Cell foundry with high product specificity and catalytic activity for 21-deoxycortisol biotransformation

Abstract

Background21-deoxycortisol (21-DF) is the key intermediate to manufacture pharmaceutical glucocorticoids. Recently, a Japan patent has realized 21-DF production via biotransformation of 17-hydroxyprogesterone (17-OHP) by purified steroid 11β-hydroxylase CYP11B1. Due to the less costs on enzyme isolation, purification and stabilization as well as cofactors supply, whole-cell should be preferentially employed as the biocatalyst over purified enzymes. No reports as so far have demonstrated a whole-cell system to produce 21-DF. Therefore, this study aimed to establish a whole-cell biocatalyst to achieve 21-DF transformation with high catalytic activity and product specificity.ResultsIn this study, Escherichia coli MG1655(DE3), which exhibited the highest substrate transportation rate among other tested chassises, was employed as the host cell to construct our biocatalyst by co-expressing heterologous CYP11B1 together with bovine adrenodoxin and adrenodoxin reductase. Through screening CYP11B1s (with mutagenesis at N-terminus) from nine sources, Homo sapiens CYP11B1 mutant (G25R/G46R/L52 M) achieved the highest 21-DF transformation rate at 10.6 mg/L/h. Furthermore, an optimal substrate concentration of 2.4 g/L and a corresponding transformation rate of 16.2 mg/L/h were obtained by screening substrate concentrations. To be noted, based on structural analysis of the enzyme-substrate complex, two types of site-directed mutations were designed to adjust the relative position between the catalytic active site heme and the substrate. Accordingly, 1.96-fold enhancement on 21-DF transformation rate (to 47.9 mg/L/h) and 2.78-fold improvement on product/by-product ratio (from 0.36 to 1.36) were achieved by the combined mutagenesis of F381A/L382S/I488L. Eventually, after 38-h biotransformation in shake-flask, the production of 21-DF reached to 1.42 g/L with a yield of 52.7%, which is the highest 21-DF production as known.ConclusionsHeterologous CYP11B1 was manipulated to construct E. coli biocatalyst converting 17-OHP to 21-DF. Through the strategies in terms of (1) screening enzymes (with N-terminal mutagenesis) sources, (2) optimizing substrate concentration, and most importantly (3) rational design novel mutants aided by structural analysis, the 21-DF transformation rate was stepwise improved by 19.5-fold along with 4.67-fold increase on the product/byproduct ratio. Eventually, the highest 21-DF reported production was achieved in shake-flask after 38-h biotransformation. This study highlighted above described methods to obtain a high efficient and specific biocatalyst for the desired biotransformation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0720-y) contains supplementary material, which is available to authorized users.

Pregnenolone Overproduction in Yarrowia lipolytica by Integrative Components Pairing of the Cytochrome P450scc System

“…P450 scc Components Pairing by Combinatorial Screening Components Sources. As known, P450 monooxygenases from the same family often vary in expression level and catalytic activities when expressed in a particular chassis, 27 and distinct types of redox proteins can support many diverse physiological processes at different levels. 28 Thus, for a specific P450 reaction, there was no clear rules to help choose a P450 monooxygenase and select the optimal redox partner for it.…”

Strategies for microbial synthesis of high-value phytochemicals

“…Similarly, mutations were rationally incorporated into taxadiene synthase and taxadien-5α -hydroxylase (CYP725A4) in order to achieve enhanced selectivity towards the desired products taxa-4(20)-11(12)-diene and taxadien-5α -ol, respectively, which resulted in improved production of Taxol precursors in E. coli 37 . A similar rational mutation approach was applied to steroid 11β -hydroxylase (CYP11B1) to achieve enhanced selectivity towards the desired product 21-deoxycortisol 38 . In another example, protein evolution was applied to enhance the activity of 4-coumarate CoA ligase towards the synthesis of coumaroyl-CoA, with mutant variants exhibiting decreased k cat /K M for the undesired substrate ferulate and enhanced k cat /K M for coumarate 39 .…”

“…However, a recent study examined the effect of different N-terminal modifications on the activity of the CYP725A4 in E. coli, and suggested that although hydrophilic tags can enhance the solubility of P450s, the enzyme activity may decrease due to the disrupted enzyme-membrane interaction 44 . Interestingly, the recent N-terminal mutation study on CYP11B1 indicates that mutations within the N-terminus of cytochrome P450s can slightly alter the selectivity 38 .…”

Synthetic biology in China: Review and prospects

“…北京大学iGEM 团队用TFs设计了测定芳香烃污染物的传感器 [112] . 4种 [115] 、甜菊糖苷(steviolglycosides)(治疗记 忆障碍药物) [116] 、番茄红素(抗氧化保健食品) [117] , 香 叶醇江(食用香精) [118] 、苦皮藤素V(celangulin V)(蔬菜 作物驱虫剂) [119] 等; 黄酮类, 如松属素(pinocembrin)(局 部麻醉作用) [120] 、灯盏花素(breviscapine)(扩张脑血 管, 抗血凝作用) [121] 等; 植物甾体类, 如菜油甾醇(campesterol)(植物激素) [122] 、21-托皮质激素(21-deoxycortisol)(药用糖皮质激素的前体分子) [123] 、薯蓣皂苷元 (diosgenin)(甾体激素类药物的基础原料) [124] 等; 抗生 素及药物类, 如链霉素和衣霉素 [125] 、金红霉素 [126] , 尼 可霉素片段 [127] 、氨甲酰多聚草氨酸 [128] 、丹参酸A (salvianic acid A) [60] 、大麻素(cannabinoids) [129] 、羟基 酪醇(hydroxytyrosol) [130] 等. 有一些合成天然化合物的产量已经达到前所未 有的水平, 具有很强的工业价值.…”