作为细菌分裂所必需的微管蛋白类似物,丝状温度敏感蛋白Z(filamentous temperature-sensitive protein Z,FtsZ)被认为是一个具有潜力的药物作用新靶点。为了构建高纯度的FtsZ重组蛋白分离纯化体系,探讨其酶学性质,该研究利用大肠杆菌BL2...作为细菌分裂所必需的微管蛋白类似物,丝状温度敏感蛋白Z(filamentous temperature-sensitive protein Z,FtsZ)被认为是一个具有潜力的药物作用新靶点。为了构建高纯度的FtsZ重组蛋白分离纯化体系,探讨其酶学性质,该研究利用大肠杆菌BL21异源表达结核分枝杆菌FtsZ重组蛋白,通过Ni亲和层析柱和G-50层析柱纯化目的蛋白,采用孔雀石绿法和90°光散射法测定FtsZ重组蛋白的GTP酶活和蛋白聚集。研究结果表明:成功获得具有生物学活性的结核分枝杆菌FtsZ重组蛋白,其相对分子质量约为49kD;该酶最适反应温度为37℃,最适pH为6.8,金属离子Mg^(2+)和K^+对FtsZ重组蛋白酶活具有促进作用,有机溶剂DMSO和TritonX-100的体积分数分别高于0.1%和0.005%时对酶活有显著抑制作用(P<0.05)。此外,FtsZ重组蛋白在加入底物GTP诱导后,快速聚集。本实验利用基因工程技术成功获得具有生物活性的FtsZ重组蛋白,并明确了该蛋白质的酶学性质,为其进一步的研究和应用奠定了基础。展开更多
Microbial morphology engineering is a novel approach for cell factory to improve the titer of target product in bio-manufacture.Hyaluronic acid(HA),a valuable glycosaminoglycan polymerized by HA synthase(HAS),a membra...Microbial morphology engineering is a novel approach for cell factory to improve the titer of target product in bio-manufacture.Hyaluronic acid(HA),a valuable glycosaminoglycan polymerized by HA synthase(HAS),a membrane protein,is particularly selected as the model product to improve its single-cell HA-producing capacity via morphology engineering.DivIVA and FtsZ,the cell-elongation and cell division related protein,respectively,were both down/up dual regulated in C.glutamicum via weak promoter substitution or plasmid overexpression.Different from the natural short-rod shape,varied morphologies of engineered cells,i.e.small-ellipsoid-like(DivIVA-reduced),bulb-like(DivIVA-enhanced),long-rod(FtsZ-reduced)and dumbbell-like(FtsZ-enhanced),were observed.Applying these morphology-changed cells as hosts for HA production,the reduced expression of both DivIVA and FtsZ seriously inhibited normal cell growth;meanwhile,overexpression of DivIVA didn't show morphology changes,but overexpression of FtsZ surprisingly change the cell-shape into long and thick rod with remarkably enlarged single-cell surface area(more than 5.2-fold-increase).And finally,the single-cell HA-producing capacity of the FtsZ-overexpressed C.glutamicum was immensely improved by 13.5-folds.Flow cytometry analyses verified that the single-cell HAS amount on membrane was enhanced by 2.1 folds.This work is pretty valuable for high titer synthesis of diverse metabolic products with microbial cell factory.展开更多
文摘作为细菌分裂所必需的微管蛋白类似物,丝状温度敏感蛋白Z(filamentous temperature-sensitive protein Z,FtsZ)被认为是一个具有潜力的药物作用新靶点。为了构建高纯度的FtsZ重组蛋白分离纯化体系,探讨其酶学性质,该研究利用大肠杆菌BL21异源表达结核分枝杆菌FtsZ重组蛋白,通过Ni亲和层析柱和G-50层析柱纯化目的蛋白,采用孔雀石绿法和90°光散射法测定FtsZ重组蛋白的GTP酶活和蛋白聚集。研究结果表明:成功获得具有生物学活性的结核分枝杆菌FtsZ重组蛋白,其相对分子质量约为49kD;该酶最适反应温度为37℃,最适pH为6.8,金属离子Mg^(2+)和K^+对FtsZ重组蛋白酶活具有促进作用,有机溶剂DMSO和TritonX-100的体积分数分别高于0.1%和0.005%时对酶活有显著抑制作用(P<0.05)。此外,FtsZ重组蛋白在加入底物GTP诱导后,快速聚集。本实验利用基因工程技术成功获得具有生物活性的FtsZ重组蛋白,并明确了该蛋白质的酶学性质,为其进一步的研究和应用奠定了基础。
基金This work was supported by National Key R&D Program of China[2018YFA0902200]the National Natural Science Foundation of China[No.21776157].
文摘Microbial morphology engineering is a novel approach for cell factory to improve the titer of target product in bio-manufacture.Hyaluronic acid(HA),a valuable glycosaminoglycan polymerized by HA synthase(HAS),a membrane protein,is particularly selected as the model product to improve its single-cell HA-producing capacity via morphology engineering.DivIVA and FtsZ,the cell-elongation and cell division related protein,respectively,were both down/up dual regulated in C.glutamicum via weak promoter substitution or plasmid overexpression.Different from the natural short-rod shape,varied morphologies of engineered cells,i.e.small-ellipsoid-like(DivIVA-reduced),bulb-like(DivIVA-enhanced),long-rod(FtsZ-reduced)and dumbbell-like(FtsZ-enhanced),were observed.Applying these morphology-changed cells as hosts for HA production,the reduced expression of both DivIVA and FtsZ seriously inhibited normal cell growth;meanwhile,overexpression of DivIVA didn't show morphology changes,but overexpression of FtsZ surprisingly change the cell-shape into long and thick rod with remarkably enlarged single-cell surface area(more than 5.2-fold-increase).And finally,the single-cell HA-producing capacity of the FtsZ-overexpressed C.glutamicum was immensely improved by 13.5-folds.Flow cytometry analyses verified that the single-cell HAS amount on membrane was enhanced by 2.1 folds.This work is pretty valuable for high titer synthesis of diverse metabolic products with microbial cell factory.
