Expression of endoglucanases in Pichia pastoris under control of the GAP promoter.
Expression of endoglucanases in Pichia pastoris under control of the GAP promoter.
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DOI：
10.1186/1475-2859-13-57
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发表时间：
2014-04-18
影响因子：
6.4
通讯作者：
Eijsink VG
中科院分区：
文献类型：
--
作者：
Várnai A;Tang C;Bengtsson O;Atterton A;Mathiesen G;Eijsink VG
植物衍生生物质是化石原料的潜在替代品，可促进绿色经济。生物质的酶促糖化已经被广泛研究，并且已经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。巴斯德毕赤酵母广泛用于外源蛋白的表达，可用于真菌内切葡聚糖酶的生产。最近上市的PichiaPink ™表达系统允许快速克隆选择，并采用甲醇诱导型AOX1启动子以确保高蛋白表达水平。然而，甲醇是有毒的，并造成火灾危险，这些问题在工业规模上变得更加重要。有可能消除这些风险，并通过切换到组成型GAP启动子仍然保持高生产率。在本研究中，为PichiaPink ™创建了携带组成型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的表达，来自烟曲霉的AfCel12A和来自金黄色嗜热菌的TaCel5A。在试管和小型生物反应器中的初始实验表明，与AOX1启动子相比，用组成型启动子获得的AfCel12A的水平相似或更高，而TaCel5A的水平略低。在15-l生物反应器中优化培养条件后，利用GAP启动子的重组巴斯德毕赤酵母菌株产生约1000mg/L的蛋白。3-5 g/l的总分泌蛋白，其中CMCase活性相当于1200 nkat/ml AfCel12A和170 nkat/ml TaCel5A。我们提出了在新型PichiaPink ™系统中组成型重组蛋白表达的策略。AfCel12A和TaCel5A在GAP启动子下在巴斯德毕赤酵母中成功地组成型表达。通过优化培养条件，达到了合理的蛋白水平。
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.
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