维生素K2的生物合成及其甲萘醌基团合成酶的功能分析

doi:10.11949/0438-1157.20221016

摘要/Abstract

摘要：

维生素K2(VK2)作为2-甲基-1，4-萘醌(简称甲萘醌)家族的衍生物，在预防骨质疏松和心血管钙化方面发挥着重要作用，随着其市场需求的日益增加，实现VK2产业化并降低生产成本成为大家关注的焦点。首先概述了VK2传统发酵工艺现状及面临的问题，之后详细分析了VK2生物合成途径及其基因工程改造策略，可以将枯草芽孢杆菌产量提高至1549.6 mg·L^-1，产量是传统发酵菌株的7倍以上。此外，还围绕合成途径中经典甲萘醌骨架合成酶的功能及其蛋白结构进行汇总分析，通过对酶的蛋白结构、催化特异性和机理进行分析，揭示各个功能酶在VK2合成途径中发挥的作用及其内在关系。最后从合成生物学技术发展角度对VK2途径设计、构建与应用进行了展望，为提升VK2工业化产量提供理论参考。

关键词: 维生素K2, 生物合成, 甲萘醌, 蛋白结构

Abstract:

Vitamin K2 (VK2), a derivative of the 2-methyl-1,4-naphthoquinone (menadione) family, plays an important role in the prevention of osteoporosis and cardiovascular calcification, and its increasing demand has aroused great interest to improve VK2 synthesis and reduce its manufacturing cost for industrial production. We first reviewed the status and some issues confronted in traditional microbial production of VK2. Then we presented its synthesis pathway and engineering efforts performed in recent studies and found that VK2 production could be greatly improved to 1549.6 mg·L^-1 in Bacillus subtilis, which was more than 7 times higher than traditional microbial fermentation. Focusing on the synthesis pathway of 2-methyl-1,4-naphthoquinone, which constitutes as the conserved structure of VK2, we summarized the involved enzymes and analyzed their protein structure, catalytic specificity, and mechanism to reveal their roles in synthesis pathway and their internal relationship. Finally, the design, construction and application of VK2 pathway are prospected from the perspective of synthetic biology technology development, which provides a theoretical reference for improving the industrial production of VK2.

Key words: vitamin K2, biosynthesis, methylnaphthoquinone, protein structure

中图分类号:

Q 568

张秋华, 刘曼路, 王峥, 张一鸣, 苏海佳. 维生素K2的生物合成及其甲萘醌基团合成酶的功能分析[J]. 化工学报, 2023, 74(1): 342-354.

Qiuhua ZHANG, Manlu LIU, Zheng WANG, Yiming ZHANG, Haijia SU. Biosynthesis of vitamin K2 and functional analysis of the biosynthetic enzymes involved in its menadione moiety[J]. CIESC Journal, 2023, 74(1): 342-354.

图/表 6

表1 天然VK2生产菌株发酵产量提高的传统策略

Table 1 Traditional strategies for increasing vitamin K2 fermentation titer

菌株	发酵形式	策略	产物	发酵水平/(mg·L^-1)	文献
Bacillus licheniformis	液体	卡那霉素抗性突变	MK-7	0.25 mg·(g DCW)^-1	[19]
Bacillus subtilis natto	固体	采用中心复合面设计研究，优化碳源和氮源	MK-7	67	[20]
Bacillus subtilis natto	液体	分批补料甘油	MK-7	86.48	[21]
Escherichia sp.	液体	添加甜菜碱和不同的表面活性剂	MK-4	47.6	[22]
Bacillus subtilis natto	液体	采用响应面分析葡萄糖、酵母提取物和酪蛋白浓度的影响	MK-7	20.5	[23]
Bacillus amyloliquefaciens	液体	室温等离子体(ARTP)诱变和碳源、氮源优化	MK-7	61.3	[24]
Bacillus subtilis natto	液体	生物反应器设计有网格状时尚 PCS 结构	MK-7	14.7	[25]

表2 VK2生产菌株产量提升的基因工程策略

Table 2 Genetic engineering strategies for increasing vitamin K2 fermentation titer

菌株	发酵形式	策略	产物	发酵水平	文献
Escherichia coli	液体	UbiCA基因的缺失，过表达MenA和MenD	MK-8	290 mg·(g DCW)^-1	[11]
Escherichia coli	液体	优化MVA途径和过表达HepPPS酶	MK-7	8.8 mg·L^-1	[30]
Elizabethkingia meningoseptica	液体	UbiA突变，Dxr、MenA和UbiE的过表达，以及前体的补充	MK-7	29.63 mg·(g DCW)^-1	[13]
Bacillus subtilis 168	液体	使用强启动子P43和过表达 crtE, MenA和MenG酶	MK-4	(145.0±2.8) mg·L^-1	[31]
	液体	过表达了MEP途径的5个基因	MK-7	360 mg·L^-1	[32]
	液体	构建了丙酮酸和丙二酰辅酶A抑制型的基因回路，联级动态调控中心代谢模块、前体IPP反应模块和产物合成模块	MK-7	1549.6 mg·L^-1	[33]

图1 VK2的生物合成途径Shikimate pathway(莽草酸途径):Gly(甘油)；G3P(甘油醛-3-磷酸)；E4P(赤藓糖-4-磷酸)；DAHP(3-脱氧-阿拉伯-庚酮酸酯 7-磷酸盐)；CHA(分支酸)；MK pathway(甲萘醌途径):ICHA(异分支酸)；SEPHCHC(2-琥珀酰基-5-烯醇丙酮酸-6-羟基-3-环己烯-1-羧酸酯)；SHCHC[(1R,6R)-2-琥珀酰基-6-羟基-2,4-环己二烯-1-羧酸酯]；OSB(2-琥珀酰苯甲酸)；OSB-CoA(2-琥珀酰苯甲酸辅酶A)；DHNA-CoA(1,4-二羟基-2-萘酰辅酶A); DHNA(1,4-二羟基-2-萘酸)；DMK(萘醌)；MK-7(甲萘醌)；MenF(异分支酸合酶)；MenD(2-琥珀酰-5-烯醇丙酮酰-6-羟基-3-环己烯-1-羧酸合酶)；MenH(2-琥珀酰-6-羟基-2,4-环己二烯-1-羧酸合酶)；MenC(邻琥珀酰苯甲酸合酶)；MenE(邻琥珀酰苯甲酸-辅酶A连接酶)；MenB(1,4-二羟基-2-萘甲酰-CoA合酶)；MenI(1,4-二羟基-2-萘甲酰-CoA水解酶)；MenA(1,4-二羟基-2-萘甲酸七异戊二烯转移酶)；MenG(去甲基甲萘醌甲基转移酶)；MEP pathway(2-C-甲基-D-赤藓糖醇-4-磷酸途径)：Pyr(丙酮酸盐)；DXP(1-脱氧木酮糖-5-磷酸)；MEP(甲基赤藓糖醇-4-二磷酸)；CDP-ME(4-二磷酸胞苷-2-C-甲基-D-赤藓糖)；CDP-MEP(2-磷酸-4-二磷酸胞苷-2-C-甲基-D-赤藓糖)；MEC(2-C-甲基赤藓糖-2,4-环焦磷酸)；HMBPP(1-羟基-2-甲基-2-丁烯基-4-焦磷酸)；DMAPP(二甲基烯丙基二磷酸)；IPP(异戊二烯焦磷酸)；GPP(牻牛儿基焦磷酸)；FPP(法呢基焦磷酸)；Hepta-PP(全反式聚异戊烯二磷酸)；Dxs(1-脱氧木酮糖-5-磷酸合酶)；Dxr(1-脱氧木酮糖-5-磷酸还原异构酶)；YacM(2-C-甲基赤藓糖醇-4-磷酸胞苷酰转移酶)；IspE(4-二磷酸胞苷-2-C-甲基赤藓糖醇激酶)；YacN(2-C-甲基赤藓糖醇-2,4-环二磷酸合酶)；Yqfp(4-羟基-3-甲基丁-2-烯基二磷酸合酶)；YpgA(异戊烯基焦磷酸异构酶)；YqiD(法呢基焦磷酸合酶)；HepS/HepT(七异戊二烯基焦磷酸合成酶)；MVA pathway(甲羟戊酸途径): AcCoA(乙酰辅酶A)；AtoB(乙酰辅酶A C-乙酰转移酶);AACoA(乙酰乙酰辅酶A); HMG-CoA(3-羟甲基戊二酰辅酶A); MVA(甲羟戊酸); MVAP(甲羟戊酸-5-磷酸)；MVAPP(甲羟戊酸-5-焦磷酸)；Erg10(乙酰辅酶A乙酰转移酶)；Erg13(羟甲基戊二酰辅酶A合酶)；Hmg1/Hmg2(羟甲基戊二酰辅酶A还原酶)；Erg12(甲羟戊酸激酶)；Erg8(磷酸戊酸激酶)；Erg19(甲羟戊酸焦磷酸脱羧酶)；Futalosine pathway: MqnA(分支酸脱水酶)；MqnE(氨基脱氧富他洛辛合酶)；MqnF(氨基脱氧氟他嘧啶脱氨酶)；MqnB(富他洛辛水解酶)；MqnC(环状脱氧黄嘌呤富他罗辛合酶)；MqnD(1,4-二羟基-6-萘酸合酶)

Fig.1 Biosynthetic pathway of VK2

图2 VK2的生物合成调控策略

Fig.2 Strategies of controlling biosynthetic pathway of VK2

表3 Men功能酶的酶学信息

Table 3 Enzymological information on Menase

名称	酶编号	蛋白大小/kDa	底物	(k_cat/K_m)/(L·s^-1·μmol^-1)	辅因子
MenF	5.4.4.2	52.812	—	—	—
MenD	2.2.1.9	64.092	—	—	Mg²⁺
MenH	4.2.99.20	27.128	—	—	—
MenC	4.2.1.113	41.629	(1R,6R)-6-羟基-2-琥珀环己烷基-2,4-二烯-1-羧酸酯	2	—
MenE	6.2.1.26	50.185	2-邻琥珀酰苯甲酸	0.77	Mg²⁺
MenB	4.1.3.36	31.633	4-(2'-羧苯基)-4- 氧代丁酰-CoA	24	HCO₃^-
MenI	3.1.2.28	14.945	1-羟基-2-萘甲酰基-CoA	1.85	—
MenA	2.5.1.74	33.838	—	—	—
MenG	2.1.1.163	27.128	—	—	—

表4 Men酶对MK-7表达的影响

Table 4 Effect of Menase on MK-7 expression

Men酶	片段大小/bp	甘油消耗速率	生物量	酶活	MK-7产量
MenA	936	+++	+	+++	+++
MenB	819	—	—	—	—
MenC	1116	++	++	+	+
MenD	1737		+	+	++
MenE	1464	+	+	++	+
MenG	702	—	—	—	—
MenH	825	*	++	+	*

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