Biosynthesis of steviol glycosides in planta proceeds via two cytochrome P450 enzymes (CYPs): kaurene oxidase (KO) and kaurenoic acid hydroxylase (KAH). KO and KAH function in succession with the support of a NADPH-dependent cytochrome P450 reductase (CPR) to convert kaurene to steviol. This work describes a platform for recombinant production of steviol glucosides (SGs) in Saccharomyces cerevisiae, demonstrating the full reconstituted pathway from the simple sugar glucose to the SG precursor steviol. With a focus on optimization of the KO-KAH activities, combinations of functional homologues were tested in batch growth. Among the CYPs, novel KO75 (CYP701) and novel KAH82 (CYP72) outperformed their respective functional homologues from Stevia rebaudiana, SrKO (CYP701A5) and SrKAH (CYP81), in assays where substrate was supplemented to culture broth. With kaurene produced from glucose in the cell, SrCPR1 from S. rebaudiana supported highest turnover for KO-KAH combinations, besting two other CPRs isolated from S. rebaudiana, the Arabidopsis thaliana ATR2, and a new class I CPR12. Some coexpressions of ATR2 with a second CPR were found to diminish KAH activity, showing that coexpression of CPRs can lead to competition for CYPs with possibly adverse effects on catalysis.

中文翻译：

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一个组合的方法来研究细胞色素P450酶的从头在面包酵母甜菊糖苷生产

甜菊糖苷的生物合成在植物中进行经由贝壳杉烯氧化酶（KO）和贝壳杉烯酸羟化酶（KAH）：2种细胞色素P450酶（CYP同工酶）。KO和KAH在NADPH依赖性细胞色素P450还原酶（CPR）的支持下连续运行，以将贝壳杉烯转化为甜菊醇。这项工作描述了在酿酒酵母中重组生产甜菊醇葡萄糖苷（SGs）的平台，展示了从简单糖葡萄糖到SG前体甜菊醇的完整重构途径。以优化KO-KAH活性为重点，在批次生长中测试了功能同源物的组合。在CYP中，新型KO75（CYP701）和新型KAH82（CYP72）优于各自的功能同源物。甜叶菊，SrKO（CYP701A5）和SrKAH（CYP81），在将底物添加到培养液中的试验中。与来自葡萄糖在细胞中产生贝壳杉烯，SrCPR1从甜叶菊支持KO-KAH组合最高的营业额，击败分离自另外两个方案建议甜叶菊中，拟南芥ATR2，和一个新的I类CPR12。发现一些ATR2与第二CPR的共表达会降低KAH活性，表明CPR的共表达可导致CYP竞争，可能对催化产生不利影响。