School of Biological Engineering, Dalian Polytechnic University, Dalian, 116034, China.
Biotechnol Lett. 2024 Feb;46(1):29-35. doi: 10.1007/s10529-023-03442-3. Epub 2023 Nov 16.
To investigate the effect of subcellular localization on the transformation efficiency of heterologous expressed functional P450s in yeast. Microbial biotransformation offers a promising substitute for the direct extraction of natural products, but its viability in industrial applications depends on achieving high transformation efficiencies. To investigate the influence of subcellular microenvironments on the activity of heterologously expressed P450s, Catharanthus roseus tabersonine 16-hydroxylase (T16H) was chosen, and its subcellular localization was regulated by fusing organelle-localization signals. Interestingly, this manipulation had no effect on the gene expression levels of T16H, but resulted in varying conversion rates from tabersonine to 16-hydroxy tabersonine. Notably, the highest transformation efficiency was observed in yeast cells expressing peroxisome-localized T16H. Given the alkaline pH optimum for P450s, the alkaline peroxisomal lumen could be a suitable compartment for P450s reactions to achieve high transformation efficiency using yeast cells. Different organelle-localization of T16H in yeast cells resulted in varying conversion rates, suggesting that compartmentalizing the expression of target enzymes could be a viable approach to increase transformation efficiency in yeast.
为了研究亚细胞定位对异源表达功能 P450 转化效率的影响。微生物生物转化为天然产物的直接提取提供了一种很有前途的替代方法，但它在工业应用中的可行性取决于能否实现高转化效率。为了研究亚细胞微环境对异源表达 P450 活性的影响，选择了长春花塔博辛 16-羟化酶（T16H），并通过融合细胞器定位信号来调节其亚细胞定位。有趣的是，这种操作对 T16H 的基因表达水平没有影响，但导致塔博辛到 16-羟基塔博辛的转化率不同。值得注意的是，过氧化物酶体定位的 T16H 在酵母细胞中的表达观察到最高的转化效率。鉴于 P450 最适碱性 pH 值，碱性过氧化物酶体腔可能是 P450 反应的合适隔室，可使用酵母细胞实现高转化效率。T16H 在酵母细胞中的不同细胞器定位导致转化率不同，这表明将靶酶的表达分隔在不同的隔室内可能是提高酵母转化效率的一种可行方法。
