云南单针藻产油关键基因与产油影响因素的相关性研究

By relating molecular mechanism, the environmental conditions can regulate and control the expressions and activities of enzymes of the lipid biosynthesis pathway to cause differences in lipid content and fatty acids profiles from microalgae. ACCase catalyzes a key metabolic step in the synthesis of fatty acid in microalgae. In some microalgae, down-regulating the PEPCase encoding gene can increase lipid yield. In order to explore dynamic mechanisms of lipid biosynthesis in oleaginous Monoraphidium from plateau fresh-water lakes in Yunnan Province, based on the previous study results of 4 strains Monoraphidium, by means of optimizations of culture conditions, this project will aim at studying dynamic mechanisms of microalgae lipid accumulation and composition caused by ACCase and PEPCase, revealing intrinsical relations between lipid accumulation and ACCase and PEPCase due to changes of culture conditions and clarifying reasons for enhancement of lipid biosynthesis flux in cells metabolism of microalgae. Moreover, in this project, differences in genes and amino acid sequences, homology and phylogenetic relationships of ACCase and PEPCase will be studied comparatively as theoretical basis for genetic regulation and construction of genetic engineering rich-lipid microalgae.

环境因素通过某些分子机理，实现对微藻脂类代谢相关酶的表达及活性的调控，从而影响油脂的含量和组成。ACCase 是脂肪酸生物合成途径的关键限速酶；在一些微藻中，下调PEPCase基因，有利于脂质合成量的提高。为探索云南高原湖泊中产油单针藻的动态产油机制，项目组拟在已有4株产油单针藻的研究基础上，通过对培养条件的改变与优化，研究ACCase 和PEPCase对油脂积累和油脂组成影响的动态机制，揭示培养条件改变油脂积累与ACCase 和PEPCase可能的内在联系，阐明微藻细胞代谢中油脂合成流量提高的原因；借助对4株单针藻ACCase 和PEPCase基因的克隆和分析，比较研究这些ACCase 和PEPCase基因、ACCase 和PEPCase氨基酸序列的差异性、同源性及进化关系，为产油微藻的遗传调控和构建高油脂工程藻奠定理论基础。

探寻、研究新的能源微藻一直是近些年来微藻生物质能研究的主要方向，而富油微藻的筛选及工程菌株的构建是将微藻生物柴油推向产业化应用的重要保证，利用基因工程技术获得微藻油脂大量积累已成为微藻生物技术应用的重要组成部分。. 为探究云南高原湖泊中产油单针藻的动态产油机制，奠定产油微藻的遗传调控和构建高油脂工程藻的理论基础，本研究在从云南高原湖泊中筛选到4株产油单针藻的研究基础上，通过改变其外部物理或化学环境条件，初探ACCase与PEPCase酶活动态变化；根据上述单因素的研究结果，采用响应面分析法优化微藻产油脂培养条件，结果表明NaNO3浓度为8.0 mM、柠檬酸铁胺浓度为0.07 mM、MgSO4•7H2O 浓度为0.13 mM，获得油脂产率值为20.39±2.4 mg L-1d-1 (n=3)，与预测值相对误差为14.86 %，相比正交实验最优水平下的油脂产率理论值（14.304 mg L-1d-1），产量提高了1.42倍。对优化后的ACCase 和PEPCase的进行分离纯化，并分析理化性质，结果表明50μM的Mg2+和Fe3+能够显著上调ACCase的活性而下调PEPCase的活性；此外，借助对4株单针藻ACCase 和PEPCase基因的克隆和分析，比较研究了ACCase 和PEPCase基因、ACCase 和PEPCase氨基酸序列的差异性、同源性及进化关系，为产油微藻的遗传调控和构建高油脂工程藻奠定理论基础。