Mormeneo María, Andrés Isabel, Bofill Cristina, Díaz Pilar, Zueco Jesús
Unidad de Microbiología. Facultad de Farmacia, Burjassot, Valencia, Spain.
Appl Microbiol Biotechnol. 2008 Sep;80(3):437-45. doi: 10.1007/s00253-008-1549-4. Epub 2008 Jul 15.
Both the secretion and the cell surface display of Bacillus subtilis lipase A (Lip A) in Saccharomyces cerevisiae was investigated using different domains of the cell wall protein Pir4 as translational fusion partners. LipA gene minus its leader peptide was fused inframe in two places of PIR4 to achieve cell wall targeting, or substituting most of the PIR4 sequence, after the signal peptide and the Kex2 processed subunit I of Pir4 to achieve secretion to the growth medium. Expression of the recombinant fusion proteins was investigated in a standard and a glycosylation-deficient strain of S. cerevisiae, grown in selective or rich medium. Fusion proteins intended to be retained at the cell wall were secreted to the growth medium, most likely as result of the degradation of the Pir4 moiety containing the cell wall retention domain, giving low levels of lipase activity. However, the fusion intended for secretion was efficiently secreted in a percentage of close to 90% and remained stable even in rich medium at high cell density cultures, yielding values of over 400 IU of lipase activity per milliliter of cell supernatant. This is, to our knowledge, the first report of the efficient production, as a secreted protein, of lipase A of B. subtilis in baker's yeast.
利用细胞壁蛋白Pir4的不同结构域作为翻译融合伙伴，对枯草芽孢杆菌脂肪酶A（Lip A）在酿酒酵母中的分泌和细胞表面展示进行了研究。去除LipA基因的前导肽后，在PIR4的两个位置进行读码框融合以实现细胞壁靶向，或者在信号肽和经Kex2处理的Pir4亚基I之后替换大部分PIR4序列，以实现向生长培养基的分泌。在标准型和糖基化缺陷型酿酒酵母菌株中，于选择性培养基或丰富培养基中培养，研究重组融合蛋白的表达情况。旨在保留在细胞壁上的融合蛋白被分泌到生长培养基中，这很可能是由于含有细胞壁保留结构域的Pir4部分降解所致，导致脂肪酶活性水平较低。然而，用于分泌的融合蛋白以接近90%的比例有效分泌，甚至在高细胞密度培养的丰富培养基中也保持稳定，每毫升细胞上清液产生超过400 IU的脂肪酶活性。据我们所知，这是首次报道在面包酵母中高效生产枯草芽孢杆菌脂肪酶A作为分泌蛋白的情况。
