Huang Peng-Wei, Xu Ying-Shuang, Sun Xiao-Man, Shi Tian-Qiong, Gu Yang, Ye Chao, Huang He
School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, China.
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, China.
Front Nutr. 2021 Dec 14;8:795651. doi: 10.3389/fnut.2021.795651. eCollection 2021.
sp. HX-308 is a marine microalga with fast growth and high lipid content, which has potential as microbial cell factories for lipid compound biosynthesis. It is significant to develop efficient genetic editing tool and discover molecular target in sp. HX-308 for lipid compound biosynthesis. In this study, we developed an efficient gene editing tool in HX-308 which was mediated by AGL-1. Results showed that the random integration efficiency reached 100%, and the homologous recombination efficiency reached about 30%. Furthermore, the metabolic pathway of lipid and terpenoid biosynthesis were engineered. Firstly, the acetyl-CoA -acetyltransferase was overexpressed in HX-308 with a strong constitutive promoter. With the overexpression of acetyl-CoA c-acetyltransferase, more acetyl-CoA was used to synthesize terpenoids, and the production of squalene, β-carotene and astaxanthin was increased 5.4, 1.8, and 2.4 times, respectively. Interestingly, the production of saturated fatty acids and polyunsaturated fatty acids also changed. Moreover, three Acyl-CoA oxidase genes which catalyze the first step of β-oxidation were knocked out using homologous recombination. Results showed that the production of lipids increased in the three knock-out strains. Our results demonstrated that the -mediated transformation method will be of great use for the study of function genes, as well as developing sp. as a strong cell factory for producing high value products.
HX-308藻株是一种生长迅速且脂质含量高的海洋微藻，具有作为脂质化合物生物合成的微生物细胞工厂的潜力。开发高效的基因编辑工具并发现HX-308藻株中脂质化合物生物合成的分子靶点具有重要意义。在本研究中，我们在HX-308中开发了一种由AGL-1介导的高效基因编辑工具。结果表明，随机整合效率达到100%，同源重组效率达到约30%。此外，对脂质和萜类生物合成的代谢途径进行了工程改造。首先，用强组成型启动子在HX-308中过表达乙酰辅酶A-乙酰转移酶。随着乙酰辅酶A-乙酰转移酶的过表达，更多的乙酰辅酶A用于合成萜类化合物，角鲨烯、β-胡萝卜素和虾青素的产量分别提高了5.4倍、1.8倍和2.4倍。有趣的是，饱和脂肪酸和多不饱和脂肪酸的产量也发生了变化。此外，利用同源重组敲除了催化β-氧化第一步的三个酰基辅酶A氧化酶基因。结果表明，在这三个敲除菌株中脂质产量增加。我们的结果表明，这种介导的转化方法将对功能基因的研究以及将HX-308藻株开发成为生产高价值产品的强大细胞工厂具有很大的用途。
