Table Of Content

GENE EXPRESSION IN RECOMBINANT MICROORGANISMS BIOPROCESTSE CHNOIX)GY Series mitor W. Courtney McGregor Xoma Corporation Berkeley, California 1. Membrane Separations in Biotechnology, edited by W. Courtney McGregor 2. Commercial Production of Monoclonal Antibodies: A Guide for Scale- Up, edited by Sally S. Seaver 3. Handbook on Anaerobic Fermentations, edited by Larry E. Erickson and Daniel Yee-Chak Fung 4. Fermentation Process Developmento f Industrial Organisms, edited by Justin 0. Neway 5. Yeast: Biotechnology and Biocatalysis, edited by Hubert Verachtert and Rend De Mot 6. Sensors in Bioprocess Control, edited by John V. Twork and Alexan- der M. Yacy n ych 7. Fundamentals of Protein Biotechnology, edited by Stanley Stein 0. Yeast Strain Selection, edited by Chandra J. Panchal 9. Separation Processes in Biotechnology, edited by Juan A. Asenjo 10. Large-scale Mammalian Cell Culture Technology, edited by Anthony S. Lubiniecki 11. Extractive Bioconversions, edited by Bo Mattiasson and Olle Holst 12. Purification and Analysis of Recombinant Proteins,e dited by Ramnath Seetharam and Satish K. Sharma 13. Drug Biotechnology Regulation: Scientific Basis and Practices, edited by Yuan- yuan H. Chiua nd John L. Gueriguian 14. Protein Immobilization: Fundamentals and Applications, edited by Richard F. Taylor 15. BiosensorP rinciples and Applications, edited by Loi'c J. Blum and Pierre R. Coulet 16. Industrial Application of Immobilized Biocatalysts, edited by Atsuo Tanaka, Tetsuya Tosa, and Takeshi Kobayashi 17. Insect Cell Culture Engineering, edited by Mattheus F. A. Goosen, Andrew J. Daugulis, and Peter Faulkner 18. Protein Purification Process Engineering, edited by Roger G. Harrison 19. Recombinant Microbes for Industrial and Agricultural Applications, edited by Yoshikatsu Murooka and Tada yuki lmanaka 20. Cell Adhesion: Fundamentals and Biotechnological Applications, edited by Martin A. Hjortso and Joseph W. Roos 21. Bioreactor System Design, edited by Juan A. Asenjo and Josd C. Merchuk 22. GeneE xpressioni nR ecombinantM icroorganisms, edited by Alan Smith ADDITIONAL VOLUMES IN PREPARATION GENE EXPRESSION IN RECOMBINANT MICROORGANISMS edited by Alan Smith CIBA-GEIGY AG Basel, Switzerland MarceDl ekker, Inc. NewY orkB. asel HongK ong Library of Congress Cataloging-in-Publication Data Gene expression in recombinant microorganismlse dited by Alan Smith. p.c m. - (Bioprocesst echnology ; 22) Includes bibliographical references and index. ISBN 0-8247-9543-1 1. Microbial genetics. 2. Recombinant microorganisms. 3. Gene expression.4 .G enetice ngineering-Methodology.I .S mith,A lan. II. Series. QH434.G149 49 4 660’.65-d~20 94-35435 CP The publisher offers discounts on this bowokh en ordered in bulk quanti- ties. For more information, writtoe Special Sales/Professional Marketing at the address below. This book is printed on acid-free paper. Copyright 6 1995 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor anyp art may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Marcel Dekker, Inc. 270 Madison Avenue, New York, New York 10016 Current printing (last digit): l 0 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA Series Introduction Bioprocess technology encompasses all oft he basic and applied sciences as well as the engineering requiredto fully exploit living systems and bring their productst o the marketplace. The technologtyh at develops is even- tually expressed in various methodologies and types of equipment and instruments builtu p along a bioprocess stream. Typically in commercial production, the streamb egins at the bioreactor, which can be a classical fermentor, ac ell culture perfusion system, oarn enzyme bioreactor. Then comes separation of the product from the living sysatnedm/os r their com- ponents followed by an appropriate number of purification steps. The stream ends with bioproduct finishing, formulation, and packaging. A given bioprocess stream may have some tributaries or outlets and may be overlaid with a variety of monitoring devices and control systems. with any stream, it will both shape and be shaped with time. Docu- As menting the evolutionary shapinogf bioprocess technology is the purpose of this series. Now that several products from recombinant DNA and cell fusion techniques are on the market, the new era of bioprocess technology is well established and validated. Books of this series represent developments ili iv Series Introduction in various segments of bioprocessing that have paralleled progress in ltihfee sciences. For obvious proprietary reasons, some developments in industry, although validated, mayb e published only lateri,f at all. Therefore, our continuing series willf ollow the growtho f this fielda s it is available from both academia and industry. W. Courtney McGregor Preface The last decade has witnessed a remarkable growth in the development of recombinant DNA technology. Applications inclutdhee use of geneti- cally engineered microorganismfos r the productiono f biopharmaceuticals such as insulin and interferon; the early diagnosis of disease; gene ther- apy for inherited diseases; and the creation of transgenic animals for food production, for production of therapeutics, and for use in screen- ing for new biologically active substances. Agricultural applications include the development of new plant strains, of plants resistant to pests and pesticides, and the deliberate release of engineered organisms for crop protection. The technology has also been exploitedt o further our understanding of basic metabolism and its regulation. This in turn has led to new approaches int he production of substances derivefdr om mul- tigene pathways such as antibiotics. Slowlyb,u t inevitably, the once im- penetrable barriers between species have been broken down. This rapidly developing field has also been accompanied ab yp ublic debate about the ethics of gene manipulation and about environmental and safety aspects. It is not the intention of this bookt o address thew hole field of gene technology, but rather to focus on one of the least contentious areas-gene V vi Preface expression in recombinant microorganisms. Neverthelesths,e chapter on regulatory and safety aspects will confront the environmental question and attempt to put into perspective the risksa nd benefits of this exciting technology now at our disposal. Scientists with international reputations have been recruited to com- pile up-to-date reviews on gene expression in recombinant microorganisms. Each chapter provides a specialist’s account of gene expression in one of the important classes of microorganism usteod e xpress both homologous and heterologous genes. Each chapter describes the state-of-the- art for the expression system, provides examples of the expression of homologous and heterologous genes, documents the industriaaln d commercial exploitation of the system, and speculates on future prospects and developments. More precisely, the features discussed include host strain characteristics, selectable markers, protease-deficient strains, trans- formation systems, plasmid vectors, integrating vectors, copy number, stability, constitutivea nd regulated promoters, activators, secretion sig- nals, posttranslational modification, glycosylation, S -S bridges, and protein folding. Where appropriate, the authors describe aspects of meta- bolic regulation and fermentation process technology. The individual chapters are “validated”b y extensive referencet o the published literature. The chapter on regulatory and safety aspects reviews current legisla- tion in the United States, England, Europaen, d elsewhere. The measures taken for large-scale operation are also addressed, including the evolu- tion of good industrial large-scale practice (GILSP), as regulatory au- thorities seek to achieve a balance between safety requirements and regulatory excess. The gram-negative bacteriumE scherichia colih as been thef ront-runner in the commercial application of rDNA technologayn d there are highly developed fermentation processes operating on a large scale for well- established products. Joan Stader’s chapter describes how this leading position is based on the expanding knowledgaeb out the basic biology of this organism. The availability of a range of regulated promoters provides numerous options for efficient gene expression. In particular, the system basedo n the T7-RNA polymerase ais p owerful tool for high-level expression of heterologous genes. However, this organism brings with it a number of problems. Among thema re the difficulties protein secre- of tion across the dual membrane system of the gram-negative bacterium, the contamination of products with endotoxins, and the reluctance with which correctly folded proteins containing disulfide bridges are produced. The synthesis of fusion proteins is one way to overcome some of these