生物合成法生产麦角硫因的研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2023-0663

摘要/Abstract

摘要：

麦角硫因（ergothioneine, ERG）作为一种稀有的天然含硫组氨酸衍生物，已被证明具有强大的抗氧化性和诸多生物学功能。因此，ERG受到越来越多研究人员和产品开发人员的关注。目前，ERG已被广泛应用于食品、化妆品和医疗等行业。研究表明只有少数细菌和真菌可体内合成ERG，植物、动物和人类自身均不能直接合成ERG，只能从其他来源获取。ERG可通过生物提取法、化学合成法以及生物合成法获得，但由于传统生产方式（生物提取法和化学合成法）存在产量低、生产效率差和生产成本较高等问题，限制了该产品的规模化生产和应用。因此，亟需开发一种高效、经济且安全、可靠的ERG生产方式以满足市场的需求。近年来合成生物学快速发展，利用基因工程、蛋白质工程和代谢工程等技术提高生物合成法生产ERG的能力已逐渐成为研究热点。本文将论述ERG的生物学活性和功能，介绍ERG生物合成途径和ERG在食品、化妆品和医疗等行业的应用前景，比较ERG主要的生产方式，总结并梳理近年来采取各种工程策略通过生物合成法生产ERG的研究进展；并就如何利用基因工程、蛋白质工程和代谢工程提高ERG产量提出几点工程策略，以期为生物合成法高产ERG提供理论参考和研究思路。

关键词: 麦角硫因, 生物合成法, 生物合成途径, 基因工程, 蛋白质工程, 代谢工程, 发酵

Abstract:

Ergothioneine（ERG）, a rare natural sulfur-containing histidine derivative, has been proved to have strong antioxidant property and many biological functions. Therefore, ERG has been received much more attention from researchers and product developers. Currently, ERG has been widely used in food, cosmetics and medical industries. Research shows that ERG only can be synthesized by a few bacteria and fungi. Plants, animals and humans cannot synthesize ERG directly, but it could be obtained by other sources. ERG can be obtained by bioextraction, chemical synthesis, and biosynthesis. However, because of the low yield and poor production efficiency of the traditional methods（bioextraction and chemical synthesis）, the large-scale production and application of ERG is limited. Therefore, there is an urgent need for an efficient, economical, safe and reliable ERG synthesis method to meet market needs. With the rapid development of synthetic biology, the use of genetic engineering, protein engineering and metabolic engineering to improve the ability of ERG biosynthesis has gradually become an increasingly favored method. This paper will elaborate the biological characteristics and functions, briefly introduce the biosynthetic pathways of ERG and application prospects of ERG in food, cosmetics and medical industries, compare the main production methods of ERG, and summarize and sort the research progress of adopting various engineering strategies to produce ERG by biosynthesis in recent years, and propose several engineering strategies on how to use genetic engineering, protein engineering and metabolic engineering to increase the yield of ERG, which aims to provide theoretical reference and research ideas for biosynthesis high-yield of ERG.

Key words: ergothioneine, biosynthesis, biosynthetic pathway, genetic engineering, protein engineering, metabolic engineering, fermentation

李亮, 徐姗姗, 姜艳军. 生物合成法生产麦角硫因的研究进展[J]. 生物技术通报, 2024, 40(1): 86-99.

LI Liang, XU Shan-shan, JIANG Yan-jun. Research Progress in the Production of Ergothioneine by Biosynthesis[J]. Biotechnology Bulletin, 2024, 40(1): 86-99.

图/表 6

参考文献 59

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生产方式 Production method	分类 Classification	优点 Advantages	缺点 Disadvantages	参考文献 Reference
生物提取法 Bioextraction	回流提取法 Reflux extraction	针对性强，收率较高 High pertinence, and high yield	相对耗时Relatively time-consuming	[13]
	酶解提取法 Enzymatic extraction	提取速度快，条件温和 Fast extraction speed, and moderate conditions	酶活范围较窄，提取条件苛刻 Narrow enzyme activity range, hash extraction condition	[13]
	超声微波联合法 Ultrasonic and microwave extraction	减少萃取溶剂和能耗，提取效率高 Lower extraction solvent and energy consumption, and high extraction efficiency	产量低 Low yield	[13]
化学合成法 Chemical synthesis	路线（1）和（2）Route（1）and（2）	——	路线冗长复杂，反应温度较高，资源浪费，收率低 Long and complex route, high reaction temperature, waste of resource, and low yield	[14]
	路线（3）Route（3）	收率高于路线（1）和（2）Yields higher than that in route（1）and（2）	原料昂贵；中间体纯化使用两次反向层析柱，导致成本增加；毫克级别High feedstock cost; intermediate purification uses two reverse chromatography columns, which increases cost; milligram level	[14]
	路线（4）Route（4）	“一锅法”制备，路线短；无中间纯化过程 Prepared by one-pot method, short route; no intermediate purification	原料昂贵且来源少 Expensive and few feedstock	[14]
	路线（5）Route（5）	操作简单，原料低廉易得，步骤简短，条件相对温和可控，产量较高 Simple operation, cheap and readily available feedstock, short route, relatively moderate and controllable conditions, and high yield	使用具有危害性的化学试剂，增加废液和废物处理成本 Use hazardous chemical reagents, increase waste liquid and waste disposal costs	[14]
生物合成法 Biosynthesis	微生物液体发酵Microbial liquid fermentation	可食用菌发酵，天然属性，安全性高 Edible mushroom fermentation, natural properties and high safety	发酵周期长，产率低 Long fermentation period, and low yield	[15]
	生物转化 Biotransformation	直接以前体氨基酸作为底物，原料成本低；工艺简单；产品浓度较高 Direct precursor amino acids as substrates, low feedstock cost; simple technology; higher product concentration	产率低；表达复合酶体外催化，经济适用性差 Low yield, in vitro catalysis of expression complex enzymes, and low economic practicality	[16]
	ERG工程菌株的发酵Fermentation of ERG engineered strains	以常见菌株作为底盘菌，方便获得；减少外源添加，原料成本低；无有害试剂，环境友好；发酵周期短；操作简单 Common strains are chassis bacteria, which are readily available; reduce exogenous additions, low feedstock cost; no hazardous chemical reagents, environment-friendly; short fermentation period; simple operation	-	[17]

生产方式 Production method	分类 Classification	优点 Advantages	缺点 Disadvantages	参考文献 Reference
生物提取法 Bioextraction	回流提取法 Reflux extraction	针对性强，收率较高 High pertinence, and high yield	相对耗时Relatively time-consuming	[13]
	酶解提取法 Enzymatic extraction	提取速度快，条件温和 Fast extraction speed, and moderate conditions	酶活范围较窄，提取条件苛刻 Narrow enzyme activity range, hash extraction condition	[13]
	超声微波联合法 Ultrasonic and microwave extraction	减少萃取溶剂和能耗，提取效率高 Lower extraction solvent and energy consumption, and high extraction efficiency	产量低 Low yield	[13]
化学合成法 Chemical synthesis	路线（1）和（2）Route（1）and（2）	——	路线冗长复杂，反应温度较高，资源浪费，收率低 Long and complex route, high reaction temperature, waste of resource, and low yield	[14]
	路线（3）Route（3）	收率高于路线（1）和（2）Yields higher than that in route（1）and（2）	原料昂贵；中间体纯化使用两次反向层析柱，导致成本增加；毫克级别High feedstock cost; intermediate purification uses two reverse chromatography columns, which increases cost; milligram level	[14]
	路线（4）Route（4）	“一锅法”制备，路线短；无中间纯化过程 Prepared by one-pot method, short route; no intermediate purification	原料昂贵且来源少 Expensive and few feedstock	[14]
	路线（5）Route（5）	操作简单，原料低廉易得，步骤简短，条件相对温和可控，产量较高 Simple operation, cheap and readily available feedstock, short route, relatively moderate and controllable conditions, and high yield	使用具有危害性的化学试剂，增加废液和废物处理成本 Use hazardous chemical reagents, increase waste liquid and waste disposal costs	[14]
生物合成法 Biosynthesis	微生物液体发酵Microbial liquid fermentation	可食用菌发酵，天然属性，安全性高 Edible mushroom fermentation, natural properties and high safety	发酵周期长，产率低 Long fermentation period, and low yield	[15]
	生物转化 Biotransformation	直接以前体氨基酸作为底物，原料成本低；工艺简单；产品浓度较高 Direct precursor amino acids as substrates, low feedstock cost; simple technology; higher product concentration	产率低；表达复合酶体外催化，经济适用性差 Low yield, in vitro catalysis of expression complex enzymes, and low economic practicality	[16]
	ERG工程菌株的发酵Fermentation of ERG engineered strains	以常见菌株作为底盘菌，方便获得；减少外源添加，原料成本低；无有害试剂，环境友好；发酵周期短；操作简单 Common strains are chassis bacteria, which are readily available; reduce exogenous additions, low feedstock cost; no hazardous chemical reagents, environment-friendly; short fermentation period; simple operation	-	[17]

菌株 Strain	关键策略 Key strategy	发酵时间 Fermentation period/h	产量 Yield	生产效率Production efficiency/（mg·L^-1·h^-1）	参考文献 Reference
大肠杆菌E. coli BW25113	过表达egtBCDE_Ms和gshA基因	72	24 mg/L	0.3	[17]
大肠杆菌E. coli MG1655	优化表达egtABCDE基因	60	437.6 mg/L	7.3	[30]
大肠杆菌E. coli BW25113	过表达egtDE_Ms 和egtB_Mp基因；表达cysE^、serA^ 和ydeD 基因；敲除metJ 基因	192	657 mg/L	3.4	[31]
大肠杆菌E. coli BL21（DE3）	表达egtBCDE_Ms、egt1_Sp 和egtA基因；过表达thrA 和serA^T410STOP基因	108	710.53 mg/L	6.6	[32]
大肠杆菌E. coli BL21（DE3）	过表达egtBCDE_Ms、egt1_Sp、egtA、thrA 和serA^T410STOP基因	108	1.1 g/L	10.2	[33]
大肠杆菌E. coli BW25113	过表达egtABCDE_Ms 基因；表达gshA、cysE^、serA^ 和ydeD基因；敲除metJ 基因	216	1.31 g/L	6.1	[34]
大肠杆菌E. coli BL21（DE3）	表达 egtBCDE_Ms 和egt1_Sp 基因；过表达egtA_Ms、thrA 和serA^T410STOP基因	108	2.01 g/L	18.6	[35]
大肠杆菌E. coli MG1655	表达egtBCDE_Ms、egtB^*_Ms、egt2_Nc 和hisG^* 基因；双拷贝表达gshA；过表达hisDBCHAFI基因	52	2.9 g/L	55.8	[36]
大肠杆菌E. coli BW25113	过表达tregt1和tregt2 基因	143	4.34 g/L	30.4	[37]
大肠杆菌E. coli BL21（DE3）	表达egtE_Ms 基因；半理性设计和随机突变EgtD 和^TNcEgt1	96	5.4 g/L	56.3	[38]
酿酒酵母Saccharomyces cerevisiae	过表达Poegt1、Peegt1 和Ptegt1 基因	—	2.5 mg/L	—	[39]
酿酒酵母 S. cerevisiae	过表达egt1_Gf和egt2_Gf 基因	168	20.61 mg/L	0.1	[40]
酿酒酵母 S. cerevisiae	共表达双拷贝 egt1_Nc 和egt2_Cp 基因	84	598 mg/L	7.1	[41]
圆红冬孢酵母 Rhodotorula toruloides	表达egt1_Nc基因	96	1.5 g/L	15.6	[42]
解脂耶氏酵母 Yarrowia lipolytica	共表达双拷贝egt1_Nc和egt2_Cp基因	220	1.63 g/L	7.4	[43]
酿酒酵母 S. cerevisiae	共表达双拷贝egt1_Nc和egt2_Cp 基因；过表达 MET14；敲除spe2 基因	160	2.4 g/L	15	[44]
粟酒裂殖酵母Schizosaccharomyces pombe	经过多轮紫外线和氯化锂突变处理	148	12.5 g/L	84.5	[45]
谷氨酸棒状杆菌 C. glutamicum	过表达egtDE_Ms 和egtB_Mp基因	336	100 mg/L	0.3	[46]
谷氨酸棒状杆菌 C. glutamicum	表达egt1_Sp 和egt2_Sp基因；过表达cysE、cysK和cysR 基因；加强硫同化和磷酸戊糖途径；敲除sdaA基因	36	264 mg/L	7.3	[47]
甲基杆菌属Methylobacterium aquaticum	过表达egtBD_Ms基因；敲除hutH 基因	168	7.0 mg/g 干重 Dry weight	—	[48]
米曲霉 Aspergillus oryzae	过表达egt1_Nc 和egt2_Nc基因	—	231 mg/kg 培养基 Medium	—	[49]
蛹虫草 Cordyceps militaris	过表达EgtD_Ms、CmE1B和CmEgt2	—	2.5 g/kg 干重 Dry weight	—	[50]
枯草芽孢杆菌 Bacillus subtilis	优化表达egtABCDE 基因	60	568.4 mg/L	9.5	[30]
新金色分枝杆菌 Mycolicibacterium neoaurum	过表达egtABCDE_Mn、hisG、hisC 和allB1 基因；敲除假定裂解酶基因 Mn_3042；过表达metK 和ahcY 基因	216	1.56 g/L	7.2	[51]

菌株 Strain	关键策略 Key strategy	发酵时间 Fermentation period/h	产量 Yield	生产效率Production efficiency/（mg·L^-1·h^-1）	参考文献 Reference
大肠杆菌E. coli BW25113	过表达egtBCDE_Ms和gshA基因	72	24 mg/L	0.3	[17]
大肠杆菌E. coli MG1655	优化表达egtABCDE基因	60	437.6 mg/L	7.3	[30]
大肠杆菌E. coli BW25113	过表达egtDE_Ms 和egtB_Mp基因；表达cysE^、serA^ 和ydeD 基因；敲除metJ 基因	192	657 mg/L	3.4	[31]
大肠杆菌E. coli BL21（DE3）	表达egtBCDE_Ms、egt1_Sp 和egtA基因；过表达thrA 和serA^T410STOP基因	108	710.53 mg/L	6.6	[32]
大肠杆菌E. coli BL21（DE3）	过表达egtBCDE_Ms、egt1_Sp、egtA、thrA 和serA^T410STOP基因	108	1.1 g/L	10.2	[33]
大肠杆菌E. coli BW25113	过表达egtABCDE_Ms 基因；表达gshA、cysE^、serA^ 和ydeD基因；敲除metJ 基因	216	1.31 g/L	6.1	[34]
大肠杆菌E. coli BL21（DE3）	表达 egtBCDE_Ms 和egt1_Sp 基因；过表达egtA_Ms、thrA 和serA^T410STOP基因	108	2.01 g/L	18.6	[35]
大肠杆菌E. coli MG1655	表达egtBCDE_Ms、egtB^*_Ms、egt2_Nc 和hisG^* 基因；双拷贝表达gshA；过表达hisDBCHAFI基因	52	2.9 g/L	55.8	[36]
大肠杆菌E. coli BW25113	过表达tregt1和tregt2 基因	143	4.34 g/L	30.4	[37]
大肠杆菌E. coli BL21（DE3）	表达egtE_Ms 基因；半理性设计和随机突变EgtD 和^TNcEgt1	96	5.4 g/L	56.3	[38]
酿酒酵母Saccharomyces cerevisiae	过表达Poegt1、Peegt1 和Ptegt1 基因	—	2.5 mg/L	—	[39]
酿酒酵母 S. cerevisiae	过表达egt1_Gf和egt2_Gf 基因	168	20.61 mg/L	0.1	[40]
酿酒酵母 S. cerevisiae	共表达双拷贝 egt1_Nc 和egt2_Cp 基因	84	598 mg/L	7.1	[41]
圆红冬孢酵母 Rhodotorula toruloides	表达egt1_Nc基因	96	1.5 g/L	15.6	[42]
解脂耶氏酵母 Yarrowia lipolytica	共表达双拷贝egt1_Nc和egt2_Cp基因	220	1.63 g/L	7.4	[43]
酿酒酵母 S. cerevisiae	共表达双拷贝egt1_Nc和egt2_Cp 基因；过表达 MET14；敲除spe2 基因	160	2.4 g/L	15	[44]
粟酒裂殖酵母Schizosaccharomyces pombe	经过多轮紫外线和氯化锂突变处理	148	12.5 g/L	84.5	[45]
谷氨酸棒状杆菌 C. glutamicum	过表达egtDE_Ms 和egtB_Mp基因	336	100 mg/L	0.3	[46]
谷氨酸棒状杆菌 C. glutamicum	表达egt1_Sp 和egt2_Sp基因；过表达cysE、cysK和cysR 基因；加强硫同化和磷酸戊糖途径；敲除sdaA基因	36	264 mg/L	7.3	[47]
甲基杆菌属Methylobacterium aquaticum	过表达egtBD_Ms基因；敲除hutH 基因	168	7.0 mg/g 干重 Dry weight	—	[48]
米曲霉 Aspergillus oryzae	过表达egt1_Nc 和egt2_Nc基因	—	231 mg/kg 培养基 Medium	—	[49]
蛹虫草 Cordyceps militaris	过表达EgtD_Ms、CmE1B和CmEgt2	—	2.5 g/kg 干重 Dry weight	—	[50]
枯草芽孢杆菌 Bacillus subtilis	优化表达egtABCDE 基因	60	568.4 mg/L	9.5	[30]
新金色分枝杆菌 Mycolicibacterium neoaurum	过表达egtABCDE_Mn、hisG、hisC 和allB1 基因；敲除假定裂解酶基因 Mn_3042；过表达metK 和ahcY 基因	216	1.56 g/L	7.2	[51]