Zhu Jie, Lin Jyun-Liang, Palomec Leidy, Wheeldon Ian
Department of Biochemistry, University of California, Riverside, USA, 92521.
Department of Chemical and Environmental Engineering, University of California, Riverside, USA, 92521.
Microb Cell Fact. 2015 Mar 17;14:35. doi: 10.1186/s12934-015-0221-9.
A key pathway for ester biosynthesis in yeast is the condensation of an alcohol with acetyl-CoA by alcohol-O-acetyltransferase (AATase). This pathway is also prevalent in fruit, producing short and medium chain volatile esters during ripening. In this work, a series of six AATases from Saccharomyces and non-Saccharomyces yeasts as well as tomato fruit were evaluated with respect to their activity, intracellular localization, and expression in Saccharomyces cerevisiae and Escherichia coli cell hosts. The series of AATases includes Atf1 and Atf2 from S. cerevisiae, as well as AATases from S. pastorianus, Kluyveromyces lactis, Pichia anomala, and Solanum lycopersicum (tomato).
When expressed in S. cerevisiae, Atf1, Atf2, and an AATase from S. pastorianus localized to lipid droplets, while AATases from non-Saccharomyces yeasts and tomato fruit did not localize to intracellular membranes and were localized to the cytoplasm. All AATases studied here formed intracellular aggregates when expressed in E. coli, and western blot analysis revealed that expression levels in E. coli were upwards of 100-fold higher than in S. cerevisiae. Fermentation and whole cell lysate activity assays of the two most active AATases, Atf1 from S. cerevisiae and an AATase from tomato fruit, demonstrated that the aggregates were enzymatically active, but with highly reduced specific activity in comparison to activity in S. cerevisiae. Activity was partially recovered at lower expression levels, coinciding with smaller intracellular aggregates. In vivo and in vitro activity assays from heterologously expressed Atf1 from S. cerevisiae, which localizes to lipid droplets under homologous expression, demonstrates that its activity is not membrane dependent.
The results of these studies provide important information on the biochemistry of AATases under homologous and heterologous expression with two common microbial hosts for biochemical processes, S. cerevisiae and E. coli. All studied AATases formed aggregates with low enzymatic activity when expressed in E. coli and any membrane localization observed in S. cerevisiae was lost in E. coli. In addition, AATases that were found to localize to lipid droplet membranes in S. cerevisiae were found to not be membrane dependent with respect to activity.
酵母中酯生物合成的关键途径是醇通过醇-O-乙酰基转移酶（AATase）与乙酰辅酶A缩合。该途径在果实中也很普遍，在果实成熟过程中产生短链和中链挥发性酯。在本研究中，对来自酿酒酵母和非酿酒酵母以及番茄果实的一系列六种AATase进行了活性、细胞内定位以及在酿酒酵母和大肠杆菌细胞宿主中的表达评估。该系列AATase包括酿酒酵母的Atf1和Atf2，以及巴氏酵母、乳酸克鲁维酵母、异常毕赤酵母和番茄（茄属番茄）的AATase。
当在酿酒酵母中表达时，Atf1、Atf2和来自巴氏酵母的一种AATase定位于脂滴，而非酿酒酵母和番茄果实的AATase不定位于细胞内膜，而是定位于细胞质。这里研究的所有AATase在大肠杆菌中表达时都会形成细胞内聚集体，蛋白质印迹分析表明其在大肠杆菌中的表达水平比在酿酒酵母中高100倍以上。对两种活性最高的AATase，即酿酒酵母的Atf1和番茄果实的一种AATase进行发酵和全细胞裂解物活性测定，结果表明这些聚集体具有酶活性，但与在酿酒酵母中的活性相比，比活性大幅降低。在较低表达水平下活性部分恢复，这与较小的细胞内聚集体一致。对在同源表达下定位于脂滴的酿酒酵母Atf1进行异源表达的体内和体外活性测定，结果表明其活性不依赖于膜。
这些研究结果提供了关于AATase在与生物化学过程的两种常见微生物宿主酿酒酵母和大肠杆菌进行同源和异源表达时的生物化学的重要信息。所有研究的AATase在大肠杆菌中表达时都会形成酶活性较低的聚集体，并且在大肠杆菌中失去了在酿酒酵母中观察到的任何膜定位。此外，发现在酿酒酵母中定位于脂滴膜的AATase在活性方面不依赖于膜。
