Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (1): 86-99.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0663
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LI Liang(), XU Shan-shan, JIANG Yan-jun()
Received:
2023-07-12
Online:
2024-01-26
Published:
2024-02-06
Contact:
JIANG Yan-jun
E-mail:liangli@hebut.edu.cn;yanjunjiang@hebut.edu.cn
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.
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 | [ |
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 | [ | |
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 | [ | |
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 | [ |
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 | [ | |
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 | [ | |
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 | [ | |
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 | [ |
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 | [ | |
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 | - | [ |
Table 1 Comparison of ergothioneine production methods
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 | [ |
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 | [ | |
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 | [ | |
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 | [ |
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 | [ | |
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 | [ | |
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 | [ | |
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 | [ |
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 | [ | |
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 | - | [ |
Fig. 3 Biosynthetic pathway of ergothioneine SAM: S-adenosylmethionine; γ-GC:γ-glutamyl-cysteine; HER: hercynine; γGC-HER: hercynyl-γ-glutamyl-cysteine sulfoxide; Cys-HER: hercynyl-cysteine sulfoxide; MetK: S-adenosylmethionine synthetase. Black represents two or more public biosynthetic pathways. Red represents biosynthetic pathways of ERG in M. smegmatis(EgtA: γ-glutamyl cysteine synthase; EgtB: mononuclear non-heme iron enzyme; EgtC: amidotransferase; EgtD: SAM-dependent histidine methyltransferase; EgtE: PLP-mediated C-S lyase). Purple represents biosynthetic pathways of ERG in N. crassa[Egt1: Bifunctional enzymes(SAM-dependent histidine methyltransferase and mononuclear non-heme iron enzyme); Egt2: PLP-mediated C-S lyase]. Orange represents biosynthetic pathways of ERG in Methylobacterium(EgtB: Similar to fungi Egt1). Pink represents biosynthetic pathways of ERG in anaerobic bacteria(EanA: Methyltransferase; EanB: rhodanese-like sulfur transferase). Green represents the participation of L-Glu in M. smegmatis biosynthetic pathway
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egtBCDEMs和gshA基因 | 72 | 24 mg/L | 0.3 | [ |
大肠杆菌E. coli MG1655 | 优化表达egtABCDE基因 | 60 | 437.6 mg/L | 7.3 | [ |
大肠杆菌E. coli BW25113 | 过表达egtDEMs 和egtBMp基因;表达cysE*、serA* 和ydeD 基因;敲除metJ 基因 | 192 | 657 mg/L | 3.4 | [ |
大肠杆菌E. coli BL21(DE3) | 表达egtBCDEMs、egt1Sp 和egtA基因;过表达thrA 和serA T410STOP 基因 | 108 | 710.53 mg/L | 6.6 | [ |
大肠杆菌E. coli BL21(DE3) | 过表达egtBCDEMs、egt1Sp、egtA、thrA 和serAT410STOP 基因 | 108 | 1.1 g/L | 10.2 | [ |
大肠杆菌E. coli BW25113 | 过表达egtABCDEMs 基因;表达gshA、cysE*、serA* 和ydeD基因;敲除metJ 基因 | 216 | 1.31 g/L | 6.1 | [ |
大肠杆菌E. coli BL21(DE3) | 表达 egtBCDEMs 和egt1Sp 基因;过表达egtAMs、thrA 和serA T410STOP基因 | 108 | 2.01 g/L | 18.6 | [ |
大肠杆菌E. coli MG1655 | 表达egtBCDEMs、egtB*Ms、egt2Nc 和hisG* 基因;双拷贝表达gshA;过表达hisDBCHAFI基因 | 52 | 2.9 g/L | 55.8 | [ |
大肠杆菌E. coli BW25113 | 过表达tregt1和tregt2 基因 | 143 | 4.34 g/L | 30.4 | [ |
大肠杆菌E. coli BL21(DE3) | 表达egtEMs 基因;半理性设计和随机突变EgtD 和TNcEgt1 | 96 | 5.4 g/L | 56.3 | [ |
酿酒酵母Saccharomyces cerevisiae | 过表达Poegt1、Peegt1 和Ptegt1 基因 | — | 2.5 mg/L | — | [ |
酿酒酵母 S. cerevisiae | 过表达egt1Gf 和egt2Gf 基因 | 168 | 20.61 mg/L | 0.1 | [ |
酿酒酵母 S. cerevisiae | 共表达双拷贝 egt1Nc 和egt2Cp 基因 | 84 | 598 mg/L | 7.1 | [ |
圆红冬孢酵母 Rhodotorula toruloides | 表达egt1Nc基因 | 96 | 1.5 g/L | 15.6 | [ |
解脂耶氏酵母 Yarrowia lipolytica | 共表达双拷贝egt1Nc 和egt2Cp 基因 | 220 | 1.63 g/L | 7.4 | [ |
酿酒酵母 S. cerevisiae | 共表达双拷贝egt1Nc和egt2Cp 基因;过表达 MET14;敲除spe2 基因 | 160 | 2.4 g/L | 15 | [ |
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 | [ |
谷氨酸棒状杆菌 C. glutamicum | 过表达egtDEMs 和egtBMp基因 | 336 | 100 mg/L | 0.3 | [ |
谷氨酸棒状杆菌 C. glutamicum | 表达egt1Sp 和egt2Sp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 | [ |
甲基杆菌属Methylobacterium aquaticum | 过表达egtBDMs 基因;敲除hutH 基因 | 168 | 7.0 mg/g 干重 Dry weight | — | [ |
米曲霉 Aspergillus oryzae | 过表达egt1Nc 和egt2Nc基因 | — | 231 mg/kg 培养基 Medium | — | [ |
蛹虫草 Cordyceps militaris | 过表达EgtDMs、CmE1B和CmEgt2 | — | 2.5 g/kg 干重 Dry weight | — | [ |
枯草芽孢杆菌 Bacillus subtilis | 优化表达egtABCDE 基因 | 60 | 568.4 mg/L | 9.5 | [ |
新金色分枝杆菌 Mycolicibacterium neoaurum | 过表达egtABCDEMn、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 | [ |
Table 2 Fermentation levels of genetically engineered strains for ergothioneine
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egtBCDEMs和gshA基因 | 72 | 24 mg/L | 0.3 | [ |
大肠杆菌E. coli MG1655 | 优化表达egtABCDE基因 | 60 | 437.6 mg/L | 7.3 | [ |
大肠杆菌E. coli BW25113 | 过表达egtDEMs 和egtBMp基因;表达cysE*、serA* 和ydeD 基因;敲除metJ 基因 | 192 | 657 mg/L | 3.4 | [ |
大肠杆菌E. coli BL21(DE3) | 表达egtBCDEMs、egt1Sp 和egtA基因;过表达thrA 和serA T410STOP 基因 | 108 | 710.53 mg/L | 6.6 | [ |
大肠杆菌E. coli BL21(DE3) | 过表达egtBCDEMs、egt1Sp、egtA、thrA 和serAT410STOP 基因 | 108 | 1.1 g/L | 10.2 | [ |
大肠杆菌E. coli BW25113 | 过表达egtABCDEMs 基因;表达gshA、cysE*、serA* 和ydeD基因;敲除metJ 基因 | 216 | 1.31 g/L | 6.1 | [ |
大肠杆菌E. coli BL21(DE3) | 表达 egtBCDEMs 和egt1Sp 基因;过表达egtAMs、thrA 和serA T410STOP基因 | 108 | 2.01 g/L | 18.6 | [ |
大肠杆菌E. coli MG1655 | 表达egtBCDEMs、egtB*Ms、egt2Nc 和hisG* 基因;双拷贝表达gshA;过表达hisDBCHAFI基因 | 52 | 2.9 g/L | 55.8 | [ |
大肠杆菌E. coli BW25113 | 过表达tregt1和tregt2 基因 | 143 | 4.34 g/L | 30.4 | [ |
大肠杆菌E. coli BL21(DE3) | 表达egtEMs 基因;半理性设计和随机突变EgtD 和TNcEgt1 | 96 | 5.4 g/L | 56.3 | [ |
酿酒酵母Saccharomyces cerevisiae | 过表达Poegt1、Peegt1 和Ptegt1 基因 | — | 2.5 mg/L | — | [ |
酿酒酵母 S. cerevisiae | 过表达egt1Gf 和egt2Gf 基因 | 168 | 20.61 mg/L | 0.1 | [ |
酿酒酵母 S. cerevisiae | 共表达双拷贝 egt1Nc 和egt2Cp 基因 | 84 | 598 mg/L | 7.1 | [ |
圆红冬孢酵母 Rhodotorula toruloides | 表达egt1Nc基因 | 96 | 1.5 g/L | 15.6 | [ |
解脂耶氏酵母 Yarrowia lipolytica | 共表达双拷贝egt1Nc 和egt2Cp 基因 | 220 | 1.63 g/L | 7.4 | [ |
酿酒酵母 S. cerevisiae | 共表达双拷贝egt1Nc和egt2Cp 基因;过表达 MET14;敲除spe2 基因 | 160 | 2.4 g/L | 15 | [ |
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 | [ |
谷氨酸棒状杆菌 C. glutamicum | 过表达egtDEMs 和egtBMp基因 | 336 | 100 mg/L | 0.3 | [ |
谷氨酸棒状杆菌 C. glutamicum | 表达egt1Sp 和egt2Sp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 | [ |
甲基杆菌属Methylobacterium aquaticum | 过表达egtBDMs 基因;敲除hutH 基因 | 168 | 7.0 mg/g 干重 Dry weight | — | [ |
米曲霉 Aspergillus oryzae | 过表达egt1Nc 和egt2Nc基因 | — | 231 mg/kg 培养基 Medium | — | [ |
蛹虫草 Cordyceps militaris | 过表达EgtDMs、CmE1B和CmEgt2 | — | 2.5 g/kg 干重 Dry weight | — | [ |
枯草芽孢杆菌 Bacillus subtilis | 优化表达egtABCDE 基因 | 60 | 568.4 mg/L | 9.5 | [ |
新金色分枝杆菌 Mycolicibacterium neoaurum | 过表达egtABCDEMn、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 | [ |
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