, 25-hydroxycholesterol, cholesterol sulfate) 3-oxo-Δ⁴ sterols (4-cholesten-3-one, 7α-hydroxy-4-cholesten-3-one, 24-hydroxy-4-cholesten-3-one, 25-hydroxy-4-cholesten-3-one)	C27-Hydroxylation and further oxidation	[57]
II. CYP450s used for heterologous biosynthesis of bile acids
CYP107D1 (OleP)	Streptomyces antibioticus, heterologously expressed in Escherichia coli	Lithocholic acid, deoxycholic acid	C6β-Hydroxylation	[59]
CYPg3484	Fusarium sporotrichioides, heterologously expressed in Pichia pastoris	Deoxycholic acid	C7β-Hydroxylation	[60]
P450_FG	Fusarium graminearum, heterologously expressed in Pichia pastoris	Deoxycholic acid	C7β-Hydroxylation	[60]
P450_FE	Fusarium equiseti HG18, heterologously expressed in Pichia pastoris	Lithocholic acid	C7β-Hydroxylation	[61]
P450_FL	Fusarium longipes, heterologously expressed in Pichia pastoris	Lithocholic acid	C7β-Hydroxylation	[62]
P450_AA	Allokutzneria albata, heterologously expressed in Escherichia coli	Lithocholic acid	C7β-Hydroxylation	[63]

CYP450羟化酶在胆汁酸生物合成中的应用

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马杉池 , 孙文涛 , 魏茜 , 胡友财 ^*

药学学报 | 综述 2026,61(5): 1484-1497

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药学学报 |综述 2026 , 61 (5) : 1484 -1497

CYP450羟化酶在胆汁酸生物合成中的应用

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马杉池, 孙文涛, 魏茜, 胡友财^*

作者信息

中国医学科学院北京协和医学院, 药物研究所, 天然药物活性物质与功能国家重点实验室, 北京 100050

通讯作者:

胡友财, Tel: 86-10-50927869, E-mail: huyoucai@imm.ac.cn

作者简介:

作者贡献:

马杉池负责文献调研整理和文章撰写; 孙文涛、魏茜负责文章审阅和校对; 胡友财提出文章选题、构思和框架, 全程指导论文撰写和修改。

Application of CYP450 hydroxylases in the biosynthesis of bile acids

Shan-chi MA, Wen-tao SUN, Qian WEI, You-cai HU^*

Affiliations

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China

出版时间: 2026-05-12 doi: 10.16438/j.0513-4870.2026-0121

文章导航

摘要

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胆汁酸在胆固醇稳态维持、脂质代谢调节中发挥核心作用, 具有广泛药用价值。由于提取分离和化学合成难以满足当前工业化生产需要, 生物催化成为极具前景的胆汁酸生产替代方案。细胞色素P450 (cytochrome P450, CYP450) 催化惰性C(sp³)-H键的羟基化, 是胆汁酸生物合成途径的核心酶。通过CYP450酶的分子改造、氧化还原伴侣工程及催化体系优化等策略, 显著提升其催化效率, 已实现多种胆汁酸的高效生物合成。本文综述胆汁酸的特性、合成策略与CYP450羟化酶应用的优化, 为胆汁酸的绿色生产与相关药物研究提供参考。

关键词

胆汁酸 / 生物合成 / CYP450 / 羟基化反应 / 酶工程

Abstract

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Bile acids play a central role in maintaining cholesterol homeostasis and regulating lipid metabolism, exhibiting extensive medicinal value. However, conventional extraction, separation, and chemical synthesis methods are unable to meet the demands of current industrial production, rendering biocatalysis a highly promising alternative for bile acid manufacturing. Cytochrome P450 (CYP450) enzymes, which catalyze the hydroxylation of inert C(sp³)-H bonds, serve as the core enzymes in the bile acid biosynthetic pathway. Through strategies such as enzyme design, redox partner engineering, and optimization of catalytic systems, the catalytic efficiency of CYP450s has been significantly enhanced, enabling the efficient biosynthesis of various bile acids. This review summarizes the characteristics and synthetic strategies of bile acids, as well as the optimized application of CYP450 hydroxylases, providing a reference for the green production of bile acids and related drug research.

Key words

bile acid / biosynthesis / CYP450 / hydroxylation reaction / enzyme engineering

引用本文

马杉池, 孙文涛, 魏茜, 胡友财. CYP450羟化酶在胆汁酸生物合成中的应用. 药学学报, 2026 , 61 (5) : 1484 -1497 . DOI: 10.16438/j.0513-4870.2026-0121

Shan-chi MA, Wen-tao SUN, Qian WEI, You-cai HU. Application of CYP450 hydroxylases in the biosynthesis of bile acids[J]. Acta Pharmaceutica Sinica, 2026 , 61 (5) : 1484 -1497 . DOI: 10.16438/j.0513-4870.2026-0121

正文

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胆汁酸是胆汁的主要活性成分, 在传统中医领域, 猪胆、牛胆、熊胆、蛇胆等44种动物胆已有2 500多年的应用历史, 具有清热、润燥、解毒、镇痉等功效^[1]。胆汁酸由环戊烷多氢菲的甾体母核(图1A) 与C17位的脂肪族侧链组成, 通常含有一个或多个羟基取代基, 迄今为止已发现100多种, 根据其结构可分为游离型胆汁酸和结合型胆汁酸(图1C)^[2]。人体内典型的24碳游离型胆汁酸包括胆酸(cholic acid, CA)、鹅去氧胆酸(chenodeoxycholic acid, CDCA)、熊去氧胆酸(ursodeoxycholic acid, UDCA)、去氧胆酸(deoxycholic acid, DCA)、石胆酸(lithocholic acid, LCA) 等。胆汁酸-CoA与牛磺酸或甘氨酸结合, 形成结合型胆汁酸, 在体内通常以钠盐形式存在, 比游离型胆汁酸更稳定、水溶性更强^[3]。此外, 27碳的胆汁酸, 如varanic acid, 在低等原始脊椎动物中更为丰富^[2], 在西印度海牛中则发现存在27碳的trichechol类胆汁醇^[4]。

胆汁酸结构具有两亲性: 甲基朝向的β面具有疏水性, α面由于α-羟基取代和侧链羧基或氨基酸缀合具有亲水性(图1B)^[5]。因此, 对于胆汁酸, 不同区域和立体选择性的羟基可能显著影响分子的药代动力学性质与生物活性, 也成为合成途径中较为关键的结构修饰。

胆汁酸是体内胆固醇分解的主要去向, 其可以乳化脂肪球供脂肪酶消化, 同时与磷脂、胆固醇等不溶性脂质形成胶束, 促进脂质和脂溶性维生素的溶解和运输, 对维持胆固醇稳态极为重要^[3]。现代医学研究表明, 胆汁酸通过激活胃肠道和肝脏内的核受体FXR、G蛋白偶联受体TGR5以及多种细胞信号传导途径, 参与调节葡萄糖、脂肪酸、胆固醇的合成、运输和代谢, 有望用于代谢综合征、2型糖尿病、非酒精性脂肪肝等的治疗^[6-9]。此外, 通过多种机制和作用靶点直接或间接作用, 胆汁酸还表现出抗病毒、抑制消化道细菌感染、缓解炎症反应、维持肠道免疫稳态等广泛的生物活性^[10-12]。

目前, CDCA作为孤儿药获欧洲药品管理局(European Medicines Agency, EMA) 批准, 用于治疗罕见的常染色体隐性遗传性疾病脑腱黄瘤病; 在elafibranor获批前, CDCA结构类似物奥贝胆酸(obeticholic acid, OCA) 与UDCA是美国食品药品监督管理局(Food and Drug Administration, FDA) 批准用于治疗原发性胆汁性胆管炎的仅有两款药物; CA是FDA批准用于治疗单酶缺乏导致的胆汁酸合成障碍的首款药物, 惠及罕见胆汁酸合成障碍患者; 猪去氧胆酸(hyodeoxycholic acid, HDCA) 在中国批准用于高脂血症的治疗; DCA是唯一获FDA批准治疗局部颏下脂肪增多的溶脂产品^[13-15]。牛磺熊去氧胆酸(tauroursodeoxycholic acid, TUDCA) 不仅能抑制细胞凋亡级联反应, 还可以减少氧化应激、保护线粒体、抗神经炎, 并作为化学伴侣维持蛋白质稳定和正确折叠, 发挥细胞保护活性, 有望用于神经退行性疾病治疗^{[16, 17]}。因而, 胆汁酸作为更多疾病的候选药物和先导化合物, 极具研究价值, 其规模化生产成为亟待解决的问题。

1　胆汁酸的合成策略

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对于常见的胆汁酸, 家畜家禽肉类加工过程中回收胆囊是较为经济的提取来源, 但其工艺复杂、制备周期长, 存在含有动物病原体的风险, 且易因加工过程中技术条件和卫生规程不达标, 造成环境污染^[2]。而UDCA作为我国传统珍贵动物药熊胆的活性成分, 历史上主要通过无管引流技术从黑熊胆汁中提取, 天然资源供给有限, 且因动物福利问题存在争议^[18]。故此, 目前正开展胆汁酸的人工合成研究, 基于化学合成和生物催化的合成路径已有较多探索, 以满足工业化生产的需要。

1.1　化学合成

化学合成作为生产胆汁酸的替代方法, 合成策略主要有两种。一种是以CA、CDCA等天然含量丰富的胆汁酸为起始原料, 选择性引入或脱去羟基, 获得UDCA等其他具有药用价值的胆汁酸^{[19, 20]}, 但原料成本较高, 获取方式存在局限性。另一种是以价格低廉的豆甾醇等植物甾醇及其发酵降解产物为原料, 包括双降醇、9α-羟基双降醇、脱氢表雄酮等, 经过十余步反应半合成制备^[21-25]。

在以9α-羟基双降醇等为原料合成DCA的途径中(图2A), 中间体烯丙位(即C12位) 的氧化为关键步骤, 通常采用先氧化为羰基再选择性还原的方式引入C12α-OH。在该位点引入羰基通常利用三氧化铬氧化体系, 毒性大、污染重^[26]。优化后的叔丁基过氧化氢/助氧化剂体系效率较低, 如需提高收率, 仍需使用氯铬酸吡啶鎓盐进一步氧化, 反应过程复杂^{[27, 28]}。赖开智等^[29]开发出以亚氯酸钠为氧化剂的制备方法, 环境友好, 但该步收率仍不超过70% (图2B)。可见, 胆汁酸的化学合成, 尤其是羟基的选择性引入仍面临诸多挑战。

1.2　生物催化

生物催化包括全细胞转化、细胞裂解物或纯酶催化, 相较于传统有机合成更为绿色环保。对于现有的胆汁酸化学合成途径, 关键步骤引入生物催化, 可提高反应选择性和收率。黄显等^[30]以鸡胆汁来源的CA为原料, 首先利用酶催化C7位特异性氧化, 再进行黄鸣龙反应, 实现从低浓度(含量35%以上) CA出发的DCA化学-酶法合成。

虽已经过工艺优化, 但高温还原等化学合成步骤仍具有一定的危险性。生物催化反应条件温和, 研究发现一些微生物具有转化和合成胆汁酸的能力。Eyssen等^[31]从大鼠肠道微生物群中分离出一种革兰阳性杆菌, 其能通过C7β-OH脱羟基和C6β-OH的差向异构化, 将β-鼠胆酸(β-muricholic acid, β-MCA) 转化为HDCA。蒋红平等^[32]利用分枝杆菌Mycobacterium sp. NRRL B-3805诱变菌种, 以工业废水废渣为原料进行生物发酵, 从植物甾醇一步构建胆汁酸的羰基侧链, 获得4-胆烯酸-3-酮, 结合化学合成制备LCA、CDCA、7-酮石胆酸(7-ketolithocholic acid, 7-KLCA)。以CDCA为原料, 脆弱拟杆菌(Bacteroides fragilis)、嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia) 等已被用于全细胞催化生产UDCA, 产率最高可达95%^[18]。然而, 这些微生物通常培养条件严苛, 且具有一定的致病性, 不利于药品安全生产。随着对合成途径的阐明和其中关键酶的挖掘, 利用大肠杆菌、酵母等模式菌种构建重组工程菌是一种更优策略。

各种含酮基的胆汁酸是由多种羟基甾体脱氢酶(hydroxysteroid dehydrogenase, HSDH) 催化的氧化产物^[33]。在重组大肠杆菌中, 7α-HSDH催化CDCA氧化脱氢生成7-KLCA, 再由7β-HSDH催化加氢还原, 可高效生成CDCA的差向异构体UDCA (图2C)。Yang等^[34]通过对7β-HSDH定点迭代饱和突变, 筛选得到五突变体, 活性较野生型提高了5.79倍。通过“一锅”中的两步反应, 50 g·L^-1 CDCA转化为UDCA的产率达94.3%, 为迄今报道的最高转化率。

由于HSDH具有高区域和立体选择性, 可催化的反应有限, 更多位点的修饰仍需依靠其他氧化反应。C(sp³)-H键的选择性氧官能团化是有机化学中最具挑战性的反应之一^[35]。然而, 多数传统合成方法依赖于底物的固有反应性, 限制了氧化发生的位点, 并且可能需要额外的保护/脱保护步骤^[36]。对于化学合成难以引入的羟基, 生物催化的羟基化反应步骤简略, 具有广阔的应用前景^[37]。

生物催化甾体惰性C(sp³)-H键的羟基化, 尤其是位于B环和C环的氧化已有较多报道。如Wei等^[38]发现人体肠道真菌小孢根霉(Rhizopus microsporus) 可以催化CDCA的C12、C15、C16、C18-羟基化和进一步修饰。黄素依赖性单加氧酶、非特异性过氧化酶、非血红素铁和α-酮戊二酸依赖性加氧酶等都有催化羟基化的活性^[39], 而细胞色素P450 (cytochrome P450, CYP450) 家族尤其作为羟化酶被广为研究, 其亦在胆汁酸的生物合成途径中发挥核心作用。

2　CYP450羟化酶参与的胆汁酸合成

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2.1　CYP450的结构与羟基化催化机制

CYP450是血红素依赖性单加氧酶, 其中原卟啉Ⅸ结合亚铁, 与保守的轴向半胱氨酸共价连接, 亚铁与一氧化碳结合在450 nm处产生特殊Soret吸收峰, 因而得名^[40]。CYP450可催化羟基化、环氧化、脱烷基等多种氧化反应^[41], 目前已挖掘到30余万种^[42]。不同家族的CYP450之间的序列相似性极低, 但整体结构高度保守, 包括A~L共12个α螺旋和β1~5折叠(图3A)^[43]。活性口袋周围的底物识别位点是参与底物识别和结合的区域, 具有高度可变序列, 决定了CYP450的底物选择性^[44]。比如, 胆汁酸合成途径中的C12α-羟化酶CYP8B1, I螺旋的W281位于血红素-铁上方, 形成活性位点的低天花板, 限制配体大小。底物7α-羟基-4-胆甾烯-3-酮的C7α-OH朝向W281, 其相互作用可能显著影响底物定位。此外, F螺旋的D210分别与W281的吲哚侧链和β4的R479存在相互作用, 该三联体之间的氢键网络可以稳定3个二级结构, 使CYP8B1发挥正常功能。相应地, W281F突变使酶对底物的亲和力下降1/2, 催化效率仅为野生型的1/6^[45]。

CYP450催化羟基化的循环(图3B) 始于与静息态Fe^Ⅲ配位的水分子被底物取代, Fe^Ⅲ从低自旋态转变为高自旋态。由于氧化还原电位降低, 在氧化还原伴侣的作用下, Fe^Ⅲ接受NAD(P)H传递的第一个电子, 被还原为Fe^Ⅱ。其结合氧分子形成铁-超氧, 第二次电子转移导致进一步被还原, 随后质子化, 产生铁-过氧化氢中间体(Cpd 0)。第二次质子化脱去水, 形成高度亲电的氧铁(Ⅳ) 卟啉π-阳离子自由基(Cpd Ⅰ)。Cpd Ⅰ通过均裂从底物中拔取氢, 产生Cpd Ⅱ。随后氧与底物自由基重新结合, 释放羟基化产物, 同时恢复Fe^Ⅲ静息态。一些CYP450还能通过利用H₂O₂而非氧分子的过氧化氢旁路, 结合底物后直接产生Cpd 0, 但其反应活性显著降低^[39]。

2.2　胆汁酸合成途径中的CYP450羟化酶

胆固醇为胆汁酸的生物合成提供合成砌块, CYP450通过经典(中性) 途径和替代(酸性) 途径参与初级胆汁酸的合成(图4)。在人体中, 经典途径始于限速酶CYP7A1催化生成7α-羟基胆固醇。随后的酶促步骤包括CYP8B1催化的C12α-羟基化以及CYP27A1催化的C27-羟基化, 主要产生CA和CDCA, 占胆汁酸总产量的75%以上^[46]。在替代途径中, 胆固醇的侧链首先被CYP27A1氧化, 随后由CYP7B1催化C7α-羟基化, 主要产生CDCA。在猪中, 替代途径产生的初级胆汁酸以HCA为主, 由CYP4A21催化CDCA的C6α-羟基化产生^[47]。此外, 胆固醇还可被CYP46A1和胆固醇25-羟化酶修饰, 生成24(S)-羟基胆固醇和25-羟基胆固醇, 并分别由CYP39A1和CYP7B1催化进一步发生C7α-羟基化, 但这两条途径对人体胆汁酸的合成贡献不大^[46]。初级胆汁酸CA和CDCA进入胃肠道, 经过微生物的脱氢、7α-脱羟基和差向异构化等反应, 产生DCA、LCA、氧代石胆酸(oxolithocholic acid, OLCA) 等次级胆汁酸, 肠道代谢进而产生UDCA、磺基石胆酸(sulfated lithocholic acid, SLCA) 等三级胆汁酸^{[48, 49]}。

疏水性较强的胆汁酸具有较大的肝脏毒性。在经典途径中, CYP8B1催化7α-羟基-4-胆甾烯-3-酮的C12α-羟基化, 最终生成CA, 通过调控CA与CDCA的比例控制胆汁酸池的整体疏水性, 稳定肠道脂肪吸收^[50]。对于LCA、DCA、UDCA和CDCA, 其游离形式及其与牛磺酸和甘氨酸的结合形式都是CYP3A4的底物^[51]。通过催化不同位置的羟基化, CYP3A4将其转化为更亲水的分子被代谢, 调节胆汁酸稳态^[52]。

CYP450催化的选择性羟基化赋予胆汁酸结构和活性多样性。对不同催化活性CYP450结构与功能的研究不仅为代谢相关疾病提供诊断标志物与治疗靶点, 将之应用于胆汁酸绿色生物制造也能缩短合成路径, 通过选择性结构修饰减少副产物积累, 提高生产效率。基于对这些羟化酶的理解, 已开展生物催化胆汁酸异源合成的相关研究, 破解合成难题(表1)^{[47, 52-63]}。

3　CYP450用于生物合成胆汁酸的优化策略

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在胆汁酸异源生物合成途径中, 不同物种来源的CYP450在结构特性与表达需求上存在差异, 需选择适宜的底盘宿主构建细胞工厂。细菌来源的CYP450多为可溶性胞质蛋白^[64], 如来源于抗生素链霉菌(Streptomyces antibioticus) 的CYP107D1 (OleP)^[59]和来源于巨大芽孢杆菌(Bacillus megaterium) 的P450 BM3 (CYP102A1)^[65], 在大肠杆菌中构建催化体系操作简便, 相较于真核系统具有蛋白产量高、生长快速、生产成本低的优势^[66]。而真菌及动物来源的CYP450多为膜结合蛋白, 需要相应的膜环境才能正常发挥功能^[64], 在大肠杆菌中的可溶性表达需通过截短疏水性跨膜锚定区、分子伴侣协助折叠等方法^[67], 因此更适合在酿酒酵母、毕赤酵母等真核底盘中异源表达, 以保证酶的正确定位、折叠及与氧化还原伴侣的电子传递^[68]。在此基础上, 综合CYP450酶的分子改造、氧化还原伴侣工程、催化体系优化等策略, 胆汁酸合成的整体效率实现进一步提升。

3.1　酶的分子改造

自然界中能够羟基化胆汁酸合成底物的CYP450不断被发现。然而, 野生型CYP450往往存在区域和立体选择性不佳、活性低、底物范围窄、热稳定性差等问题^[69]。为此, 蛋白质工程已成为改进酶的催化特性、甚至创造新功能的强大工具。

2018年诺贝尔化学奖得主Frances H. Arnold^{[70, 71]}最早将“酶的定向进化”概念付诸实践, 即通过人工选择模拟自然界适者生存的进化机制, 引入突变后筛选目标性质优化的酶, 将其作为下一轮进化的模板进行多轮筛选, 直至达到预期水平。近年来, 随着酶的结构解析信息和辅助计算工具的日益丰富, 筛选量更小、更为理性的酶工程策略不断发展, 对于CYP107D1、P450 BM3等CYP450的改造卓有成效(图5)。

3.1.1　CYP107D1

CYP107D1对于睾酮具有C6β、C7β、C12β、C15β的多位点羟基化活性^[72], 然而其能特异性催化LCA和DCA单一发生C6β-羟基化, 大肠杆菌静息细胞24 h催化产率分别为19.5%和35%^[59]。对此, Grobe等^[73]利用3DM数据库和分子对接分析决定选择性的关键残基, 通过丙氨酸扫描和构建“小而精”的突变文库, 结合比色测定法高效筛选, 突变体的羟基化选择性转变为C7β, 实现LCA到UDCA的一步生物转化。基于残基突变偏好性分析的进一步筛选获得三突变体F84Q/S240A/V291G (OlePs), 使UDCA产量较初始突变体提高10倍。

为进一步提高CYP107D1的催化活性和特异性, 毛淑红等^[74]在突变体OlePs的基础上对电子传递链、催化活性中心的关键氨基酸残基M58、T86、M197进行改造。其中, 通过接连引入M58W和T86L突变, 大肠杆菌全细胞催化产量较报道值分别提高33%和67.3%。

Song等^[75]则采用无细胞酶催化方法, 以OlePs为模板, 通过半理性设计迭代组合突变, 筛选得到优化的四突变体G294A/N236H/F321W/V297A, 1 mmol·L^-1 LCA可产生约0.68 mmol·L^-1 UDCA, 催化活性较模板提升50%, 区域选择性提升40%。分子动力学模拟显示, 突变一方面稳定疏水催化微环境, 增强与活性空腔开闭动力学相关的关键区域的灵活性; 另一方面直接与底物相互作用, 从而提升底物两端的稳定性。

3.1.2　P450 BM3

P450 BM3是自然界一种自给自足的CYP450, 其具有包含FMN和FAD的还原酶结构域用于电子传递, 兼具高稳定性和强催化效率, 极具改造潜力。通过实验室进化, 已形成可羟基化甾体C1、C2、C6、C7、C15和C16等位点的突变体库^[76]。通过聚焦诱变策略, 结合基于DNA组装和尿嘧啶特异性切除试剂USER酶克隆的新型蛋白质文库构建技术, 筛选得到的高活性突变体LG-23可催化六种甾体的C7β-羟基化, 具有显著的区域和立体选择性^[77]。

LG-23对LCA仅有13.9%的转化率, 且仍具有C1β、C6β、C7β等多种羟基化选择性。为提高转化率和C1β-羟基化的选择性, Li等^[78]基于半理性设计策略构建突变文库, 分别引入小体积氨基酸和疏水性氨基酸, 以重塑底物通道和结合口袋, 增加蛋白与底物的氢键和疏水作用。经过四轮叠加突变, 获得最优突变体G87A/W72T/A74L/L181M, 对LCA的转化率达68.1%, C1β-羟基化选择性达99.4%, 转换数(turnover number, TON) 较LG-23提高了26倍。最终羟基化产物产量较模板LG-23提高21.5倍, 首次实现LCA在A环的高效羟基化修饰。

对于C6β-羟基化选择性的优化, Deng等以LG-23为模板, 首先通过丙氨酸/甘氨酸/苯丙氨酸扫描鉴定活性口袋关键残基, 发现T260G突变使转化率和选择性显著提高。再以此为模板开展两轮突变, 优化活性口袋空间。突变体L82V/T260G/G328A与底物的疏水作用和氢键增强, 不仅表现出更优的催化活性, 转化率提高8.5倍, 选择性也显著提升至96.3%。分子动力学模拟显示, 突变体的血红素Cpd I与LCA的C6平均距离缩短21.5%; TON值是LG-23的3.8倍, 表明突变体能更稳定地催化LCA生成鼠去氧胆酸(murideoxycholic acid, MDCA)。

3.1.3　其他工程化CYP450

DCA发生C7β-羟基化生成熊胆酸(ursocholic acid, UCA) 是由DCA工业合成UDCA的第一步。拟枝孢镰刀菌(Fusarium sporotrichioides) 的贝壳杉烯氧化酶CYPg3484可催化该步反应, 但副产物较多。加尔迪等^[60]首先筛选其相似序列, 确定以活性与选择性更优的源自禾谷镰刀菌(Fusarium graminearum) 的CYP450 (本文暂命名为P450_FG) 为起点, 进行连续突变, 以提高毕赤酵母催化体系的收率。由于C12-OH与蛋白之间的氢键促进C7β-羟基化, 对该区域残基进行随机诱变和饱和突变, R113V/A368I突变组合使收率由49%提高至65%。在此基础上, 引入E110S/L365S突变以调整酶的三维结构, 一方面减少DCA与蛋白第二键合口袋间的相互作用, 减少过氧化副反应; 另一方面促进DCA进入活性位点, 增加蛋白对底物的亲和力, 有效降低底物抑制。最终实现90%的收率, UCA产量超过2 g·L^-1。

Zhou等^[61]结合基因组和转录组测序, 从木贼镰刀菌(Fusarium equiseti) HG18中鉴定了一种新型CYP450 (P450_FE) 及其还原酶。其能催化LCA的C7β-羟基化, 为首个真菌来源的该功能酶。但在重组毕赤酵母中, UDCA产率仅5.2%。结合同源建模、分子对接与保守氨基酸序列分析, 并通过定点突变验证, 揭示Q112、V362和L363是调控C7β-羟基化活性和选择性的关键残基。其中, V362I突变体的UDCA产率较野生型提高2.6倍, 选择性由61.2%提高至90.4%。此外, 以P450_FE为模板, 通过系统发育分析, 从长柄镰刀菌(Fusarium longipes) 挖掘到具有同样催化活性的P450_FL。基于活性位点饱和突变/迭代组合突变和计算机辅助设计, 增强酶与底物的氢键、疏水作用和静电相互作用, 最终获得最优四突变体M4 (T116N/V364I/L488I/E268K), UDCA产率由11.5%提高至94.2%。重组毕赤酵母经补料分批级联反应, 实现3.9 g·L^-1的高产^[62]。

来源于白色异库茨涅尔氏菌(Allokutzneria albata) 的CYP450 (本文暂命名为P450_AA) 同样能C7β-羟基化LCA, 底物浓度为50 mmol·L^-1时, 24 h产物摩尔收率达95.9%, 光学纯度为99% (R)。为进一步提高底物浓度用于工业生产, 石金田等^[63]以此为模板设计突变体L84Q/D93A, 其催化活力为5.6 U·mg^-1, 是野生型的42.4倍, 并且底物浓度提升至500 mmol·L^-1时, LCA仍能在24 h内完全转化。

这些研究基于酶的晶体结构、理化性质进行理性和半理性设计, 或对于未充分表征的酶构建随机突变体文库广泛筛选, 并通过虚拟筛选和高通量检测方法减少验证的工作量, 丰富了酶改造的工具箱; 同时加深了对CYP450活性口袋结构与催化功能间关系的理解, 为机器学习辅助的理性设计积累高质量的数据样本。

3.2　氧化还原伴侣工程

CYP450可通过不同的氧化还原系统进行电子传递(图3C)。Ⅰ型常见于某些细菌和真核生物线粒体中, 电子通过黄素依赖性铁氧还蛋白还原酶(ferredoxin reductase, FdR) 和含铁硫簇[Fe-S]的铁氧还蛋白(ferredoxin, Fdx) 传递; Ⅱ型利用含有FAD和FMN的CYP450还原酶(cytochrome P450 reductase, CPR), 有时另一种膜结合血红素蛋白, 即细胞色素b₅也会参与电子传递^[79]。近年来, 其他特殊的电子传递系统不断被发现。一些CYP450因本身含有氧化还原酶结构域而能自给自足, 如P450 BM3, 以及融合含FMN与[Fe-S]结构域的CYP116家族^{[80, 81]}。I型三组分系统中, Fdx有时会被含FMN的黄素氧还蛋白Fld取代, 且可以与CYP450融合^[82]。P450nor是目前唯一发现的可溶性真核CYP450, 可以直接利用NADH作为电子供体^[83]。

CYP450的催化循环需要氧化还原伴侣向血红素-铁反应中心传递电子, 其效率对催化活性至关重要。对于P450_FE, 当单独表达CYP450时, LCA生成UDCA的转化率仅为1.1%; 而与同源还原酶feCPR共表达时, 转化率提高至10.2%^[84]。因此, 筛选合适的氧化还原伴侣并进一步工程化改造, 优化电子传递链, 有助于羟基化反应循环的高效运转。

3.2.1　氧化还原伴侣的适配

对于酵母全细胞催化体系, 因其具有翻译后修饰能力和内膜系统, 其内源CPR可以参与异源CYP450的电子传递^[85]。为了提高效率, 往往需要过表达内源CPR或共表达异源CPR。同一物种来源的CYP450与氧化还原伴侣形成的电子传递链通常较为高效, 但有时该物种的氧化还原伴侣尚未鉴定, 或者其他物种来源的在异源宿主中反而更为适配。

为筛选CYP107D1的氧化还原伴侣, Sun等^[86]基于双分子荧光互补技术(bimolecular fluorescence complementation, BiFC), 设计了sfGFP传感器。Fdx融合至sfGFP-1-10的N端, CYP107D1与sfGFP-11的C端融合, 当二者相互作用强烈时, sfGFP-1-10和sfGFP-11形成发色团, 可检测到绿色荧光, 使得筛选更灵敏简便, 最终获得最优氧化还原伴侣PetH/PetF。相较于最初使用的CamA/CamB, 该组合使全细胞催化DCA的C6β-羟基化转化率由34.8%显著提升至89.2%, 展现了氧化还原伴侣的高效筛选对于提升CYP450催化效率的强大潜力。

3.2.2　构建融合蛋白

自然界中自给自足的CYP450具有较高的催化效率, 如P450 BM3催化肉豆蔻酸盐ω-2羟基化的转化频率可达每分钟4 000^[87]。受此启发, 将氧化还原伴侣与CYP450连接形成融合蛋白, 有望提高电子传递效率, 促进反应进行。

连接肽的长度及其柔性对融合蛋白的活性有重要影响。由小体积氨基酸组成的柔性连接肽, 如(GGGGS)_n, 应用较多。一方面甘氨酸更具灵活性, 连接的结构域可进行一定程度的移动; 另一方面丝氨酸与水分子形成氢键, 维持连接肽在水溶液中的稳定性。n的取值需要保证蛋白的正确折叠, 结构域之间既保持一定距离, 又能相互作用^[88]。当多个结构域的空间分离对蛋白正常发挥功能较为关键时, 刚性连接肽则是更佳选择, 通常采用α螺旋或包含多个Pro残基, 以保持融合蛋白的稳定性和生物活性^[89]。

基于CYP107D1突变体OlePs, 吕迪^[90]构建了多酶融合表达体系, 以(PG)_n为基本单元的刚性连接肽或柔性连接肽(GGGGS)₃连接OlePs及其氧化还原伴侣PDR/PDX, 发现使用(PG)₄连接肽的效率最高, 产物得率为53.88%。

但蛋白的融合也可能导致催化活性的降低甚至丧失。对于CYP107D1, 与PDR/PDX融合表达的催化效率不及多酶共表达体系^[90]; 与氧化还原伴侣PetH/PetF的融合可能反而影响其自身的功能, 融合酶虽然能高水平表达, 但完全失活^[91]。对于融合表达如何影响蛋白活性, 其机制尚未完全明确。

3.2.3　氧化还原伴侣的改造

CYP7A1是胆汁酸经典合成途径的限速酶, 催化胆固醇C7α-羟基化, 生成初级胆汁酸的关键前体。然而CYP7A1及其CPR为膜结合蛋白, 微生物异源表达存在溶解度低、电子传递低效等瓶颈。Shang等^[92]通过密码子优化和N端截短, 增强CYP7A1及其CPR的可溶性表达, N端截短27个残基的CPR使胆固醇转化率达到全长CPR组合的2倍。通过共表达分子伴侣蛋白辅助蛋白折叠, 实现人源CYP7A1在大肠杆菌中的功能性表达。

针对CYP107D1催化LCA生成MDCA的电子传递链, Sun等^[91]对氧化还原伴侣PetF进行半理性设计。基于同源序列比对划分5个蛋白相互作用区域, 结合残基保守性分析和FoldX自由能计算, 确定7个核心残基。虚拟饱和突变初步筛选后, 利用MDCA经75%硫酸-乙醇处理产生的特征吸收峰进行高通量筛选, 获得突变体PetF_F64D, NADH耦合效率从26.9%提升至46.4%。分子动力学模拟显示, 突变体的D64与CYP107D1的R105形成新的稳定盐桥作用, 缩短了电子传递距离, 电子传递速率提升2.9倍, 使MDCA转化率从32.5%提高至80.9%, TON值从406.2提高至1 617.9。

氧化还原伴侣作为电子传递的核心媒介, 其功能优化可突破电子转移导致的催化限速瓶颈。工程化改造的探索也为阐明CYP450与氧化还原伴侣相互作用的分子机制积累经验, 建立普适性的互作界面设计原则, 以期通过精准改造实现反应的定向调控。

3.3　催化体系的优化

结合CYP450的催化特性, 通过构建高效辅因子再生系统、提升底物利用效率等策略进一步优化催化体系, 为生物催化提供稳定环境, 兼顾效率提升和成本控制。

3.3.1　辅因子再生系统的构建

CYP450催化循环的正常进行需要辅因子的足量供应。在酵母中, NADPH主要通过磷酸戊糖途径再生, 共底物葡萄糖首先磷酸化为葡萄糖-6-磷酸, 随后由ZWF1基因编码的葡萄糖-6-磷酸脱氢酶催化脱氢反应, 产生NADPH^[93]。在全细胞催化体系中, 基于脱氢酶构建额外的辅因子再生系统, 有望增强细胞内辅因子的循环效率, 从而促进底物转化。

为促进6β-OH DCA的高效合成, Sun等^[86]在大肠杆菌中构建了内源NADPH再生系统。通过共表达NAD⁺激酶nadK或膜结合转氢酶pntAB, 转化率从71.2%分别提升至90.3%和77.6%。最终构建的全细胞催化体系无需外源添加NADPH, 转化率达到99.1%。

3.3.2　提高底物利用

甾体底物的水溶性较差, 在培养基中可能析出而无法被细胞摄取, 大规模发酵时易产生泡沫, 限制了其转化效率。为了促进底物被微生物利用, 添加助溶剂是一种有效策略(图6)。环糊精(cyclodextrin, CD) 是6、7或8个葡萄糖形成α-1,4-葡萄糖单元的环状低聚物, 分别称为α-、β-、γ-环糊精。其兼具亲水表面和疏水空腔, 能与腔内亲脂性化合物形成水溶性包合物, 从而提高其溶解度、稳定性和生物利用度^[94]。

此外, 底物必须跨膜运输进细胞才能被酶催化, 提高细胞壁和细胞膜的通透性可以增强底物向细胞内的转运, 促进高效转化。Tween 80是常用的水包油(O/W) 型乳化剂, 一方面由于两亲性, 有助于难溶性物质在溶液中均匀分散; 另一方面作为非离子表面活性剂, 能够溶解细胞膜而不影响其完整性, 改变细胞壁组成, 提高菌体细胞通透性, 从而利于底物进入细胞^[95]。多粘菌素B (polymyxin B) 是从多黏芽孢杆菌(Bacillus polymyxa) 分离得到的抗生素, 通过结合革兰阴性菌外膜上的脂多糖, 可增强膜的通透性^[96]。

在大肠杆菌胆固醇C7α-羟基化催化体系中, 32 μg·mL^-1多粘菌素B使胆固醇转化率从42.99%显著提升至60.96%; 45%羟丙基-β-环糊精促进胆固醇的高效溶解, 转化率达到61%^[92]。然而, 过高浓度的助渗透物质可能对细胞膜的破坏能力过强, 使得细胞形态难以维持, 反而造成活性显著丧失^[97]。

总体而言, 由于CYP450家族的多样性以及底盘细胞复杂的调控网络, 催化体系的优化策略因酶而异。对于反应条件需要精细化调控, 摸索最优组合, 以最大限度地激发酶的活性; 同时在保证效率的前提下兼顾成本, 匹配工业化应用场景。

4　总结与展望

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胆汁酸作为一类活性广泛的甾体化合物, 不仅在人体代谢过程中扮演着不可或缺的角色, 更为多种疑难病症的治疗提供新方向。CYP450羟化酶的结构修饰功能调控着体内胆汁酸池, 对于胆汁酸的高效生产也已表现出强大的应用潜力。通过酶的挖掘和改造, 选择性专一、活性显著的候选酶库日益丰富, 设计CYP450的底物识别位点能够适配不同胆汁酸合成前体, 实现特定选择性的羟基化修饰。综合多种催化条件的优化策略, CYP450的催化效率进一步提升, 为高价值胆汁酸的规模化生物合成奠定了坚实基础。

尽管CYP450生物催化体系的研究已取得诸多进展, 但构建高效微生物细胞工厂仍存在一些瓶颈: 部分工程酶的催化效率与工业应用要求仍有差距, 高底物浓度下存在底物抑制现象; 氧化还原伴侣与CYP450的匹配机制仍需深入解析, 以进一步提升电子传递效率; 从实验室验证到工业化生产, 催化体系的稳定性与重复性有待验证, 并探索更为经济绿色的合成条件。

未来研究可从以下角度重点推进: 一是聚焦临床急需胆汁酸的定向合成, 结合人工智能与结构生物学技术, 针对UDCA、TUDCA等探索CYP450羟化酶的定向设计与精准改造, 进一步优化酶的选择性与催化效率, 降低生产成本, 满足临床应用需求; 二是创新催化体系与形式, 开发高效电子传递系统, 构建多酶复合体, 应用固定化酶、细胞表面展示等技术, 进一步提高催化体系的可重复利用性与稳定性, 为药用胆汁酸的工业化生产提供技术支撑; 三是拓展胆汁酸的结构修饰空间, 随着对胆汁酸构效关系的深入研究, 通过CYP450进行更多位点的羟基化, 开发新型胆汁酸衍生物, 筛选低毒性、高活性的药物先导化合物。基于酶工程、代谢工程、合成生物学等多学科的交叉融合, CYP450羟化酶介导的胆汁酸生物合成将不仅助力现有药品的稳定供应, 更能推动新型药物的研发与转化, 彰显胆汁酸独特药用价值的广阔应用前景。

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2026年第61卷第5期

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doi: 10.16438/j.0513-4870.2026-0121

接收时间：2026-01-28
首发时间：2026-09-21
出版时间：2026-05-12

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中国医学科学院北京协和医学院, 药物研究所, 天然药物活性物质与功能国家重点实验室, 北京 100050

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胡友财, Tel: 86-10-50927869, E-mail: huyoucai@imm.ac.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Name	Source	Substrate(s)	Reaction(s)	Ref
I. CYP450s in the natural biosynthetic pathway of bile acids
CYP7A1	Vertebrates	Cholesterol	C7α-Hydroxylation	[53]
CYP8B1	Vertebrates	7α-Hydroxycholest-4-en-3-one, 5β-cholestane-3α,7α-diol, 3α,7α-dihydroxy-5β-cholestanoic acid, chenodeoxycholic acid, 7α-hydroxycholesterol, 7α,26-dihydroxycholest-4-en-3-one, 7α-hydroxy-3-oxo-4-cholestenoic acid	C12α-Hydroxylation	[54-56]
CYP7B1	Vertebrates	27-Hydroxycholesterol, 25-hydroxycholesterol, pregnenolone, dehydroepiandrosterone	C7α-Hydroxylation	[58]
5α-Androstane-3β,17β-diol	C6α-, C7α-Hydroxylation
CYP4A21	Sus scrofa	Chenodeoxycholic acid, taurochenodeoxycholic acid	C6α-Hydroxylation	[47]
CYP3A4	Higher vertebrates	Lithocholic acid	C3-Dehydrogenation; C1β-, C6α-hydroxylation	[52]
Deoxycholic acid	C3-Dehydrogenation; C1β-hydroxylation
Chenodeoxycholic acid	C3-Dehydrogenation; C6α-hydroxylation
Ursodeoxycholic acid	C3-Dehydrogenation; C1β-, C22-hydroxylation
Cholic acid	C3-Dehydrogenation
CYP27A1	Vertebrates	5β-Cholestane-3α,7α-diol, 5β-cholestane-3α,7α,12α-triol, 3β-hydroxy-Δ⁵ sterols (cholesterol, 7α-hydroxycholesterol, 24-hydroxycholesterol, 25-hydroxycholesterol, cholesterol sulfate) 3-oxo-Δ⁴ sterols (4-cholesten-3-one, 7α-hydroxy-4-cholesten-3-one, 24-hydroxy-4-cholesten-3-one, 25-hydroxy-4-cholesten-3-one)	C27-Hydroxylation and further oxidation	[57]
II. CYP450s used for heterologous biosynthesis of bile acids
CYP107D1 (OleP)	Streptomyces antibioticus, heterologously expressed in Escherichia coli	Lithocholic acid, deoxycholic acid	C6β-Hydroxylation	[59]
CYPg3484	Fusarium sporotrichioides, heterologously expressed in Pichia pastoris	Deoxycholic acid	C7β-Hydroxylation	[60]
P450_FG	Fusarium graminearum, heterologously expressed in Pichia pastoris	Deoxycholic acid	C7β-Hydroxylation	[60]
P450_FE	Fusarium equiseti HG18, heterologously expressed in Pichia pastoris	Lithocholic acid	C7β-Hydroxylation	[61]
P450_FL	Fusarium longipes, heterologously expressed in Pichia pastoris	Lithocholic acid	C7β-Hydroxylation	[62]
P450_AA	Allokutzneria albata, heterologously expressed in Escherichia coli	Lithocholic acid	C7β-Hydroxylation	[63]

Name

Source

Substrate(s)

Reaction(s)

Ref

I. CYP450s in the natural biosynthetic pathway of bile acids

CYP7A1

Vertebrates

Cholesterol

C7α-Hydroxylation

[53]

CYP8B1

Vertebrates

7α-Hydroxycholest-4-en-3-one, 5β-cholestane-3α,7α-diol, 3α,7α-dihydroxy-5β-cholestanoic acid, chenodeoxycholic acid, 7α-hydroxycholesterol, 7α,26-dihydroxycholest-4-en-3-one, 7α-hydroxy-3-oxo-4-cholestenoic acid

C12α-Hydroxylation

[54-56]

CYP7B1

Vertebrates

27-Hydroxycholesterol, 25-hydroxycholesterol, pregnenolone, dehydroepiandrosterone

C7α-Hydroxylation

[58]

5α-Androstane-3β,17β-diol

C6α-, C7α-Hydroxylation

CYP4A21

Sus scrofa

Chenodeoxycholic acid, taurochenodeoxycholic acid

C6α-Hydroxylation

[47]

CYP3A4

Higher vertebrates

Lithocholic acid

C3-Dehydrogenation; C1β-, C6α-hydroxylation

[52]

Deoxycholic acid

C3-Dehydrogenation; C1β-hydroxylation

Chenodeoxycholic acid

C3-Dehydrogenation; C6α-hydroxylation

Ursodeoxycholic acid

C3-Dehydrogenation; C1β-, C22-hydroxylation

Cholic acid

C3-Dehydrogenation

CYP27A1

Vertebrates

5β-Cholestane-3α,7α-diol, 5β-cholestane-3α,7α,12α-triol, 3β-hydroxy-Δ⁵ sterols (cholesterol, 7α-hydroxycholesterol, 24-hydroxycholesterol, 25-hydroxycholesterol, cholesterol sulfate) 3-oxo-Δ⁴ sterols (4-cholesten-3-one, 7α-hydroxy-4-cholesten-3-one, 24-hydroxy-4-cholesten-3-one, 25-hydroxy-4-cholesten-3-one)

C27-Hydroxylation and further oxidation

[57]

II. CYP450s used for heterologous biosynthesis of bile acids

CYP107D1 (OleP)

Streptomyces antibioticus, heterologously expressed in Escherichia coli

Lithocholic acid, deoxycholic acid

C6β-Hydroxylation

[59]

CYPg3484

Fusarium sporotrichioides, heterologously expressed in Pichia pastoris

Deoxycholic acid

C7β-Hydroxylation