异柠檬酸脱氢酶在高山被孢霉脂质合成中的作用和调控机制

Oleaginous fungus Mortierella alpina possesses high lipid-producing capacity and accumulates functional high value polyunsaturated fatty acids such as arachidonic acid (ARA) and eicosapntemacnioc acid (EPA). In oleaginous fungi, isocitrate dehydrogenase (IDH) usually exists in both mitochondria and cytoplasm, the mitochondrial IDH can regulate the substrate acetyl-CoA supply for lipid biosynthesis, and the cytosolic IDH can provide the reducing power NADPH for lipid biosynthesis. Bioinformatics analysis showed that IDH in M. alpina have different cellular locations and different coenzyme dependence. Transcriptome and proteome study indicated that IDH is closely associated with lipid accumulation, and metabolome analysis also illustrated that IDH affects the flow of carbon in lipid biosynthesis pathway. Yet, the mechanism of IDH regulating lipid accumulation is not clear due to the differences in localization and coenzyme dependence. In this proposed study, we will investigate the subcellular localization, molecular modification, biochemical characteristics, and pathway of regulating lipid biosynthesis of IDH in M. alpina, and elucidate the effects and regulatory mechanism of IDH on lipid accumulation. This study will not only enhance our understanding of the molecular mechanism of lipid accumulation in oleaginous fungi, but also promote the industrialized production of polyunsaturated fatty acids by microbial fermentation.

产油真菌高山被孢霉具有很强的脂质合成能力，能够合成花生四烯酸（ARA）、二十碳五烯酸（EPA）等高价值的功能性多不饱和脂肪酸。在产油真菌中，异柠檬酸脱氢酶（IDH）通常在线粒体内和胞质中均存在，线粒体内的IDH能够调控脂质合成底物乙酰辅酶A的供应，而胞质中的IDH能够为脂质合成提供还原力NADPH。高山被孢霉的生物信息学分析发现其存在不同位置和不同辅酶依赖的IDH，转录组和蛋白组结果显示IDH与脂质积累密切相关，代谢组分析也表明IDH影响了脂质合成碳的流向，但由于IDH在胞内定位和辅酶依赖存在差异，其对脂质合成的具体调控机制还不明确。本项目拟对高山被孢霉中不同IDH进行亚细胞定位、分子改造、生化特性研究、作用途径解析，阐释不同定位和辅酶依赖的IDH在脂质合成中的作用和调控机制。本研究将提高对产油真菌脂质积累分子机制的认识，也为利用微生物发酵法工业化生产多不饱和脂肪酸提供理论基础。

不同异柠檬酸脱氢酶（IDH）能够影响胞内碳源流向或者提供还原力NADPH，对脂质合成具有调控作用，本项目针对能够积累高价值多不饱和脂肪酸的真菌高山被孢霉，研究了该真菌中不同亚细胞定位和辅酶依赖的IDH在脂质合成中的作用，分析了关键IDH的酶学性质，并对IDH调节脂质积累途径中的柠檬酸转运蛋白进行了探究，提高了对产油真菌脂质积累调控机制的认识，为微生物发酵法生产功能性多不饱和脂肪酸提供了基础。主要研究结果如下：.（1）高山被孢霉中存在6个MaIDH同工酶，其中MaIDH1/2/3为NAD+依赖型MaIDH，定位于线粒体；MaIDH4/5/6为NADP+依赖型MaIDH，定位于细胞质；过表达3个NAD+-MaIDH或干扰NADP+-MaIDH4使高山被孢霉脂肪酸含量降低了3.7%~12.5%；过表达3个NADP+-MaIDH或干扰NAD+-MaIDH3使脂肪酸含量提高了3.4%~8.2%；其中NAD+-MaIDH3和NADP+-MaIDH4对高山被孢霉脂肪酸含量影响较为显著；过表达NAD+-MaIDH显著降低了胞内柠檬酸含量，而过表达NADP+-MaIDH提高了胞内NADPH的含量。.（2）构建了MaIDH3和MaIDH4在毕赤酵母系统的异源表达菌株，明确了MaIDH3和MaIDH4毕赤酵母重组菌的诱导条件，MaIDH3在2.5%的甲醇培养基中诱导60 h可获得最佳的酶活性和蛋白表达量，而MaIDH4是在2.5%的甲醇培养基中诱导36 h；确定了MaIDH4适合的表达宿主，相比于大肠杆菌表达系统，毕赤酵母表达系统可获得更高MaIDH4酶产量和纯酶比活力；酶学性质研究发现MaIDH4是一种专一性依赖于NADP+，受Mn2+激活，具有嗜热和嗜碱性质的酶。.（3）在高山被孢霉基因组中筛选出4个编码柠檬酸转运蛋白的基因，在3个编码柠檬酸-苹果酸类转运蛋白基因（MaCT1/MaCT2/MaTCT）中，过表达MaCT2使高山被孢霉脂肪酸含量最高提高了29.5%，其转录水平也随着脂质合成速度的下降而降低；编码编码柠檬酸-酮戊二酸转运蛋白的MaYHM过表达使高山被孢霉的脂肪酸含量最高提高了30.4%，代谢物水平和NAD+-IDH的酶活变化发现MaYHM的过表达可能促进碳源从GABA途径/谷氨酸脱氢酶途径进入TCA循环，使更多碳源流向脂质合成，从而提高脂质积累。