Lau, Shong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 124-129). / Abstracts in English and Chinese. / Abstract of thesis --- p.i / 碩士論文摘要 --- p.iii / Acknowledgement --- p.iv / Declaration --- p.v / Abbreviations --- p.vi / Genetic symbols --- p.viii / Table of content --- p.ix / List of figures --- p.xiii / List of tables --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Thesis outline --- p.1 / Chapter 1.2 --- A. chrysogenum --- p.3 / Chapter 1.3 --- Antibiotic industry --- p.4 / Chapter 1.4 --- Cephalosporins --- p.4 / Chapter 1.5 --- CPC biosynthetic pathway --- p.5 / Chapter 1.6 --- DAC --- p.8 / Chapter 1.7 --- Aims of study --- p.10 / Chapter Chapter 2 --- Construction of transformation cassettes --- p.11 / Chapter 2.1 --- Introduction --- p.11 / Chapter 2.2 --- Materials and Methods --- p.13 / Chapter 2.2.1 --- Construction of cassette RTCAH --- p.13 / Chapter 2.2.1.1 --- Cultivation of R. toruloides --- p.13 / Chapter 2.2.1.2 --- Preparation of R. toruloides genomic DNA --- p.13 / Chapter 2.2.1.3 --- PCR of R. toruloides CAH gene --- p.14 / Chapter 2.2.1.4 --- Construction of pRTCAHhyr --- p.16 / Chapter 2.2.2 --- Construction of cassette GHG --- p.17 / Chapter 2.3 --- Results and Discussion --- p.18 / Chapter 2.3.1 --- pGHG construction --- p.18 / Chapter 2.3.2 --- pRTCAHhyr construction --- p.18 / Chapter Chapter 3 --- Optimization of integrative transformation of A. chrysogenum --- p.22 / Chapter 3.1 --- Introduction --- p.22 / Chapter 3.2 --- Materials and Methods --- p.24 / Chapter 3.2.1 --- Strain and culture medium --- p.24 / Chapter 3.2.2 --- Reagents --- p.24 / Chapter 3.2.3 --- Standard transformation procedures --- p.25 / Chapter 3.2.3.1 --- Cell cultivation --- p.25 / Chapter 3.2.3.2 --- Protoplast preparation --- p.25 / Chapter 3.2.3.3 --- PEG mediated protoplast fusion --- p.27 / Chapter 3.2.4 --- Examination of transformation parameters --- p.28 / Chapter 3.3 --- Results and Discussion --- p.29 / Chapter 3.3.1 --- Cell growth period --- p.30 / Chapter 3.3.2 --- DNA concentration --- p.32 / Chapter 3.3.3 --- PEG molecular weight --- p.35 / Chapter 3.3.4 --- Transformation additives --- p.37 / Chapter 3.3.5 --- Modified transformation protocol --- p.39 / Chapter Chapter 4 --- Metabolic engineering of A. chrysogenum --- p.42 / Chapter 4.1 --- Introduction --- p.42 / Chapter 4.2 --- Materials and Methods --- p.43 / Chapter 4.2.1 --- Transformation of A. chrysogenum --- p.43 / Chapter 4.2.2 --- Screening of transformants --- p.43 / Chapter 4.2.3 --- HPLC analysis --- p.43 / Chapter 4.2.4 --- A. chrysogenum genomic DNA preparation --- p.44 / Chapter 4.2.5 --- Genotyping by PCR --- p.45 / Chapter 4.2.5.1 --- GHG transformants --- p.45 / Chapter 4.2.5.2 --- RTCAH transformants --- p.47 / Chapter 4.2.6 --- Genotyping of GHG transformants by Southern hybridization --- p.47 / Chapter 4.2.6.1 --- Preparation of DIG-labeled GHG probe by PCR --- p.48 / Chapter 4.2.6.2 --- PCR preparation of DIG-labeled Cef-EF probe --- p.48 / Chapter 4.2.6.3 --- Restriction digestion of genomic DNA of GHG transformants --- p.49 / Chapter 4.2.6.4 --- Agarose gel electrophoresis and DNA transfer to positively charged nylon membrane --- p.50 / Chapter 4.2.6.5 --- Pre-hybridization and hybridization --- p.52 / Chapter 4.2.6.6 --- Membrane washing and blocking --- p.52 / Chapter 4.2.6.7 --- Membrane detection --- p.53 / Chapter 4.2.7 --- Genotyping of RTCAH transformants by Southern hybridization --- p.53 / Chapter 4.2.7.1 --- PCR preparation of DIG-labeled RTCAH probe --- p.54 / Chapter 4.2.7.2 --- Restriction digestion of genomic DNA of RTCAH transformants --- p.54 / Chapter 4.2.7.3 --- Agarose gel electrophoresis and Southern hybridization of RTCAH transformants --- p.55 / Chapter 4.3 --- Results and Discussion --- p.56 / Chapter 4.3.1 --- Hygromycin B screening and HPLC analysis of GHG transformants --- p.56 / Chapter 4.3.2 --- Genotyping of GHG232 by PCR --- p.56 / Chapter 4.3.3 --- Genotyping of GHG232 by Southern hybridization --- p.58 / Chapter 4.3.4 --- Hygromycin B screening and HPLC analysis of RTCAH transformant --- p.61 / Chapter 4.3.5 --- Genotyping of RTCAH transformants by PCR --- p.61 / Chapter 4.3.6 --- Genotyping of RTCAH transformants by Southern hybridization --- p.63 / Chapter Chapter 5 --- Characterization of modified strains and fermentation study --- p.65 / Chapter 5.1 --- Introduction --- p.65 / Chapter 5.2 --- Materials and Methods --- p.67 / Chapter 5.2.1 --- Comparison of DAC production in GHG232 and RTCAH transformants by small-scale fermentation --- p.67 / Chapter 5.2.2 --- CPC conversion assay --- p.67 / Chapter 5.2.3 --- Evaluation of fermentation media --- p.68 / Chapter 5.2.4 --- Factorial design for medium formulation --- p.68 / Chapter 5.3 --- Results and Discussion --- p.71 / Chapter 5.3.1 --- Evaluation of DAC production profiles of GHG232 and RTCAH transformants --- p.71 / Chapter 5.3.2 --- CPC conversion assay --- p.79 / Chapter 5.3.3 --- Medium formulation --- p.81 / Chapter 5.3.4 --- Factorial design for medium formulation --- p.85 / Chapter 5.3.4.1 --- CSL and NH4OAc --- p.85 / Chapter 5.3.4.2 --- CaC03 --- p.91 / Chapter 5.3.4.3 --- Sucrose --- p.94 / Chapter 5.3.4.4 --- Starch and soy oil --- p.97 / Chapter 5.3.4.5 --- Methionine --- p.99 / Chapter Chapter 6 --- Conclusive remarks --- p.101 / Appendix: Study of reusability of commercial plasmid extraction kit --- p.103 / Chapter A1 --- Introduction --- p.103 / Chapter A2 --- Materials and Methods --- p.105 / Chapter A2.1 --- Preparation of competent E. coli DH5a cells --- p.105 / Chapter A2.2 --- Transformation and cultivation ofE. coli DH5a cells --- p.106 / Chapter A2.3 --- Column and solutions for plasmid DNA preparation --- p.107 / Chapter A2.4 --- Plasmid preparation --- p.108 / Chapter A2.5 --- Regeneration and storage of DNA extraction column --- p.109 / Chapter A2.6 --- Yield and quality assessment of the prepared plasmid DNA --- p.109 / Chapter A3 --- Results and Discussion --- p.111 / Chapter A3.1 --- Plasmid DNA yield and purity comparison --- p.111 / Chapter A3.2 --- Column regeneration and EtOH storage --- p.111 / Chapter A3.2.1 --- DNA yield after column regeneration --- p.111 / Chapter A3.2.2 --- Purity of plasmid DNA --- p.112 / Chapter A3.2.3 --- Restriction digestion --- p.117 / Chapter A3.2.4 --- E. coli DH5α cells transformation --- p.121 / Chapter A4 --- Conclusive remarks --- p.123 / Bibliography --- p.124