Várnai Anikó, Tang Campbell, Bengtsson Oskar, Atterton Andrew, Mathiesen Geir, Eijsink Vincent G H
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO Box 5003Chr, Magnus Falsens vei 1, N-1432 Ås, Norway.
Microb Cell Fact. 2014 Apr 18;13(1):57. doi: 10.1186/1475-2859-13-57.
Plant-derived biomass is a potential alternative to fossil feedstocks for a greener economy. Enzymatic saccharification of biomass has been studied extensively and endoglucanases have been found to be a prerequisite for quick initial liquefaction of biomass under industrial conditions. Pichia pastoris, widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. The recently marketed PichiaPink™ expression system allows for rapid clone selection, and employs the methanol inducible AOX1 promoter to ensure high protein expression levels. However, methanol is toxic and poses a fire hazard, issues which become more significant at an industrial scale. It is possible to eliminate these risks and still maintain high productivity by switching to the constitutive GAP promoter.
In the present study, a plasmid carrying the constitutive GAP promoter was created for PichiaPink™. We then studied expression of two endoglucanases, AfCel12A from Aspergillus fumigatus and TaCel5A from Thermoascus aurantiacus, regulated by either the AOX1 promoter or the GAP promoter. Initial experiments in tubes and small bioreactors showed that the levels of AfCel12A obtained with the constitutive promoter were similar or higher, compared to the AOX1 promoter, whereas the levels of TaCel5A were somewhat lower. After optimization of cultivation conditions using a 15-l bioreactor, the recombinant P. pastoris strains utilizing the GAP promoter produced ca. 3-5 g/l of total secreted protein, with CMCase activity equivalent to 1200 nkat/ml AfCel12A and 170 nkat/ml TaCel5A.
We present a strategy for constitutive recombinant protein expression in the novel PichiaPink™ system. Both AfCel12A and TaCel5A were successfully expressed constitutively in P. pastoris under the GAP promoter. Reasonable protein levels were reached after optimizing cultivation conditions.
植物源生物质是实现更绿色经济的化石原料的潜在替代品。生物质的酶促糖化已得到广泛研究，并且发现内切葡聚糖酶是在工业条件下生物质快速初始液化的先决条件。广泛用于异源蛋白表达的毕赤酵母可用于生产真菌内切葡聚糖酶。最近上市的毕赤酵母粉色表达系统允许快速克隆筛选，并采用甲醇诱导型AOX1启动子以确保高蛋白表达水平。然而，甲醇有毒且有火灾隐患，这些问题在工业规模上变得更加突出。通过切换到组成型GAP启动子，可以消除这些风险并仍保持高生产力。
在本研究中，为毕赤酵母粉色表达系统构建了携带组成型GAP启动子的质粒。然后，我们研究了由AOX1启动子或GAP启动子调控的两种内切葡聚糖酶，烟曲霉的AfCel12A和橙色嗜热子囊菌的TaCel5A的表达。在试管和小型生物反应器中的初步实验表明，与AOX1启动子相比，组成型启动子获得的AfCel12A水平相似或更高，而TaCel5A的水平略低。使用15升生物反应器优化培养条件后，利用GAP启动子的重组毕赤酵母菌株产生约3-5克/升的总分泌蛋白，其CMCase活性相当于1200纳卡特/毫升AfCel12A和170纳卡特/毫升TaCel5A。
我们提出了一种在新型毕赤酵母粉色表达系统中组成型重组蛋白表达的策略。AfCel12A和TaCel5A均在GAP启动子下在毕赤酵母中成功组成型表达。优化培养条件后达到了合理的蛋白水平。
