目的传统β-熊果苷生产依赖植物提取与化学合成,存在原料供应不稳定、环境污染等问题。该研究旨在开发绿色高效的生物合成新路径。方法运用基因工程手段,构建糖基转移酶itUGT2与蔗糖合酶GmSuSy协同体系,实现UDPG的循环再生,建立β-熊果...目的传统β-熊果苷生产依赖植物提取与化学合成,存在原料供应不稳定、环境污染等问题。该研究旨在开发绿色高效的生物合成新路径。方法运用基因工程手段,构建糖基转移酶itUGT2与蔗糖合酶GmSuSy协同体系,实现UDPG的循环再生,建立β-熊果苷的酶法合成工艺。结果单酶催化的最适温度和pH分别为35℃与7.0。经优化,双酶体系最佳反应条件为:温度40℃、pH 6.5,itUGT2与GmSuSy酶量比1:2(20μg:40μg),底物HQ与蔗糖浓度比1:4。此外,添加0.5 mM MgSO_(4)或β-环糊精可显著提高产率,可能通过稳定酶结构、改善底物溶解性发挥作用。结论该研究成功开发的酶法合成技术,解决了传统工艺的弊端,实现辅因子原位再生,具有反应条件温和、绿色环保、易于产业化等优势,为β-熊果苷工业化生产提供了创新解决方案,具备显著的科学价值与应用潜力。展开更多
UGPase （UDP-glucose pyrophosphorylase）, one of the primary enzymes concerned with carbohydrate metabolism, catalyzes the formation of UDPG. By inserting the UGPase cDNA fragment cloned from Saccharum officinarum int...UGPase （UDP-glucose pyrophosphorylase）, one of the primary enzymes concerned with carbohydrate metabolism, catalyzes the formation of UDPG. By inserting the UGPase cDNA fragment cloned from Saccharum officinarum into PQE-30, the prokaryotic expression vector of PQE-UGP was successfully constructed. Then the vector plasmid of PQE-UGP was transformed into host bacteria M 15 and the expression of target gene was induced by Isopropyl β-D-1-Thiogalactopyranoside （IPTG）. The research laid foundation for study on the prokaryotic expression of UGPase.展开更多
Hydroxysalidroside is an important natural phenylethanoid glycoside with broad application prospects in the food and pharmaceutical fields.However,its low concentration in plants and complex extraction hinder its prod...Hydroxysalidroside is an important natural phenylethanoid glycoside with broad application prospects in the food and pharmaceutical fields.However,its low concentration in plants and complex extraction hinder its production.Despite being a promising way to synthesize hydroxysalidroside in Escherichia coli,glycosylation remains the limiting factor for its production.A de novo biosynthetic pathway for hydroxysalidroside was successfully constructed in E.coli via the screening of glycosyltransferase,overexpressing phosphoglucomutase(pgm)and UDP-glucose pyrophosphorylase(galU)to ensure a sufficient supply of UDP-glucose(UDPG).Additionally,a semi-rational design of UGT85A1 was conducted to expand the acceptor-binding pocket to eliminate steric hindrance interfering with the binding of hydroxytyrosol.The endogenous genes ushA and otsA were knocked out to further reduce the consumption of UDPG.Finally,a titer of 5837.2 mg/L was achieved in a 5 L fermenter by optimizing the feeding times of carbon sources.This laid the foundation for the subsequent biosynthesis of phenylethanoid glycosides.展开更多
文摘目的传统β-熊果苷生产依赖植物提取与化学合成,存在原料供应不稳定、环境污染等问题。该研究旨在开发绿色高效的生物合成新路径。方法运用基因工程手段,构建糖基转移酶itUGT2与蔗糖合酶GmSuSy协同体系,实现UDPG的循环再生,建立β-熊果苷的酶法合成工艺。结果单酶催化的最适温度和pH分别为35℃与7.0。经优化,双酶体系最佳反应条件为:温度40℃、pH 6.5,itUGT2与GmSuSy酶量比1:2(20μg:40μg),底物HQ与蔗糖浓度比1:4。此外,添加0.5 mM MgSO_(4)或β-环糊精可显著提高产率,可能通过稳定酶结构、改善底物溶解性发挥作用。结论该研究成功开发的酶法合成技术,解决了传统工艺的弊端,实现辅因子原位再生,具有反应条件温和、绿色环保、易于产业化等优势,为β-熊果苷工业化生产提供了创新解决方案,具备显著的科学价值与应用潜力。
文摘UGPase （UDP-glucose pyrophosphorylase）, one of the primary enzymes concerned with carbohydrate metabolism, catalyzes the formation of UDPG. By inserting the UGPase cDNA fragment cloned from Saccharum officinarum into PQE-30, the prokaryotic expression vector of PQE-UGP was successfully constructed. Then the vector plasmid of PQE-UGP was transformed into host bacteria M 15 and the expression of target gene was induced by Isopropyl β-D-1-Thiogalactopyranoside （IPTG）. The research laid foundation for study on the prokaryotic expression of UGPase.
基金supported by the National Key Research and Development Program of China(2022YFC2106100)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China(32021005).
文摘Hydroxysalidroside is an important natural phenylethanoid glycoside with broad application prospects in the food and pharmaceutical fields.However,its low concentration in plants and complex extraction hinder its production.Despite being a promising way to synthesize hydroxysalidroside in Escherichia coli,glycosylation remains the limiting factor for its production.A de novo biosynthetic pathway for hydroxysalidroside was successfully constructed in E.coli via the screening of glycosyltransferase,overexpressing phosphoglucomutase(pgm)and UDP-glucose pyrophosphorylase(galU)to ensure a sufficient supply of UDP-glucose(UDPG).Additionally,a semi-rational design of UGT85A1 was conducted to expand the acceptor-binding pocket to eliminate steric hindrance interfering with the binding of hydroxytyrosol.The endogenous genes ushA and otsA were knocked out to further reduce the consumption of UDPG.Finally,a titer of 5837.2 mg/L was achieved in a 5 L fermenter by optimizing the feeding times of carbon sources.This laid the foundation for the subsequent biosynthesis of phenylethanoid glycosides.
