模式硅藻三角褐指藻支链氨基酸的代谢路径研究

Diatoms are the major primary producer and believed to be responsible for 40% of the total carbon fixation in the oceans. More importantly, under certain circumstances, some diatoms can store carbon and energy in the form of lipids (predominantly triacylglycerols [TAGs]), suggesting the possibility of cultivating diatoms for biodiesel production. Phaeodactylum tricornutum is one of the most widely utilized model systems for studies on ocean diatoms. Recently we have found that branched-chain amino acids (BCAAs) degradation contributes to TAGs accumulation in P. tricornutum, but valine (Val) and isoleucine (Ile) degradation pathways are not clear. Based on the genomic data of sequenced organisms, we find that most of the photosynthetic organisms have no methylmalonyl-CoA epimerase (MCEE) encoding gene, besides green algae and higher plants don’t have methylmalonyl-CoA mutase (MCM) encoding gene. Therefore, it is impossible for propionyl-CoA, degradation product of Ile and Val, to use propionyl-CoA carboxylase to produce succinyl-CoA and enter the TCA cycle. It was indicated that catabolism of Val takes place first through conversion to leucine (Leu) in Arabidopsis thaliana and P. tricornutum. Photosynthetic organisms can synthesize BCAAs de novo and their degradation may also be related with each other, which is different from the pathway in metazoan. HIBCH, the 3-hydroxyisobutyryl-CoA hydrolase encoding gene involved in the Ile and Val degradation, will be knockouted or overexpressed in P. tricornutum, to interpret its function. According to the results of the gene expression profile, enzyme activity and metabolite analysis in the mutants, the pathway of BCAAs metabolism in diatom will be proposed with an attempt to provide candidate genes for improving oil yield in this diatom.

硅藻占海洋固碳量的40%，同化产物主要是油，被认为是优质的生物柴油原料。三角褐指藻是硅藻研究的模式种，以其为对象，本实验室发现支链氨基酸（BCAA）降解对硅藻油脂积累有重要贡献，但具体路径尤其是缬氨酸（Val）和异亮氨酸（Ile）降解路径尚不清楚。基于已测序生物的基因组数据，我们发现绝大多数光合生物无甲基丙二酰CoA消旋酶基因，绿藻和高等植物还无甲基丙二酰CoA变位酶基因，使得Ile和Val降解产生的丙酰辅酶A不能经由琥珀酰辅酶A进入三羧酸循环。对拟南芥及三角褐指藻的研究表明Val降解主要经由亮氨酸进行。与后生动物各自独立的路径不同，光合生物自身能合成BCAA，其降解也可能相互联系。本研究拟通过对涉及在Ile和Val降解路径的关键基因3-羟基异丁酰CoA水解酶的功能研究，根据基因表达谱、酶活及代谢组分析结果，揭示硅藻乃至光合生物BCAA代谢路径，应用上为操控BCAA降解提高油含量提供依据。

硅藻占海洋固碳量的40%，同化产物主要是油，被认为是优质的生物柴油原料。三角褐指藻是硅藻研究的模式种，以其为对象，本实验室发现支链氨基酸（BCAA）降解对硅藻油脂积累有重要贡献，但具体路径尤其是缬氨酸（Val）和异亮氨酸（Ile）降解路径尚不清楚。本研究通过对涉及在Ile和Val降解路径的关键基因3-羟基异丁酰CoA水解酶（HIBCH）进行敲除或过量表达并进行详细的表型分析，发现HIBCH过量表达对外源BCAAs的利用和外源BCAAs为唯一氮源培养条件下的生物量和细胞中性脂积累无影响，HIBCH基因敲除显著影响了藻细胞对外源Ile和Val的利用速率，三种BCAAs为氮源培养时HIBCH基因敲除藻株的生物量都受到明显抑制，细胞内中性脂的积累都显著增加。基因表达和代谢组分析发现，HIBCH基因敲除不仅仅阻碍了Ile的降解，还影响了其他两种BCAAs的代谢，并且对细胞内多种物质的代谢都有影响，改变了细胞内的碳氮分配，使更多碳骨架流向了中性脂（主要是TAGs）的合成。硝酸盐和蛋白胨组合氮源的使用对于硅藻在生物柴油生产方面有实践意义。