title: A recombinant CYP11B1 dependent Escherichia coli biocatalyst for selective cortisol production and optimization towards a preparative scale.
abstract: BACKGROUND
  Human mitochondrial CYP11B1 catalyzes a one-step regio- and stereoselective 11β-hydroxylation of 11-deoxycortisol yielding cortisol which constitutes not only the major human stress hormone but also represents a commercially relevant therapeutic drug due to its anti-inflammatory and immunosuppressive properties. Moreover, it is an important intermediate in the industrial production of synthetic pharmaceutical glucocorticoids. CYP11B1 thus offers a great potential for biotechnological application in large-scale synthesis of cortisol. Because of its nature as external monooxygenase, CYP11B1-dependent steroid hydroxylation requires reducing equivalents which are provided from NADPH via a redox chain, consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx).
  
  
  RESULTS
  We established an Escherichia coli based whole-cell system for selective cortisol production from 11-deoxycortisol by recombinant co-expression of the demanded 3 proteins. For the subsequent optimization of the whole-cell activity 3 different approaches were pursued: Firstly, CYP11B1 expression was enhanced 3.3-fold to 257 nmol∗L(-1) by site-directed mutagenesis of position 23 from glycine to arginine, which was accompanied by a 2.6-fold increase in cortisol yield. Secondly, the electron transfer chain was engineered in a quantitative manner by introducing additional copies of the Adx cDNA in order to enhance Adx expression on transcriptional level. In the presence of 2 and 3 copies the initial linear conversion rate was greatly accelerated and the final product concentration was improved 1.4-fold. Thirdly, we developed a screening system for directed evolution of CYP11B1 towards higher hydroxylation activity. A culture down-scale to microtiter plates was performed and a robot-assisted, fluorescence-based conversion assay was applied for the selection of more efficient mutants from a random library.
  
  
  CONCLUSIONS
  Under optimized conditions a maximum productivity of 0.84 g cortisol∗L(-1)∗d(-1) was achieved, which clearly shows the potential of the developed system for application in the pharmaceutical industry.
authors.affiliation: Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. lina.schiffer@uni-saarland.de.
authors.forename: Lina
authors.initials: L
authors.lastname: Schiffer
authors.affiliation: Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. s.anderko@mx.uni-saarland.de.
authors.forename: Simone
authors.initials: S
authors.lastname: Anderko
authors.affiliation: Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. anna.hobler@gmx.de.
authors.forename: Anna
authors.initials: A
authors.lastname: Hobler
authors.affiliation: Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. f.hannemann@mx.uni-saarland.de.
authors.forename: Frank
authors.initials: F
authors.lastname: Hannemann
authors.affiliation: Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. norio.kagawa@med.nagoya-u.ac.jp.
authors.forename: Norio
authors.initials: N
authors.lastname: Kagawa
authors.affiliation: Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. ritabern@mx.uni-saarland.de.
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
mesh_terms: D000325:Adrenodoxin / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006854:Hydrocortisone / Q000096:biosynthesis*; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013252:Steroid 11-beta-Hydroxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D000325:Adrenodoxin; D013252:Steroid 11-beta-Hydroxylase; D006854:Hydrocortisone