酵母细胞催化合成18α-甘草酸

doi:10.11949/0438-1157.20240310

摘要/Abstract

摘要：

18α-甘草酸（18α-Glycyrrhizin， 18α-GL）是一种齐墩果烷型皂苷，18α-GL比其差向异构体18β-GL极性小、亲脂性好，更强的抗毒抗炎作用和更高的肝脏靶向性使18α-GL成为保肝护肝领域的主要药物成分。但目前18α-GL的制备方法污染大，效率低，亟需开发一种绿色简单地合成18α-GL的方法。本研究利用酵母细胞异源表达了糖基转移酶，通过全细胞催化的方式鉴定出可催化18α-甘草次酸（18α-Glycyrrhetinic acid，18α-GA）特异性合成18α-单葡萄糖醛酸甘草次酸（18α-Glycyrrhetinic acid 3-O-monoglucuronide，18α-GAMG）的糖基转移酶cGuCSyGT和催化18α-GAMG 特异性合成18α-GL的糖基转移酶GgUGT1。进一步运用了蛋白质结构预测和分子动力学模拟探究了cGuCSyGT对18α-GA的催化活性比18β-GA低的原因。最后采用优化底物添加浓度、底盘细胞、底物添加时间、反应时间、培养基成分补加和底物溶剂的策略构建了酵母催化合成18α-GAMG和18α-GL的最优工艺，使18α-GAMG和18α-GL的产量分别达到36.38（±1.87）mg/L和39.32（±0.75）mg/L。本研究首次实现了18α-GAMG和18α-GL的微生物催化合成，为将来18α-GL的微生物全合成提供了理论基础和技术支撑。

关键词: 甘草, 18α-甘草酸, 18α-单葡萄糖醛酸甘草次酸, 糖基转移酶, 酵母细胞催化

Abstract:

18α-Glycyrrhizin (18α-GL), an oleanane-type saponin, is less polar and more lipophilic than its epimer 18β-GL, and has stronger antitoxic resistance. The inflammatory effect and higher hepar targeting make 18α-GL the main pharmaceutical ingredient in the field of hepar protection. However, the current preparation method of 18α-GL is highly polluting and has low efficiency. Therefore, there is an urgent need to develop a green and simple method to synthesize 18α-GL. Glycosyltransferases were heterologously expressed in yeast cells. Through whole-cell catalysis, glycosyltransferases cGuCSyGT was identified as being able to catalyze the specific synthesis of 18α-Glycyrrhetinic acid 3-O-monoglucuronide (18α-GAMG) from 18α-Glycyrrhetinic acid (18α-GA) and GgUGT1 was identified as being able to catalyze the specific synthesis of 18α-glycyrrhizin (18α-GL) from 18α-GAMG. We further employed protein structure prediction and molecular dynamics simulation to explore the reason why cGuCSyGT has lower catalytic activity for 18α-GA than 18β-GA. Finally, an optimal process for yeast catalytic synthesis of 18α-GAMG and 18α-GL was constructed by optimizing various parameters, including substrate addition concentration, chassis host cells, substrate addition time, catalytic time, medium component addition and substrate solvent. Thus, the production of 18α-GAMG and 18α-GL reached 36.38(±1.87)mg/L and 39.32(±0.75)mg/L, respectively. This research achieved the microbial catalytic synthesis of 18α-GAMG and 18α-GL for the first time, which will provide a theoretical basis and technical support for the future total microbial synthesis of 18α-GL.

Key words: licorice, 18α-glycyrrhizic acid, 18α-glycyrrhetinic acid 3-O-monoglucuronide, glycosyltransferase, yeast cell catalysis

中图分类号:

Q 814

邢登雪, 张良, 李文强, 梁建华, 秦磊, 张根林, 李春. 酵母细胞催化合成18α-甘草酸[J]. 化工学报, DOI: 10.11949/0438-1157.20240310.

Dengxue XING, Liang ZHANG, Wenqiang LI, Jianhua LIANG, Lei QIN, Genlin ZHANG, Chun LI. Synthesis of 18α-glycyrrhizic acid by yeast cells[J]. CIESC Journal, DOI: 10.11949/0438-1157.20240310.

图/表 10

参考文献 29

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菌株名称	基因型	来源
BY4742	S. cerevisiae MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0	实验室保藏
BY4741	S. cerevisiae MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0	实验室保藏
CEN.PK2-1C	S. cerevisiae Matα ura3-52 trp1-289 leu2-3 112 his3Δ1; MAL2-8c; SUC2	实验室保藏
CEN.PK2-1D	S. cerevisiae Matα ura3-52 trp1-289 leu2-3 112 his3Δ1; MAL2-8c; SUC2	实验室保藏
SynV	BY4741.MATa his3Δ1 leu2 met15Δ ura3-52 with synthetic chromosome V	赠予
pPHR3	GS115-His4::P _GAP-hCas9; His4::P _TEF1 -ScRAD52; Int11::P _GAP -ScRAD59; Int20::P _TEF1 -ScMRE11	赠予
GAMG_X00	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -cGuCSyGT-T _ADH1; Δgal80	本研究构建
GAMG_X01	BY4742::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -cGuCSyGT-T _ADH1; Δgal80	本研究构建
GAMG_X02	BY4741::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -cGuCSyGT-T _ADH1;Δgal80	本研究构建
GAMG_X03	CEN.PK2-1C::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -cGuCSyGT-T _ADH1; Δgal80	本研究构建
GAMG_X04	CEN.PK2-1D::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -cGuCSyGT-T _ADH1; Δgal80	本研究构建
GAMG_X05	pPHR3::Int1::P _GAP -UGDH-T _AOX1; Int33::P _GAP -cGuCSyGT- T _AOX1	本研究构建
GL_X00	GAMG_X00::HO::P _GAL7 -GgUGT1-T _PGK1	本研究构建
GL_X01	GAMG_X01::HO::P _GAL7 -GgUGT1-T _PGK1	本研究构建
GL_X02	GAMG_X02::HO::P _GAL7 -GgUGT1-T _PGK1	本研究构建
GL_X03	GAMG_X03::HO::P _GAL7 -GgUGT1-T _PGK1	本研究构建
GL_X04	GAMG_X04::HO::P _GAL7 -GgUGT1-T _PGK1	本研究构建
GL_X05	pPHR3:: Int1::P _GAP -UGDH- T _AOX1; Int33::P _GAP -cGuCSyGT- T _AOX1 -P _TEF -GgUGT1-T ₀₅₄₇	本研究构建
GAMG_S01	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -UGT73F15-T _ADH1; Δgal80	本研究构建
GAMG_S02	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -UGT73F17-T _ADH1; Δgal80	本研究构建
GAMG_S04	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -UGT73F24-T _ADH1; Δgal80	本研究构建
GAMG_S05	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -UGT73C11-T _ADH1; Δgal80	本研究构建
GAMG_S06	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -UGT73C33-T _ADH1; Δgal80	本研究构建
GAMG_S07	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -UGT109A1-T _ADH1; Δgal80	本研究构建
GAMG_S08	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -Yojk-T _ADH1; Δgal80	本研究构建
GAMG_S09	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -BsYjic-T _ADH1; Δgal80	本研究构建
GL_S01	SynV:: YPRC3::P _GAL10 -HsUGDH-T _TPI1;HO::P _GAL7 -UGT73P12-T _PGK1; Δgal80	本研究构建
GL_S02	SynV:: YPRC3::P _GAL10 -HsUGDH-T _TPI1;HO::P _GAL7 -GgUGT1-T _PGK1; Δgal80	本研究构建

菌株名称	基因型	来源
BY4742	S. cerevisiae MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0	实验室保藏
BY4741	S. cerevisiae MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0	实验室保藏
CEN.PK2-1C	S. cerevisiae Matα ura3-52 trp1-289 leu2-3 112 his3Δ1; MAL2-8c; SUC2	实验室保藏
CEN.PK2-1D	S. cerevisiae Matα ura3-52 trp1-289 leu2-3 112 his3Δ1; MAL2-8c; SUC2	实验室保藏
SynV	BY4741.MATa his3Δ1 leu2 met15Δ ura3-52 with synthetic chromosome V	赠予
pPHR3	GS115-His4::P _GAP-hCas9; His4::P _TEF1 -ScRAD52; Int11::P _GAP -ScRAD59; Int20::P _TEF1 -ScMRE11	赠予
GAMG_X00	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -cGuCSyGT-T _ADH1; Δgal80	本研究构建
GAMG_X01	BY4742::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -cGuCSyGT-T _ADH1; Δgal80	本研究构建
GAMG_X02	BY4741::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -cGuCSyGT-T _ADH1;Δgal80	本研究构建
GAMG_X03	CEN.PK2-1C::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -cGuCSyGT-T _ADH1; Δgal80	本研究构建
GAMG_X04	CEN.PK2-1D::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -cGuCSyGT-T _ADH1; Δgal80	本研究构建
GAMG_X05	pPHR3::Int1::P _GAP -UGDH-T _AOX1; Int33::P _GAP -cGuCSyGT- T _AOX1	本研究构建
GL_X00	GAMG_X00::HO::P _GAL7 -GgUGT1-T _PGK1	本研究构建
GL_X01	GAMG_X01::HO::P _GAL7 -GgUGT1-T _PGK1	本研究构建
GL_X02	GAMG_X02::HO::P _GAL7 -GgUGT1-T _PGK1	本研究构建
GL_X03	GAMG_X03::HO::P _GAL7 -GgUGT1-T _PGK1	本研究构建
GL_X04	GAMG_X04::HO::P _GAL7 -GgUGT1-T _PGK1	本研究构建
GL_X05	pPHR3:: Int1::P _GAP -UGDH- T _AOX1; Int33::P _GAP -cGuCSyGT- T _AOX1 -P _TEF -GgUGT1-T ₀₅₄₇	本研究构建
GAMG_S01	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -UGT73F15-T _ADH1; Δgal80	本研究构建
GAMG_S02	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -UGT73F17-T _ADH1; Δgal80	本研究构建
GAMG_S04	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -UGT73F24-T _ADH1; Δgal80	本研究构建
GAMG_S05	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -UGT73C11-T _ADH1; Δgal80	本研究构建
GAMG_S06	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -UGT73C33-T _ADH1; Δgal80	本研究构建
GAMG_S07	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -UGT109A1-T _ADH1; Δgal80	本研究构建
GAMG_S08	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -Yojk-T _ADH1; Δgal80	本研究构建
GAMG_S09	SynV::YPRC3::P _GAL10 -HsUGDH-T _TPI1 -P _GAL2 -BsYjic-T _ADH1; Δgal80	本研究构建
GL_S01	SynV:: YPRC3::P _GAL10 -HsUGDH-T _TPI1;HO::P _GAL7 -UGT73P12-T _PGK1; Δgal80	本研究构建
GL_S02	SynV:: YPRC3::P _GAL10 -HsUGDH-T _TPI1;HO::P _GAL7 -GgUGT1-T _PGK1; Δgal80	本研究构建

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