(–)-α-Bisabolol is naturally occurring in many plants and has great potential in health products and pharma-ceuticals.However,the current extraction method from natural plants is unsustainable and cannot fulfil the ...(–)-α-Bisabolol is naturally occurring in many plants and has great potential in health products and pharma-ceuticals.However,the current extraction method from natural plants is unsustainable and cannot fulfil the increasing requirement.This study aimed to develop a sustainable strategy to enhance the biosynthesis of(–)-α-bisabolol by metabolic engineering.By introducing the heterologous gene MrBBS and weakening the competitive pathway gene ERG9,a de novo(–)-α-bisabolol biosynthesis strain was constructed that could produce 221.96 mg/L(–)-α-bisabolol.Two key genes for(–)-α-bisabolol biosynthesis,ERG20 and MrBBS,were fused by a flexible linker(GGGS)3 under the GAL7 promoter control,and the titer was increased by 2.9-fold.Optimization of the mevalonic acid pathway and multi-copy integration further increased(–)-α-bisabolol production.To promote product efflux,overexpression of PDR15 led to an increase in extracellular production.Combined with the optimal strategy,(–)-α-bisabolol production in a 5 L bioreactor reached 7.02 g/L,which is the highest titer reported in yeast to date.This work provides a reference for the efficient production of(–)-α-bisabolol in yeast.展开更多
目的研究α-红没药醇(α-bisabolol)对胶质母细胞瘤细胞U251和U87迁移和侵袭的影响并探讨其可能的机制。方法体外培养人脑胶质母细胞瘤细胞U251和U87,CCK8比色法检测不同浓度(0μmol/L、1.25μmol/L、2.5μmol/L、5μmol/L、10μmol/L)...目的研究α-红没药醇(α-bisabolol)对胶质母细胞瘤细胞U251和U87迁移和侵袭的影响并探讨其可能的机制。方法体外培养人脑胶质母细胞瘤细胞U251和U87,CCK8比色法检测不同浓度(0μmol/L、1.25μmol/L、2.5μmol/L、5μmol/L、10μmol/L)α-红没药醇作用24 h后细胞存活率的变化;细胞划痕实验、Transwell实验、Western blotting检测不同浓度(0μmol/L、1.25μmol/L、2.5μmol/L)α-红没药醇对U251和U87细胞迁移、侵袭能力及对细胞MMP-2、MMP-9、c-Met蛋白表达量的影响;U251细胞分为A组(空载质粒组)、B组(空载质粒+2.5μmol/Lα-红没药醇组)、C组(过表达c-Met质粒组)、D组(过表达c-Met质粒+2.5μmol/Lα-红没药醇组),转染质粒后24 h,B、D组加入2.5μmol/Lα-红没药醇,细胞划痕实验、Transwell实验、Western blotting分别检测4组细胞迁移、侵袭能力及c-Met、MMP-2、MMP-9蛋白表达量。结果 5μmol/L、10μmol/L组细胞存活率较0μmol/L、1.25μmol/L、2.5μmol/L组下降(U251:61.22%±5.08%、29.48%±4.84%vs 100%±0.00%、98.16%±5.71%、96.89±7.30%,P=0.00;U87:55.72%±8.17%、19.66%±4.82%vs100%±0.00%、97.86%±5.41%、95.31%±5.42%,P=0.00)。0μmol/L、1.25μmol/L、2.5μmol/L组划痕愈合百分比分别为U251:49.36%±5.44%、35.08%±3.79%、23.89%±4.51%,U87:46.64%±4.83%、33.42%±3.10%、22.35%±3.62%,侵袭到下室的细胞数量分别为U251:248.67±14.94、171.11±17.91、87.11±15.49,U87:202.44±16.98、145.44±11.91、71.98±9.32,三组间差异均有统计学意义(P均=0.000)。c-Met、MMP-2、MMP-9蛋白表达量分别为:U251(c-Met:1.00±0.00、0.70±0.09、0.33±0.08;MMP-2:1.00±0.00、0.70±0.08、0.32±0.10;MMP-9:1.00±0.00、0.69±0.09、0.24±0.07);U87(c-Met:1.00±0.00、0.71±0.08、0.27±0.08;MMP-2:1.00±0.00、0.71±0.10、0.29±0.04;MMP-9:1.00±0.00、0.72±0.08、0.23±0.04),三组间差异均有统计学意义(P=0.000)。转染c-Met过表达质粒后D组较B组划痕愈合百分比明显升高(35.61%±4.70%vs 13.11%±2.99%),D组侵袭到下室的细胞数量较B组明显升高(209.44±18.13 vs 91.33±14.46),D组较B组c-Met、MMP-2、MMP-9蛋白表达量明显升高(c-Met:0.60±0.04 vs 0.39±0.04;MMP-2:0.71±0.10 vs 0.49±0.08,MMP-9:0.73±0.11 vs 0.45±0.07,P=0.000)。结论α-红没药醇可以通过下调c-Met抑制胶质母细胞瘤细胞迁移和侵袭。展开更多
基金supported by the National Natural Science Foundation of China(22208123)the Jiangsu Postdoctoral Research Funding Pro-gram(2021K589C)the Starry Night Science Fund of Zhejiang University Shanghai Institute for Advanced Study(Grant No.SN-ZJU-SIAS-0013).
文摘(–)-α-Bisabolol is naturally occurring in many plants and has great potential in health products and pharma-ceuticals.However,the current extraction method from natural plants is unsustainable and cannot fulfil the increasing requirement.This study aimed to develop a sustainable strategy to enhance the biosynthesis of(–)-α-bisabolol by metabolic engineering.By introducing the heterologous gene MrBBS and weakening the competitive pathway gene ERG9,a de novo(–)-α-bisabolol biosynthesis strain was constructed that could produce 221.96 mg/L(–)-α-bisabolol.Two key genes for(–)-α-bisabolol biosynthesis,ERG20 and MrBBS,were fused by a flexible linker(GGGS)3 under the GAL7 promoter control,and the titer was increased by 2.9-fold.Optimization of the mevalonic acid pathway and multi-copy integration further increased(–)-α-bisabolol production.To promote product efflux,overexpression of PDR15 led to an increase in extracellular production.Combined with the optimal strategy,(–)-α-bisabolol production in a 5 L bioreactor reached 7.02 g/L,which is the highest titer reported in yeast to date.This work provides a reference for the efficient production of(–)-α-bisabolol in yeast.
文摘目的研究α-红没药醇(α-bisabolol)对胶质母细胞瘤细胞U251和U87迁移和侵袭的影响并探讨其可能的机制。方法体外培养人脑胶质母细胞瘤细胞U251和U87,CCK8比色法检测不同浓度(0μmol/L、1.25μmol/L、2.5μmol/L、5μmol/L、10μmol/L)α-红没药醇作用24 h后细胞存活率的变化;细胞划痕实验、Transwell实验、Western blotting检测不同浓度(0μmol/L、1.25μmol/L、2.5μmol/L)α-红没药醇对U251和U87细胞迁移、侵袭能力及对细胞MMP-2、MMP-9、c-Met蛋白表达量的影响;U251细胞分为A组(空载质粒组)、B组(空载质粒+2.5μmol/Lα-红没药醇组)、C组(过表达c-Met质粒组)、D组(过表达c-Met质粒+2.5μmol/Lα-红没药醇组),转染质粒后24 h,B、D组加入2.5μmol/Lα-红没药醇,细胞划痕实验、Transwell实验、Western blotting分别检测4组细胞迁移、侵袭能力及c-Met、MMP-2、MMP-9蛋白表达量。结果 5μmol/L、10μmol/L组细胞存活率较0μmol/L、1.25μmol/L、2.5μmol/L组下降(U251:61.22%±5.08%、29.48%±4.84%vs 100%±0.00%、98.16%±5.71%、96.89±7.30%,P=0.00;U87:55.72%±8.17%、19.66%±4.82%vs100%±0.00%、97.86%±5.41%、95.31%±5.42%,P=0.00)。0μmol/L、1.25μmol/L、2.5μmol/L组划痕愈合百分比分别为U251:49.36%±5.44%、35.08%±3.79%、23.89%±4.51%,U87:46.64%±4.83%、33.42%±3.10%、22.35%±3.62%,侵袭到下室的细胞数量分别为U251:248.67±14.94、171.11±17.91、87.11±15.49,U87:202.44±16.98、145.44±11.91、71.98±9.32,三组间差异均有统计学意义(P均=0.000)。c-Met、MMP-2、MMP-9蛋白表达量分别为:U251(c-Met:1.00±0.00、0.70±0.09、0.33±0.08;MMP-2:1.00±0.00、0.70±0.08、0.32±0.10;MMP-9:1.00±0.00、0.69±0.09、0.24±0.07);U87(c-Met:1.00±0.00、0.71±0.08、0.27±0.08;MMP-2:1.00±0.00、0.71±0.10、0.29±0.04;MMP-9:1.00±0.00、0.72±0.08、0.23±0.04),三组间差异均有统计学意义(P=0.000)。转染c-Met过表达质粒后D组较B组划痕愈合百分比明显升高(35.61%±4.70%vs 13.11%±2.99%),D组侵袭到下室的细胞数量较B组明显升高(209.44±18.13 vs 91.33±14.46),D组较B组c-Met、MMP-2、MMP-9蛋白表达量明显升高(c-Met:0.60±0.04 vs 0.39±0.04;MMP-2:0.71±0.10 vs 0.49±0.08,MMP-9:0.73±0.11 vs 0.45±0.07,P=0.000)。结论α-红没药醇可以通过下调c-Met抑制胶质母细胞瘤细胞迁移和侵袭。
