毕赤酵母中表达透明颤菌血红蛋白提高重组脂肪酶的表达

Expression of Vitreoscilla hemoglobin improves recombinant lipase production in Pichia pastoris

摘要:

解脂耶氏酵母胞外脂肪酶Lip2 (YlLip2) 是一种具有广泛应用前景的工业酶。为了改善高密度发酵生产YlLip2过程中的溶氧限制，提高YlLip2的表达量，将YlLip2基因lip2和透明颤菌血红蛋白 (VHb) 基因vgb分别置于AOX1启动子和PsADH2启动子的调控之下，进行YlLip2和VHb在毕赤酵母中的共表达。PsADH2启动子来源于树干毕赤酵母Pichia stipitis，在低氧条件下能被激活。SDS-PAGE和CO-差式光谱分析表明，YlLip2和VHb在重组菌中成功实现了共表达。在氧限制性条件下，VHb表达的细胞 (VHb+，GS115/9Klip2-pZPVT) 与对照细胞 (VHb?，GS115/9Klip2) 相比，摇瓶和10 L发酵罐中YlLip2表达量分别提高了25％和83％。此外，在低氧条件下，VHb+细胞在10 L发酵罐中的生物量也比VHb?细胞高。文中也获得了一株表达了VHb的并携带有多个lip2基因拷贝的克隆子GS115/9Klip2-pZPVTlip2 49#，在低氧条件下，该克隆子在10 L发酵罐中的最高脂肪酶水解活力达33 900 U/mL。因此，在毕赤酵母中用PsADH2启动子表达VHb，同时增加lip2基因的拷贝数是提高YlLip2表达量的一种有效策略。

Abstract:

Yarrowia lipolytica lipase Lip2 (YlLip2) is an important industrial enzyme with many potential applications. To alleviate the dissolved oxygen (DO) limitation and improve YlLip2 production during high-cell density fermentation, the YlLip2 gene lip2 and Vitreoscilla hemoglobin (VHb) gene vgb were co-expressed in Pichia pastoris under the control of AOX1 and PsADH2 promoter, respectively. The PsADH2 promoter from Pichia stipitis could be activated under oxygen limitation. The SDS-PAGE and CO-difference spectrum analysis indicated that VHb and YlLip2 had successfully co-expressed in recombinant strains. Compared with the control cells (VHb?, GS115/9Klip2), the expression levels of YlLip2 in VHb-expressing cells (VHb+, GS115/9Klip2-pZPVT) under oxygen limitation were improved 25% in shake-flask culture and 83% in a 10 L fermentor. Moreover, the VHb+ cells displayed higher biomass than VHb? cells at lower DO levels in a 10 L fermentor. In this study, we also achieved a VHb-expressing clone harboring multicopy lip2 gene (GS115/9Klip2-pZPVTlip2 49#), which showed the maximum lipolytic activity of 33 900 U/mL in a 10 L fermentor under lower DO conditions. Therefore, it can be seen that expression of VHb with PsADH2 promoter in P. pastoris combined with increasing copies of lip2 gene is an effective strategy to improve YlLip2 production.