新型高产β-胡萝卜素巴斯德毕赤酵母表达宿主菌的构建

Construction of a New Pichia pastoris Engineering Strain for High-level β-carotene Production

分泌型巴斯德毕赤酵母(Pichia pastoris)作为高效表达宿主菌已被广泛应用,通过高密度培养工艺用于多种酶制剂(如植酸酶、甘露聚糖酶等)的规模生产。然而,在生产过程中产生的大量菌体利用率低,多数被废弃掩埋,或只能作为廉价的菌体蛋白,造成大量的资源浪费。为提高酵母发酵副产物的利用率,向毕赤酵母宿主菌GS115中导入来源于红发夫酵母(Xanthophyllomyces dendrorhous)β-胡萝卜素合成途径中的关键基因(idi、crt E、crt YB和crt I),获得了胞内高产β-胡萝卜素的工程菌株P.pastoris GS115-CARO,实现工业生产中的菌体蛋白的高附加值利用。同时以来源于嗜热篮状菌(Talaromyces leycettanus JCM12802)的甘露聚糖酶基因Man5T作为参照,验证了宿主菌P.pastoris GS115和P.pastoris GS115-CARO在表达外源蛋白的差异。结果表明:Man5T基因的导入并没有影响,两株宿主菌的甘露聚糖酶表达量(分别为280 U/m L和286 U/m L,P>0.05),但宿主菌P.pastoris GS115-CARO胞内β-胡萝卜素产量高达到31.27 mg/g(菌体干重dry cell weight,dcw)。构建的工程菌株不仅可以实现外源酶制剂的高效表达,有效地提升酵母菌体资源的利用价值,同时也在一定程度上了解决了发酵菌体对环境的污染问题。

As a high-level efficient expressive host bacteria,methylotrophic yeast Pichia pastoris is widely used to produce various feed enzymes like phytase and mannanase by high-density culture. However,the large amount of yeast cells produced during the fermentation process are discarded or just used as microbial protein with low values.To improve the utilization efficiency of these by-products,this study intended to introduce the key gene（ idi,crt E,crt YB and crt I） involved in the β-carotene biosynthesis of Xanthophyllomyces dendrorhous into P. pastoris GS115 to gain an engineering strain P. pastoris GS115-CARO with high β-carotene yield and to realize utilization of mycoprotein with high additional value in industrial production. An extracellular mannanase gene Man5 T from Talaromyces leycettanus JCM12802 was as a reference to prove the differences of host bacteria P. pastoris GS115 and P. pastoris GS115-CARO in expression of exogenous protein. The results showed that the mannanase yields had no significant difference（ 280 U/m L vs. 286 U/m L） in both strains,but the yield of intracellular β-carotene was up to 31.27 mg/g（ dcw） in P. pastoris GS115-CARO. The constructed strain herein could not only achieve high-level expression of exogenous enzymes and efficiently upgrade the utilization of yeast resources,but also alleviate to certain extent the environment pollution by yeast by-products.