氧化还原伴侣工程：P450低效问题的解决方案之一

doi:10.13560/j.cnki.biotech.bull.1985.2022-0985

摘要/Abstract

摘要：

细胞色素P450酶（CYPs或P450s）可将O₂的一个原子插入有机底物同时将另一个原子还原为水，广泛参与各种合成代谢和分解代谢过程，所以一直以来都是生物技术领域关注的焦点。在催化循环底物的氧化依赖于氧化还原伴侣向血红素铁传递电子，因此电子转移是P450s催化过程中的限速步骤。利用不同方法优化蛋白质-蛋白质相互作用以提高P450系统的电子转移效率，被称为“氧化还原伴侣工程”，是目前工程化P450s的重要手段之一，并取得了卓有成效的进展。本文将着重介绍关于氧化还原伴侣组分替换组装、P450酶与氧化还原伴侣融合及P450酶与氧化还原伴侣作用界面修饰等方面的进展，期望为未来该方面的工作提供一定的指导作用。

关键词: 细胞色素P450酶, 氧化还原伴侣, 电子转移, 组件替换, 融合酶

Abstract:

Cytochrome P450 enzymes（CYPs or P450s）can insert one atom of O₂ into organic substrates and reduce another atom to water. As P450s are widely involved in various anabolic and catabolic processes, they have always been a focus of biotechnology. Oxidation of substrates in the catalytic cycle relies on the transfer of electrons from redox partners to the heme iron, suggesting electron transfer as a rate-limiting step in the catalytic process of P450s. Optimization of protein-protein interactions to improve the electron transfer efficiency of the P450 system, known as “redox partner engineering”, is one of the important means to engineer P450s, and has achieved fruitful progress. This paper will focus on the progress in the replacement and assembly of redox partners, fusion of P450 enzymes with redox partners, and interface modification between P450 enzymes and redox partners, aiming to provide some guidance for future work in this area.

Key words: cytochrome P450 enzymes, redox partners, electron transfer, component replacement, fusion enzymes

张岩峰, 叶丽丹, 于洪巍. 氧化还原伴侣工程：P450低效问题的解决方案之一[J]. 生物技术通报, 2023, 39(4): 10-23.

ZHANG Yan-feng, YE Li-dan, YU Hong-wei. Redox Partner Engineering: A Solution to the Low Catalytic Efficiency of P450s[J]. Biotechnology Bulletin, 2023, 39(4): 10-23.

图/表 4

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RPs	P450酶 Enzyme P450	底物Substrate	组装结果Assembly results	实例Example
P. putida Pdx/PdR	StreptomycesCYP154C3s	孕酮	支持对10种类固醇底物的反应活性	[11]
P. putida Pdx/PdR	S. avermitilisCYP107X1	孕酮	支持孕酮的16α-羟基化	[12]
E. coli Fld/Fpr	牛CYP17A1	孕酮	支持孕酮的17α-羟基化	[13-14]
E. coli Fld/Fpr	S. cellulosumCYP264A1 B. megateriumCYP106A2	4-甲基-3-苯基香豆素，11-脱氧皮质酮	Adx/Fpr比Adx/AdR 对CYP264A1和CYP106A2的反应初始速率分别提升52%和33%	[15]
菠菜Fdx/FdR	R.wratislaviensis CYP108N7	苯甲硫醚	比其他RPs的转化率高5倍以上	[18]
哺乳动物Adx/AdR	B. megateriumCYP106A2	11-脱氧皮质酮	支持转化速率达到1 mmol/（L·d）	[18]
哺乳动物Adx/AdR	人CYP11B1	11-脱氧皮质醇	支持皮质醇产量达0.84 g/（L·d）	[21]
S. pombe Etp1^fd/Arh1	S. griseolusCYP105A1	磺酰脲类	支持3-环己基羟基化活性	[25]
S. pombe Etp1^fd/Arh1	牛CYP21A2	Madrane	比同源CPR的时空产率高1.8倍	[24]
S. elongatusSelFdx1499/SelFdR0978	S. elongatusADO（非P450酶）	正十五烷	比菠菜Fdx/FdD产率高26.6%	[26]
S. elongatusSelFdx1499/SelFdR0978	S. benihana CYP-sb21	环孢菌素	比其他RPs支持的转化率高1.4倍以上	[27]
B. megaterium Fdx2 /BmCPR	B. megateriumCYP106A1	11-酮基-β-乳香酸	Fdx2/Arh1支持最高的转化率（79.6±6.9）%	[28]
B. megaterium Fdx2 /BmCPR	牛CYP21A2	孕酮	BmCPR比同源CPR催化效率高1.7倍	[29]
S. coelicolor Fdx4/ FDR1	S. coelicolorCYP105D5	脂肪酸	比其他同源和异源RPs催化效率均高	[30]
S. Cellulosum Fdxs/FdR_B	S. CellulosumCYP260A1	诺卡酮	Fdx2/FdR_B比其他同源RPs转化率均高	[31]
S. Cellulosum Fdxs/FdR_B	S. CellulosumCYP109D1	脂肪酸	Fdx8/FdR_B比Fdx2/FdR_B有效，但产物生成效率比牛Adx/AdR低3倍	[32]
Synechocystis SynFdx/C. reinhardtii FNR	S. cellulosumP450 EpoK	埃博霉素D	SynFdx/ FNR比其他RPs组合转化率高8-11倍	[33]
S. cerevisiae CPR	Aspergillus oryzaeCYPs	7-乙氧基香豆素	支持多个P450酶的催化活性	[34]
T. cuspidata CPR	T. cuspidata 10β-羟化酶	紫杉烯-5α-乙酸酯	比酵母CPR羟化活性提升7倍	[35]
C. apicola CPR	B. subtilisCYP109B1，T. fusca CYP154E1	肉豆蔻酸，香叶醇	支持CYP109B1和CYP154E1的羟化活性	[36]

RPs	P450酶 Enzyme P450	底物Substrate	组装结果Assembly results	实例Example
P. putida Pdx/PdR	StreptomycesCYP154C3s	孕酮	支持对10种类固醇底物的反应活性	[11]
P. putida Pdx/PdR	S. avermitilisCYP107X1	孕酮	支持孕酮的16α-羟基化	[12]
E. coli Fld/Fpr	牛CYP17A1	孕酮	支持孕酮的17α-羟基化	[13-14]
E. coli Fld/Fpr	S. cellulosumCYP264A1 B. megateriumCYP106A2	4-甲基-3-苯基香豆素，11-脱氧皮质酮	Adx/Fpr比Adx/AdR 对CYP264A1和CYP106A2的反应初始速率分别提升52%和33%	[15]
菠菜Fdx/FdR	R.wratislaviensis CYP108N7	苯甲硫醚	比其他RPs的转化率高5倍以上	[18]
哺乳动物Adx/AdR	B. megateriumCYP106A2	11-脱氧皮质酮	支持转化速率达到1 mmol/（L·d）	[18]
哺乳动物Adx/AdR	人CYP11B1	11-脱氧皮质醇	支持皮质醇产量达0.84 g/（L·d）	[21]
S. pombe Etp1^fd/Arh1	S. griseolusCYP105A1	磺酰脲类	支持3-环己基羟基化活性	[25]
S. pombe Etp1^fd/Arh1	牛CYP21A2	Madrane	比同源CPR的时空产率高1.8倍	[24]
S. elongatusSelFdx1499/SelFdR0978	S. elongatusADO（非P450酶）	正十五烷	比菠菜Fdx/FdD产率高26.6%	[26]
S. elongatusSelFdx1499/SelFdR0978	S. benihana CYP-sb21	环孢菌素	比其他RPs支持的转化率高1.4倍以上	[27]
B. megaterium Fdx2 /BmCPR	B. megateriumCYP106A1	11-酮基-β-乳香酸	Fdx2/Arh1支持最高的转化率（79.6±6.9）%	[28]
B. megaterium Fdx2 /BmCPR	牛CYP21A2	孕酮	BmCPR比同源CPR催化效率高1.7倍	[29]
S. coelicolor Fdx4/ FDR1	S. coelicolorCYP105D5	脂肪酸	比其他同源和异源RPs催化效率均高	[30]
S. Cellulosum Fdxs/FdR_B	S. CellulosumCYP260A1	诺卡酮	Fdx2/FdR_B比其他同源RPs转化率均高	[31]
S. Cellulosum Fdxs/FdR_B	S. CellulosumCYP109D1	脂肪酸	Fdx8/FdR_B比Fdx2/FdR_B有效，但产物生成效率比牛Adx/AdR低3倍	[32]
Synechocystis SynFdx/C. reinhardtii FNR	S. cellulosumP450 EpoK	埃博霉素D	SynFdx/ FNR比其他RPs组合转化率高8-11倍	[33]
S. cerevisiae CPR	Aspergillus oryzaeCYPs	7-乙氧基香豆素	支持多个P450酶的催化活性	[34]
T. cuspidata CPR	T. cuspidata 10β-羟化酶	紫杉烯-5α-乙酸酯	比酵母CPR羟化活性提升7倍	[35]
C. apicola CPR	B. subtilisCYP109B1，T. fusca CYP154E1	肉豆蔻酸，香叶醇	支持CYP109B1和CYP154E1的羟化活性	[36]

RPs	P450酶Enzyme P450	底物Substrate	融合结果Fusion results	实例Example
B. megateriumP450 BM3还原酶（BMR）	哺乳动物肝脏CYPs	多种脂肪酸和药物	支持多个P450酶的催化活性，真核P450酶的溶解度明显提升	[46-51]
	A. OryzaeCYP57B3	大豆苷元	支持6-OH大豆苷元最大产量为9.1 mg/L	[52]
	JeotgalicoccusP450 OleTJE	饱和脂肪酸	比Pdx/PdR的周转率高8.9倍	[53]
B. subtilisCYP102A3还原酶	B. megateriumP450 BM3血红素域	12-对硝基苯基十二烷酸	比天然酶的热稳定性明显提升	[54]
RhodococcusCYP116B2还原酶（RhFRED）	S. venezuelaeP450 PikC	12元环大环内酯YC-17和14元环大环内酯那博霉素	比菠菜Fdx/FdR的催化活性提高约4倍	[57]
	StreptomycesP450 TamI	去氧糖胺糖苷修饰的碳环	替代菠菜Fdx/FdR实现了制备级规模的C10羟化反应	[58]
	A. orientalisP450_Prava	紧缩素	支持在产黄青霉中产生超过6 g/L的普伐他汀	[59]
	M. griseorubidaP450 MycG	麦新米星	支持P450 MycG的常规羟化反应	[37]
R. ruberCYP116B3还原酶（PFOR）	M. aquaeoleiCYP153AM.aq	十二烷酸	比融合RhFRED的反应初始速率高3倍	[61]
T. thermophilesCYP116B46还原酶	R. coprophilusTC-2 P450tol	甲苯	比融合RhFRED的半衰期和活性分别提高20倍和2倍以上	[62]
大鼠CPR	大鼠CYP1A1	7-乙氧基香豆素	融合酶比无CPR的CYP1A1催化活性高4倍	[63]
ArabidopsisCPR	P. ginseng P450 PPDS	达马烯二醇	比共表达CPR的催化活性高约4.5倍	[65]
T. cuspidataCPR	T. cuspidataCYP725A4	紫衫二烯	比共表达CPR的催化活性显著降低	[66]
H. tuberosusCPR	H. tuberosusCYP76B1	苯脲类除草剂	比只存在CYP76B1的除草剂耐受性降低一半以上	[67]
人Adx/AdR	人CYP11A1	胆固醇	比共表达Adx/AdR的表观V_max增加5倍以上	[68]
人Adx/AdR	人CYP11B1	11-脱氧皮质酮	比共表达Adx/AdR的转化率低	[69]
P. putidaPdx/PdR	P. putidaP450cam	樟脑	比共表达Pdx/PdR的催化速率略低	[70]
P. putidaPdx/PdR	P. putidaP450cam	樟脑	比天然系统的稳态NADH氧化速率提高两倍	[71]
E. coli Fld/Fpr	B. subtilisCYP109B1	肉豆蔻酸	融合形式与非融合形式的催化活性相似	[73]

RPs	P450酶Enzyme P450	底物Substrate	融合结果Fusion results	实例Example
B. megateriumP450 BM3还原酶（BMR）	哺乳动物肝脏CYPs	多种脂肪酸和药物	支持多个P450酶的催化活性，真核P450酶的溶解度明显提升	[46-51]
	A. OryzaeCYP57B3	大豆苷元	支持6-OH大豆苷元最大产量为9.1 mg/L	[52]
	JeotgalicoccusP450 OleTJE	饱和脂肪酸	比Pdx/PdR的周转率高8.9倍	[53]
B. subtilisCYP102A3还原酶	B. megateriumP450 BM3血红素域	12-对硝基苯基十二烷酸	比天然酶的热稳定性明显提升	[54]
RhodococcusCYP116B2还原酶（RhFRED）	S. venezuelaeP450 PikC	12元环大环内酯YC-17和14元环大环内酯那博霉素	比菠菜Fdx/FdR的催化活性提高约4倍	[57]
	StreptomycesP450 TamI	去氧糖胺糖苷修饰的碳环	替代菠菜Fdx/FdR实现了制备级规模的C10羟化反应	[58]
	A. orientalisP450_Prava	紧缩素	支持在产黄青霉中产生超过6 g/L的普伐他汀	[59]
	M. griseorubidaP450 MycG	麦新米星	支持P450 MycG的常规羟化反应	[37]
R. ruberCYP116B3还原酶（PFOR）	M. aquaeoleiCYP153AM.aq	十二烷酸	比融合RhFRED的反应初始速率高3倍	[61]
T. thermophilesCYP116B46还原酶	R. coprophilusTC-2 P450tol	甲苯	比融合RhFRED的半衰期和活性分别提高20倍和2倍以上	[62]
大鼠CPR	大鼠CYP1A1	7-乙氧基香豆素	融合酶比无CPR的CYP1A1催化活性高4倍	[63]
ArabidopsisCPR	P. ginseng P450 PPDS	达马烯二醇	比共表达CPR的催化活性高约4.5倍	[65]
T. cuspidataCPR	T. cuspidataCYP725A4	紫衫二烯	比共表达CPR的催化活性显著降低	[66]
H. tuberosusCPR	H. tuberosusCYP76B1	苯脲类除草剂	比只存在CYP76B1的除草剂耐受性降低一半以上	[67]
人Adx/AdR	人CYP11A1	胆固醇	比共表达Adx/AdR的表观V_max增加5倍以上	[68]
人Adx/AdR	人CYP11B1	11-脱氧皮质酮	比共表达Adx/AdR的转化率低	[69]
P. putidaPdx/PdR	P. putidaP450cam	樟脑	比共表达Pdx/PdR的催化速率略低	[70]
P. putidaPdx/PdR	P. putidaP450cam	樟脑	比天然系统的稳态NADH氧化速率提高两倍	[71]
E. coli Fld/Fpr	B. subtilisCYP109B1	肉豆蔻酸	融合形式与非融合形式的催化活性相似	[73]