Anila N, Simon Daris P, Chandrashekar Arun, Ravishankar G A, Sarada R
Department of Botany, St. Xavier's College For Women (Affiliated to Mahatma Gandhi University), Aluva, 683101, India.
Plant Cell Biotechnology Department, Central Food Technological Research Institute (A Constituent Laboratory of the Council of Scientific and Industrial Research, CSIR), Mysore, Karnataka, 570 020, India.
Photosynth Res. 2016 Mar;127(3):321-33. doi: 10.1007/s11120-015-0188-8. Epub 2015 Sep 3.
Dunaliella is a commercially important marine alga producing high amount of β-carotene. The use of Dunaliella as a potential transgenic system for the production of recombinant proteins has been recently recognized. The present study reports for the first time the metabolic engineering of carotenoid biosynthesis in Dunaliella salina for ketocarotenoid production. The pathway modification included the introduction of a bkt gene from H. pluvialis encoding β-carotene ketolase (4,4'β-oxygenase) along with chloroplast targeting for the production of ketocarotenoids. The bkt under the control of Dunaliella Rubisco smaller subunit promoter along with its transit peptide sequence was introduced into the alga through standardized Agrobacterium-mediated transformation procedure. The selected transformants were confirmed using GFP and GUS expression, PCR and southern blot analysis. A notable upregulation of the endogenous hydroxylase level of transformants was observed where the BKT expression was higher in nutrient-limiting conditions. Carotenoid analysis of the transformants through HPLC and MS analysis showed the presence of astaxanthin and canthaxanthin with maximum content of 3.5 and 1.9 µg/g DW, respectively. The present study reports the feasibility of using D. salina for the production of ketocarotenoids including astaxanthin.
杜氏盐藻是一种具有重要商业价值的海洋藻类，能产生大量的β-胡萝卜素。近年来，杜氏盐藻作为生产重组蛋白的潜在转基因系统已得到认可。本研究首次报道了对盐生杜氏藻中类胡萝卜素生物合成进行代谢工程改造以生产酮类胡萝卜素。途径改造包括引入来自雨生红球藻的编码β-胡萝卜素酮酶（4,4'-β-加氧酶）的bkt基因，并进行叶绿体靶向以生产酮类胡萝卜素。将受杜氏盐藻Rubisco小亚基启动子控制的bkt及其转运肽序列通过标准化的农杆菌介导转化程序导入该藻类。使用绿色荧光蛋白（GFP）和β-葡萄糖醛酸酶（GUS）表达、聚合酶链反应（PCR）和Southern印迹分析对所选转化体进行了确认。在营养限制条件下，当BKT表达较高时，观察到转化体内源羟化酶水平有显著上调。通过高效液相色谱（HPLC）和质谱（MS）分析对转化体的类胡萝卜素进行分析，结果显示存在虾青素和角黄素，其最大含量分别为3.5和1.9 μg/g干重。本研究报道了利用盐生杜氏藻生产包括虾青素在内的酮类胡萝卜素的可行性。
