Jiao Xiang, Sun Wenyi, Zhang Yue, Liu Xiangjian, Zhang Qi, Wang Qian, Zhang Sufang, Zhao Zongbao Kent
Division of Biotechnology, Dalian Institute of Chemical Physics, CAS Dalian 116023 PR China.
University of Chinese Academy of Sciences Beijing 100049 PR China.
RSC Adv. 2018 Oct 12;8(61):34967-34972. doi: 10.1039/c8ra06510a. eCollection 2018 Oct 10.
The yeast serves as a promising host for the production of a wide range of chemical compounds and fuels. Currently, simultaneous expression of several genes could be achieved the use of 2A viral peptides, yet detailed characterizations to assess the discrepancy of different orders of genes linked by 2A peptides are rarely sufficient. In this study, we investigated the effects of the order of genes linked by porcine teschovirus-1 2A (P2A) peptide on the metabolic pathway in . A heterologous carotenoid biosynthetic system involving nine kinds of polycistronic expression of codon-optimized carotenogenic genes , and from was introduced into . The order of genes in the polycistronic segment was exchanged; β-carotene production by engineered yeasts was significantly different. The highest β-carotene yield was achieved in transformants carrying the plasmid, with as the first gene in the polycistronic construct regardless of the location of , . In addition, we found that β-carotene production was coupled with the growth in engineered strain with the highest β-carotene content during the shake flask fermentation and fed-batch fermentation. A novel microbial heterologous carotenoid production system was established by optimizing the order of genes linked by P2A peptide sequences in a polycistronic expression construct. The observation of the importance of the order in a polycistronic construct may be used to increase yields in other P2A peptide-containing expression systems.
酵母是生产多种化合物和燃料的理想宿主。目前，利用2A病毒肽可以实现多个基因的同时表达，但对于评估由2A肽连接的不同基因顺序差异的详细表征却很少。在本研究中，我们研究了猪捷申病毒-1 2A（P2A）肽连接的基因顺序对酿酒酵母代谢途径的影响。将一个涉及来自欧文氏菌的九个密码子优化的类胡萝卜素生成基因crtE、crtB和crtI的多顺反子表达的异源类胡萝卜素生物合成系统引入酿酒酵母。多顺反子片段中的基因顺序被交换；工程酵母产生的β-胡萝卜素显著不同。携带该质粒的转化体中β-胡萝卜素产量最高，无论crtB和crtI的位置如何，crtE作为多顺反子构建体中的第一个基因。此外，我们发现在摇瓶发酵和补料分批发酵过程中，β-胡萝卜素的产生与β-胡萝卜素含量最高的工程菌株的生长相关。通过优化多顺反子表达构建体中由P2A肽序列连接的基因顺序，建立了一种新型的微生物异源类胡萝卜素生产系统。多顺反子构建体中顺序重要性的观察结果可用于提高其他含P2A肽的表达系统的产量。
