Dissertations / Theses on the topic 'Acremonium chrysogenum'

Orientador: João Lucio de Azevedo
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Doutorado
Doutor em Genetica

Spontaneous chlorate resistant mutants of Penicillium chrysogenum and Cephalosporium acremonium have been isolated. Putative genotypes of the P. chrysogenum mutants were identified using the phenotypic characterization of Cove (1979). This analysis was also attempted with C. acremonium, however it was found that the wild type organism could not grow on minimal media containing glucose and hypoxanthine and thus cnx and niaD mutants could not be differentiated using this method. Following the distinction of cnx and niaD mutants using cytochrome C reductase and purine hydroxylase I assays, a simple plate test using minimal media containing quinic acid as carbon source and inosine as nitrogen source was developed to analyse cnx and niaD mutants. Nonreverting (at less than 1 in 108) niaD mutants were isolated for later experiments. P. chrysogenum niaD mutant niaD19 was transformed to nitrate utilization at a frequency of up to 20 transformants/mug DNA using the Aspergillus nidulans niaD gene, up to nine/mug DNA with the A. niger niaD gene and up to three/mug DNA using the A. oryzae niaD gene. Vector constructs carrying the A. nidulans ans-1 sequence with the A niger niaD gene did not show increased transformation efficiency. Linearization of the A. niger niaD containing plasmid resulted in a two to three times increase in transformation frequency. Southern blot hybridization analysis demonstrated that vector sequences had integrated into the recipient genome. The control of heterologous niaD gene expression generally agreed with that found in the wild type strain, that is, induction by nitrate and repression in the presence of ammonium. The A. nidulans, A. niger and A. oryzae genes failed to transform C. acremonium niaD mutants. A DNA fragment containing the C. acremonium niaD gene was isolated by cross hybridization to the A. nidulans niaD gene. This fragment was sub-cloned into pUC18 (designated pSTA700), and showed back hybridization to C. acremonium wild type DNA exhibiting the expected hybridization pattern. When partially sequenced, it showed nucleotide and determined amino acid homology to the A. nidulans niaD gene and protein. The clone pSTA700 transformed a C. acremonium niaD mutant, CSG116 and P. chrysogenum STP19 to nitrate utilization at a frequency of up to 47 and six transformants/mug DNA, respectively. When heat shock was applied to C. acremonium protoplasts 10mins prior to the addition of DNA, transformation frequencies of up to 137/mug DNA were obtained. Southern analysis of transformants revealed multiple integration of vector sequences, with no detectable preference for the homologous niaD site. Some C. acremonium transformants showed a greatly increased nitrate reductase activity (up to 10 times wild type activity) when induced with nitrate. The niaD transformation system was successfully used to introduce unselected markers into C. acremonium such as hygromycin B and benomyl resistance. The co-transformation frequency was up to 25% when equal molar ratios of plasmids were used. Antibiotic biosynthetic genes isolated from C. acremonium (pcbC and cefEF) and A. nidulans (pcbC and penE) were introduced into C. acremonium CSG116 by co-transformation with the C. acremonium niaD gene or the construction of vectors containing both the C. acremonium niaD gene and antibiotic biosynthetic genes. Isolates transformed with the C. acremonium pcbC and cefEF genes exhibited an increase in antibacterial activity (greater than 20% compared to wild type) at frequencies of up to 10% and six percent of transformants, respectively. When A. nidulans constructs containing the pcbC and penE genes, but not the pcbC gene alone, were co-transformed into C. acremonium, up to 18% of transformants exhibited an increase in antibacterial activity. The presence of penicillin antibiotics could not be found in these transformants and thus the exact cause of this increase in antibacterial activity could not be determined.

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