[christina smolke] the metabolic pathway engineeri(bookfi.org)

THE METABOLIC PATHWAY

ENGINEERING HANDBOOK

Fundamentals

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The Metabolic Pathway Engineering Handbook, 1st Edition

The Metabolic Pathway Engineering Handbook: Fundamentals

The Metabolic Pathway Engineering Handbook: Tools and Applications

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THE METABOLIC PATHWAY

ENGINEERING HANDBOOK

Edited by

Christina D. Smolke

Fundamentals

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CRC PressTaylor & Francis Group6000 Broken Sound Parkway NW, Suite 300Boca Raton, FL 33487-2742

2010 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business

No claim to original U.S. Government worksPrinted in the United States of America on acid-free paper10 9 8 7 6 5 4 3 2 1

International Standard Book Number-13: 978-1-4398-0296-0 (Hardcover)

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Library of Congress Cataloging-in-Publication Data

The metabolic pathway engineering handbook : fundamentals / editor, Christina D. Smolke.p. ; cm.

Includes bibliographical references and index.ISBN 978-1-4398-0296-0 (hardcover : alk. paper)1. Genetic engineering--Handbooks, manuals, etc. 2. Biosynthesis--Handbooks, manuals, etc. I.

Smolke, Christina D. II. Title.[DNLM: 1. Genetic Engineering--methods. 2. Metabolic Networks and Pathways. 3. Biological

Products--metabolism. 4. Biotechnology--methods. 5. Models, Biological. QU 450 M5871 2010]

TP248.6.M478 2010660.65--dc22 2008051635

Visit the Taylor & Francis Web site athttp://www.taylorandfrancis.comand the CRC Press Web site athttp://www.crcpress.com

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vContents

Introduction .............................................................................................................................. ixEditor .......................................................................................................................................... xvContributors ........................................................................................................................... xvii

I Cellular MetabolismSeCtIon

Andy Ekins and Vincent J.J. Martin 1 Solute Transport Processes in the Cell .................................................................... 1-1

Adelfo Escalante, Alfredo Martnez, Manuel Rivera, and Guillermo Gosset

2 Catabolism and Metabolic Fueling Processes ....................................................... 2-1Olubolaji Akinterinwa and Patrick C. Cirino

3 Biosynthesis of Cellular Building Blocks: The Prerequisites of Life ............... 3-1Zachary L. Fowler, Effendi Leonard, and Mattheos Koffas

4 Polymerization of Building Blocks to Macromolecules: Polyhydroxyalkanoates as an Example ................................................................... 4-1Si Jae Park, Soon Ho Hong, and Sang Yup Lee

5 Rare Metabolic ConversionsHarvesting Diversity through Nature .............................................................................................................. 5-1Manuel Ferrer and Peter N. Golyshin

II Balances and Reaction ModelsSeCtIon

Walter M. van Gulik 6 Growth Nutrients and Diversity ............................................................................... 6-1

Joseph J. Heijnen

7 Mass Balances, Rates, and Experiments ................................................................. 7-1Joseph J. Heijnen

8 Data Reconciliation and Error Detection ............................................................... 8-1Peter J.T. Verheijen

9 Black Box Models for Growth and Product Formation ...................................... 9-1Joseph J. Heijnen

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vi Contents

10 Metabolic Models for Growth and Product Formation .................................... 10-1Walter M. van Gulik

11 A Thermodynamic Description of Microbial Growth and Product Formation ...................................................................................................... 11-1Joseph J. Heijnen

III Bacterial transcriptional Regulation of SeCtIon Metabolism

James C. Liao12 Transcribing Metabolism Genes: Lessons from a Feral Promoter................. 12-1

Alan J. Wolfe

13 Regulation of Secondary Metabolism in Bacteria .............................................. 13-1Wenjun Zhang, Joshua P. Ferreira, and Yi Tang

14 A Synthetic Approach to Transcriptional Regulatory Engineering .............. 14-1Wilson W. Wong and James C. Liao

IV Modeling tools for Metabolic engineeringSeCtIon

Costas D. Maranas15 Metabolic Flux Analysis ............................................................................................ 15-1

Maria I. Klapa

16 Metabolic Control Analysis ...................................................................................... 16-1Joseph J. Heijnen

17 Structure and Flux Analysis of Metabolic Networks ........................................ 17-1Kiran Raosaheb Patil, Prashant Madhusudan Bapat, and Jens Nielsen

18 Constraint-Based Genome-Scale Models of Cellular Metabolism ................ 18-1Radhakrishnan Mahadevan

19 Multiscale Modeling of Metabolic Regulation ................................................... 19-1C.A. Leclerc and Jeffrey D. Varner

20 Validation of Metabolic Models ..............................................................................20-1Sang Yup Lee, Hyohak Song, Tae Yong Kim, and Sung Bum Sohn

V Developing Appropriate Hosts for SeCtIon Metabolic engineering

Jens Nielsen21 Escherichia coli as a Well-Developed Host for

Metabolic Engineering ............................................................................................... 21-1Eva Nordberg Karlsson, Louise Johansson, Olle Holst, and Gunnar Lidn

22 Metabolic Engineering in Yeast .............................................................................. 22-1Maurizio Bettiga, Marie F. Gorwa-Grauslund, and Brbel Hahn-Hgerdal

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Contents vii

23 Metabolic Engineering of Bacillus subtilis ........................................................... 23-1John Perkins, Markus Wyss, Hans-Peter Hohmann, and Uwe Sauer

24 Metabolic Engineering of Streptomyces ................................................................24-1Irina Borodina, Anna Eliasson, and Jens Nielsen

25 Metabolic Engineering of Filamentous Fungi ..................................................... 25-1Mikael Rrdam Andersen, Kanchana Rucksomtawin, Gerald Hofmann, and Jens Nielsen

26 Metabolic Engineering of Mammalian Cells .......................................................26-1Lake-Ee Quek and Lars Keld Nielsen

Index .......................................................................................................................................... I-1

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ix

Introduction

Progression of Biological Synthesis Methods toward Commercial Relevance

The advent of recombinant DNA in the 1970s brought transformative technologies for the synthesis and manipulation of artificial genetic material. The ability to amplify, cut, and piece together fragments of DNA outside of a cell and to get (or transform) that DNA into a cell of interest resulted in a set of molec-ular cloning tools that enabled the field of genetic engineering. In genetic engineering, foreign DNA that encodes for new or altered functions or traits is inserted into an organism of interest. Many early applications of recombinant DNA technology focused on heterologous protein production in microbial hosts. The first medicine made through recombinant DNA technology that was approved by the United States Federal Drug Administration was the synthesis of synthetic human insulin in Escherichia coli. This was an important early application of recombinant DNA technology, as the success of producing a safe and effective synthetic hormone in a bacteria led to the widespread acceptance of the technology and significant resources and funding to be directed to its support and advancement.

As the technologies in support of synthesizing and manipulating artificial DNA matured and advanced, so did the applications to which they were applied. The early successful applications of recom-binant DNA technology resulted in alternative routes to the synthesis of medicines, such as insulin, human growth factor, and erythropoietin, vaccines, and even genetically modified organisms, includ-ing crops that exhibit more desirable traits. Technologies were developed for the manipulation of artifi-cial DNA in both prokaryotic and eukaryotic host organisms, including mammalian and plant cells. In addition, inspired by the diversity of natural products, chemicals, and materials synthesized by biologi-cal systems that are observed in the natural world, researchers began to look beyond applications that were limited to the synthesis of a single heterologous protein product in a cellular host to more com-plicated engineering feats. In particular, these new applications focused on the manipulation of sets or combinations of proteins, or enzymes, that acted in conjunction in a cell, within metabolic pathways, to convert energy and precursor chemicals into desired natural and non-natural products.

The production of chemicals, materials, and energy through biology presents an alternative to tra-ditional chemical synthesis routes. While the development of chemical synthesis methods for the production of valuable chemicals and small molecule pharmaceuticals is a more mature field and has demonstrated significant successes, many chemicals remain difficult to be synthesized through such strategies, particularly those with many chiral centers. Biological catalysts, or enzymes, have dem-onstrated remarkable adeptness at the synthesis of very complex molecules. In addition, cellular bio-synthesis strategies offer several advantages over traditional chemical synthesis strategies in that the former is often conducted under less harsh conditions, thereby enabling green synthesis strategies that are associated with the production of fewer toxic by-products. In addition, cellular biosynthesis

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x Introduction

takes advantage of the cells natural ability to replenish enzymes and cofactors and to provide precursors from often inexpensive and renewable starting materials. Such advantages are particularly compelling in light of the global challenges we face today in energy, the environment, and sustainability.

However, new challenges are presented when manipulating the metabolic pathways in cellular hosts that link energy sources and starting materials to products of commercial interest. The unique challenges faced in engineering metabolic pathways, when compared to the early genetic engineering applications of heterologous protein production, require the development of new enabling technologies, spanning experimental and analytical techniques and computational tools.

the Field of Metabolic engineering

Metabolic engineering is a field that includes the construction, redirection, and manipulation of cellular metabolism through the alteration of endogenous and/or heterologous enzyme activities and levels to achieve the biosynthesis or biocatalysis of desired compounds. Researchers in metabolic engineering often view the biological system as a chemical factory that is converting starting materials to different value-added products. Because the yield or productivity of the process is linked to its commercial viabil-ity, the ability to precisely regulate the flow of energy and materials through different cellular pathways becomes critical to the optimization of the overall process, drawing parallels to the more traditional engineering discipline of chemical process design.

The basic tenet of metabolic engineering, the use of biology as a technology for the conversion of energy, chemicals, and materials to value-added products, has a long history. Early applications can be cited, even prior to the development of recombinant DNA technology, in the food and beverage industry where more traditional methods of strain development based on evolution, mating, and selection strate-gies were used to develop more desired production hosts for particular applications. However, recom-binant DNA technology enabled the capability to introduce new enzymatic activities and pathways into production hosts allowing access to different energy resources and starting materials and to the production of different chemicals and materials. Such technologies support the forward design of more complex synthetic pathways in host organisms or the targeted manipulation of endogenous pathways, enabling more directed manipulation of the cellular host. Current metabolic engineering efforts are focused on the synthesis of products such as chemical commodities, small molecule drugs, and alterna-tive energy sources including biofuels. In addition, significant effort is also directed to the engineering of host metabolisms to utilize renewable, low cost energy resources.

Many of the challenges faced in metabolic engineering are related to the engineering of energy and material flow within complex systems. More specifically, metabolic pathways make up complex inter-connected networks in cells, which can rarely be manipulated in isolation of the rest of the network. Highlighting the interconnections between cellular metabolites is the fact that all metabolites are made from a set of 12 common precursors. In addition, the flow of metabolites through a network of enzymes, and in the background of other cellular enzymes that may exhibit activity on these metabolites, is often controlled through layered processes that act at different time scales, implement dynamic feedback con-trol, and utilize localization and transport. Metabolic engineering requires a breadth of skill sets to tackle different points of system design and as a result has developed into a very interdisciplinary field. Researchers with expertise spanning a variety of disciplines, including chemical engineering, biological engineering, environmental engineering, biochemistry, molecular biology, cell biology, bioinformatics, and control theory, are working in different areas of metabolic engineering. However, as an academic endeavor, metabolic engineering has remained an interdisciplinary research discipline with courses covering aspects of the field depending on the expertise of the department in which it is taught.

As it has matured, metabolic engineering has gained greater industrial significance. Initial industrial interest was directed to the synthesis of chemical commodities in microorganisms largely at groups within larger chemical companies. However, many smaller startup companies have developed in recent years that are focused on the synthesis of specialty chemicals such as pharmaceuticals and biofuels, on

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Introduction xi

the development of computational and modeling programs to direct metabolic engineering efforts, and on the discovery and development of new enzyme activities in support of engineering new synthetic pathways into host organisms. The intersection of metabolic engineering, with other emerging areas of systems and synthetic biology, presents exciting opportunities to develop solutions to many of the global challenges we face in energy, the environment, health and medicine, resources, and sustainability, and will likely continue to fuel a significant sector of the biotechnology industry in future years.

An overview of the Metabolic Pathway engineering Handbook

The purpose of The Metabolic Pathway Engineering Handbook is to provide a thorough overview of the field of metabolic engineering. Each section provides an overview of different aspects of a particular topic that is a central component of the field by experts in that area. Sections are introduced by section editors to provide a perspective on the topic and a description of how the chapters in that section link together to form an integrated overview of that particular topic. The sections are split into two books, where the content of the first book focuses on fundamentals or basic principles of metabolic engineer-ing and the second book focuses on tools and applications in metabolic engineering. Due to its orga-nization, the handbook can be used as a reference book and read for individual sections or chapters, or it can be used as a book for advanced courses in metabolic engineering.

Section I in The Metabolic Pathway Engineering Handbook: Fundamentals provides an overview of the basic processes that support cellular metabolism. The boundary of a cell is defined by its cellu-lar membrane, which acts to separate cellular constituents from the environment. Metabolism begins with systems that allow the import of nutrients and starting materials across the cellular membrane and efforts to engineer transport systems for particular chemicals have been important strategies in enabling cells to convert those chemicals to desired products. Once inside the cell, nutrients are broken down into common precursors for metabolic syntheses, which provide the energy and reducing power necessary for cell survival. In addition, precursors are channeled into the synthesis of important build-ing blocks that the cell then utilizes to build larger macromolecules, including lipids, nucleic acids, and proteins. An understanding of the central metabolic pathways and the general flow of metabolism through a small number of common precursors and carriers is critical to being able to effectively link new synthetic nutrient or product pathways to endogenous metabolisms. Finally, the wealth of untapped diversity in nature, particularly in the microbial biosphere, provides significant opportunities in har-vesting new enzymatic activities from nature that can be applied to the production of new chemicals and materials in engineered hosts.

Section II provides an overview of mass balances and reaction models applied to predicting product formation and microbial growth in fermentation processes. Various models have been proposed and utilized in the field that exhibit varying levels of detail to provide predictions of product yield and cell growth. Conversion rates are calculated from mass balances and rate equations that take into account the basic nutrients and constituents of cellular systems. Different models, such as those based on ther-modynamic or metabolic network constraints, can be utilized to predict product yield and cell growth in fermentation processes. Different models may be more or less appropriate based on the specifics of the fermentation. The application of such models to experimental systems can allow minimization of error in detection strategies resulting in optimized control schemes for fermentations based on such experimental measurements.

Section III provides an overview of transcriptional regulation of metabolic pathways in bacterial systems. Bacterial cells use a variety of mechanisms to regulate the transcription of enzymes involved in primary and secondary metabolisms. Transcriptional regulatory strategies exist that regulate a small set of genes in response to specific environmental chemicals, such as operon-specific regulation and two-component systems. However, other strategies exist that regulate larger sets of genes in response to significant environmental changes such as heat shock or nitrogen starvation, through sigma factors and global transcriptional factors. An understanding of the strategies used to regulate the expression of

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xii Introduction

enzymes in a cellular host is critical in metabolic engineering to developing effective strategies to alter the expression of endogenous enzymes and to design synthetic systems that exhibit more sophisticated regulatory schemes to balance and coordinate the expression of multiple enzymes to ultimately opti-mize flux through desired pathways.

Section IV is an overview of modeling tools that have been developed for metabolic engineering applications. Earlier modeling and computation efforts that resulted in tools for metabolic flux analysis (MFA) and metabolic control analysis (MCA) have been very powerful for the elucidation of fluxes and control strategies in metabolic networks given partial sets of data. Computation tools based on network and graph concepts have enabled structure and flux analyses that provide optimization tools for meta-bolic engineering. In addition, metabolic network reconstruction and modeling efforts have resulted in genome-scale models of cellular metabolism for specific organisms based on sets of constraints that enable prediction of flux distributions under different conditions. Whereas multi-scale modeling tools are extending current predictive capabilities by integrating stoichiometry, kinetics, and regulatory and control responses in metabolic networks, such multi-scale tools can be utilized by metabolic engineers to predict the dynamic metabolic response.

Section V provides an overview of common cellular hosts that are used in metabolic engineering appli-cations. In particular, the bacterial hosts Escherichia coli, Bacillus subtilis, and Streptomyces have been utilized in various metabolic engineering applications, with E. coli being the most well-developed and utilized host largely due to the genetic tools available for manipulating pathways in this host organ-ism. In addition, two lower eukaryotic hosts, yeast and filamentous fungi, have been utilized in various metabolic engineering applications for the production of natural products or for pathway enzymes that are more readily expressed in functional forms in eukaryotic organisms. Finally, much effort has also been put toward the development of mammalian cell culture hosts for the production of metabolites and products that are more readily produced in mammalian cells. Each host may present advantages and disadvantages in the synthesis of a desired chemical based on the genetic tools available for manipulating pathways and the endogenous metabolism and processing pathways present in that organism, such that the selection of a suitable host is driven largely by the properties of the pathway of interest.

Section I in The Metabolic Pathway Engineering Handbook: Tools and Applications provides an over-view of the evolutionary tools widely in use in the engineering of metabolic enzymes and networks. Evolutionary strategies have been traditionally used in metabolic engineering to select for desired phe-notypes in host organisms. As biological organisms naturally undergo processes of evolution and selec-tion, design strategies that integrate evolutionary engineering objectives with metabolic engineering objectives may result in a more robustly performing engineered cellular system. Directed evolution is a laboratory tool that is used to mimic the evolutionary process in a test tube, by generating diversity in cellular components and then screening or selecting through this diversity for optimized component properties. Various experimental strategies have been utilized for generating and screening through component diversity. In addition, computational tools have been developed that optimize the design of laboratory evolution strategies. These experimental and computational tools have been applied to the directed evolution of enzymes, regulatory systems, pathways, and whole genomes for the optimization of flux through targeted metabolic pathways.

Section II provides an overview of gene expression tools that have been utilized in metabolic engi-neering applications. Various tools have been developed that regulate DNA copy number and enable chromosomal engineering in host organisms. In addition, a variety of other genetic tools have been developed that precisely regulate gene expression levels through post-transcriptional and translational mechanisms. Still other tools have been developed that regulate the activity of enzymes through post-translational engineering strategies. The application of the tools described in this section is critical to balancing the expression of multiple enzymes, such that individual conversion steps do not limit prod-uct yield, toxic intermediates do not accumulate, and cellular resources and energy are efficiently uti-lized by the host cell. Several examples exist of engineered systems that have utilized such genetic tools for the optimization of flux through metabolic pathways.

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Introduction xiii

Section III provides an overview of emerging technologies and their application to metabolic engi-neering. Genome-wide technologies that allow global profiling of cellular transcripts, proteins, metabo-lites, and phenotypes are critical for efficient troubleshooting and debugging of engineered systems. Bioinformatics tools that allow for management and analysis of the vast amounts of data collected from these techniques are also critical. As these technologies mature and become more available, their imple-mentation as standard techniques in metabolic engineering will improve our understanding of the engi-neered system response and result in efficient troubleshooting and optimization strategies.

Section IV provides an overview of key future prospects in metabolic engineering. The integration of new computational tools, such as genome-scale models, and new technologies for analyzing and under-standing complex systems, such as systems biology, with metabolic engineering are rapidly advancing the success with which metabolic networks can be forward engineered. In addition, alternative strate-gies to cellular biosynthesis that remove complications associated with engineering living, evolving sys-tems, such as cell-free synthesis systems, have demonstrated impressive successes. Finally, the modeling and optimization of engineered metabolic pathways in silico, prior to construction and characterization, will significantly transform the field of metabolic engineering and integrate advances in computational modeling, systems biology, and engineering design.

Section V provides an overview of common tools that are utilized to determine flux through meta-bolic pathways. Various types of isotope flux labeling strategies have been widely used to monitor flux through metabolic pathways, where the data from such experiments are typically integrated into the modeling tools described in Section IV. In addition, various analytical strategies are utilized to profile cellular metabolites, where current and future efforts have been focused on developing strategies to profile and quantify global metabolite levels.

Section VI provides an overview of various metabolic engineering application areas. One broad application area is focused on the engineering and regulation of the energy state, cofactor supply, and redox balance of cellular hosts. This is a challenge that affects most if not all metabolic engineering applications, where the introduction of new pathways or the manipulation of endogenous pathways can result in imbalances in cellular pathways and stress responses. Metabolic engineering applications are generally directed toward the synthesis of commercially relevant molecules including specialty or com-modity chemicals, small molecule drugs, or alternative energy sources. Each of these application areas of metabolic engineering presents distinct challenges that must be addressed in the process design based on chemical and pathway complexity, market cost of the product, volume demand of the product, end use of the product, and purity requirements.

Metabolic engineering: Looking toward the Future

Metabolic engineering as a field has evolved significantly over the past 10 to 15 years in large part due to the scientific and technological advances made during this time frame in support of this application area. The future prospects of metabolic engineering are extremely exciting, and as other supporting scientific and engineering fields mature it is likely to see transformative advances that direct it further toward an engineering discipline. There are several key supporting fields that will aid in directing this transformation.

First, enzyme engineering and enzyme discovery will be critical to expanding the diversity of natural and non-natural products that can be produced in engineered organisms. Much of the living world has not been cultured and characterized. Even in those organisms that have been cultured, we do not have genome sequence information, have not mapped functions to many of the sequenced genes, or have not characterized many of the enzyme activities in these organisms. For example, many pathways in plants responsible for the synthesis of diverse pharmacologically relevant molecules have not been elucidated, although many of these activities and their corresponding genes are currently present in large expressed sequence tag (EST) libraries. Because we cannot forward design enzymes to exhibit specific catalytic

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xiv Introduction

activities, the existing limitations in characterized enzyme activities severely limit the pathways that we can reconstruct in organisms. In addition, programs that will allow us to predict and design enzyme function from sequence will be critically enabling for the design of new activities that have not been recovered from natural systems.

Second, because metabolic engineering is largely a systems engineering challenge, continued advances in systems biology will provide important insights into the function of biological systems that will inform engineering design and strategies directed at manipulating metabolic pathways. Many analytic techniques in support of systems biology, including strategies that allow global profiling of transcript, protein, and metabolite levels, are providing vast amounts of information regarding levels of cellular constituents under different conditions. In addition, computational tools are being developed to process the vast amounts of data coming from these techniques. Newer and future efforts in systems biology must focus on taking the information coming from these techniques and abstracting from it the organizing principles governing cellular metabolism and regulation. An understanding of how cells generally layer metabolic pathways with different regulatory strategies will allow engineers to design more robustly performing synthetic pathways that are better integrated with the endogenous metabolic pathways. In addition, such understanding will allow better identification of manipulation points in endogenous networks to alter flux through pathways.

Third, the integration of information theory and control theory with systems biology and metabolic engineering will likely have a significant impact on our understanding of biological systems. Such tools will enable a deeper understanding of architectures and properties of complex networks that support robustness, evolvability, and fragility of the system, providing a conceptual framework to systems biol-ogy. In addition, such tools will allow researchers to more quantitatively examine models of control schemes around metabolic pathways to better elucidate the design principles around regulating flux through metabolic pathways. Such tools can also be used to examine synthetic network and control scheme designs and guide the more effective design of engineered systems.

Finally, metabolic engineering is seeing a transformation with the emerging field of synthetic biology. Synthetic biology is the design, construction, and characterization of biological systems using engineer-ing design principles. To support a framework for engineering biology, synthetic biology is rooted in foundational technologies that enable the construction of more complex, heterologous networks in liv-ing systems. With advances in DNA sequencing and synthesis it is becoming common practice to syn-thesize entire genes and pathways from scratch, no longer limiting researchers to the physical DNA that they obtain from natural organisms. In addition, abstraction frameworks have been proposed to enable rapid assembling and reassembling of basic biological components (or parts) into larger networks (or devices) and systems, supporting the rapid prototyping and troubleshooting and reliable construction of complex metabolic pathways in cellular hosts (or chassis). An example of a synthetic biology approach to the rapid prototyping of a metabolic pathway in Escherichia coli was recently described (http://parts.mit.edu/wiki/index.php/MIT_2006). There are also efforts directed to the engineering of specific chas-sis, or cellular hosts, optimized for metabolic engineering applications. Finally, enabling genetically encoded technologies are being developed for use in precise and quantitative manipulation of pathway components such as enzymes.

Christina D. SmolkeEditor-in-Chief

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xvxv

Editor

Christina Smolke is an assistant professor in the Department of Bioengineering at Stanford University. She graduated with a BS in chemical engineering with a minor in biology from the University of Southern California in 1997. She conducted her graduate training as a National Science Foundation Fellow in the Chemical Engineering Department at the University of California at Berkeley and earned her PhD in 2001. Christina conducted her postdoctoral training as a National Institutes of Health Fellow in cell biology at UC Berkeley. She started her independent research program as an assistant professor in the Division of Chemistry and Chemical Engineering at the California Institute of Technology from 20032008. She has pioneered a research program in developing foundational technologies for the design and construction of engineered ligand-responsive RNA-based regulatory molecules, their integration into molecular computation and signal integration strategies, and their reliable implementation into diverse cellular engineering applications. These technologies are resulting in scaleable platforms for the construction of molecular tools that work across many cellular systems and allow regulation of targeted gene expression levels in response to diverse endogenous or exogenous molecular ligands. Her research is rapidly advancing current capabilities of noninvasive detection of cellular state and programming cellular function. In particular, her laboratory is examining the application of these tools to the optimi-zation of metabolic pathway engineering strategies in organisms such as yeast.

Dr. Smolkes innovative research program has recently been recognized with the receipt of a National Science Foundation CAREER Award, a Beckman Young Investigator Award, an Alfred P. Sloan Research Fellowship, and the listing of Dr. Smolke as one of Technology Reviews Top 100 Young Innovators in the World. She is also a member and adjunct faculty of the Comprehensive Cancer Centers Cancer Immunotherapeutics Program at the City of Hope, where she has several translationally oriented col-laborative projects exploring the clinical applications of these technologies. She is the inventor of over nine patents and serves on the Scientific Advisory Board of Codon Devices. Dr. Smolke is currently serving as the President of the Institute of Biological Engineering. She is a member of AIChE, ACS, the RNA Society, and IBE.

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xvii

Contributors

olubolaji AkinterinwaDepartment of Chemical

EngineeringPennsylvania State UniversityUniversity Park, Pennsylvania

Mikael Rrdam Andersen

Center for Microbial Biotechnology

BioCentrum-DTUTechnical University of DenmarkLyngby, Denmark

Prashant Madhusudan Bapat



Maurizio BettigaDepartment of Applied

MicrobiologyLund UniversityLund, Sweden

Irina BorodinaCenter for Microbial

BiotechnologyBioCentrum-DTUTechnical University of DenmarkLyngby, Denmark

Patrick C. CirinoDepartment of Chemical

EngineeringPennsylvania State UniversityUniversity Park, Pennsylvania

Andy ekinsDepartment of BiologyCentre for Structural and

Functional GenomicsConcordia UniversityMontreal, Quebec, Canada

Anna eliassonCenter for Microbial


Adelfo escalanteCellular Engineering Biocatalysis

Department Biotechnology InstituteNational Autonomous University

of MexicoCuernavaca, Mexico

Joshua P. FerreiraDepartment of Chemical and

Biomolecular EngineeringUniversity of CaliforniaLos Angeles, California

Manuel FerrerDepartment of BiocatalysisInstitute of CatalysisConsejo Superior de

Investigaciones CientficasMadrid, Spain

Zachary L. FowlerDepartment of Chemical and

Biological EngineeringState University of New York

at BuffaloBuffalo, New York

Peter n. GolyshinDepartment of Environmental

MicrobiologyHZI-Helmholtz Centre for

Infection ResearchBraunschweig, Germany

Marie F. Gorwa-Grauslund

Department of Applied Microbiology

Lund UniversityLund, Sweden

Guillermo GossetCellular Engineering Biocatalysis



Brbel Hahn-HgerdalDepartment of Applied

MicrobiologyLund UniversityLund, Sweden

Joseph J. HeijnenBioprocess Technology GroupDepartment of BiotechnologyDelft University of TechnologyDelft, the Netherlands

Gerald HofmannCenter for Microbial

Biotechnology, BioCentrum-DTUTechnical University of DenmarkLyngby, Denmark

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xviii Contributors

Hans-Peter HohmannDSM Nutritional Products LtdBasel, Switzerland

olle HolstDepartment of BiotechnologyLund UniversityLund, Sweden

Soon Ho HongDepartment of Chemical

Engineering and Bioengineering

University of UlsanUlsan, Republic of Korea

Louise JohanssonDepartment of Chemical

EngineeringLund UniversityLund, Sweden

eva nordberg KarlssonDepartment of BiotechnologyLund UniversityLund, Sweden

tae Yong KimDepartment of Chemical and

Biomolecular EngineeringCenter for Systems and Synthetic

BiotechnologyInstitute for the BioCenturyKorea Advanced Institute of

Science and TechnologyDaejeon, Korea

Maria I. KlapaDepartment of Chemical and

Biomolecular EngineeringInstitute of Chemical Engineering

and High-Temperature Chemical Processes

Foundation for Research and Technology-Hellas

Patras, Greece

Mattheos KoffasDepartment of Chemical and



C.A. LeclercDepartment of Chemical

EngineeringMcGill UniversityMontreal, Quebec, Canada

Sang Yup LeeDepartment of Chemical and




effendi LeonardDepartment of Chemical and



James C. LiaoChemical and Biomolecular

Engineering DepartmentUniversity of CaliforniaLos Angeles, California

Gunnar LidnDepartment of Chemical

EngineeringLund UniversityLund, Sweden

Radhakrishnan Mahadevan

Department of Chemical Engineering and Applied Chemistry

Institute of Biomaterials and Biomedical Engineering

University of TorontoToronto, Ontario, Canada

Costas D. MaranasDepartment of Chemical

EngineeringPennsylvania State UniversityFenske LaboratoryUniversity Park, Pennsylvania

Vincent J.J. MartinDepartment of BiologyCentre for Structural and

Functional GenomicsConcordia UniversityMontreal, Quebec, Canada

Alfredo MartnezCellular Engineering Biocatalysis



Jens nielsenSystems BiologyDepartment of Chemical

and Biological Engineering

Chalmers University of Technology

Gothenburg, Sweden

and



Lars Keld nielsenAustralian Institute for

Bioengineering and Nanotechnology

The University of QueenslandBrisbane, Australia

Si Jae ParkCorporate R&DLG Chem, Ltd.Daejeon, Republic of Korea

Kiran Raosaheb PatilCenter for Microbial


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Contributors xix

John PerkinsDSM Nutritional Products LtdBasel, Switzerland

Lake-ee QuekAustralian Institute for

Bioengineering and Nanotechnology

The University of QueenslandBrisbane, Australia

Manuel RiveraCellular Engineering Biocatalysis



Kanchana Rueksomtawin



Uwe SauerInstitute for Molecular

Systems BiologyETH ZrichZrich, Switzerland

Christina D. SmolkeDivision of Chemistry and

Chemical EngineeringCalifornia Institute of

TechnologyPasadena, California

Seung Bum SohnDepartment of Chemical and




Hyohak SongDepartment of Chemical and




Yi tangDepartment of Chemical

and Biomolecular Engineering

University of CaliforniaLos Angeles, California

Walter M. van GulikBioprocess Technology GroupDepartment of BiotechnologyDelft University of TechnologyDelft, the Netherlands

Jeffrey D. VarnerDepartment of Chemical and

Biomolecular EngineeringCornell UniversityIthaca, New York

Peter J.t. VerheijenDepartment of BiotechnologyDelft University of TechnologyDelft, the Netherlands

Alan J. WolfeDepartment of Microbiology and

ImmunologyLoyola University at ChicagoStritch School of MedicineMaywood, Illinois

Wilson W. WongChemical and Biomolecular

Engineering DepartmentUniversity of CaliforniaLos Angeles, California

Markus WyssDSM Nutritional Products LtdBasel, Switzerland

Wenjun ZhangDepartment of Chemical and

Biomolecular EngineeringUniversity of CaliforniaLos Angeles, California

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I-1

ICellular MetabolismAndy Ekins and Vincent J.J. MartinConcordia University

1 Solute Transport Processes in the Cell Adelfo Escalante, Alfredo Martnez, Manuel Rivera, and Guillermo Gosset .....................................................................................1-1Introduction Structure and Function of the Bacterial Membrane The Transporter Classification (TC) System

2 Catabolism and Metabolic Fueling Processes Olubolaji Akinterinwa and Patrick C. Cirino ..........................................................................................................................2-1Introduction Classification of Organisms Thermodynamics of Fueling Processes Products of Fueling Processes Redox Potentials and Mobile Electron Carriers Examples of Catabolic Processes in Different Organisms Concluding Remarks

3 Biosynthesis of Cellular Building Blocks: The Prerequisites of Life Zachary L. Fowler, Effendi Leonard, and Mattheos Koffas .....................................3-1Introduction Amino Acid Biosynthesis Nucleotides as Building Blocks Synthesis of Carbohydrates for Building Cells Cell Synthesis of Lipids

4 Polymerization of Building Blocks to Macromolecules: Polyhydroxyalkanoates as an Example Si Jae Park, Soon Ho Hong, and Sang Yup Lee ................................................................................................................................4-1Introduction PHAs PHA Synthases Metabolic Engineering of Microorganisms for PHA Production Conclusion

5 Rare Metabolic ConversionsHarvesting Diversity through Nature Manuel Ferrer and Peter N. Golyshin .......................................................5-1Introduction How Diverse Are Functional Groups? Diversity of Enyzmes and Current Frontiers for Bioconversions Main Chemical Conversions Mediated by Enzymes: Putative Rare Conversions How Can New Catalytic Functions Be Achieved? Recent Advances in Metagenomics: The Untapped Reservoir of Proteins from Unculturable Microbes

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I-2 Cellular Metabolism

THE FIELD OF METABOLIC ENGINEERING has advanced over time and will undoubtedly continue to do so, based on a solid scientific foundation and continued research and innovation. Of critical importance is a solid understanding of the metabolism of the cell which will ulti-mately be manipulated through various techniques to produce a desired end product, or alternatively, remove or breakdown an undesirable one.

This section describes the well defined knowledge of how, in particular Escherichia coli, is able to transport a variety of nutrients and use such nutrients, through a variety of metabolic pathways, to derive energy and synthesize the wide spectrum of cellular components required for the maintenance of a cell. In some instances, the alteration of such metabolic pathways of a particular cell may lead to the production of a desired product, while in others the synthesis of the desired product may require the introduction of foreign genes, isolated and characterized from other organisms, in order to allow synthesis to proceed. Furthermore, with the advent of metagenomics, one can sift out genes allowing unique metabolic conversions which have yet to have been described in cultured microorganisms.

While the organism of choice for many metabolic engineering studies is E. coli, all cells are, by defi-nition, enveloped by a membrane which separates cellular components from the extracellular environ-ment. Highly efficient transport and export systems have evolved to allow exchange across this barrier. A sound knowledge of the transport systems which import the nutrients required for cell growth and drive the metabolism of the cell is essential in order to ensure that the desired metabolic pathway receives the necessary precursors and energy required for the production of a selected compound. As the outer membrane of E. coli is only capable of allowing the passive diffusion of molecules with a molecular weight less than approximately 600 Da, it is of utmost importance to determine if the import of precur-sors, for instance, can cause a bottleneck in the synthesis of a particular compound. Additionally, the type of transport system present in the cell can have an impact on the carbon flux within a cell. As an example, modification of the E. coli phosphotransferase system has been a strategy successfully applied to metabolically engineered strains (Gosset, 2005). Manipulation of the transport systems can increase the diversity of nutrients imported while manipulation of the regulatory systems of the cell can allow the simultaneous import and use of multiple carbon sources, as is the case for carbon catabolite repres-sion mutants for example (Dien et al., 2002).

As nutrients are catabolized, precursors for metabolic syntheses are generated along with the energy and reducing power required to drive the synthesis of all the components required by the cell. Solid knowledge of the metabolic pathways within the cell allows one to ensure that the proper pre-cursors, reducing power and energy are in ample supply in order to produce the molecule of interest. Furthermore, culture components and conditions can be altered to enhance the efficiency of a particular metabolic conversion.

In some instances, one may wish to overproduce a compound in an organism that does not naturally produce said compound. One such example is the production of polyhydroxyalkanoates (PHAs) in E. coli, an organism that does not naturally produce PHAs. It is therefore necessary to express PHA synthase genes isolated from foreign organisms. Additionally, in order to increase production of the desired PHA, it is crucial to evaluate the metabolic flux of the host organism and perhaps amplify certain endogenous pathways to increase the availability of precursors and reducing power, without decreasing the overall health of the expressing organism. In the case of poly (3-hydroxybutyrate) [P(3HB)] production, it was found that over expression of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydroge-nase led to an increase in the NADPH/NADP+ ratio and a subsequent rise in the concentration of P(3HB), there was however a detrimental effect on the producing cells and the observed increase in P(3HB) produc-tion was due to a lower cell concentration (Lim et al., 2002). In another experiment, 2-D gel electrophoresis and metabolic flux analysis was performed on E. coli producing P(3HB) and it was revealed that there was an increase in certain glycolytic pathway enzymes. Subsequently, amplification of the glycolytic pathway enzymes led to increased production of acetyl-CoA, which could subsequently be used to increase yields of P(3HB) (Park and Lee, unpublished results).

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Cellular Metabolism I-3

The pursuit of rare conversions have, up to this point, focused on the ability of cultured organisms and their enzymes to perform such functions. The products of such rare conversions are invaluable to a variety of industries spanning the agricultural, pharmaceutical, food additive and bioremediation fields, as examples. While many advances have been made in the realm of protein engineering using techniques such as directed evolution, it is reasonable to assume that an even greater diversity exists within the genomes of unculturable microorganisms. The diversity of the cultured microbial world has led to the discovery of many rare conversions, there remains, however, a large pool of untapped gen-etic material within the many unculturable microorganisms that are currently estimated to represent close to 99% of the microbial world (Ftterer et al., 2004). With the knowledge that each sequenced microbial genome yields on average 3050% of genes with unknown function (Bode and Mller, 2005) and the recent shotgun sequencing of DNA isolated from the Sargasso Sea revealed greater than 1.2 mil-lion genes of unknown function (Venter et al., 2004) it would appear reasonable to assume that there exists a vast pool of untapped genetic resources that can be applied to all realms of biotechnology.

References

Bode, H.B., and Mller, R. The impact of bacterial genomics on natural product research. Angew. Chem. Int. Ed. Engl., 44, 6828, 2005.

Dien, B.S., Nichols, N.N., and Bothast, R.J. Fermentation of sugar mixtures using Escherichia coli catabo-lite repression mutants engineered for production of L-lactic acid. J. Ind. Microbiol. Biotechnol., 29, 221, 2002.

Ftterer, O., et al. Genome sequence of Picrophilus torridus and its implications for life around pH 0. Proc. Natl. Acad. Sci. USA., 101, 9091, 2004.

Gosset, G. Improvement of Escherichia coli production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system. Microb. Cell. Fact., 4, 14, 2005.

Lim, S.J. et al. Amplification of the NADPH-related genes zwf and gnd for the oddball biosynthesis of PHB in an E. coli transformant harboring a cloned phbCAB operon. J. Biosci. Bioeng. 93, 543, 2002.

Venter, J.C. et al. Environmental genome shotgun sequencing of the Sargasso Sea. Science, 304, 66, 2004.

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1-1

1.1 Introduction .......................................................................................1-11.2 Structure and Function of the Bacterial Membrane ....................1-2 Structure of the Cellular Membrane Functions Kinetics of

Transport Processes1.3 The Transporter Classification (TC) System .................................1-8 Channels and Pores Electrochemical Potential-Driven

Transporters Primary Active Transporters Group Translocators Transmembrane Electron Flow Systems

References ....................................................................................................1-19

1.1 Introduction

The cell membrane constitutes a hydrophobic barrier that isolates the cytoplasm from the external medium. The entry and exit of most of the nutrients required for cell growth and the byproducts gener-ated by metabolism are highly restricted by this cellular structure. However, to sustain high growth rates, microbes require a high rate of nutrient import. The presence of specialized transport proteins in the mem-brane allows the cell to circumvent the permeability restrictions imposed by this barrier. Analyses of micro-bial genomes have revealed that approximately 10% of the genes encode proteins involved in transport [1]. These transport systems participate in the import and export of different classes of molecules and also in other important cellular functions. They allow the entry of nutrients to sustain metabolism and ion species to maintain concentration gradients leading to membrane potential and energy generation. Transporters allow the excretion of metabolite by-products and other toxic substances, like drugs or certain metal ions. Transport systems also participate in the secretion of lipids, carbohydrates, and proteins into membrane(s) or the external medium. They enable the transfer of nucleic acids between organisms, contributing to microbial diversity. Finally, transporters participate in the uptake of different types of signaling molecules like alarmones and hormones, among others, thus allowing cellular communication [2].

Solute transport and metabolism are linked processes in the cell. Genetic organization in bacteria frequently reflects this functional coupling by the clustering of genes encoding both transport and met-abolic activities in transcriptional units. This association is generally observed in operons encoding catabolic pathways for carbon sources [3]. Transport and regulatory systems participate in the process whereby the bacterial cell can select from a mixture of nutrients those that afford the highest growth rate [4]. In addition, the differential expression of genes encoding distinct transporters for a specific substrate allow the cell to select the transport mechanism according to the physiological state and envi-ronmental conditions [5].

Transport systems are potential targets for modification with the aim of microbial production strain improvement. Metabolic engineering efforts usually focus on modifying metabolic enzyme activities.

1Solute Transport

Processes in the Cell

Adelfo Escalante, Alfredo Martnez,Manuel Rivera, and Guillermo GossetNational Autonomous University of Mexico

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1-2 Cellular Metabolism

However, it can be envisioned that high performance production strains will also require the modifica-tion of other cellular functions, including transport. Modification of transport systems can result in the improvement of several cellular properties including: (a) increasing the range of carbon source uti-lization [6]; (b) increasing metabolic precursor availability for the synthesis of amino acids, shikimate pathway intermediates, TCA cycle intermediates, and fermentation products like ethanol [710]; (c) increasing the efficiency in sugar mixture utilization by partial disruption of catabolic repression [11]; and (d) controlling overflow metabolism, thus reducing acetate production [12].

1.2 Structure and Function of the Bacterial Membrane

1.2.1 Structure of the Cellular Membrane

The cell membrane, also known as the cytoplasmic membrane, plasma membrane, or cell surface mem-brane, is a thin structure that surrounds the cell. It is the barrier that defines the boundaries of the cell, separating the cytoplasm from its environment. If the membrane is damaged, the integrity of the cell is altered and the cytoplasm leaks into the environment, causing cell death. The general structure of prokaryotic and eukaryotic cell membranes (and the outer membranes of Gram-negative bacteria) is a bilayer composed of phospholipids, which contain both hydrophobic (fatty acid) and hydrophilic (glycerol-phosphate) components. It can exist in many chemical forms as a consequence of a diversity of compounds attached to the glycerol backbone. As phospholipids aggregate in aqueous solution they spontaneously organize to form two parallel rows, known as a lipid bilayer.

Phospholipid molecules align with the fatty acids pointing inward toward each other to form a hydro-phobic environment, whereas the hydrophilic portions face both the external side and the internal or cytoplasmic side of the membrane. The bilayer structure represents the most stable arrangement of the lipid molecules in an aqueous environment. The whole structure of the plasma membrane is stabi-lized by hydrogen bonds and hydrophobic interactions. In addition, cations such as Mg2+and Ca2+help to stabilize the membrane due to ionic interactions with the negative charges of the phospholipids. A model of the structures of the bacterial cell membranes of Gram-positive and Gram-negative bacteria is shown in Figure 1.1 [1315].

An important amount of protein and other materials is partially or completely embedded in the membrane layer. A typical bacterial membrane contains up to 200 different kinds of proteins (approxi-mately 75% of the mass of the membrane). Protein molecules in the membrane are arranged in a variety of ways. Some proteins are fully embedded in the membrane and are thus called integral or transmem-brane proteins. They can be removed from the membrane only after disrupting the lipidic bilayer. Some of these proteins are channels that have a pore, through which substances enter and exit the cell. Other proteins, called peripheral, are easily removed from the membrane by mild treatments and are firmly associated with the inner or outer surface of the membrane. They may function as enzymes that catalyze chemical reactions, as scaffolds for support of cell components, and as mediators of changes in mem-brane shape during movement. Some peripheral membrane proteins contain a lipid tail on the amino terminus that anchors the protein to the membrane. These proteins are called lipoproteins and interact directly with integral proteins in important cellular processes such as energy metabolism.

Many proteins and some of the lipids on the outer membrane of the plasma membrane have carbohy-drates attached to them. These structures are known as glycoproteins and glycolipids, respectively. Both of these structures help to protect the cell and are involved in cell-to-cell interactions.

Sterols and related molecules are present in eukaryotic membranes. They are rigid and planar molecules, whereas fatty acids are flexible; their presence stabilizes and makes membranes less flexible. Sterols are absent in prokaryotic cellular membranes, except for methanotrophs and mycoplasms. Polycyclic com-pounds known as hopanes (derivatives of pentacyclic triterpenoides) are widely distributed among bacteria, and it is proposed that they may play a role in maintaining membrane rigidity (Figure 1.2). One widely dis-tributed hopane is the C30 hopanoid diploptene. Hopanes are not present in species of Archaea [16].

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Solute Transport Processes in the Cell 1-3

Lipopolysaccahride

Gram-positive

Gram-negative O-specific side chains

Outermembrane

Cytoplasmicmembrane

Periplasmic spaceand cell wall

Transmembrane proteinsPeripheral proteins

Phospholipids

Cytoplasmicmembrane

Murein lipoprotein

Murein

FIguRE 1.1 Cell membranes of Gram-positive and Gram-negative bacteria. Schematic representation of the inner and outer membrane lipid bilayers of Gram-negative bacteria (upper panel) and Gram-positives (lower panel). Several structures associated to cell membranes such as porins, integral or transmembrane and peripheral proteins, and cell wall components are shown.

HO

CH3

CH3

CH

CH3

H2CH2CH2CC

CH3

H3C

H

(a)

CH3 CH3

CH3

CH3

CH3CH3OH OH

OH OH(b)

FIguRE 1.2 Structure of membrane sterols and hopanoids. (a) Structure of the cholesterol molecule, a typical sterol present in cell membranes of eukaryotic cells, methanotrophic bacteria, and mycoplasmas. (b) Structure of a hopane, a polyterpenoid present in prokaryotic cell membranes.

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Membranes have a viscosity similar to that of light-grade oil. Experimental evidence has demon-strated that at temperatures that permit growth, membrane molecules are not static but move quite freely within the membrane surface. Individual lipid molecules are also generally free to exchange places with another lipid in the membrane, resembling a two dimensional fluid. It is proposed that this movement is most probably associated to the functions of the plasma membrane. These dynamics of phospholipids and proteins are known as the fluid mosaic model [17]. However, it is also proposed that some membrane regions have considerable order, because some lipids molecules are not free due to their relationship with specific membrane proteins and some other components [18].

The phospholipids of the cell membrane from bacteria contain ester linkages bonding the fatty acids to glycerol whereas in Archaea the membrane lacks fatty acids (Figure 1.3). Instead, their side chains are composed of repeating units of the five carbon hydrocarbon isoprene that is linked to glycerol by an ether bond; however, the overall architecture of the cytoplasmic membrane of Archaea, forming an inner and outer hydrophilic surfaces with a hydrophobic interior, is the same as in bacteria.

Glycerol diethers and glycerol tetraethers are the major lipids present in membranes from Archaea. In the tetramer molecule, the phytanyl side chains (composed of four linked isoprenes) from each glyc-erol molecule are covalently bonded together (Figure 1.4), leading a lipid monolayer instead of a bilayer cytoplasmic membrane. This structure is widely distributed among hyperthermophilic Archaea helping to maintain the membrane architecture at high temperatures [1920].

1.2.2 Functions

The most important function of the cell membrane is to serve as a selective barrier through which mate-rial enters and exits the cell. The cytoplasm consists of an aqueous solution containing salts, sugars, amino acids, nucleotides, vitamins, coenzymes, proteins, and a variety of other soluble materials. The hydrophobic nature of the internal region of the plasma membrane constitutes a tight diffusion barrier with selective permeability, allowing certain molecules and ions to pass through and blocking passage to others. Some smaller molecules, such as water, oxygen, carbon dioxide, and some simple sugars, usually pass freely through the membrane by diffusion (Table 1.1). This is also the case for molecules that are dissolved easily in lipids (oxygen, carbon dioxide, and nonpolar organic molecules). In contrast, hydrophilic and small charged molecules such as the hydrogen ion (H+) do not pass through the membrane but instead must be specifi-cally transported. Water is a molecule that freely crosses the membrane, because it is sufficiently small to pass through the phospholipid bilayer. However, water transport through the membrane can be accelerated

H2C

HC

H2C

O

O

C

P

O

O

O

HC

H2C

O

O

C

P

O

O

R

O

H2CC

CH3

CH

CH2

O C

R

O

O

RH2C O C R

(a) (b) (c)Ester

Ether

FIguRE 1.3 Chemical diversity of lipidic bonds in cell membranes. (a) An ester linkage found in lipids of bacteria and eukaryotic cells. (b) An ether linkage of lipids of cell membrane of Archaea. (c) Structure of isoprene, the parent structure of the hydrophobic side chains present in Archaea.

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by specific transport proteins called aquaporins. The movement of most hydrophilic solutes across plasma membranes depends on transporter molecules, which will be described in the following sections [21].

1.2.3 Kinetics of transport Processes

A solute transport system consists of integral membrane proteins that can be regarded as membrane-bound enzymes. However, instead of catalyzing the conversion of substrates to products, they mediate

(a) Glycerol diether

Lipid bilayer

C

H

C

H O

C

H O

CH2

CH2

H

H O R

C

H

C

HO

C

HO

CH2

CH2

H

HOR

Ether linkage Phytanyl

PhytanylCH3 group

Glycerolphosphate

(b) Diglycerol tetraether

Lipid monolayer

C

H

C

H O

C

H O

CH2

H

H O H

CH2

C

H

C

HO

C

HO

H

H

HOCH2

CH2

Biphytanyl

FIguRE 1.4 Structures of the Archaea cell membranes. (a) Schematic representation of bilayers of isoprenoids linked to glycerol by ether bonds. (b) Structure of the monolayers of the isoprenoid biphytanyl glycerol ether.

TaBLE 1.1 Comparison of Diffusion-Controlled and Carrier Mediated Solute Fluxes across Bacterial Plasma Membranes

Typical Transfer Rate [mol min-1 (g Dry Mass) -1]

Diffusion-Controlled at a Concentration Difference of

Transported Solute 10 M 10 mM Carrier Mediated (Vmax)

Potassium ion (K+) 0.00002 0.02 100Glutamate


the transfer of solutes between compartments separated by a membrane. Each transport system displays an affinity and specificity toward particular substrate(s). It is not uncommon to find in one organ-ism more than one transporter for a solute, each having a different affinity and specificity, a situation analogous to that of isozymes in metabolism. In addition, there is considerable diversity with regard to energetic coupling mechanisms that drive active transport. Although transport processes are not fully understood, some models are helpful to understand basic molecular events [22,23]. The function of a transporter can be defined in three basics steps, analogous to those of enzyme activity: binding, trans-location, and release of solute. The translocation step can involve a major conformational change of the transporter protein, thus defining either of two functional conformations.

Diffusion plays an important role in solute transport across the lipid bilayer membrane. This pro-cess can occur either in the absence of or mediated by specific protein transporters. By measuring the transport rate of a solute it is possible to determine if the diffusion process is transporter mediated. In a transporter-independent process, there is a linear increase in the rate of diffusion with an increase in solute concentration, as shown in Figure 1.5. In contrast, in transporter mediated diffusion, a maximum value is reached. This response is explained considering that the transporter proteins become saturated once the solute substrate reaches a specific high concentration.

The processes that govern the simple diffusion of an electrically neutral molecule were studied by Adolf Eugen Fick in the 1800s and is applied to the special case of a cell membrane. The measurement of the concentrations of a solute Sx outside and inside a cell allows us to predict if it is in equilibrium across the cell membrane or whether Sx would tend to passively move into the cell or out of it. As long as the movement of Sx is not coupled to the movement of another substance or to some biochemical reaction, the only factor determining the direction of the net transport is the difference in concentration. The ability to predict the movement of Sx is independent of any detailed knowledge of the actual transport pathway mediating its passive transport.

When the concentration of an external solute Sx ([Sx]o) is greater than its internal concentration ([Sx]i), the net movement of Sx will be into the cell. The movement of Sx is described by its flux (Jsx), namely, the number of moles of Sx crossing a unit area of membrane (typically 1 cm2) per unit of time (s), i.e., moles/(cm2s). High Sx solubility in the membrane lipids (the higher the lipidwater partition coefficient of Sx) will correspond to a higher flux through the membrane barrier. The flux of Sx will also be greater if Sx moves more readily once it is in the membrane (higher diffusion coefficient) and if the distance that it must traverse is short (membrane thickness). These three factors form part of the parameter called the perme-ability coefficient of Sx (Psx). Finally, the flux of Sx will be greater as the difference in [Sx] between the two sides of the membranes increases (gradient). All these concepts are integrated in the equation known as Fick s law:

Substrate concentration (S)

Rate

of d

iffus

ion

Carrier mediated transport plus diffusion

Carrier mediated transport

Diffusion

FIguRE 1.5 Comparison of solute transport kinetics in the presence and absence of a transporter.

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Js Ps Sx Sxx x o i= -( )[ ] [ ] (1.1)The net f lux of Sx can be decomposed into a unidirectional inf lux (Jsxoi) and a unidirectional

eff lux (Jsxio). The net f lux of Sx, as shown in Equation 1.1, is simply the difference between the unidirectional f luxes. Thus, the unidirectional inf lux is proportional to the outside concentration, the unidirectional eff lux is proportional to the inside concentration, and the net f lux is propor-tional to the concentration difference; in all cases the proportionality constant is PSx. The following equation allows the calculation of the electrochemical energy difference or electrochemical poten-tial of Sx (Sx). This parameter combines values of concentration and voltage gradients across a membrane:

Sx=RT ln([Sx]i/[Sx]o)+zXF(io) (1.2)

where zX is the charge of Sx, T is the absolute temperature, R is the gas constant, and F is the Faraday constant; this part of the equation defines the electrical energy difference. The term RT ln([Sx]i/[Sx]o) describes the energy (joules/mole) change as Sx moves across the membrane; it is a measure of the chemical energy difference. The term zXF(io) describes the energy change as a mole of charged par-ticles (each with a valence of zX) moves across the membrane. The difference (i0) is a voltage differ-ence across the membrane (membrane potential difference, Vm). By definition, Sx is at equilibrium when the electrochemical potential difference for Sx across the membrane is zero (Sx=0).

When Sx is not zero, its value represents the net driving force, causing Sx to either enter or leave the cell, provided that a pathway exists for it to cross the membrane. It is important to consider a couple of spe-cial cases for the equilibrium state equation (Equation 1.2). In the first case, when either the chemical or the electrical term in this equation is zero, for instance, when Sx is uncharged (zX=0), as in the case of glucose, then equilibrium occurs only when [Sx] is equal at both sides of the membrane. Alternatively, when Sx is charged as in the case of the Na+, and the electrical potential difference and thus Vm are zero, equilibrium likewise occurs only when [Sx] is equal on both sides of the membrane. The second case is when neither the chemical nor the electrical term in Equation 1.2 is zero; equilibrium occurs when the two terms are equal but of opposite signs. This relationship is the Nernst equation, when Sx from Equation 1.2:

Vm=EX=(RT/zXF) ln ([Sx]i/[Sx]o) (1.3)

Hence, the Nernst equation describes the conditions when an ion is in equilibrium across a membrane. Given values for [Sx]i and [Sx]o, Sx can be in equilibrium only when the voltage difference across the membrane equals the equilibrium potential (EX), also know as the Nernst potential [24].

Transport processes can be studied by applying some of the tools employed for enzyme kinetics anal-yses. Transport can be regarded as a reaction, where substrate location, and not its structure, is changed. Mathematical/graphical analyses of experimental data, similar to those used in enzyme kinetics, can be performed. Plots such as velocity versus substrate concentration (Michaelis and Menten model; Equation 1.4), as shown in Figure 1.6 and Equation 1.4, and reciprocal plots (LineweaverBurk) are used to characterize kinetics and the type of inhibition.

v=Vmax [Sx]/(Km+[Sx]) (1.4)

The values for Km can vary considerably from one transporter to another, and for a particular trans-porter with different solutes. For transport studies, the definition of Km is the solute concentration that

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results in half-maximal velocity for the transport reaction. An equivalent way of stating this is that Km represents the substrate concentration at which half of the transporter is occupied by solute molecules in steady state. Hence, the constant Km can be used as a relative measure of substrate binding affinity.

1.3 the transporter Classification (tC) System

The phylogenetic and functional analyses of transporter proteins from many different organisms have provided the basis for the development of a transporter classification (TC) system [25]. In this system, permeases are classified according to both function and phylogeny. The TC system is an International Union of Biochemistry and Molecular Biology (IUBMB) approved system of nomenclature for trans-port protein classification. The systematic classification of solute transporters is based on several criteria including mode of transport, energy-coupling source, and molecular phylogeny. A specific five-digit TC number classifies each transporter according to five criteria. The first digit is a num-ber referring to the class, which defines the mode of transport and energy-coupling mechanism; the second digit is a letter indicating the subclass as defined by the type of transporter and the energy coupling mechanism; the third digit is a number indicating the superfamily or family; the fourth digit is a number indicating a phylogenetic cluster within a family or a family within a superfamily; and the last digit is a number indicating the substrate or range of substrates transported and the polar-ity of transport. For example, the TC number for the galactose:H+symporter form E. coli (GalP) is 2.A.1.1.1. This number indicates that GalP is a member of the 2.A.1.1. sugar porter family from the 2.A.1. major facilitator superfamily of the 2.A. porters subclass of the 2. electrochemical potential-driven class of transporters.

The functional and phylogenetic TC system taxonomy can be accessed in the transporter classifica-tion database (TCDB). This is a Web accessible (http://www.tcdb.org) relational database containing a wealth of information about transport systems. It is a curated database that continuously updates and classifies structural, functional, evolutionary, and sequence information. The TCDB compiles infor-mation from over 10,000 references and includes approximately 3,000 different proteins organized in approximately 400 transporter families. The Web interface provides several methods for accessing data and searching the database. It also includes several bioinformatic tools designed to analyze transport proteins [26].

Table 1.2 shows an outline of TC according to permease type and energy source. There are four known classes of transporters: (1) channels, (2) porters, (3) primary active transporters, and (4) group translocators. An additional class (8) includes auxiliary transport proteins and class includes

Substrate concentration (S)

Tran

spor

t rat

e Vmax

Vmax

Km

FIguRE 1.6 Michaelis and Menten kinetics of a transport-mediated solute diffusion process.

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uncharacterized sequences that are homologous to transporters and characterized transporters whose sequences are not known. Categories 67 are reserved for yet to be discovered novel types of transporters.

Figure 1.7 shows the known classes and mechanisms of solute transporters found in bacteria that include: (a) channel and pore-mediated passive diffusion; (b) carrier-mediated solute-H+symport; (c) carrier-mediated solute-H+symport with an external solute-recognition receptor; (d) primary active uptake ABC transporter driven by ATP hydrolysis; and (e) group translocating permease of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Each of these classes will be presented in detail in the following sections.

1.3.1 Channels and Pores

The channel or pore is one of the simplest structures for the transport of solutes (Figure 1.7a). In this case, facilitated diffusion is not coupled to the use of metabolic energy, and hence it cannot generate concentration gradients of the transported substrate across the membrane. In this class of transporters, the solute passes by a diffusion-limited process from one side of the membrane to the other via a chan-nel or pore that is coated by amino acyl residue moieties of the constituent protein(s) that recognizes hydrophilic, hydrophobic, or amphipathic substrates.

In Gram-negative bacteria some small solutes that in principle can cross the membrane relatively without restraint may need an additional transport system to sustain a high enough flux for physi-ological processes. Channel type proteins, called porins, that mediate the passive transfer of solutes, perform transport of molecules such as carbohydrates, amino acids, and simple ions across the outer membrane. A bacterium like E. coli can have up to 105 copies of porins OmpF, OmpC, or PhoE,

TaBLE 1.2 Classes and Subclasses of Transporters According to the TC Systema

1. Channels and Pores1.A -Type channels1.B -Barrel porins1.C Pore-forming toxins (proteins and peptides)1.D Nonribosomally synthesized channels

2. Electrochemical Potential-Driven Transporters2.A Porters (uniporters, symporters, antiporters)2.B Nonribosomally synthesized porters2.C Ion-gradient-Driven energizers

3. Primary Active Transporters3.A P-P-bond hydrolysis-Driven transporters3.B Decarboxylation-Driven transporters3.C Methyltransfer-Driven transporters3.D Oxidoreduction-Driven transporters3.E Light absorption-Driven transporters

4. Group Translocators4.A Phosphotransfer-Driven group translocators

5. Transmembrane Electron Flow Systems5.A Two-electron carriers5.B One-electron carriers

8. Accessory Factors Involved in Transport8.A Auxiliary transport proteins

9. Incompletely Characterized Transport Systems9.A Recognized transporters of unknown biochemical mechanism9.B Putative but uncharacterized transport proteins9.C Functionally characterized transporters lacking identified sequences

a Categories 67 are reserved for yet to be discovered novel types of transporters.

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which tend to form a barrel of antiparallel -sheets, consisting of trimeric complexes of identical subunits of ca. 35 kDa, with an approximate diameter of 1 nm that allows the passage for molecules up to a mass of about 600 Da.

1.3.1.1 -type ChannelsThis class of channel proteins is composed mainly of -helical spanners (sometimes -strands play a part in the channel) that catalyze the movement of solutes using transmembrane aqueous pores by an energy-independent mechanism and are found in bacteria and eukaryotes.

1.3.1.2 -Barrel PorinsThese porin proteins are mainly -barrels constituted by -strands in the transmembrane region, which also allow the transport of solutes by an energy-independent mechanism. They are found in the outer membranes of Gram-negative bacteria, mitochondria, and plastids.

IICP~

H+

H++

H++

H+

ATP

Pi + ADP

IIC

IIBIIA

PEP

PYR

EI HPr

(a)

(b)

(c)

(d)

(e)

FIguRE 1.7 Classes and mechanisms of solute transporters. (a) Channel and pore-mediated passive diffusion. (b) Carrier-mediated solute-H+symport. (c) Carrier-mediated solute-H+symport with an external solute-recognition receptor. (d) Primary active uptake ABC transporter driven by ATP hydrolysis. (e) Group translocating permease of the phosphoenolpyruvate:sugar phosphotransferase system.

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1.3.1.3 Pore Forming toxins (Proteins and Peptides)

This category of transmembrane pores includes proteins or peptides ribosomally synthesized by one cell and secreted for insertion into the membrane of another cell. They are composed mainly of -strands. The pores formed by these proteins allow the free flow of electrolytes and other small molecules across the membrane of the host, or they may allow entry into the target cell cytoplasm of a toxin protein that ultimate kills or controls the cell.

1.3.1.4 nonribosomally Synthesized Channels

These oligomeric transmembrane ion channels are chains of L- and D-amino acids, or small polymers of hydroxylactate or -hydroxybutirate, in which the arrangement of the pore structure is stimulated by voltage changes, and are made by bacteria and fungi as agents of biological warfare that confer a selec-tive advantage.

1.3.2 electrochemical Potential-Driven transporters

Chemical, light, or electrochemical energy is used by the cells to transport and accumulate solutes inside the cell. Several transport phenomena are driven by electrochemical potential gradients, such as proton and sodium gradients. Concentration gradients of solutes power transporter-mediated facilitated diffusion; it does not require the expenditure of metabolic energy in the form of ATP. This mechanism permits the transfer of solutes across a membrane against a concentration gradient. This type of transport is also known as secondary transport and is catalyzed by uniporters, symporters, and antiporters (Figure 1.7b and c). In contrast to transporters driven by ATP, electrochemical transport-ers are relatively simple in composition, generally consisting of a single protein that transverses the membrane with several loops.

1.3.2.1 Porters (Uniporters, Symporters, and Antiporters)

Some sugars, amino acids, nucleosides, and small molecules, such as Na+, are transported by uniporter proteins, which move one solute across a membrane down a concentration gradient from an area of greater concentration to one of lesser concentration. Selective conformational changes are induced by interactions between the solute and the uniporter, which enables the uniporter to transport the solute across the cytoplasmic membrane.

Some sugars, amino acids, and ions (e.g., sulfate and phosphate) are co-transported by symporter pro-teins, which use the proton motive force to move a solute against a concentration gradient, i.e., symport-ers simultaneously transport two solutes across the membrane in the same direction. Antiport involves a tightly coupled process where two or more solutes are transported in opposite directions. Na+can be transported by antiporter proteins. In this instance, a gradient of protons generates the potential energy, and simultaneously the solute (Na+) is transported trough the membrane in the opposite direc-tion against a concentration gradient.

1.3.2.2 nonribosomally Synthesized Porters

These transmembrane porters are peptides or small polymers of nonpeptide nature. They complex solutes (like a cation) in their hydrophilic interior and make possible the translocation of the complex across the membrane by exposing its hydrophobic exterior and moving from one side of the membrane bilayer to the other. Transport is electrophoretic if the porter in the uncomplexed form can cross the membrane and electroneutral (one charged solute is exchanged for another) if only the complexed form can cross the membrane.

1.3.2.3 Ion-Gradient-Driven energizers

This is a family of auxiliary proteins, like the TonB family, that mediate active transport using an outer membrane receptor, which is energized to accumulate its solutes inside the periplasm against large

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concentration gradients. These energizers make use of protons or sodium ions fluxes, i.e., the proton motive force, through themselves to energize outer membrane receptors or porins. Conformational changes of receptors allow electrophoretic transport of protons.

1.3.3 Primary Active transporters

These systems consist of transporters that use a primary source of energy to drive the active transport of a solute against a concentration gradient. Primary energy sources known to be coupled to transport include chemical, electrical, and light.

1.3.3.1 P-P-Bond-Hydrolysis-Driven transporters

These transport systems hydrolyze the diphosphate bond of inorganic pyrophosphate, ADP, ATP, or another nucleoside triphosphate, to drive the active uptake and/or extrusion of a solute or solutes. The transport protein may or may not be transiently phosphorylated, but the substrate is not phos-phorylated during the process. This subclass is the most abundant of the primary active transporters and comprises three superfamilies and 18 families dis

[christina smolke] the metabolic pathway engineeri(bookfi.org)

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