Accessing Chemo- and Regioselective Benzylic and Aromatic Oxidations by Protein Engineering of an Unspecific Peroxygenase,ACS Catalysis

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Accessing Chemo- and Regioselective Benzylic and Aromatic Oxidations by Protein Engineering of an Unspecific Peroxygenase
ACS Catalysis ( IF 11.3 ) Pub Date : 2021-06-07 , DOI: 10.1021/acscatal.1c00847
Anja Knorrscheidt ₁ , Jordi Soler ₂ , Nicole Hünecke ₁ , Pascal Püllmann ₁ , Marc Garcia-Borràs ₂ , Martin J Weissenborn _{1,

3}

Affiliation

Unspecific peroxygenases (UPOs) enable oxyfunctionalizations of a broad substrate range with unparalleled activities. Tailoring these enzymes for chemo- and regioselective transformations represents a grand challenge due to the difficulties in their heterologous productions. Herein, we performed protein engineering in Saccharomyces cerevisiae using the MthUPO from Myceliophthora thermophila. More than 5300 transformants were screened. This protein engineering led to a significant reshaping of the active site as elucidated by computational modelling. The reshaping was responsible for the increased oxyfunctionalization activity, with improved k_cat/K_m values of up to 16.5-fold for the model substrate 5-nitro-1,3-benzodioxole. Moreover, variants were identified with high chemo- and regioselectivities in the oxyfunctionalization of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with enantioselectivities of up to 95% ee. The proposed evolutionary protocol and rationalization of the enhanced activities and selectivities acquired by MthUPO variants represent a step forward toward the use and implementation of UPOs in biocatalytic synthetic pathways of industrial interest.

中文翻译：

通过非特异性过氧化酶的蛋白质工程获得化学和区域选择性苄基氧化和芳香氧化

非特异性过氧化酶 (UPO) 能够以无与伦比的活性对广泛的底物范围进行氧化功能化。由于异源生产的困难，为化学和区域选择性转化定制这些酶是一项巨大的挑战。在此，我们使用来自嗜热毁丝霉的Mth UPO在酿酒酵母中进行了蛋白质工程。筛选了 5300 多个转化体。正如计算模型所阐明的那样，这种蛋白质工程导致了活性位点的显着重塑。重塑是增加氧官能化活性的原因，k _cat / K _m有所提高模型底物 5-nitro-1,3-benzodioxole 的值高达 16.5 倍。此外，在芳烃和苄基碳的氧官能化中分别鉴定出具有高化学选择性和区域选择性的变体。证明苄基羟基化以高达 95% ee 的对映选择性进行。Mth UPO 变体所获得的增强活性和选择性的拟议进化方案和合理化代表了 UPO 在具有工业价值的生物催化合成途径中的使用和实施的一步。

更新日期：2021-06-18

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