异养黄丝藻利用低值外源脂肪酸合成棕榈油酸的关键调控机制研究

Palmitoleic acid (C16:1 Δ 9) is one of omega-7 monounsaturated fatty acids, which is taken as a novel adipose-derived lipid hormones. It is widely used in aquatic feed, biofuels, chemical products, healthcare products and pharmaceuticals. Filamentous microalgae Tribonema is a newfound and significant potential source of enriched palmitoleic acid oil. Besides photo-autotrophy, Tribonema could use organic carbon sources such as glucose to fast heterotrophy with high biomass density. However, the lipid content by heterotrophy is much lower than autotrophy. Our previous researches showed that the exogenous fatty acids supplied in Tribonema heterotrophy could greatly boost the lipids accumulation, which also providing an efficient pathway to transform low valued saturated fatty acids to high valued palmitoleic acid. But how does Tribonema use exogenous fatty acids and what the transformation pathway and its regulation mechanism are unclear. . This proposal will focus on accumulation enhancement of enriched palmitoleic acid oil by heterotrophic fermentation with exogenous fatty acids. Firstly, the metabolic flux of exogenous fatty acids in heterotrophic Tribonema will be elucidated by using GC/MS quantitative analysis techniques of isotope. Then, on the transcriptomic level, the key genes and responses to environmental factors associated with biosynthesis of palmitoleic acid will be screened to understand its regulation mechanism. Finally, with the help of regulation mechanism, efficient regulation strategy by fermentation environmental factors will be constructed and optimized to enhance the oil and palmitoleic acid accumulation supplying of exogenous fatty acids in Tribonema heterotrophic fermentation. Results will help the construction of palmitoleic acid production technology through low valued exogenous fatty acids by Tribonema fermentation, and provide referable information of such similar microbe oil process.

棕榈油酸是一种ω-7单不饱和脂肪酸，被称为一种新型非蛋白类荷尔蒙，在水产饵料、能源化工和医药健康领域有重要应用价值。黄丝藻是富棕榈油酸的脂质新资源，除了光合自养，其可利用葡萄糖进行快速高密度异养发酵，但在异养下细胞油脂含量远低于自养。前期研究发现，黄丝藻异养时添加外源廉价脂肪酸可大幅度提高其油脂含量。但对黄丝藻如何利用外源脂肪酸，其油脂转化合成途径及调控因子尚不清楚。.项目针对异养黄丝藻，借助GC/MS同位素定量分析技术从脂质代谢组层面探明黄丝藻利用外源脂肪酸合成自身油脂的代谢流向，从转录组水平分析黄丝藻利用外源脂肪酸合成棕榈油酸的关键基因及对环境因素的响应，从而揭示其分子调控机制；基于该调控机制建立环境调控方法，实现黄丝藻对外源脂肪酸向棕榈油酸的定向高效转化。研究成果将为建立外源脂肪酸强化黄丝藻脂质/棕榈油酸合成的工业过程奠定理论技术基础， 也对其他微生物油脂的生物合成与强化有借鉴作用。

棕榈油酸是ω-7单不饱和脂肪酸的一种，其在医药、健康、水产饵料和能源化工领域有重要应用价值。黄丝藻是新近发现的富棕榈油酸的脂质新资源，其可利用葡萄糖进行快速高密度异养发酵，但在异养下细胞油脂含量远低于自养。前期研究发现，黄丝藻可利用外源添加的廉价脂肪酸大幅度提高自身油脂含量，但对其外源脂肪酸利用机制以及相关油脂转化合成途径及调控因子尚缺乏研究。本研究针对异养的黄丝藻，一方面从脂质代谢组层面探明了黄丝藻利用外源脂肪酸合成自身油脂的代谢流向，一方面从转录组水平分析了黄丝藻利用外源脂肪酸合成棕榈油酸的关键基因及对环境因素的响应，从而揭示了其分子调控机制。研究结果表明，在优化条件下，14.6%的外源肉豆蔻酸经脱饱和作用后形成了肉豆蔻油酸，11.2%经碳链延长并脱饱和后形成了棕榈油酸，仍有近70%的外源肉豆蔻酸在黄丝藻细胞内没有发生转化，依旧以肉豆蔻酸的形式存在；从外源肉豆蔻酸的脂质流向来看，68.0%用于了甘油三酯的合成，19.5%继续以游离脂肪酸的形式存在，只有3.4%和0.9%分别被用于糖脂和磷脂的形成。此外，研究还发现，黄丝藻β烯酯酰-ACP还原酶和溶血磷脂酸酰基转移酶表达水平的高低或是影响外源脂肪酸在胞内向棕榈油酸等高效转化的关键限速节点。研究成果将为建立通过外源脂肪酸添加法强化黄丝藻脂质/棕榈油酸的生物合成的工业化应用过程奠定理论依据和技术基础，同时也对其他微生物油脂的研究和生产起到借鉴作用。