MICROBIAL GENETICS APPLIED TO BIOTECHNOLOGY PRINCIPLES AND TECHNIQUES OF GENE TRANSFER AND MANIPULATION MICROBIAL GENETIC5 APPLIED TO BlOT ECH NOLOGV PRINCIPLES AND TECHNIQUES OF GENE TRANSFER AND MANIPULATION VENETIA A. SAUNDERS AND JON R. SAUNDERS CROOM HELM London & Sydney © 1987 Venetia A. Saunders and Jon R. Saunders Croom Helm Ltd, Provident House, Burrell Row, Beckenham, Kent BR3 1AT Croom Helm Australia, 44-50 Waterloo Road, North Ryde, 2113, New South Wales British Library Cataloguing in Publication Data Saunders, Venetia A. Microbial genetics applied to biotechnology. 1. Microbial genetic engineering 2. Biotechnology I. Title II. Saunders, Jon R. 660' .62 TP248.6 ISBN-13 : 978-1-4615-9798-8 e-ISBN-13 : 978-1-4615-9796-4 DOl : 10.1007/978-1-4615-9796-4 Typeset in Times Roman by Leaper & Gard Ltd, Bristol, England For our son MARK Contents Preface 1. Introduction 1 2. In Vivo Genetic Manipulation 3 3. In Vitro Genetic Manipulation 54 4. In Vivo and In Vitro Mutagenesis 163 5. Optimisation of Expression of Cloned Genes 219 6. Microbial Strain Improvement and Novel Products 265 7. Medical and Veterinary Applications 306 8. Plant Technology 333 9. Environmental Biotechnology 384 10. Conclusion 407 Index 411 Preface This book describes techniques of microbial genetics and how they may be applied to biotechnology. The text is concerned largely with the application of these techniques to microbial technology. We have therefore utilised illustrative material that is given in our own courses in applied micro biology. The book assumes in the reader a basic knowledge of microbial genetics and industrial microbiology. We hope it will prove useful to under graduates, postgraduates and others taking courses in applied micro biology. We would like to thank various colleagues, including John Carter, Julian Davies, Gordon Dougan, David Hopwood, Gwyn Humphreys, Alan McCarthy, David O'Connor, Tony Hart, Steve Oliver, Roger Pickup, Hilary Richards, Bob Rowlands, David Sherratt, Peter Strike, Richard Sykes and Liz Wellington, all of whom provided information at various stages during the writing of this book. Many thanks are also due to Linda Marsh for patiently typing the many drafts of the manuscript. 1 Introduction Natural genetic variation has always been exploited by man to improve the properties of microbial strains. Spontaneous mutations that arise in micro bial populations and that have properties advantageous to man have been gradually selected over centuries of use. However, it is only since the development of modem genetic techniques that more rational approaches have been possible. Such newer technologies have permitted the tailoring of microorganisms, plant or animal cells to manufacture specific products of commercial or social benefit and to manage the environment. It is the intention of this book to demonstrate how fundamental princi ples of microbial genetics have been applied to biotechnology. Microorgan isms have a wide range of physiological capabilities not encountered in higher organisms. Bacteria and fungi can also normally be grown in culture under controlled conditions. Furthermore, microorganisms possess rela tively simple genetic organisations and are more amenable to manipulation than higher organisms. Genetics is a discipline par excellence for breaking down and analysing complex biological problems and for using the inform ation so obtained for the rational manipulation of biological processes. Fundamental studies in genetics have provided both direct and indirect benefits to biotechnology by permitting a better understanding of the biology of microorganisms. This has generated an infrastructure of know ledge, particularly for bacteria, such as Escherichia coli, and fungi, such as Saccharomyces cerevisiae, that has, in tum, promoted the development of modem manipulative techniques. During recent years much emphasis has been placed on the role of in vitro recombinant DNA technology (see Chapters 3, 4 and 5) in stimulating a resurgence of biotechnology. Although it is true that in vitro techniques do provide a radical new ingredient in the exploitation of biological systems, it should not be forgot ten that many commercially important applications of microbial genetics involve the use oftraditional in vivo gene manipulations (see Chapters 6, 7, 8 and 9). Moreover, the development of much in vitro genetic manipula- 1 INTRODUCfION tion technology has been strictly dependent on 'conventional' in vivo techniques (Chapters 2 and 4). }The ability to exploit microbial genetics has depended to a great extent on gene transfer and other evolutionary mechanisms found in natural populations of microorganisms. Crucial to the development of genetic technologies has been an understanding of the biology of plasmid and virus genomes. Such extrachromosomal elements, which are widespread in both bacteria and fungi, provide important vehicles for the natural exchange of genes both within and between species (see Chapter 2). These elements are relatively small, autonomously replicating, nucleic acid molecules, and are particularly amenable to manipulation by both in vivo and in vitro genetic techniques. Furthermore, many extrachromosomes are present in host cells at copy numbers greater than chromosomes. This provides a means of amplifying genes that the element happens to carry. Plasmids may be classified into incompatibility (Inc) groups based on the ability of pairs of plasmids to coexist in the same cell. Those plasmids that fail to coexist belong to the same Inc group. Incompatible plasmids share homologous systems for controlling replication and hence copy number (see Chapter 5). The diversity of Inc groups in different bacteria and fungi has permitted the construction of a variety of useful genetic vectors, of both broad and narrow host range, for gene manipulation (see Chapters 2, 3 and 5). This book describes in vivo and in vitro genetic manipulation methodol ogies and considers examples of their application to biotechnology. Genetics can be used for the deliberate enhancement of existing attributes of microorganisms (see Chapter 6) and for their manipulation to provide novel properties or products (see, for example, Chapters 6 and 7). Examples of the application of the principles of microbial genetics to veterinary and human medicine (Chapter 7), to agriculture (Chapter 8) and to the management of the environment (Chapter 9) are also consid ered. 2 2 In Vivo Genetic Manipulation Genes can be shuffled within and between microbial species by a variety of mechanisms. Such mechanisms lead to the formation of new genotypes by bringing together and reassorting genes from different organisms. In bacteria new combinations of genes may be generated by using one of the three natural processes of gene transfer, namely transformation (section 2.2), conjugation (section 2.3) or transduction (section 2.4). In fungi, genetic exchange can be effected through the agency of the sexual (section 2.5) or parasexual (section 2.6) cycle. Transformation systems are also available for gene transfer in yeasts and certain filamentous fungi. Proto plast fusion (section 2.7) provides a further route for combining groups of genes from different strains and modifying the genetic constitution of microorganisms. These various processes for manipulating genomes enable the formation of innumerable genetic combinations, in tum producing variability within microbial populations. Transposable genetic elements (section 2.1) provide another source of variability among microorganisms. These elements can insert into and excise from a variety of replicons and promote a number of genome rearrangements. Transposable elements can cause mutation when they interrupt the coding sequence of a gene. Various properties of these elements make them useful for manipulating genomes. This chapter describes mechanisms for gene manipulation in vivo. 2.1 TRANSPOSABLE GENETIC ELEMENTS 2.1.1 Properties of transposable elements Transposable genetic elements are segments of DNA that are capable of inserting as discrete nonpermuted DNA sequences at various sites within a genome. These elements have been found in the genomes of a variety of microorganisms, including bacteria, fungi and bacteriophages, as well as in 3