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GENETIC MANIPULATION of ZYMOMONAS MOBILIS: Gene Transfer Systems, Cloning and Expression of Genes Involved in Cellulosic Bioconversion

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GENETIC MANIPULATION of ZYMOMONAS MOBILIS: Gene Transfer Systems, Cloning and Expression of Genes Involved in Cellulosic Bioconversion GENETIC MANIPULATION OF ZYMOMONAS MOBILIS: Gene Transfer Systems, Cloning and Expression of Genes Involved in Cellulosic Bioconversion A thesis submitted for the degree of DOCTOR OF PHILOSOPHY by PING SU January, 1989 Department of Biotechnology University of New South Wales UNIVERSITY CF N.S.W. 2 8 MAR 1990 B'OMED’CAL LIBRARY ACKNOWLEDGEMENTS I wish to thank Professor Pamela Rickard for allowing me to undertake a Ph.D. in the Department and for her interest in my progress. I am indebted to my supervisors, Dr.Stephen Delaney and Associate Professor Peter Rogers, for their valuable time, advice and encouragement. Special thanks are due to Associate Professor Noel Dunn and Dr. Amanda Goodman for their helpful suggestions and advice. I would also like to thank all the academic staff and students of the Department, especially Liu, Rachel, Russell, Paul, Aneta, Pittaya, Glenda, Wolf, Janette, Geoff, Phil, Baha, Rose, John and Cho for their help and friendship. I am grateful for the Postgraduate Scholarships provided by Mr. Martial Lawson of Lily vale Mushrooms Pty. Ltd. and by the University of New South Wales. Finally, I thank Jenny for her excellent typing of this thesis. w I ABSTRACT Zymomonas mobilis has been evaluated as being "an ideal ethanol producer" if the problem of its narrow substrate range could be overcome. Cellobiose is one of the main products of cellulase-catalysed breakdown of cellulose, which is the most abundant organic compound in the world. This thesis reports the development of efficient recombinant DNA techniques in Z. mobilis and construction of Zymomonas strains capable of converting cellobiose to ethanol. A rapid procedure achieving high transformation frequencies of Z. mobilis ZM6 by a range of plasmids was established. Using a hybrid plasmid, pNSW301, the highest efficiency of transformation obtained was 1.8 x 105 transformants per jig plasmid DNA. High frequency transformation was also achieved with a native Z. mobilis plasmid marked with a transposon, with large broad-host-range IncP-1 and IncW plasmids, and with small IncW cloning vectors. The kinetics of transformation and behaviour of the small IncW plasmids in Z. mobilis was investigated. A method for preparing Z. mobilis spheroplasts was developed and fusion of auxotrophic Z. mobilis ZM4 strains was achieved. A pKT230 gene bank of the genome from the cellulolytic bacterium, Xanthomonas albilineans, was screened for p-glucosidase-producing clones. The p-glucosidase from one such clone which harboured the plasmid, pNSW904, and which grew efficiently on cellobiose was partially characterised. Transfer of the p-glucosidase gene to Z. mobilis ZM6 and ZM6100 was achieved by subcloning the p-glucosidase gene onto the small broad-host-range plasmid, pRK404, followed by three-way mating involving the helper plasmid, pRK2013. Enzyme assays showed that p- glucosidase was produced by the recombinant strains ZM6901, ZM6902 and II ZM6903. Thin layer and gas chromatography of ZM6901 extracts indicated that cellobiose was consumed and glucose formed simultaneously. The glucose was further converted to ethanol yielding 13.3 mM ethanol from 5 mM cellobiose. Intact cells of ZM6901 were capable of producing 132 mM ethanol from 110 mM cellobiose after 11 days. Genes encoding p-glucosidase and endoglucanase from X. albilineans were linked on the same vector, pRK404, and then transferred to E. coli HB101 and Z. mobilis ZM6. Simultaneous expression of p-glucosidase and endoglucanase in Z. mobilis was confirmed, thus completing a further step of construction a novel cellulytic pathway in Z. mobilis. Ill LIST OF PUBLICATIONS JOURNALS Su, P. and Goodman, A.E. (1987) High frequency transformation of Zymomonas mobilis by plasmid DNA. J. Biotechnol. 6, 247-258. Su, P., Delaney, S.F. and Rogers, P.L. (1988) Kinetics of plasmid transformation in Zymomonas mobilis. J. Biotechnol. 8, 317-320. Su, P., Delaney, S.F. and Rogers, P.L. (1989) Cloning and expression of a p- glucosidase gene from Xanthomonas albilineans in Escherichia coli and Zymomonas mobilis. J. Biotechnol., in press. CONFERENCE Su, P., Delaney, S.F. and Rogers, P.L. (1989) Cloning and expression of a p- glucosidase gene from Xanthomonas albilineans in Escherichia coli and Zymomonas mobilis. Proc. 8th Aust. Biotechnol. Conf. IV TABLE OF CONTENTS Page ACKNOWLEDGEMENTS I ABSTRACT II LIST OF PUBLICATIONS IV TABLE OF CONTENTS V LIST OF TABLES X LIST OF FIGURES XI CHAPTER 1: INTRODUCTION 1 1.1 Introduction to Zymomonas mobilis 1 1.1.1 Characteristics of Z. mobilis 1 1.1.2 Carbohydrate metabolism in Z. mobilis 2 1.1.3 Ethanol production by Z. mobilis and its industrial potential 6 1.2 Development of genetic techniques forZ. mobilis 10 1.2.1 Strain selection 10 1.2.2 Strain improvement 10 1.2.3 Cloning vehicles 11 1.2.4 Systems of gene transfer 13 1.3 Conversion of cellulosic materials to ethanol and other products 15 1.4 Cloning and expression of cellulase genes 20 1.4.1 Cellulose - degrading microorganisms 20 V 1.4.2 The cellulase enzyme system 21 1.4.3 Strategies for cloning and expressing cellulase genes 23 1.4.4 Survey of cloned cellulase genes 25 1.5 Major objectives of the investigation 33 CHAPTER 2: MATERIALS AND METHODS 34 2.1 General equipment 34 2.2 Reagents, solutions and media 35 2.2.1 Reagents 35 2.2.2 Solutions 36 2.2.2.1 Saline 36 2.2.22 Saline phosphate buffer (SPB) 36 2.2.2.3 Carbon sources 37 2.2.2.4 Mcllvaine’s buffer 37 2.2.2.5 DNS reagent 37 2.22.6 Gel electrophoresis buffer (TAE) 37 2.22.1 TBE buffer 38 V 2.2.2.8 TE buffer 38 2.2.2.9 Saline sodium citrate (SSC) 38 2.2.2.10 Dilution fluid 38 2.2.3 Media 38 2.2.3.1 Luria broth (LB) 39 2.2.3.2 Minimal medium (MM) 39 2.2.3.3 Rich medium (RM) 39 2.2.3.4 Basal medium (BM) 40 2.2.3.5 Nutrient yeast broth (NYB) 40 2.2.3.6 PM medium 40 223.1 Spheroplast regeneration medium 42 2.2.3.8 Esculin plates 42 2.23.9 Carboxymethyl cellulose (CMC) - Congo red plates 42 2.2.3.10 X-Gal plates 43 2.3 Biological materials 43 2.4 Microbiological techniques 49 2.4.1 Preparation of standard bacterial inocula for enzyme work 49 2.4.2 Growth of cultures 49 2.4.3 Antibiotic supplementation of media 50 2.4.4 Estimation of bacterial concentrations 52 2.4.5 Storage of bacterial cultures 52 2.4.6 Patching 52 2.4.7 Replica plating 53 2.4.8 Stability testing of introduced plasmids 53 2.5 Recombinant DNA techniques 54 2.5.1 Plasmid isolation 54 2.5.1.1 Plasmid isolation from Z. mobilis 54 2.5.1.2 Plasmid isolation from E. coli 55 2.5.2 Electrophoresis 55 2.5.3 Determination of plasmid DNA concentration 55 2.5.4 Size determination of DNA fragments - 56 2.5.5 Restriction endonuclease digests 56 2.5.6 Physical mapping 56 2.5.7 Ligations 57 2.5.8 Electroelution of DNA from gels 57 2.5.9 Southern blotting and hybridization 58 2.5.9.1 Southern transfer 58 2.5.9.2 Preparation of hybridization probes 58 2.5.9.3 Hybridization 59 2.5.10 Transformation 59 2.5.10.1 Transformation of E. coli 59 2.5.10.2 Transformation of Z. mobilis 59 2.5.10.3 Storage of competent cells 61 2.5.11 Conjugation 61 2.5.11.1 Spot mating 61 2.5.11.2 Filter mating 61 2.5.12 Formation of Z. mobilis spheroplasts and cell wall regeneration 62 VI 2.6 Analytical procedures 62 2.6.1 Quantitative analysis of cellobiose by high pressure liquid chromatography (HPLC) 62 2.6.2 Thin layer chromatography (TLC) 63 2.6.3 Gas chromatography (GC) 64 2.6.4 Enzyme preparation for assay 64 2.6.5 p-Glucosidase assay 65 2.6.6 Cellobiase assay 66 2.6.7 Endoglucanase assay 66 2.6.8 Total protein determination 66 CHAPTER 3: HIGH FREQUENCY TRANSFORMATION OF ZYMOMONAS M OBI US BY PLASMID DNA 68 3.1 Transformation of Z. mobilis ZM6 with pNSW301 68 3.2 Effect of various factors on transformation frequency 73 3.2.1 State of the recipient cells 73 3.2.2 Effect of DNA exposure time 76 3.2.3 The effect of one or two heat shocks on transformation frequency 78 3.2.4 Effect of storage time of competent cells on cell viability and transformation 80 3.3 Kinetics of plasmid transformation in Zymomonas mobilis 82 3.4 Transformation of Z. mobilis ZM6 by other plasmids 82 3.5 Behaviour of a set of plasmids derived from Sa in Z. mobilis 91 3.5.1 Stability test of pSal52, pSa727 and pSa747 in Z. mobilis 91 3.5.2 Re-arrangement of pSal52 and pSa747 in ZM6 after long periods of growth under selective conditions 95 3.5.3 Characterisation of pNSW901 and pNSW902 97 3.6 Discussion 99 CHAPTER 4: SPHEROPLAST FUSION IN ZYMOMONAS MOBILIS 102 4.1 Spheroplast formation and regeneration 102 4.2 Optimization of lysozyme treatment for spheroplast formation 105 4.2.1 Effect of incubation time with lysozyme on spheroplast formation and regeneration in ZM6 105 4.2.2 Effect of lysozyme concentration on spheroplast formation and regeneration in ZM6 107 4.3 Effect of different hypertonic stabilizers on spheroplast regeneration in ZM6 107 4.4 Spheroplast fusion in ZM4 109 4.5 Discussion 109 VII CHAPTER 5: CLONING AND EXPRESSION OF A p-GLUCOSIDASE GENE FROM XANTHOMONAS ALBIUNEANS IN ESCHERICHIA COU 111 5.1 Selection and screening procedures 112 5.1.1 Selection via Pseudomonas 112 5.1.2 Sequential selection procedure in E.
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