TU Wien, Institute of Chemical, Environmental and Bioscience Engineering, Gumpendorfer Straße 1a, Vienna, 1060, Austria.
Yeast. 2020 Feb;37(2):217-226. doi: 10.1002/yea.3441. Epub 2020 Jan 6.
Cytochrome P450s comprise one of the largest protein superfamilies. They occur in every kingdom of life and catalyse a variety of essential reactions. Their production is of utmost interest regarding biotransformation and structure-function elucidation. However, they have proven hard to express due to their membrane anchor, their complex co-factor requirements and their need for a redox-partner. In our study, we investigated and compared different yeast strains for the production of the plant cytochrome P450 chalcone 3-hydroxylase. To our knowledge, this is the first study evaluating different yeasts for the expression of this abundant and highly significant protein superfamily. Saccharomyces cerevisiae and three different strains of Pichia pastoris expressing chalcone 3-hydroxylase were cultivated in controlled bioreactor runs and evaluated regarding physiological parameters and expression levels of the cytochrome P450. Production differed significantly between the different strains and was found highest in the investigated P. pastoris MutS strain KM71H where 8 mg P450 per gram dry cell weight were detected. We believe that this host could be suitable for the expression of many eukaryotic, especially plant-derived, cytochrome P450s as it combines high specific product yields together with straightforward cultivation techniques for achieving high biomass concentrations. Both factors greatly facilitate subsequent establishment of purification procedures for the cytochrome P450 and make the yeast strain an ideal platform for biotransformation as well.
细胞色素 P450 是最大的蛋白质超家族之一。它们存在于生命的每个领域，并催化各种必需的反应。它们的产生对于生物转化和结构功能阐明至关重要。然而，由于它们的膜锚定、复杂的辅因子要求以及需要氧化还原伴侣，它们的表达一直很困难。在我们的研究中，我们研究并比较了不同酵母菌株用于生产植物细胞色素 P450 查尔酮 3-羟化酶。据我们所知，这是第一项评估不同酵母表达这种丰富且具有重要意义的蛋白质超家族的研究。我们在控制的生物反应器中培养酿酒酵母和表达查尔酮 3-羟化酶的三种不同毕赤酵母菌株，并根据生理参数和细胞色素 P450 的表达水平对其进行评估。不同菌株之间的产量存在显著差异，在所研究的毕赤酵母 MutS 菌株 KM71H 中发现最高，每克干细胞重量检测到 8 毫克 P450。我们认为这种宿主可能适合表达许多真核生物，特别是植物来源的细胞色素 P450，因为它结合了高特异性产物产率和简单的培养技术，可实现高生物量浓度。这两个因素极大地促进了随后对细胞色素 P450 的纯化程序的建立，并使酵母菌株成为生物转化的理想平台。
