Laboratory of Marine Environmental Microbiology, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan.
Appl Biochem Biotechnol. 2018 Jan;184(1):80-91. doi: 10.1007/s12010-017-2521-5. Epub 2017 Jun 13.
Production of functional carotenoids using microalgae may facilitate the commercialization of anti-aging nutritional supplements. The green alga Chlamydomonas reinhardtii uses a non-mevalonate (MEP) pathway for isopentenyl diphosphate (IPP) synthesis. Two enzymes thought to play important roles in this MEP pathway to IPP synthesis are 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and reductase (DXR). DnaJ-like chaperone (Orange protein) is thought to support phytoene synthase, a key enzyme in plant carotenoid synthesis. Genes for Orange (OR), DXS, and DXR were overexpressed via nuclear transformation into C. reinhardtii. CDS of OR, DXS, and DXR were amplified and connected with dual promoters of heat-shock protein 70A and ribulose bisphosphate carboxylase small chain 2. Compared with the parental strain, transformant CrOR#2 produced increased lutein and β-carotene (1.9-fold and 1.7-fold per cell, respectively). Transformant CrDXS#1 produced lutein and β-carotene at lower per-cell abundances than those for the parental strain. CrDXR#2 transformant produced lutein and β-carotene at higher per-cell abundances than their parental counterpart; however, these transformants produced lutein and β-carotene at lower per-medium abundances than their parental counterparts. These results suggest that OR protein supports phytoene synthase in C. reinhardtii and that the phytoene synthesis step is rate-limiting in carotenoid synthesis.
利用微藻生产功能性类胡萝卜素可能有助于抗衰老营养补充剂的商业化。绿藻莱茵衣藻（Chlamydomonas reinhardtii）使用非甲羟戊酸（MEP）途径合成异戊烯二磷酸（IPP）。有两种酶被认为在这个 MEP 途径到 IPP 合成中起着重要作用，分别是 1-脱氧-D-木酮糖 5-磷酸合酶（DXS）和还原酶（DXR）。推测 DnaJ 样伴侣（橙色蛋白）支持类胡萝卜素合成的关键酶——八氢番茄红素合酶。通过核转化将橙色（OR）、DXS 和 DXR 基因过表达到莱茵衣藻中。扩增了 OR、DXS 和 DXR 的 CDS，并与热休克蛋白 70A 和核酮糖二磷酸羧化酶小链 2 的双重启动子连接。与亲本菌株相比，转化体 CrOR#2 产生的叶黄素和β-胡萝卜素分别增加了 1.9 倍和 1.7 倍（每个细胞）。转化体 CrDXS#1 产生的叶黄素和β-胡萝卜素的每个细胞丰度低于亲本菌株。CrDXR#2 转化体产生的叶黄素和β-胡萝卜素的每个细胞丰度高于其亲本，但这些转化体在培养基中的叶黄素和β-胡萝卜素产量低于其亲本。这些结果表明，OR 蛋白在莱茵衣藻中支持八氢番茄红素合酶，并且类胡萝卜素合成中的番茄红素合成步骤是限速步骤。
