Hilberath Thomas, Raffaele Alessandra, Windeln Leonie M, Urlacher Vlada B
Institute of Biochemistry, Heinrich-Heine University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany.
Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629HZ, Delft, The Netherlands.
AMB Express. 2021 Dec 4;11(1):162. doi: 10.1186/s13568-021-01319-0.
Cytochromes P450 catalyze oxidation of chemically diverse compounds and thus offer great potential for biocatalysis. Due to the complexity of these enzymes, their dependency of nicotinamide cofactors and redox partner proteins, recombinant microbial whole cells appear most appropriate for effective P450-mediated biocatalysis. However, some drawbacks exist that require individual solutions also when P450 whole-cell catalysts are used. Herein, we compared wet resting cells and lyophilized cells of recombinant E. coli regarding P450-catalyzed oxidation and found out that lyophilized cells are well-appropriate as P450-biocatalysts. E. coli harboring CYP105D from Streptomyces platensis DSM 40041 was used as model enzyme and testosterone as model substrate. Conversion was first enhanced by optimized handling of resting cells. Co-expression of the alcohol dehydrogenase from Rhodococcus erythropolis for cofactor regeneration did not affect P450 activity of wet resting cells (46% conversion) but was crucial to obtain sufficient P450 activity with lyophilized cells reaching a conversion of 72% under the same conditions. The use of recombinant lyophilized E. coli cells for P450 mediated oxidations is a promising starting point towards broader application of these enzymes.
细胞色素P450催化多种化学性质不同的化合物的氧化反应，因此在生物催化方面具有巨大潜力。由于这些酶的复杂性、对烟酰胺辅因子和氧化还原伴侣蛋白的依赖性，重组微生物全细胞似乎最适合用于有效的P450介导的生物催化。然而，即便使用P450全细胞催化剂时也存在一些缺点，需要针对具体情况逐一解决。在此，我们比较了重组大肠杆菌的湿静息细胞和冻干细胞在P450催化氧化方面的表现，发现冻干细胞非常适合作为P450生物催化剂。以携带来自天蓝色链霉菌DSM 40041的CYP105D的大肠杆菌作为模型酶，以睾酮作为模型底物。首先通过优化静息细胞的处理方式提高了转化率。来自红平红球菌的醇脱氢酶用于辅因子再生的共表达对湿静息细胞的P450活性没有影响（转化率为46%），但对于在相同条件下使冻干细胞获得足够的P450活性至关重要，此时转化率达到72%。使用重组冻干大肠杆菌细胞进行P450介导的氧化反应是这些酶更广泛应用的一个有前景的起点。
