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Microbial Transformation of Bile Acids. a Unified Scheme for Bile Acid Degradation and Hydroxylation of Bile Acids

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Microbial Transformation of Bile Acids. a Unified Scheme for Bile Acid Degradation and Hydroxylation of Bile Acids SR P T03 Form 3 CONDITIONAL THE UNIVERSITY OF NEW SOUTH WALES DECLARATION RELATING TO DISPOSITION OF PROJECT REPORT/THESIS This is to certify that I d?..^ ...being a candidate for the degree of am fully aware of the policy of the University relating to the retention and use of higher degree project reports and theses, namely that the University retains the copies submitted for examination and is free to allow them to be consulted or borrowed. Subject to the provisions of the Copyright Act, 1968, the University may issue a project report or thesis in whole or in part, in photostat or microfilm or other copying medium. I wish the following condition to attach to the use of my project f^pert/thesis: . \ ^-ri pi^^fM-i oA ...-to. .m.P^^.E d .v ^y ^ Ci^^k ^ S.t 01- ^ ^sos-. ^ .TTlk^.f^ u QMIT^. ^L.J^ I also authorize the publication by University Microfilms of a 350 word abstract in Dissertation Abstracts International (applicable to doctorates only). Witness... ..e GENETIC MANIPULATION OF PSEUDOMONADS TO PRODUCE STEROID CATABOLITES FROM BILE ACIDS. A THESIS SUBMriTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF NEW SOUTH WALES AUSTRALIA BY JOHN ALTON IDE SCHOOL OF BIOTECHNOLOGY MAY, 1989. UNIVERSITY OF N.S.W. 16 MAY 1990 CONTENTS Page ABSTRACT i DECLARATION iii ACKNOWLEDGEMENTS iv LIST OF PUBLICATIONS v LIST OF TABLES vi LIST OF FIGURES viii ABBREVIATIONS xi 1 INTRODUCTION 1 LI INTRODUCTION TO STEROID PRODUCTION. 1 L2. EARLY SYNTHETIC PROCESSES FOR THE MANUFACTURE OF STEROID DRUGS. 4 L3. CURRENT PROCESSES 8 L4. MICROBL\L TRANSFORMATION OF STEROIDS. 12 L5. ALTERNATIVE BIOTECHNOLOGICAL PROCESSES. 18 1.6. FERMENTATION PROCESSES AND ALTERNATIVES. 18 1.7. MICROBL\L DEGRADATION OF STEROLS 24 1.8. CONVERSION OF STEROLS BY MUTANTS. 27 1.9. BILE ACIDS AS AN ALTERNATIVE SUBSTRATE. 28 1.10. MICROBIAL DEGRADATION OF BILE ACIDS. 29 1.11. GENERAL PROPERTIES OF TRANSPOSONS. 40 1.12. TRANSPOSONS AS MUTAGENIC AGENTS AND VECTORS FOR THEIR INTRODUCTION INTO RECIPIENTS. 44 continued.... Contents (continued) 1.13. USE OF TRANSPOSONS IN GENE CLONING. 46 1.14. AIMS OF THIS THESIS. 50 2. MATERIALS AND METHODS. 51 2.1. BACTERIAL STRAINS AND PLASMIDS. 51 2.2. GENERAL PROCEDURES AND CHEMICALS. 51 2.2.1. Water and sterilization. 51 2.2.2. Growth conditions. 51 2.2.3. Chemicals and reagents. 51 2.3 BUFFERS AND SOLUTIONS. 57 2.3.1. Amino Acids and Nucleotide Growth Factors. 57 2.3.2. 25% Glucose. 58 2.3.3. Antibiotics. 58 2.3.4. PAS Salts Concentrate. 58 2.3.5. Vogel-Bonner Salts Concentrate. 58 2.3.6. Saline plus 10% Nutrient Broth (SNB). 58 2.3.7. Citrate Buffer. 59 2.3.8. Tris-EDTA (TE). 59 2.3.9. Tris-Acetate-EDTA (TAE). 59 2.3.10. Saline Sodium Citrate (SSC). 59 2.3.11. Denhardt's Reagent. 60 2.3.12. Pre-Hybridization Buffer. 60 2.3.13. Hybridization Buffer. 60 2.3.14. Deoxynucleotide Triphosphates. 61 2.3.15. Nick Translation Buffer. 61 2.3.16. Nick Stop Buffer. 61 2.3.17. Restriction Buffer. 62 2.3.18. Ligation Buffer. 62 continued Contents (Continued) 2.3.19. Phenol. 62 2.3.20. Chloroform. 62 2.4. MEDIA. 63 2.4.1. Nutrient Broth (NB). 63 2.4.2. Nutrient Agar (NA). 63 2.4.3. Luria Broth (LB). 63 2.4.4. PAS Minimal Media. 63 2.4.5. VB Minimal Media. 64 2.5. METHODS 64 2.5.1. Curing of Plasmids. 64 2.5.2. NTG Mutagenesis. 64 2.5.3. Filter Mate Conjugation. 65 2.5.4. Transposon Mutagenesis. 66 2.5.5. TLC Identification of Steroid Products. 66 2.5.6. Small Scale Isolation of Plasmid DNA. 67 2.5.7. Large Scale Isolation of DNA. 67 2.5.8. Isolation of Genomic DNA. 68 2.5.9. Isolation of Mu Phage DNA. 68 2.5.10. Restriction Endonuclease Digests. 69 2.5.11. Dephosphorylation of Spliced DNA. 69 2.5.12. Ligations. 69 2.5.13. Agarose Gel Electrophoresis. 70 2.5.14. Electro-elution of DNA Fragments from Agarose Gels. 70 2.5.15. Transformation of Plasmid DNA. 71 2.5.16. Labelling DNA by Nick Translation. 71 2.5.17. Southern Transfer of DNA and Hybridization. 72 continued Contents (Continued) 2.5.17.1. Transfer of DNA from Agarose Gels. 72 2.5.17.2. Colony Blotting. 73 2.5.17.3. Hybridization. 74 2.5.17.3.1. Hybridization with Southern Blots. 74 2.5.17.3.2. Hybridization with Colony Blots. 75 3. RESULTS. 76 3.1. PRELIMINARY CHARACTERIZATION OF BILE-UTILIZING PSEUDOMONADS. 76 3.1.1. Plasmid Profile. 76 3.1.2. Catabolic Properties. 76 3.1.3. Antibiotic-resistance Properties. 79 3.1.4. Isolation and Identification of Auxotrophic Mutants. 79 3.1.5. Introduction of Catabolic Plasmidsby Conjugation. 81 3.1.6. Curing of the Resident Plasmids. 81 3.2. ISOLATION OF MUTANTS BLOCKED IN STEROID BIOCONVERSIONS. 84 3.2.1. Isolation of NTG-induced Mutants Affected in Steroid Utilization. 85 3.2.2. Transposon Mutagenesis with Tn5 to Isolate Steroid Catabolic Mutants. 88 3.2.3. Transposon Mutagenesis with Tnl and Tn7 to Issolate Steroid Catabolic Mutants 100 3.2.4. Transposon Mutagenesis with TnlO. 106 3.2.5. Summary of Transposon Mutation. 107 3.2.6. Double Mutation of Bile-utilizing Strains. 108 3.2.7. Fermentation Studies of Transposon-induced Mutants. 118 Continued Contents (Continued) 3.3. MECHANISM OF TRANSPOSITION BY TN5 FROM pJB4JI. 123 3.3.1. Selection for Clones Encoding Kanamycin Resistance from pJB4JI-derived Mutants. 124 3.3.2. Restriction Mapping and Southern Hybridization Analysis of Clones isolated from pJB4JI-derived Mutants. 127 3.3.3. Southem Hybridization Analysis of Tn5-induced Mutants Derived from the Vector pJB4JI. 135 3.4. MECHANISM OF TRANSPOSITION BY Tn5 FROM pSUPlOll. 152 3.4.1. Selection for Clones Encoding Kanamycin Resistance from pSUPlOll-derived Mutants. 152 3.4.2. Restriction Mapping of pND209. 153 3.4.3. Hybridization of pND209 with Fragments of pSUPlOll. 153 3.4.4. Southem Hybridization Analysis of Tn5-induced Mutants Derived from the Vector pSUPlOll. 158 3.5. CLONING OF THE STEROID CATABOLIC PATHWAY 161 3.5.1. Hybridization of DNA from PS5-1 and PS8-1 with the Clones pND200 and pND209. 164 3.5.2. Derivation of Gene Libraries of PS5-1 and PS8-1 using the Vector pKT230. 170 3.5.3. Cloning of Pseudomonas DNA into the //mdin Site of pKT230. 171 3.5.4. Screening of the Pseudomonas Gene Libraries for Clones Encoding the Steroid Catabolic Pathway using Colony Hybridization. 173 Continued Contents (Continued) 3.5.5. Preliminary Cloning of PS8-1 and PS5-1 DNA into the Cloning Vector pBR329. 174 4. DISCUSSION. 177 5. BIBLIOQRAPHY. 203 1. ABSTRACT. The object of the project was to modify, by mutation, bile steroid catabolizing Pseudomonads so that they could accumulate steroid intermediates of the breakdown pathway. In the four Pseudomonas strains investigated, genetic information relevant to the steroid catabolic pathway appeared to be encoded on the chromosome. One NTG-induced mutant, PS5-7, accumulated secophenol and secocatechol compounds. Although speculated, the secocatechol had never previously been isolated as a degradation product. Transposon-induced mutants of PS5-7 were isolated which accumulated only the phenolic secosteroid. A total of 46 transposon-induced mutants of bile acid-catabolizing Pseudomonads (from 13000 transposed clones tested) were isolated which accumulated steroid pathway intermediates. Seven classes of intermediates were accumulated and although mutants varied in stability, individual mutants were obtained which enabled some products to be accumulated in yields approaching theoretical. The major products accumulated from cholic acid fermentations (and the yields) are as follows : 7a,12a-dihydroxy-3-oxo-l,4-pregnadiene-20-carboxylic acid (86%), 7a, 12a-dihydroxy-1,4-androstadiene-3,17-dione (97%), 7a, 12P-dihydroxy- l,4-androstadiene-3,17-dione (96%) and 3,7,12a-trihydroxy phenolic secosteroid (97%). The remaining product classes were mixtures and were not studied in detail. Introduction of a second transposon into selected strains seldom altered the products they accumulated, but often altered the stability of the clone. The mechanism of transposition by two Tn5-loaded vectors, pJB4JI and pSUPlOll, was investigated. Hybridization studies with DNA isolated from selected mutants showed that all of the mutants investigated had at least one copy u. of Tn5 in the chromosome. Clones, containing the transposon and the DNA encoding the flanking regions about that transposon, were isolated from selected mutants and investigated. The transposon Tn5 is carried within Mu DNA in pJB4JI. In 14 out of 19 pJB4JI-derived mutants, Mu was inherited as well as Tn5. The remaining 5 mutants harboured only Tn5. The one pSUPlOll- derived mutant investigated harboured only Tn5. Attempts to clone genes relevant to the steroid catabolic pathway into the cloning vector pKT230 were unsuccessful because of instability of DNA inserts in the vector. Preliminary cloning experiments have shown that DNA of the required size could be stably inserted into pBR329. Cloning of DNA encoding steroid catabolic genes appears practicable using this altemative vector. m SR.P.T10 CERTIFICATE OF ORIGINALITY I hereby declare that this thesis is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by an other person nor material which to a substantial extent has been accepted for the award of any other degree or diploma of a university or other institute of higher learning, except where due aknowledgement is made in the text of the thesis. (Signed) IV.
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