A recombinant CYP11B1 dependent Escherichia coli biocatalyst for selective cortisol production and optimization towards a preparative scale

BackgroundHuman 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).ResultsWe 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.ConclusionsUnder 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.

[1]  M. Waterman,et al.  Characterization of stable human aromatase expressed in E. coli , 2004, Steroids.

[2]  R. Bernhardt,et al.  Design and characterization of an efficient CYP105A1-based whole-cell biocatalyst for the conversion of resin acid diterpenoids in permeabilized Escherichia coli , 2013, Applied Microbiology and Biotechnology.

[3]  Application of a new versatile electron transfer system for cytochrome P450-based Escherichia coli whole-cell bioconversions , 2013, Applied Microbiology and Biotechnology.

[4]  P. White,et al.  Characterization of two genes encoding human steroid 11 beta-hydroxylase (P-450(11) beta). , 1989, The Journal of biological chemistry.

[5]  R. Bernhardt,et al.  Modulation of aldosterone and cortisol synthesis on the molecular level , 2004, Molecular and Cellular Endocrinology.

[6]  R. Bernhardt,et al.  Interaction of CYP11B1 (cytochrome P-45011 beta) with CYP11A1 (cytochrome P-450scc) in COS-1 cells. , 1999, European journal of biochemistry.

[7]  R. Bernhardt,et al.  Efficient conversion of 11-deoxycortisol to cortisol (hydrocortisone) by recombinant fission yeast Schizosaccharomyces pombe. , 2005, FEMS yeast research.

[8]  Denis Pompon,et al.  Total biosynthesis of hydrocortisone from a simple carbon source in yeast , 2003, Nature Biotechnology.

[9]  R. Bernhardt,et al.  Cytochromes P450 as versatile biocatalysts. , 2006, Journal of biotechnology.

[10]  R. Bernhardt,et al.  Coexpression of redox partners increases the hydrocortisone (cortisol) production efficiency in CYP11B1 expressing fission yeast Schizosaccharomyces pombe. , 2008, Journal of biotechnology.

[11]  C. A. Thomas,et al.  Molecular cloning. , 1977, Advances in pathobiology.

[12]  M. Tusié-Luna,et al.  The product of the CYP11B2 gene is required for aldosterone biosynthesis in the human adrenal cortex. , 1991, Molecular endocrinology.

[13]  G. Jänig,et al.  Steroid 11ß-hydroxylation by a fungal microsomal cytochrome P450 , 1992, The Journal of Steroid Biochemistry and Molecular Biology.

[14]  Siegfried S. F. Leung,et al.  Structural Characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei Bound to the Antifungal Drugs Posaconazole and Fluconazole , 2010, PLoS neglected tropical diseases.

[15]  V. Urlacher,et al.  A fluorimetric assay for cortisol , 2005, Analytical and Bioanalytical Chemistry.

[16]  R. Bernhardt,et al.  Adrenodoxin: the archetype of vertebrate-type [2Fe-2S] cluster ferredoxins. , 2011, Biochimica et biophysica acta.

[17]  J. Walker,et al.  Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. , 1996, Journal of molecular biology.

[18]  R. Bernhardt,et al.  Design of an Escherichia coli system for whole cell mediated steroid synthesis and molecular evolution of steroid hydroxylases. , 2006, Journal of biotechnology.

[19]  Andreas Schmid,et al.  Practical issues in the application of oxygenases. , 2003, Trends in biotechnology.

[20]  J. Stewart,et al.  Understanding and Improving NADPH‐Dependent Reactions by Nongrowing Escherichia coli Cells , 2008, Biotechnology progress.

[21]  A. Gilep,et al.  Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s. , 2007, Biochemical and biophysical research communications.

[22]  R. Bernhardt,et al.  The Human Steroid Hydroxylases CYP11B1 and CYP11B2 , 2002, Biological chemistry.

[23]  J. Lambeth,et al.  The participation of a second molecule of adrenodoxin in cytochrome P-450-catalyzed 11beta hydroxylation. , 1978, Journal of Biological Chemistry.

[24]  T. Sakaki.  Practical application of cytochrome P450. , 2012, Biological & pharmaceutical bulletin.

[25]  Andreas Schmid,et al.  Heme-iron oxygenases: powerful industrial biocatalysts? , 2008, Current opinion in chemical biology.

[26]  Andreas Schmid,et al.  The production of fine chemicals by biotransformations. , 2002, Current opinion in biotechnology.

[27]  J. Lancaster,et al.  Participation of the membrane in the side chain cleavage of cholesterol. Reconstitution of cytochrome P-450scc into phospholipid vesicles. , 1979, The Journal of biological chemistry.

[28]  R. Bernhardt,et al.  Molecular Evolution of a Steroid Hydroxylating Cytochrome P450 Using a Versatile Steroid Detection System for Screening , 2008, Lipids.

[29]  R. Bernhardt,et al.  CYP17- and CYP11B-dependent steroid hydroxylases as drug development targets. , 2006, Pharmacology & therapeutics.

[30]  A. Kiener,et al.  Industrial biocatalysis today and tomorrow , 2001, Nature.

[31]  B. Hamberger,et al.  Cytochrome P450-mediated metabolic engineering: current progress and future challenges. , 2014, Current opinion in plant biology.

[32]  K. Suzuki,et al.  Purification and properties of cytochrome P-450 (P-450lun) catalyzing steroid 11 beta-hydroxylation in Curvularia lunata. , 1993, Biochimica et biophysica acta.

[33]  U. Heinemann,et al.  Adrenodoxin: Structure, stability, and electron transfer properties , 2000, Proteins.

[34]  D. Engelberg,et al.  Procedure for controlling number of repeats, orientation, and order during cloning of oligonucleotides. , 2004, BioTechniques.

[35]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[36]  R. Bernhardt,et al.  Cytochrome P450 systems--biological variations of electron transport chains. , 2007, Biochimica et biophysica acta.

[37]  T. Omura,et al.  THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. I. EVIDENCE FOR ITS HEMOPROTEIN NATURE. , 1964, The Journal of biological chemistry.

[38]  M. Waterman,et al.  Purification and characterization of bovine steroid 21-hydroxylase (P450c21) efficiently expressed in Escherichia coli. , 2006, Biochemical and biophysical research communications.

[39]  N. Kagawa,et al.  Expression of human aromatase (CYP19) in Escherichia coli by N-terminal replacement and induction of cold stress response , 2003, Steroids.

[40]  J. Doehmer,et al.  Cloning of CYP11B1 and CYP11B2 from normal human adrenal and their functional expression in COS-7 and V79 Chinese hamster cells. , 1995, Endocrine Research.

[41]  V. Urlacher,et al.  Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations , 2014, Applied Microbiology and Biotechnology.

[42]  R. Bernhardt,et al.  Changing the Regioselectivity of a P450 from C15 to C11 Hydroxylation of Progesterone , 2012, Chembiochem : a European journal of chemical biology.

[43]  Gianfranco Gilardi,et al.  Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis , 2013, Biomolecules.

[44]  M. Kitagawa,et al.  Chaperone Coexpression Plasmids: Differential and Synergistic Roles of DnaK-DnaJ-GrpE and GroEL-GroES in Assisting Folding of an Allergen of Japanese Cedar Pollen, Cryj2, inEscherichia coli , 1998, Applied and Environmental Microbiology.

[45]  J. Lancaster,et al.  Steroidogenic electron transport in adrenal cortex mitochondria , 1982, Molecular and Cellular Biochemistry.

[46]  E. Gillam.  Engineering cytochrome p450 enzymes. , 2008, Chemical research in toxicology.

[47]  N. Kagawa,et al.  Human aldosterone synthase: Recombinant expression in E. coli and purification enables a detailed biochemical analysis of the protein on the molecular level , 2012, The Journal of Steroid Biochemistry and Molecular Biology.

[48]  J. Imlay.  Iron‐sulphur clusters and the problem with oxygen , 2006, Molecular microbiology.

[49]  G. Cauet,et al.  11β‐Hydroxylase Activity in Recombinant Yeast Mitochondria , 1996 .

[50]  M. Waterman,et al.  Purification and functional characterization of human 11β hydroxylase expressed in Escherichia coli , 2008, The FEBS journal.

[51]  C. Arrowsmith,et al.  Structural insights into aldosterone synthase substrate specificity and targeted inhibition. , 2013, Molecular endocrinology.

[52]  Frank McCormick,et al.  [Pharmaceutical biotechnology]. , 1992, Bollettino chimico farmaceutico.

[53]  K. Nakao,et al.  Cloning of cDNA and genomic DNA for human cytochrome P‐45011β , 1990, FEBS letters.

[54]  Vlada B Urlacher,et al.  Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. , 2012, Trends in biotechnology.

[55]  J. Peterson,et al.  A close family resemblance: the importance of structure in understanding cytochromes P450. , 1998, Structure.