Wriessnegger Tamara, Moser Sandra, Emmerstorfer-Augustin Anita, Leitner Erich, Müller Monika, Kaluzna Iwona, Schürmann Martin, Mink Daniel, Pichler Harald
ACIB - Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria; Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, Petersgasse 14/2, 8010 Graz, Austria.
ACIB - Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.
Fungal Genet Biol. 2016 Apr;89:114-125. doi: 10.1016/j.fgb.2016.02.004. Epub 2016 Feb 18.
Cytochrome P450 enzymes (CYPs) play an essential role in the biosynthesis of various natural compounds by catalyzing regio- and stereospecific hydroxylation reactions. Thus, CYP activities are of great interest in the production of fine chemicals, pharmaceutical compounds or flavors and fragrances. Industrial applicability of CYPs has driven extensive research efforts aimed at improving the performance of these enzymes to generate robust biocatalysts. Recently, our group has identified CYP-mediated hydroxylation of (+)-valencene as a major bottleneck in the biosynthesis of trans-nootkatol and (+)-nootkatone in Pichia pastoris. In the current study, we aimed at enhancing CYP-mediated (+)-valencene hydroxylation by over-expressing target genes identified through transcriptome analysis in P. pastoris. Strikingly, over-expression of the DNA repair and recombination gene RAD52 had a distinctly positive effect on trans-nootkatol formation. Combining RAD52 over-expression with optimization of whole-cell biotransformation conditions, i.e. optimized media composition and cultivation at higher pH value, enhanced trans-nootkatol production 5-fold compared to the initial strain and condition. These engineering approaches appear to be generally applicable for enhanced hydroxylation of hydrophobic compounds in P. pastoris as confirmed here for two additional membrane-attached CYPs, namely the limonene-3-hydroxylase from Mentha piperita and the human CYP2D6.
细胞色素P450酶（CYPs）通过催化区域特异性和立体特异性羟基化反应，在各种天然化合物的生物合成中发挥着至关重要的作用。因此，CYP活性在精细化学品、药物化合物或香料和香精的生产中备受关注。CYPs的工业适用性推动了广泛的研究工作，旨在提高这些酶的性能以产生强大的生物催化剂。最近，我们小组发现CYP介导的（+）-瓦伦烯羟基化是毕赤酵母中反式诺卡酮和（+）-诺卡酮生物合成的主要瓶颈。在本研究中，我们旨在通过在毕赤酵母中过表达通过转录组分析鉴定的靶基因来增强CYP介导的（+）-瓦伦烯羟基化。令人惊讶的是，DNA修复和重组基因RAD52的过表达对反式诺卡酮的形成具有明显的积极影响。将RAD52过表达与全细胞生物转化条件的优化相结合，即优化培养基组成和在较高pH值下培养，与初始菌株和条件相比，反式诺卡酮产量提高了5倍。正如在此对另外两种膜附着的CYPs，即来自薄荷的柠檬烯-3-羟化酶和人CYP2D6所证实的那样，这些工程方法似乎普遍适用于增强毕赤酵母中疏水性化合物的羟基化。
