Hapsari Rizqiya Astri, Chrysanthy Tamara, Synthiarini Vaniarta, Masduki Fifi Fitriyah, Setiawan Agus, Muranaka Toshiya
School of Pharmacy, Bandung Institute of Technology, Bandung, Indonesia.
University Centre of Excelence - Nutraceutical, Bioscience and Biotechnology Research Centre, Bandung Institute of Technology, Bandung, Indonesia.
Adv Pharm Bull. 2021 Jan;11(1):181-187. doi: 10.34172/apb.2021.019. Epub 2020 Nov 7.
Artemisinin, a secondary metabolite in Artemisia annua is one of primary choice for the treatment of malaria, it is naturally produced in low concentration from this plant. This study was aimed to clone key enzymes of artemisinin production in order to enhance its production through the semi-synthetically production in Two key enzymes in artemisinin biosynthetic pathway which are farnesyl phosphate synthase () and amorpha-4,11-diene synthase (ads) genes were transformed into using pBEVY vector. Successful transformation was checked by polymerase chain reaction (PCR) method and sequencing analysis Recombinant plasmids which are pBEVY-GU_ and pBEVY_GL_ were successfully constructed. The optimized gene was amplified using PCR with a couple of primers that are designed in order to provide the homolog recombination between gene with the expression plasmid of pBEVY-GU respectively. While the optimized gene was cloned using classical method. Transformants were grown in selective media Synthetic Defined (SD) without leucine for transformants contain plasmid pBEVY-GL_ and media without uracil for transformants contain plasmid pBEVY-GU_. Confirmation of colonies was done by PCR with primers to amplify and . DNA from yeast was isolated from positive colonies then transformed to . Plasmid from was isolated for restriction analysis and sequencing. Protein expression was induced by cultivating the yeast in the media with 2% galactose. Based on PCR, restriction and sequencing analysis, it could be concluded that and genes were successfully constructed, transformed and expressed in S. cerevisiae.
青蒿素是青蒿中的一种次生代谢产物，是治疗疟疾的主要选择之一，它在这种植物中天然产生的浓度较低。本研究旨在克隆青蒿素生产的关键酶，以便通过半合成生产来提高其产量。青蒿素生物合成途径中的两种关键酶，即法呢基磷酸合酶（FPS）和紫穗槐-4,11-二烯合酶（ADS）基因，被使用pBEVY载体转化到酿酒酵母中。通过聚合酶链反应（PCR）方法和测序分析检查成功的转化。成功构建了重组质粒pBEVY-GU_FPS和pBEVY_GL_ADS。使用PCR扩增优化的FPS基因，使用一对引物进行设计，以便分别在FPS基因与pBEVY-GU表达质粒之间提供同源重组。而优化的ADS基因则使用经典方法克隆。转化体在不含亮氨酸的合成定义（SD）选择培养基中生长，对于含有质粒pBEVY-GL_ADS的转化体，以及在不含尿嘧啶的培养基中生长，对于含有质粒pBEVY-GU_FPS的转化体。通过用引物扩增FPS和ADS来进行菌落确认。从阳性菌落中分离酵母的DNA，然后转化到大肠杆菌中。从大肠杆菌中分离质粒进行限制性分析和测序。通过在含有2%半乳糖的培养基中培养酵母来诱导蛋白质表达。基于PCR、限制性和测序分析，可以得出结论，FPS和ADS基因已成功构建、转化并在酿酒酵母中表达。
