Department of Biological Science & Technology, I-Shou University, Kaohsiung 82445, Taiwan.
Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan.
J Microbiol Methods. 2019 May;160:20-28. doi: 10.1016/j.mimet.2019.03.012. Epub 2019 Mar 16.
Astaxanthin possesses various biological properties and is used in the animal and fish feed, food, and beverage industries. In this study, we derived zeaxanthin biosynthesis genes (crtE, crtB, crtI, crtY, and crtZ) from Erwinia uredovora and crtW from Agrobacterium aurantiacum. We fused inducible and constitutive promoters to astaxanthin biosynthesis genes to construct a novel plasmid (dubbed PTP3-6) that can effectively enhance free-form astaxanthin (FFAX) production. The PTP3-6 plasmid contains one T7 promoter, driving IPTG inducible crtW expression, and three constitutive promoters (isolated from E. uredovora) driving expression of the other zeaxanthin biosynthesis genes. Escherichia coli BL21 (DE3) cells carrying the PTP3-6 plasmid produced 8.3 mg/g dry cell weight astaxanthin, which is 69.4-fold higher than has been previously reported. Using multiple promoter fusions of astaxanthin biosynthesis genes could be applied in other hosts to enhance astaxanthin production. FFAX was identified in recombinant E. coli cells through ultra-performance liquid chromatography-mass spectrometry.
虾青素具有多种生物特性，被应用于动物和鱼类饲料、食品和饮料行业。在本研究中，我们从欧文氏菌（Erwinia uredovora）中获得了玉米黄质生物合成基因（crtE、crtB、crtI、crtY 和 crtZ），从黄单胞菌（Agrobacterium aurantiacum）中获得了 crtW 基因。我们将诱导型和组成型启动子与虾青素生物合成基因融合，构建了一种新型质粒（命名为 PTP3-6），可有效提高游离态虾青素（FFAX）的产量。PTP3-6 质粒含有一个 T7 启动子，驱动 IPTG 诱导的 crtW 表达，以及三个组成型启动子（来自欧文氏菌），驱动其他玉米黄质生物合成基因的表达。携带 PTP3-6 质粒的大肠杆菌 BL21（DE3）细胞可产生 8.3mg/g 干细胞重的虾青素，比之前报道的提高了 69.4 倍。通过虾青素生物合成基因的多启动子融合，可以在其他宿主中提高虾青素的产量。通过超高效液相色谱-质谱联用技术在重组大肠杆菌细胞中鉴定出了游离态虾青素。
