三角褐指藻丙二酰单酰辅酶A:酰基载体蛋白转酰基酶的功能分析

Functional analysis of a malonyl-CoA:ACP transacylase gene from Phaeodactylum tricornutum

【背景】丙二酰单酰辅酶A:酰基载体蛋白转酰基酶(malonyl coenzyme A:acyl carrier protein transacylase,MCAT)是Ⅱ型脂肪酸合酶(fatty acid synthaseⅡ,FASⅡ)的重要亚基,与脂肪酸合成直接相关,然而关于微藻MCAT的信息却很少。【目的】验证三角褐指藻MCAT的功能。【方法】在模式硅藻三角褐指藻的全基因组序列中发现了一个可能的mcat基因序列,对其进行生物信息学分析,构建原核表达载体,并转入MCAT缺陷型大肠杆菌L48菌株中,最后利用GC-MS分析突变株脂肪酸的成分和含量。【结果】三角褐指藻MCAT主要结构为α-螺旋和无规则卷曲,与圆柱脆杆藻的亲缘关系最为接近,为protits型MCAT;三角褐指藻MCAT的表达使MCAT缺陷型大肠杆菌L48菌株恢复了合成脂肪酸的功能;对L48回复突变株的脂肪酸组成进行分析,发现该酶对C14:0具有底物偏好性,从而促进中长链脂肪酸如C16:0和C17:1的合成,这一特点与protits型MCAT的特性基本相符。【结论】三角褐指藻MCAT能促进脂肪酸的合成,这为微藻脂肪酸合成及代谢调控研究提供了新的线索,有利于微藻脂质的研究和应用。

[Background]Malonyl-CoA:ACP transacylase(MCAT),an important subunit of typeⅡfatty acid synthase(FASⅡ),is associated with fatty acid synthesis.However,little information is available on microalgal MCAT.[Objective]To validate the function of the MCAT gene from Phaeodactylum tricornutum.[Methods]We found a possible mcat gene in the whole genome sequence of the model diatom P.tricornutum and cloned this gene.We performed bioinformatic analysis of this gene and introduced it into the MCAT-deficient E.coli L48 strain.Gas chromatography-mass spectrometry(GC-MS)was employed to analyze the composition and content of fatty acids in the mutant strains.[Results]The MCAT in P.tricornutum had the secondary structure mainly composed ofα-helix and random coil and the closest genetic relationship with that in Fragilaria cylindrus,being a protits-type MCAT.To verify the function of the gene,we introduced this gene into the MCAT-deficient E.coli L48strain and found that the strain recovered the function of fatty acid synthesis.Further,we analyzed the fatty acid composition of the reverting and found that the enzyme had a substrate preference to C14:0.The MCAT promoted the synthesis of medium-and long-chain fatty acids such as C16:0 and C17:1.This feature is largely consistent with the characteristics of protits-type MCATs.[Conclusion]There is a protits-type MCAT in P.tricornutum.This study provides new clues for the study of fatty acid synthesis and metabolism in microalgae,which is beneficial to the research and application of microalgal lipids.