Docosahexaenoic acid (DHA) is an important omega-3 long-chain polyunsaturated fatty acid that has a variety of physiological functions for infant development and human health. Although metabolic engineering was previously demonstrated to be a highly efficient way to rapidly increase lipid production, metabolic engineering has seldom been previously used to increase DHA accumulation in Schizochytrium spp. Here, a sensitive β-galactosidase reporter system was established to screen for strong promoters in Schizochytrium sp. Four constitutive promoters (EF-1αp, TEF-1p, ccg1p, and ubiquitinp) and one methanol-induced AOX1 promoter were characterized by the reporter system with the promoter activity ccg1p> TEF-1p > AOX1p (induced) > EF-1αp > ubiquitinp. With the strong constitutive promoter ccg1p, Schizochytrium ATP-citrate lyase (ACL) and acetyl-CoA carboxylase (ACC) were overexpressed in Schizochytrium sp. ATCC 20888. The cells were cultivated at 28 °C and 250 rpm for 120 h with glucose as the carbon source. Shake-flask fermentation results showed that the overexpression strains exhibited growth curves and biomass similar to those of the wild-type strain. The lipid contents of the wild-type strain and of the OACL, OACC, and OACL-ACC strains were 53.8, 68.8, 69.8, and 73.0%, respectively, and the lipid yields of the overexpression strains were increased by 21.9, 30.5, and 38.3%, respectively. DHA yields of the wild-type strain and of the corresponding overexpression strains were 4.3, 5.3, 6.1, and 6.4 g/L, i.e., DHA yields of the overexpression strains were increased by 23.3, 41.9, and 48.8%, respectively. Acetyl-CoA and malonyl-CoA are precursors for fatty acid synthesis. ACL catalyzes the conversion of citrate in the cytoplasm into acetyl-CoA, and ACC catalyzes the synthesis of malonyl-CoA from acetyl-CoA. The results demonstrate that overexpression of ACL and ACC enhances lipid accumulation and DHA production in Schizochytrium sp.

中文翻译：

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通过在裂殖壶菌属中过表达 ATP-柠檬酸裂解酶和乙酰辅酶 A 羧化酶来增强二十二碳六烯酸的产生。

二十二碳六烯酸 (DHA) 是一种重要的 omega-3 长链多不饱和脂肪酸，对婴儿发育和人类健康具有多种生理功能。尽管代谢工程以前被证明是一种快速增加脂质生产的高效方法，但代谢工程以前很少用于增加裂殖壶菌属中 DHA 的积累。在这里，建立了一个敏感的β-半乳糖苷酶报告系统来筛选裂殖壶菌属中的强启动子。四个组成型启动子（EF-1αp、TEF-1p、ccg1p 和泛素）和一个甲醇诱导的 AOX1 启动子由报告系统表征，启动子活性为 ccg1p > TEF-1p > AOX1p（诱导）> EF-1αp > ubiquitinp . 使用强组成型启动子 ccg1p，裂殖壶菌 ATP-柠檬酸裂解酶 (ACL) 和乙酰辅酶 A 羧化酶 (ACC) 在裂殖壶菌属中过表达。ATCC 20888。细胞在 28 °C 和 250 rpm 下以葡萄糖为碳源培养 120 小时。摇瓶发酵结果表明，过表达菌株的生长曲线和生物量与野生型菌株相似。野生型菌株和OACL、OACC和OACL-ACC菌株的脂质含量分别为53.8、68.8、69.8和73.0%，过表达菌株的脂质产量分别增加了21.9、30.5和30.0%。分别为 38.3%。野生型菌株和相应过表达菌株的DHA产量分别为4.3、5.3、6.1和6.4g/L，即过表达菌株的DHA产量分别提高了23.3、41.9和48.8%。乙酰辅酶A和丙二酰辅酶A是脂肪酸合成的前体。ACL催化细胞质中的柠檬酸盐转化为乙酰辅酶A，ACC催化乙酰辅酶A合成丙二酰辅酶A。结果表明，ACL 和 ACC 的过表达增强了裂殖壶菌属中脂质的积累和 DHA 的产生。