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
Cyanidin-3-O-glucoside(C3G),a highly valuable flavonoid with strong antioxidant properties and potential in preventing cardiovascular diseases,was efficiently biosynthesized in E.coli using(+)-catechin as a substrate....Cyanidin-3-O-glucoside(C3G),a highly valuable flavonoid with strong antioxidant properties and potential in preventing cardiovascular diseases,was efficiently biosynthesized in E.coli using(+)-catechin as a substrate.To address UDP-glucose(UDPG)supply limitations,we compared cellobiose-based and sucrose-based pathways,identifying the cellobiose-based pathway as the superior strategy,resulting in a fourfold increase in C3G production.We then constructed three expression systems—single cistron expression cassette,bicistronic expression cassette,and fusion protein—with the bicistronic expression cassette system yielding the highest C3G output.Further refinement was achieved through RBS optimization,which fine-tuned the relative expression levels of the two key enzymes.Semi-rational design of glucosyltransferase At3GT led to a double mutant,At3GTL344V/F378G,which exhibited a 92.26%±3.58%increase in C3G yield compared to the wild-type enzyme.Integrated into the engineered system,this mutant,combined with optimized fermentation conditions,achieved a C3G titer of 1036.82±20.13 mg/L and a 71.28%±0.01%substrate conversion rate,the highest reported in E.coli.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 Natural Science Foundation of China(32371314).
文摘Cyanidin-3-O-glucoside(C3G),a highly valuable flavonoid with strong antioxidant properties and potential in preventing cardiovascular diseases,was efficiently biosynthesized in E.coli using(+)-catechin as a substrate.To address UDP-glucose(UDPG)supply limitations,we compared cellobiose-based and sucrose-based pathways,identifying the cellobiose-based pathway as the superior strategy,resulting in a fourfold increase in C3G production.We then constructed three expression systems—single cistron expression cassette,bicistronic expression cassette,and fusion protein—with the bicistronic expression cassette system yielding the highest C3G output.Further refinement was achieved through RBS optimization,which fine-tuned the relative expression levels of the two key enzymes.Semi-rational design of glucosyltransferase At3GT led to a double mutant,At3GTL344V/F378G,which exhibited a 92.26%±3.58%increase in C3G yield compared to the wild-type enzyme.Integrated into the engineered system,this mutant,combined with optimized fermentation conditions,achieved a C3G titer of 1036.82±20.13 mg/L and a 71.28%±0.01%substrate conversion rate,the highest reported in E.coli.
