Department of Life Science and Biochemical Engineering, SunMoon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.
Department of BT-Convergent Pharmaceutical Engineering, SunMoon University, 70 Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam, 31460, Republic of Korea.
Microb Cell Fact. 2019 Jan 17;18(1):7. doi: 10.1186/s12934-019-1056-6.
Multi-monocistronic and multi-variate vectors were designed, built, and tested for the improved production of cyanidin 3-O-glucoside (C3G) in Escherichia coli BL21 (DE3). The synthetic bio-parts were designed in such a way that multiple genes can be assembled using the bio-brick system, and expressed under different promoters in a single vector. The vectors harbor compatible cloning sites, so that the genes can be shuffled from one vector to another in a single step, and assembled into a single vector. The two required genes: anthocyanidin synthase (PhANS) from Petunia hybrida, and cyanidin 3-O-glucosyltransferase (At3GT) from Arabidopsis thaliana, were individually cloned under P, P, and P promoters. Both PhANS and At3GT were shuffled back and forth, so as to generate a combinatorial system for C3G production. The constructed systems were further coupled with the genes for UDP-D-glucose synthesis, all cloned in a multi-monocistronic fashion under P. Finally, the production of C3G was checked and confirmed using the modified M9 media, and analyzed through various chromatography and spectrometric analyses.
The engineered strains endowed with newly generated vectors and the genes for C3G biosynthesis and UDP-D-glucose synthesis were fed with 2 mM (+)-catechin and D-glucose for the production of cyanidin, and its subsequent conversion to C3G. One of the engineered strains harboring At3GT and PhANS under P promoter and UDP-D-glucose biosynthesis genes under P promoter led to the production of ~ 439 mg/L of C3G within 36 h of incubation, when the system was exogenously fed with 5% (w/v) D-glucose. This system did not require exogenous supplementation of UDP-D-glucose.
A synthetic vector system using different promoters has been developed and used for the synthesis of C3G in E. coli BL21 (DE3) by directing the metabolic flux towards the UDP-D-glucose. This system has the potential of generating better strains for the synthesis of valuable natural products.
为了提高大肠杆菌 BL21(DE3)中飞燕草色素 3-O-葡萄糖苷（C3G）的产量，设计、构建和测试了多单顺反子和多变量载体。合成的生物部件设计方式使得多个基因可以使用生物砖系统组装，并在单个载体中使用不同的启动子表达。载体上还带有兼容的克隆位点，因此可以在单个步骤中从一个载体到另一个载体转移基因，并将它们组装到单个载体中。两个必需的基因：来自矮牵牛的花色素苷合酶（PhANS）和来自拟南芥的飞燕草色素 3-O-葡萄糖基转移酶（At3GT），分别在 P、P 和 P 启动子下单独克隆。PhANS 和 At3GT 被来回交换，从而生成用于 C3G 生产的组合系统。构建的系统进一步与 UDP-D-葡萄糖合成基因偶联，所有基因均以 P 启动子下的多单顺反子方式克隆。最后，使用改良的 M9 培养基检查和确认 C3G 的生产，并通过各种色谱和光谱分析进行分析。
用新生成的载体和 C3G 生物合成和 UDP-D-葡萄糖合成基因赋予工程菌株，并以 2mM（+）-儿茶素和 D-葡萄糖喂养，以生产飞燕草色素，并将其转化为 C3G。在 P 启动子下携带 At3GT 和 PhANS 以及 P 启动子下携带 UDP-D-葡萄糖生物合成基因的一个工程菌株，在 36 小时的孵育期内，当系统外源添加 5%（w/v）D-葡萄糖时，可生产约 439mg/L 的 C3G。该系统不需要外源补充 UDP-D-葡萄糖。
已经开发并使用了一种使用不同启动子的合成载体系统，通过将代谢通量导向 UDP-D-葡萄糖来指导大肠杆菌 BL21(DE3)中 C3G 的合成。该系统有可能为有价值的天然产物的合成产生更好的菌株。
