Berninger G, Schmidtchen R, Casel G, Knörr A, Rautenstrauss K, Kunau W H, Schweizer E
Lehrstuhl für Biochemie, Universität Erlangen-Nürnberg, Germany.
Eur J Biochem. 1993 Sep 1;216(2):607-13. doi: 10.1111/j.1432-1033.1993.tb18180.x.
Using a Yarrowia lipolytica genomic library, several overlapping clones of the peroxisomal 3-oxoacyl-CoA-thiolase gene, POT1, were isolated. The library was prepared in the bacterial expression vector lambda gt11, thus allowing an immunological screening of recombinant bacteriophages with specific antibodies raised against purified peroxisomal thiolase. The isolated POT1 clones hybridized to a 1.4 kb RNA species, which was induced approximately 30-fold when oleate was the carbon source. A 3634-bp segment of the cloned DNA was sequenced. This segment contained, on both strands, three major overlapping open-reading frames of 678, 1122 and 1242 bp. Northern-hybridization analysis showed that only the largest of these reading frames was transcribed. It encodes a protein of 414 amino acids and molecular mass 43.059 kDa. Its deduced amino acid sequence has 30-60% identity and 50-70% sequence similarity when compared to other known thiolases. According to both the amount (68-71%) and location of conserved amino acids, the encoded protein belongs to the peroxisomal rather than the mitochondrial or cytoplasmic class of thiolases. Compared to bacterial and yeast cytosolic thiolases, the POT1 gene product contains a N-terminal extension of 25 amino acids which clearly differs from typical mitochondrial import signals. One of the isolated clones contained, in addition to the POT1 coding sequence, 784 bp of the corresponding 5' flanking region. Nevertheless, it was efficiently expressed in Escherichia coli suggesting the correct recognition of this fungal promoter by the prokaryotic transcriptional and translational machinery. The Y. lipolytica genomic POT1 gene was disrupted by replacing 120 bp of its coding sequence with 2.7 kbp of DNA including the Y. lipolytica LEU2 gene. The resulting delta pot1::LEU2 cells were free of immunologically cross-reacting thiolase. Western-blot analysis showed that the product of the non-disrupted gene had a molecular mass of approximately 42 kDa. This corresponds well to the molecular mass of purified Y. lipolytica peroxisomal thiolase. Disruption of POT1 abolished the ability of Y. lipolytica cells to grow on solid media with oleate as a carbon source. This inability to grow in the presence of oleate suggests both the catabolic function of POT1 and the absence of additional catabolic thiolases in Y. lipolytica. However, the delta pot1::LEU2 cells were unaffected in their ability to elongate externally added tridecanoic acid to its higher-chain-length homologues. Hence, another, POT1-independent and biosynthetic 3-oxoacyl-CoA thiolase must be responsible for this reaction in Y. lipolytica.
利用解脂耶氏酵母基因组文库，分离出了过氧化物酶体3-氧代酰基辅酶A硫解酶基因POT1的几个重叠克隆。该文库构建于细菌表达载体λgt11中，因此可用针对纯化的过氧化物酶体硫解酶产生的特异性抗体对重组噬菌体进行免疫筛选。分离得到的POT1克隆与一个1.4 kb的RNA杂交，当以油酸作为碳源时，该RNA的表达量可被诱导增加约30倍。对克隆DNA的一个3634 bp片段进行了测序。该片段的两条链上均包含三个主要的重叠开放阅读框，长度分别为678、1122和1242 bp。Northern杂交分析表明，这些阅读框中只有最大的一个被转录。它编码一个414个氨基酸、分子量为43.059 kDa的蛋白质。与其他已知硫解酶相比，其推导的氨基酸序列具有30%-60%的同一性和50%-70%的序列相似性。根据保守氨基酸的数量（68%-71%）和位置，该编码蛋白属于过氧化物酶体硫解酶类，而非线粒体或细胞质硫解酶类。与细菌和酵母胞质硫解酶相比，POT1基因产物含有一个25个氨基酸的N端延伸，这明显不同于典型的线粒体导入信号。其中一个分离的克隆除了包含POT1编码序列外，还包含相应5'侧翼区域的784 bp。然而，它在大肠杆菌中能有效表达，这表明原核转录和翻译机制能正确识别该真菌启动子。通过用包含解脂耶氏酵母LEU2基因的2.7 kbp DNA替换其120 bp的编码序列，破坏了解脂耶氏酵母基因组中的POT1基因。产生的Δpot1::LEU2细胞没有免疫交叉反应的硫解酶。Western印迹分析表明，未被破坏基因的产物分子量约为42 kDa。这与纯化的解脂耶氏酵母过氧化物酶体硫解酶的分子量非常吻合。POT1基因的破坏消除了解脂耶氏酵母细胞在以油酸作为碳源的固体培养基上生长的能力。在油酸存在下无法生长表明POT1具有分解代谢功能，且解脂耶氏酵母中不存在其他分解代谢硫解酶。然而，Δpot1::LEU2细胞在将外部添加的十三烷酸延长为其更高链长同系物的能力方面不受影响。因此另一种不依赖POT1的生物合成3-氧代酰基辅酶A硫解酶必定在解脂耶氏酵母中负责此反应。
