剔除3-酮類固醇9α-羥基化酶基因導致Rhodococcus equi菌株於固醇培養基累積雄二烯二酮

雄二烯二酮(Androstadienedione, ADD)是微生物代謝固醇類化合物中重要的中間產物，可作為多種類固醇藥物生產的前驅物。類固醇藥物被廣泛地用於各種疾病的治療和預防，包括內分泌科、婦科、風濕免疫科以及腫瘤科的臨床應用，大量的需求讓類固醇藥物在製藥工業上佔有重要的地位，因此發展微生物突變菌株以轉化固醇產生ADD具相當之工業效益。目前已知3-酮類固醇9α-羥基化酵素(3-ketosteroid 9α-hydroxylase, KSH)是降解ADD的關鍵酵素，KSH是由鐵氧蛋白還原酶(ferredoxin reductase, KshB)和末端加氧酶(terminal oxygenase, KshA)所組成，可參與微生物對各種類固醇環系結構之代謝。Rhodococcus equi USA-18 (原命名Arthrobacter simplex USA-18)為膽固醇氧化酶生產菌株。在本研究中，我們根據已知KshB保留區域的胺基酸序列以聚合酶連鎖反應方法從R. equi USA-18中選殖出一段kshB基因。接著利用基因同源重組方法剔除該基因，構建kshB基因缺失的突變株，得到R. equi USA-18∆B8。另外， R. equi F1601 (原生質體融合菌株)中的兩套kshB基因也經由連續同源重組操作而去除，此基因剔除菌株命名為R. equi F1601∆F12。此二突變株培養在以膽固醇為主要碳源之培養基可累積ADD，顯示突變株已缺乏KSH酵素活性。R. equi USA-18∆B8及R. equi F1601∆F12除了可累積ADD同時也會產生3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (Δ1,4-BNC)。由巨噬細胞感染試驗確認R. equi USA-18和其突變株均無法在巨噬細胞內生存。在巨噬細胞中不能持續性地生長之特性，可屏除生物安全應用上的疑慮，表明R. equi USA-18、R. equi F1601和其突變株具有工業應用的潛力。最後利用反應曲面法（response surface methodology, RSM）之Box-Behnken設計分析所篩選出的3個因子Tween 20、酵母抽出物及甘油，運用RSM分析法確定因子之間的交互作用及培養基最佳條件。結果顯示，優化之培養基為0.2％膽固醇-基礎培養基中添加0.5% Tween 20、1%酵母抽出物和1%甘油。將R. equi USA-18∆B8培養於此優化培養基中，ADD最高之累積量為0.65 mg/mL，莫耳轉換率為44.4%。此外將R. equi F1601∆F12培養於0.2%植物固醇-基礎培養基中並添加0.5% Tween 20、1%酵母抽出物和1%甘油，ADD最高之累積量為0.344 mg/mL，莫耳轉換率為25%。綜合上述結果顯示，R. equi USA-18∆B8與R. equi F1601∆F12能成功累積ADD和Δ1,4-BNC之固醇藥物合成的重要中間體，故R. equi USA-18及其衍生之突變株值得進一步研究其應用之潛力。Androstadienedione (ADD), an important intermediate of microbial steroid metabolite, is used as a starting material for the synthesis of a variety of pharmaceutical steroids, which are widely used in clinical applications for disease treatment and prevention in aspects of endocrinology, gynecology, rheumatology, and oncology. It is well known that the bacterial degradation of ADD is catalyzed firstly by a two-component 3-ketosteroid 9α-hydroxylase (KSH), consisting of a ferredoxin reductase (KshB) and a terminal oxygenase (KshA). In this study, the gene encoding the putative reductase component (KshB) of 3-ketosteroid 9α-hydroxylase was cloned from Rhodococcus equi USA-18, a cholesterol oxidase-producing strain formerly named Arthrobacter simplex USA-18, by PCR according to consensus amino acid motifs of several bacterial KshB subunits. Deletion of the gene in R. equi USA-18 by a PCR-targeted gene disruption method resulted in a mutant strain R. equi USA-18∆B8. In addition, the two copies of kshB in R. equi F1601, a protoplast fusion strain created for high expression amount of cholesterol oxidase, were deleted to create R. equi F1601∆F12. Both R. equi USA-18∆B8 and F1601∆F12 strains, could accumulate 1,4-androstadien-3,17-dione (ADD) in cholesterol-containing medium, indicating the involvement of the deleted enzyme in 9α-hydroxylation of steroids. These two mutants also accumulated 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (Δ1,4-BNC). According to macrophage infection assay, R. equi USA-18 and its derived mutant strains were unable to survive within macrophages. Inability to persistently grow in macrophages suggests that R. equi USA-18ΔB8 has an application potential in industry. Finally, the medium composition was optimized by using response surface methodology (RSM) employed with Box-Behnken design. The optimized fermentation condition obtained with RSM was a 0.2% cholesterol-minimal medium supplemented with 0.5% Tween 20, 1% yeast extract and 1% glycerol. The ADD concentration produced by R. equi USA-18∆B8 continuously increased to 0.65 mg/mL, equivalent to 44.4% molar yield, in the optimal culture conditions. The ADD yield produced by R. equi F1601∆F12 reached to 0.344 mg/mL, equivalent to 24.96% conversion ratio, when cultured in B medium supplemented with 0.2% (w/v) phytosterols, 0.5% Tween 20, 1% yeast extract and 1% glycerol. Since, ADD and Δ1,4-BNC are important intermediates for the synthesis of steroid drugs, the mutants derived from R. equi USA-18 and R. equi F1601 may deserve further investigation for its application potential.Table of contents Chinese abstract........................................i English abstract......................................iii Table of contents.......................................v List of tables........................................vii List of figures......................................viii Chapter 1: General introduction 1.1 Steroid............................................ 1 1.2 Discovery and synthesis of steroid drug............ 2 1.3 3-ketosteroid 9α-hydroxylase....................... 4 1.4 Steroid intermediate: AD/ADD....................... 6 1.5 The current challenges in microbial steroid bioconversion.......................................... 8 1.6 Main objective of this research.................... 9 Chapter 2: kshB gene cloning and reclassification of A. simplex USA-18 as R. equi USA-18 2.1 Introduction............................... .......11 2.2 Materials and methods..............................13 2.3 Results............................................16 2.4 Discussions........................................21 Chapter 3: Deletion of the gene encoding the reductase component of 3-ketosteroid 9α-hydroxylase in R. equi USA-18 disrupts sterol catabolism, leading to the accumulation of 3-oxo-23,24-bisnorchola-1,4-dien- 22-oic acid and 1,4-androstadien-3,17-dione 3.1 Introduction.......................................22 3.2 Materials and methods..............................25 3.3 Results............................................30 3.4 Discussions........................................36 Chapter 4: Restricted growth of R. equi USA-18 in macrophage 4.1 Introduction.......................................40 4.2 Materials and methods..............................44 4.3 Results............................................45 4.4 Discussions........................................47 Chapter 5: Accumulation of ADD in the fermentation broth of R. equi USA-18 ∆B8 and R. equi F1601∆F12 5.1 Introduction.......................................50 5.2 Materials and methods..............................53 5.3 Results............................................56 5.4 Discussions........................................61 Chapter 6: Conclusions.................................63 Tables.................................................65 Figures................................................77 References............................................10