Title page

Abstract

Contents

Chapter I General Introduction 24

1.1. Introduction 25

1.2. 4,5-Disubstituted aminoglycoside antibiotics and their biological action 29

1.3. Aminoglycosides antibiotics resistance genes 31

1.4. Biosynthetic study of 4, 5-disubstituted DOS containing aminoglycosides 34

1.5. Genetic studies on DOS-containing aminoglycosides 35

1.6 Objectives of this study 37

Chapter II Materials and Methods 38

2.1. Bacterial strains, vectors and recombinant plasmids 39

2.2. DNA manipulations, sequencing and hybridization 41

2.3. Construction and screening of cosmid libraries 43

2.4. Polymerase Chain Reaction (PCR) 43

2.5 Hybridization 46

2.6 Sequence analysis 46

2.7. Construction of plasmids expressing DOI synthase 47

2.7.1 pBS1 and pBS2 47

2.7.2. pDOS-2 47

2.7.3. pBS4 47

2.8. Expression of DOI synthase in E. coli 48

2.9. Expression of DOI synthase in Streptomyces lividans TK24 48

2.10. In vitro enzyme assay of DOI synthases and analyses of the products 49

2.11. Construction of plasmids expressing L-glutamine: DOI 49

2.11.1 pBS5-a and pBS5-b 50

2.11.2. pBS6 50

2.12. Expression of rbmB and neo6 in E. coli 50

2.13. Expression of rbmB in Streptomyces lividans TK24 51

2.14. Enzyme assay for L-glutamine: DOI aminotransferases 51

2.15. TLC and HPLC analyses for 2-deoxy-scyllo-inosamine 51

2.16. Construction of plasmid expressing dehydrogenase (neo5) 52

2.16.1. pBS8 52

2.17. Expression and enzyme assay for dehydrogenase 52

2.18. One pot enzyme reaction of Neo7, 6, 5 and analyses 53

2.19. Construction of plasmids for expressing nucleotidylyltransferase 53

2.19.1. pBS11 54

2.19.2. pBS12 54

2.20. Enzyme preparation and assay for nucleotidylyltransferase 54

2.21. Construction of plasmid for the expression of resistant gene 55

2.21.1. pBS3 55

2.22. Expression and assay for resistance gene 56

2.23. Construction of pGB1 for the inactivation of neo8 56

2.24. Construction of pGB3 for the inactivation of neo6 57

2.25. Construction of pGB4 for the inactivation of neo5 57

2.26. Conjugation procedure 58

2.27. Selection of mutated colonies and Southern blot hybridization 58

2.28. Construction of pGB5 and its expression in S. fradiae BS1 59

2.29. Construction of pRBMD and its expression in S. fradiae BS1 60

2.30. Protoplast transformation in S. fradiae BS1 60

2.31. Analysis of products from Streptomyces spp. 61

2.32. Accession number 61

Chapter III Ribostamycin biosynthetic gene cluster in Streptomyces ribosidificus: comparison with butirosin biosynthesis 62

3.1. Introduction 63

3.2. Results and discussions 64

3.2.1. Isolation of Rbm biosynthetic gene cluster 64

3.2.2. Comparison of ribostamycin and butirosin biosynthetic gene clusters 67

3.2.3. DOS biosynthetic genes 67

3.2.4. Nucleotidylyltransferase and glycosyltransferase gene 68

3.2.5. Resistant genes 69

3.2.6. Other genes 70

3.2.7. RbmA catalyzes the conversion of G-6-P to DOI 76

3.2.8. Functional Identification of RbmB 80

3.2.9. Identification of RacJ 83

3.2.10. Acetyltransferase gene 85

3.3. Heterologous expression of ribostamycin biosynthetic gene cluster 90

Chapter IV An analogous genetics for neomycin and ribostamycin biosynthesis from S. fradiae and S. ribosidificus: Inactivation and expression of genes involved in the biosynthesis of neomycin. 104

4.1. Introduction 105

4.2. Results 106

4.2.1. Cloning and sequencing of the neomycin biosynthetic gene cluster 106

4.2.2. Comparison of neomycin and ribostamycin gene clusters 107

4.2.2.1. Genes involved in the blosynthesis of the 2-deoxystreptamine molety 107

4.2.2.2. The proposed genes involved in the formation of neosamine and ribose 107

4.2.2.3. The proposed gene involved in the glycosylation 108

4.2.2.4. Genes involved in the resistance and regulation 112

4.2.3. Expression and inactivation of genes involved in DOS biosynthesis 115

4.2.3.1. One pot enzymatic production of DOS from glucose-6-phosphate and L-glutamine 115

4.2.4. Inactivation of neo6 122

4.2.5. Inactivation of neo5 125

4.2.6. The assay for biological activity 128

4.2.7. Functional identification of Neo16 131

4.2.8. Inactivation of neo8 133

4.2.8.1. Functional identification of Neo8 133

4.2.8.2. Construction of the pGB1 disruption vector for gene inactivation 136

4.2.8.3. Screening of mutant 138

4.2.8.4. Generation of double cross over mutant. 138

4.2.8.5. Isolated compound from the mutant and wild type S. fradiae and bioassay 140

4.2.9. Complementation experiment 142

4.3. Discussion 146

Chapter V Conclusion 151

5.1. Ribostamycin and neomycin 152

5.2. Cloning of ribostamycin and neomycin gene clusters 152

5.3. Comparison of ribostamycin and neomycin gene clusters 154

5.4. Heterologous expression of cosmid 154

5.5. One pot reaction 155

5.6. Disruption of DOS biosynthetic genes 155

5.7. Inactivation of neo8 156

Prospectives 157

개요 159

References 162

Acknowledgements 168

Publications 170

Table 2-1: Actinomycetes used in this study 40

Table 2-2: Plasmids and recombinants used in the experiments 42

Table 2-3: PCR primers used in this study 45

Table 3-1: Deduced function of ORFs in cosmid pRBM4, and their identity with Bacillus sp., other Streptomyces and Micromonospora sp. 75

Table 4-1: Acceptor binding residues of Neo8 and RbmD 110

Table 4-2: Donor binding residues in Neo8 and RbmD 111

Table 4-3: Summary of proteins identified from the Nem cluster. 114

Figure 1-1: Classification of aminoglycoside-aminocyclitols. 26

Figure 1-2: Structures of AmAcs. 27

Figure 1-3: DOS-containing AmAc antibiotics. 28

Figure 1-4: Schematic representation of Am modifying enzymes and their targets. 33

Figure 3-1: Screening of cosmids using a core sequence of DOI synthase as a probe. 65

Figure 3-2: PCR product obtained from the chromosomal DNA of positive cosmids after second screening of S. ribosidificus genomic library. 66

Figure 3-3: Butirosin and Ribostamycin biosynthetic gene clusters. Similar arrows indicate the genes encoding similar proteins. 72

Figure 3-4: Amino acid sequence alignment of RacJ homologues. 73

Figure 3-5: A proposed pathway for the formation of Ribostamycin in S. ribosidificus. 74

Figure 3-6: Construction of recombinant plasmid, pBS1, for the expression of rbmA in E. coli and to measure the activity of RbmA in vitro. 77

Figure 3-7: SDS-PAGE analysis of RbmA expressed in E. coli. 77

Figure 3-8: Construction of recombinant plasmid, pBS2, for the expression of rbmA in S. lividans TK24. 78

Figure 3-9: SDS-PAGE analysis of RbmA expressed for 3 days in S. lividans TK24. 78

Figure 3-10: HPLC analysis of oxime derivative of DOI. 79

Figure 3-11: Construction of recombinant plasmid, pBS5-a, for the expression of rbmB in E.coli and to measure the activity of RbmB in vitro. 81

Figure 3-12: SDS-PAGE analysis of RbmB expressed in E. coli. 81

Figure 3-13: Construction of recombinant plasmid, pBS5-b, for the expression of rbmB in S. lividans TK 24. 82

Figure 3-14: SDS-PAGE analysis of RbmB expressed for 3 days in S. lividans TK24. 82

Figure 3-15: Construction of recombinant plasmid, pBS11, for the expression of racJ in E. coli and to measure the activity of RacJ in vitro. 84

Figure 3-16: SDS-PAGE analysis of RacJ. 84

Figure 3-17: Construction of recombinant plasmid, pBS3, for the expression of rbmI in E. coli and to measure the activity of RbmI in vitro. 87

Figure 3-18: SDS-PAGE analysis of RbmI. 87

Figure 3-19: The activity of RbmI monitored on TLC. 88

Figure 3-20: Bioassay of RbmI acetylated AmAcs against B. subtilis. 89

Figure 3-21: TLC of isolated compound and authentic ribostamycin. 92

Figure 3-22: ESI-MS of the isolated culture broths. 94

Figure 3-23: Antibacterial assay of the metabolites against the B. subtilis: P, the metabolites from the S. lividans TK24/pOJ446; T, from the S. lividans TK24/pRBM4; W, crude ribostamycin from the wild S. ribosidificus; S, authentic ribostamycin (50 μg). 95

Figure 3-24: Reaction of ribostamycin and 9-flourenylmethyl chloroformate (FMOCl). 96

Figure 3-25: HPLC analysis of FMOCl derivatized compounds: A. derivatives of the metabolites from the S. lividans TK24/pOJ446; B. from S. lividans TK24/ pRBM4, and C. Standard ribostamycin. The thick and thin arrows point the peaks for the derivatives of ribostamyin and other impurities respectively. 98

Figure 3-26: LC-ESI-mass spectra of the compounds: A, FMOCl-derivatives of the metabolites from S. lividans/pOJ446; B, from the S. lividans/pRBM4; C authentic ribostamycin (Sigma). The peaks at 677, 899, 1121 and 1360 amu stand for [M+H+] of 3, 4, 5 and [M+H₂O+] of 6, respectively. The compounds were detected due to the incomplete derivatization of four amino groups of ribostamycin. 103

Figure 4-1: Neomycin and Ribostamycin gene cluster maps 113

Figure 4-2: Construction of recombinant plasmid, pBS4, for the expression of neo7 gene in E. coli. 117

Figure 4-3: Construction of recombinant plasmid, pBS6, for the expression of neo6 in E. coli and to measure the activity of Neo6 in vitro. 117

Figure 4-4: Construction of recombinant plasmid, pBS8, for the expression of neo5 in E. coli. 118

Figure 4-5: SDS-PAGE analysis of Neo7, Neo6 and Neo5. Lane 1, Neo7; Lane 2, Neo6; Lane3, Neo5 (+18 kDa trx tag) and Lane M, protein marker (Novagen, USA). 118

Figure 4-6: ESI-MS of the amide derivative of DOS. A. Coupling reaction product of Neo7, Neo6, and Neo5. B. Std. DOS. The arrow indicates the peak for the partial FMOCL derivative of 2-deoxystreptamine (DOS). 119

Figure 4-7: ESI mass analysis of the product (2-deoxy-scyllo-inosamine) from the coupling enzyme assay of Neo7 and Neo6 excluding the Neo5, 164.2 (M+H+). 120

Figure 4-8: A biosynthetic pathway for the formation of 2-deoxystreptamine in S. fradiae; 1. glucose-6-phosphate; 2. 2-deoxy-scyllo-inosose; 3. 2-deoxy-scyllo-inosamine; 4. 2- deoxy-3-keto-scyllo-inosamine; 5. 2-deoxystreptamine (DOS); 6. Neomycin; 7. Partial FMOCl derivative of DOS. 121

Figure 4-9: Schematic representation for the inactivation of neo6 by in-frame deletion neo7 (2-deoxy-scyllo-insose synthase) fragment was used as a probe. 123

Figure 4-10: Southern blot analysis of neo6 defective mutants. Wild-type S. fradiae (WT) and double cross-over mutants (ΔAT64, and 77,) is depicted. Genomic DNA was restricted by NcoI. 124

Figure 4-11: Schematic representation for the inactivation of neo5 by in-frame deletion 126

Figure 4-12: Southern blot analysis of neo5 defective mutants. Lane 1 and 3 represents single cross-over mutants (ΔDH1 and ΔDH2) and Lane 2 represent the wild-type S. fradiae (WT). Genomic DNA was restricted by NcoI. 127

Figure 4-13: Agarose gel picture of PCR product of tsr amplified from the total DNA of apra/tsr resistant colonies of DH mutant. Lane1-3 represents for wild type and lane 4-7 represents for mutant type. 127

Figure 4-14: The microbiological activity of neomycin production, using the wild type and ATinactived strains, was assayed against B. subtilis by the conventional agar-piece method. WT; activity of the compounds from the wild S. fradiae and other (AT38, 63, 7, 65, 73) activity of the compound from the double cross over mutants after 7 days culture plate. 129

Figure 4-15: The microbiological activity of Neomycin production, using the wild type and DHinactive strains, was assayed against B. subtilis by the conventional agar-piece method. W; activity of the compounds from the wild S. fradiae and other (DHM1 and DHM2) activity of the compound from the double cross over mutants after 7 days culture plate. 130

Figure 4-16: Construction of recombinant plasmid, pBS12, for the expression of neo16 in E. coli and to measure the activity of Neo16 in vitro. 132

Figure 4-17: SDS-PAGE analysis of Neo16. Lane 1, Insouble protein; Lane 2, Soluble protein and Lane M, protein marker (Novagen, USA). 132

Figure 4-18: Sequence homologue of Neo16 with other glycosyltransferases. RbmD S. ribosidificus, KanE and KanF, S. kanamyceticus, Gniz and GntD, M. ehinospora, TbmD, S. tenebrarius and BtrM, B. circulans. 134

Figure 4-19: Phylogenetic tree of glycosyltransferase, the multiple alignments were carried out using the Clustal W program and the figure was generated using the Tree View program. RbmD, S ribosidificus, KanE and KanF, S kanamyceticus, GntD and GntZ, M. ehinospora TobM1 and TobM2, S. tenebratius, BtrM, B. circulans. 135

Figure 4-20: Schematic representation for inactivation of neo8 by in-frame deletion. Neo7 (2- deoxy-scyllo-insose synthase) fragment was used as a probe. 137

Figure 4-21: Southern blot analysis of neo8 defective mutants. 139

Figure 4-22: Bioassay using B. subtilis as a sensitive test organism to analyze neomycin production after the isolation of products (See material and method) under identical conditions. 141

Figure 4-23: Construction of recombinant plasmid (pGB1) for the complementation of GT mutant of the S. fradiae. 143

Figure 4-24: Streptomyces fradiae (W), GT mutant (253) (GTM) and GTmutant/pSETneo8 (GTMC) growing in same plate A, 50 ㎍/㎍ tsr and B, 100 ㎍/㎍ apra and 50 ㎍/㎍ erythromycin were used. 144

Figure 4-25: S. fradiae (W), GT mutant (253) (GTM) and GTmutant/pSETneo8 (GTMC) growing in same plate with different morphology. 144

Figure 4-26: Construction of recombinant plasmid (pGB2) for the complementation of GT mutant of the S. fradiae. 145