Table of Contents

Cover

Related Titles

Title Page

Copyright
Dedication

List of Contributors

About the Series Editors

Preface

Part I: Industrial Biotechnology: From Pioneers to Visionary

Chapter 1: History of Industrial Biotechnology
1. 1.1 The Beginning of Industrial Microbiology
2. 1.2 Primary Metabolites and Enzymes
3. 1.3 The Antibiotic Era
4. 1.4 The Biotechnology Era Between 1970 and 2015
5. 1.5 How Pioneering Developments Led to Genetic Engineering
6. References
Chapter 2: Synthetic Biology: An Emerging Approach for Strain Engineering
1. 2.1 Introduction
2. 2.2 Basic Elements
3. 2.3 Functional and Robust Modules
4. 2.4 Microbial Communities
5. 2.5 Conclusions and Future Prospects
6. Acknowledgments
7. References
Chapter 3: Toward Genome-Scale Metabolic Pathway Analysis
1. 3.1 Introduction
2. 3.2 DD Method
3. 3.3 Calculating Short EFMs in Genome-Scale Metabolic Networks
4. 3.4 Conclusions
5. Acknowledgments
6. References
Chapter 4: Cell-Free Synthetic Systems for Metabolic Engineering and Biosynthetic Pathway Prototyping
1. 4.1 Introduction
2. 4.2 Background
3. 4.3 The Benefits of Cell-Free Systems
4. 4.4 Challenges and Opportunities in Cell-Free Systems
5. 4.5 Recent Advances
6. 4.6 Summary
7. Acknowledgments
8. References

Part II: Multipurpose Bacterial Cell Factories

Chapter 5: Industrial Biotechnology: Escherichia coli as a Host
1. 5.1 Introduction
2. 5.2 E. coli Products
3. 5.3 Rewiring Central Metabolism
4. 5.4 Alternative Carbon Sources
5. 5.5 E. coli Techniques and Concerns
6. 5.6 Conclusions
7. References
Chapter 6: Industrial Microorganisms: Corynebacterium glutamicum
1. 6.1 Introduction
2. 6.2 Physiology and Metabolism
3. 6.3 Genetic Manipulation of Corynebacterium glutamicum
4. 6.4 Systems Biology of Corynebacterium glutamicum
5. 6.5 Application in Biotechnology
6. 6.6 Conclusions and Perspectives
7. References
Chapter 7: Host Organisms: Bacillus subtilis
1. 7.1 Introduction and Scope
2. 7.2 Identification of Genetic Traits Pertinent to Enhanced Biosynthesis of a Value Product
3. 7.3 Traits to Be Engineered for Enhanced Synthesis and Secretion of Proteinaceous Products
4. 7.4 Engineering of Genetic Traits in Bacillus subtilis
5. 7.5 Genome Reduction
6. 7.6 Significance of Classical Strain Improvement in Times of Synthetic Biology
7. 7.7 Resource-Efficient B. subtilis Fermentation Processes
8. 7.8 Safety of Bacillus subtilis
9. 7.9 Bacillus Production Strains on the Factory Floor: Some Examples
10. Acknowledgments
11. References
Chapter 8: Host Organism: Pseudomonas putida
1. 8.1 Introduction
2. 8.2 Physiology and Metabolism
3. 8.3 Genetic Manipulation
4. 8.4 Systems Biology
5. 8.5 Application in Biotechnology
6. 8.6 Future Outlook
7. References

Part III: Exploiting Anaerobic Biosynthetic Power

Chapter 9: Host Organisms: Clostridium acetobutylicum/Clostridium beijerinckii and Related Organisms
1. 9.1 Introduction
2. 9.2 Microorganisms
3. 9.3 Bacteriophages
4. 9.4 ABE Fermentation of Solvent-Producing Clostridium Strains
5. 9.5 Genome-Based Comparison of Solvent-Producing Clostridium Strains
6. 9.6 Regulation of Solvent Formation in C. acetobutylicum
7. 9.7 Genetic Tools for Clostridial Species
8. 9.8 Industrial Application of ABE Fermentation
9. Acknowledgments
10. References
Chapter 10: Advances in Consolidated Bioprocessing Using Clostridium thermocellum and Thermoanaerobacter saccharolyticum
1. 10.1 Introduction
2. 10.2 CBP Organism Development Strategies
3. 10.3 Plant Cell Wall Solubilization by C. thermocellum
4. 10.4 Bioenergetics of C. thermocellum Cellulose Fermentation
5. 10.5 Metabolic Engineering
6. 10.6 Summary and Future Directions
7. Acknowledgments
8. References
Chapter 11: Lactic Acid Bacteria
1. 11.1 Introduction
2. 11.2 Fermented Foods
3. 11.3 Industrially Relevant Compounds
4. 11.4 Conclusions
5. Conflict of Interest
6. References

Part IV: Microbial Treasure Chests for High-Value Natural Compounds

Chapter 12: Host Organisms: Myxobacterium
1. 12.1 Introduction into the Myxobacteria
2. 12.2 Phylogeny and Classification
3. 12.3 Physiology
4. 12.4 Growth and Nutritional Requirements
5. 12.5 Genetics and Genomics
6. 12.6 Secondary Metabolism
7. 12.7 Myxococcus
8. 12.8 Sorangium
9. 12.9 Outlook
10. References
Chapter 13: Host Organism: Streptomyces
1. 13.1 Introduction
2. 13.2 Streptomyces Genome Manipulation Toolkits
3. 13.3 Hosts for Heterologous Production of Natural Products
4. Acknowledgments
5. References

Part V: Extending the Raw Material Basis for Bioproduction

Chapter 14: Extreme Thermophiles as Metabolic Engineering Platforms: Strategies and Current Perspective
1. 14.1 Introduction
2. 14.2 Bioprocessing Advantages for Extremely Thermophilic Hosts
3. 14.3 Biobased Chemicals and Fuels: Targets and Opportunities
4. 14.4 Considerations for Selecting an Extremely Thermophilic Host
5. 14.5 General Strategies for Genetic Manipulation of Extreme Thermophiles
6. 14.6 Limitations and Barriers to Genetic Modification of Extreme Thermophiles
7. 14.7 Current Status of Metabolic Engineering Efforts and Prospects in Extreme Thermophiles
8. 14.8 Metabolic Engineering of Extreme Thermophiles – Tool Kit Needs
9. 14.9 Conclusions and Future Perspectives
10. Acknowledgments
11. References
Chapter 15: Cyanobacteria as a Host Organism
1. 15.1 Introduction and Relevance: Cyanobacteria as a Host Organism
2. 15.2 General Description of Cyanobacteria
3. 15.3 Genetic Tools
4. 15.4 Improving Photosynthetic Efficiency
5. 15.5 Direct Conversion of CO₂ into Biofuels and Chemicals
6. 15.6 Conclusions
7. References
Chapter 16: Host Organisms: Algae
1. 16.1 Introduction to Algae as an Industrial Organism
2. 16.2 Algal Genetic Engineering
3. 16.3 Therapeutic and Nutraceutical Applications
4. 16.4 Bioenergy Applications
5. 16.5 Other Industrial Applications
6. 16.6 Industrial-Scale Algal Production
7. 16.7 Conclusions and Potential of Algal Platforms
8. References

Part VI: Eukaryotic Workhorses: Complex Cells Enable Complex Products

Chapter 17: Host Organisms: Mammalian Cells
1. 17.1 Introduction
2. 17.2 Basics of Cellular Structure and Metabolism
3. 17.3 The Genome of CHO Cells
4. 17.4 Molecular Biology Tools
5. 17.5 Kinetics of Growth and Product Formation
6. 17.6 Intracellular Metabolome Analysis
7. 17.7 Proteome and Gene Expression Analysis
8. 17.8 Improving Cellular Performance by Genetic and Metabolic Engineering
9. 17.9 Outlook
10. References
Chapter 18: Industrial Microorganisms: Saccharomyces cerevisiae and other Yeasts
1. 18.1 Industrial Application of Yeasts
2. 18.2 Baker's Yeast as Versatile Host for Metabolic Engineering
3. 18.3 Protein Production in Yeasts
4. 18.4 Lipid Production in Yeasts
5. 18.5 Pentose-Utilizing Yeasts
6. 18.6 Conclusions
7. Conflict of Interest
8. References
Chapter 19: Industrial Microorganisms: Pichia pastoris
1. 19.1 Physiology and Genetics of Pichia pastoris
2. 19.2 Methylotrophic Metabolism
3. 19.3 Application for the Production of Recombinant Proteins
4. 19.4 Application of P. pastoris for Metabolite Production
5. 19.5 Conclusion
6. References
Index

End User License Agreement

List of Illustrations

Chapter 1: History of Industrial Biotechnology

Figure 1.1 Basic molecular structure of biological production processes with microorganisms.

Chapter 2: Synthetic Biology: An Emerging Approach for Strain Engineering

Scheme 2.1 Overview of engineering targets for synthetic biology. Multiple levels of synthetic biology can impact strain engineering. In the bottom panel, basic elements such as synthetic genes and regulatory elements are engineered, where they are combined into synthetic pathways and circuits in the middle panel. All these synthetic modules can enable cell-cell communication as sketched in the top panel.

Chapter 3: Toward Genome-Scale Metabolic Pathway Analysis

Figure 3.1 Growth- and non-growth-coupled toy metabolic networks (panels a and b, respectively) along with their associated phenotypic yield spaces (panels c and d, respectively). Both networks consist of nine irreversible reactions (diamonds, $c03-math-017$ – $c03-math-018$ ), four internal metabolites (full rectangles), four external metabolites (checkered rectangles: $c03-math-019$ , biomass; $c03-math-020$ , product of interest; $c03-math-021$ by-product; $c03-math-022$ , substrate; note that the metabolite Q is the product of two reactions– $c03-math-023$ and $c03-math-024$ ) and five EFMs. In the phenotypic yield space EFMs are represented by full circles. Note that the point (1/0) in the phenotypic yield space of network B represents two EFMs with identical yields. The feasible yield space is bounded by the two axes and the dashed line. Growth-coupled production of P is achievable only in network A, but not in network B.
Figure 3.2 Computing the shortest EFMs with the DD method. The box with blue background color highlights the main extension of the maximum cardinality feature over common implementations of the DD method.
Figure 3.3 Comparison of execution times. Runtime comparison between maximum cardinality EFMA and the MILP based approach in various models (see Table 3.1). (a) E. coli core I [28], (b) E. coli core II, (c) E. coli core III, (d) P. tricornutum [11], (e) Blattibacteriacae cuenoti Bge [29], and (f) liver cancer [30].

Chapter 4: Cell-Free Synthetic Systems for Metabolic Engineering and Biosynthetic Pathway Prototyping

Figure 4.1 Comparison of traditional and cell-free metabolic engineering. (a) Desired biochemical pathway. (b) Methodology for metabolic engineering in vivo. (c) Methodology for metabolic engineering in vitro.
Figure 4.2 Biochemical pathways of key cell-free metabolic engineering achievements. (a) Pathway from [23]. (b) Pathway from [53]. (c) Pathway from [61].

Chapter 5: Industrial Biotechnology: Escherichia coli as a Host

Figure 5.1 Production pathways of selected products discussed in the text. Gene/protein symbols: aroF, DAHP synthase; pykF, pyruvate kinase; ldhA, lactate dehydrogenase; alaA, alanine transaminase; ilvIH, acetolactate synthase (alsS = heterologous enzyme from Bacillus subtilis); aceA, pyruvate dehydrogenase; ppc, phosphoenolpyruvate carboxylase; aspC, aspartate transaminase; gltA, citrate synthase; and gdhA, glutamate dehydrogenase.
Figure 5.2 Isobutanol production via valine pathway and 1-butanol production via CoA-dependent pathway implemented. Common enzyme abbreviations or gene symbols: ilvIH, acetolactate synthase (E. coli); alsS, acetolactate synthase (Bacillus subtilis); ilvC, acetohydroxy acid isomeroreductase (E. coli); ilvD, dihydroxy acid dehydratase (E. coli); kivd, ketoisovalerate decarboxylase (Lactoccus lactis); ADH2, alcohol dehydrogenase (Saccharomyces cerivisiae); atoB, thiolase (E. coli); hbd, hydroxybutyryl-CoA dehydrogenase (Clostridium acetobutylicum); crt, crotonase (C. acetobutylicum); ter, trans-enoyl-CoA reductase (Treponema denticola); and Bcd-etf, butyryl-CoA dehydrogenase/electron transfer complex (C. acetobutylicum) AdhE2, alcohol dehydrogenase (bifunctional, C. acetobutylicum).
Figure 5.3 Overview of some E. coli feedstocks (white text on gray background) and products (black text in black boxes) discussed in this text.

Chapter 6: Industrial Microorganisms: Corynebacterium glutamicum

Figure 6.1 Carbon core metabolism of Corynebacterium glutamicum comprising the major catabolic routes of pentose phosphate pathway and Embden-Meyerhof-Parnas pathway, tricarboxylic acid cycle, glyoxylate shunt, and anaplerotic reactions. The relevance of the individual pathways and carbon building blocks for biosynthesis of the broad product portfolio of C. glutamicum is indicated. Additionally, the extended substrate spectrum and the heterologous assimilation routes are illustrated. Natural products and substrates are highlighted in turquoise and dark green, non-natural products and substrates in yellow and light green.
Figure 6.2 Timeline of research and development of the industrial, Gram-positive soil bacterium Corynebacterium glutamicum, highlighting major discovery breakthroughs in the microbiology, genetic, omics, and synthetic biology era. The gray background indicates the number of publications that appeared as deduced from PubMed (http://www.ncbi.nlm.nih.gov/pubmed).
Figure 6.3 Maps of plasmid vectors for C. glutamicum (adapted from [100]) (a) C. glutamicum/E. coli shuttle promoter-probe vector pET2 and (b) C. glutamicum/E. coli shuttle expression vector pVWEx1. Dark areas indicate regions coming from plasmids of Corynebacteria (pBL1, pCG1, and pGA1, respectively). Arrows inside the map indicate genes. aph, Kanamycin resistance determinant; cat_PL, promoterless reporter gene coding for chloramphenicol acetyltransferase; T_CE, tandems of transcriptional terminators; rep, initiator of RC replication; per, positive effector of plasmid replication; lacI^q, lactose repressor; and P-tac, IPTG inducible promoter.

Chapter 7: Host Organisms: Bacillus subtilis

Figure 7.1 The Sec and Tat protein secretion pathways of B. subtilis. The cartoon depicts the components involved in Sec- and Tat-dependent export of proteins from the cytoplasm (IN) to the membrane, cell wall, and extracellular milieu (OUT) of the bacterium. A nascent precursor protein is schematically presented as emerging from the ribosome (R) and being bound by the SRP-FtsY complex for targeting to the membrane. Another precursor protein is shown as being translocated via the SecYEG channel in an unfolded state. On the right side, a folded precursor protein with twin-arginine signal peptide (RR) is targeted to the Tat translocase.
Figure 7.2 Schematic representation of the subcellular localization of the 10 extracytoplasmic proteases of B. subtilis. The key regulatory two-component systems CssRS and DegSU for the expression of the respective protease genes are also shown.
Figure 7.3 “Crossbreeding” by lysed protoplast transformation. (1) A competent B. subtilis cell with wild-type alleles (black dots) is mixed in a hypotonic buffer with a B. subtilis protoplast comprising a multitude of mutant alleles (black triangles). The competent cell takes up the chromosome of the burst protoplast. CSM is a counterselection marker on the chromosome of the competent wild-type B. subtilis cell and SM is a selection marker on the chromosome of the mutant B. subtilis protoplast. (2) Intensive crossing over between the chromosomes of the donor and recipient bacteria. (3) Selection and counterselection provide transformants with thoroughly mixed-up genetic traits of the recipient and the donor strains.
Figure 7.4 Regulation of pur gene expression in B. subtilis (simplified). In addition to the pur operon PurR also interferes with purAB transcription.
Figure 7.5 Riboflavin biosynthesis in B. subtilis.
Figure 7.6 Biosynthesis of (R)-pantothenic acid and the side metabolite 3-(2-hydroxy-3-methyl-butyrylamino)-propionic acid (HMBPA) in B. subtilis. Regeneration of methylene tetrahydrofolate (THF=CH₂) in the GlyA-catalyzed reaction and in the glycine cleavage cycle (GcvPA, GcvPB, and GcvT) is shown as well.
Figure 7.7 Biotin biosynthesis in B. subtilis.
Figure 7.8 Thiamine pyrophosphate (TPP) biosynthesis in B. subtilis. In addition to the de novo route, salvage reactions to recruit nonphosphorylated 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), 5-(2-hydroxyethyl)-4-methylthiazole (HET), and thiamine are shown.
Figure 7.9 Pyridoxal-5′-phosphate biosynthesis. In the upper part of the Figure the DXP route is depicted as realized, for example, in E. coli. The lower part shows the R5P (DXP-independent) route of, for example, in B. subtilis.

Chapter 8: Host Organism: Pseudomonas putida

Figure 8.1 Biochemical pathways involved in glucose catabolism in Pseudomonas putida KT2440. The metabolic network depicted is sketched around four main metabolic blocks, identified with different colors: (i) the peripheral oxidative pathways, that encompass the oxidative transformation of glucose into gluconate and 2-ketogluconate (and the corresponding phosphorylated derivatives of these metabolites); (ii) the Embden–Meyerhof–Parnas (EMP) pathway (nonfunctional, due to the absence of a 6-phosphofructokinase activity); (iii) the pentose phosphate (PP) pathway; and (iv) the Entner–Doudoroff (ED) pathway. Note that further catabolism downward acetyl-CoA (i.e., the tricarboxylic acid cycle) is represented by a wide shaded arrow. Some reactions and pools of metabolites in this scheme have been lumped to simplify the diagram. The main enzymes involved in this biochemical network are identified; in the instances in which no gene name has been assigned, the PP number is given for each open reading frame encoding the corresponding activity.
Figure 8.2 Molecular basis for the generation of knockout mutants in P. putida [56].
Figure 8.3 Architecture of pSEVA vector modules for genetic engineering of P. putida. The different restriction enzymes available for exchange of these modules are shown. Rep, replication origin and Ab^R, antibiotic resistance. The three modules are separated by three permanent regions (shared by all vectors) comprising the T0 and T1 transcriptional terminators and the oriT origin, which are intended for broad host range transfer to diverse Gram-negative recipients mediated by the RP4 conjugation machinery.
Figure 8.4 Biosynthetic potential of Pseudomonas putida. Extended carbon core metabolism of Pseudomonas putida KT2440 including the major catabolic routes of Entner–Doudoroff pathway, Embden–Meyerhof–Parnas pathway, pentose phosphate pathway, tricarboxylic acid cycle, glyoxylate shunt, anaplerotic reactions, fatty acid de novo biosynthesis, β-oxidation of fatty acids, as well as the convergent β-ketoadipate pathway for catabolism of aromatics. Known pathways for respective precursor supply for the broad product spectrum of P. putida KT2440 are indicated by light red arrows. Natural products and substrates are highlighted in black, heterologous products and substrates in red.

Chapter 9: Host Organisms: Clostridium acetobutylicum/Clostridium beijerinckii and Related Organisms

Figure 9.1 Metabolism and products of solventogenic bacteria. Ack, acetate kinase; Adc, acetoacetate decarboxylase; AdhE, AdhE2, aldehyde/alcohol dehydrogenase; BdhA, BdhB, alcohol dehydrogenase; Bcd, butyryl-CoA dehydrogenase; Buk, butyrate kinase; Crt, crotonase; CtfA/B, CoA transferase; EtfA/B, electron-transfer flavoproteins A and B; Hbd, 3-hydroxybutyryl-CoA dehydrogenase; NADH, reduced nicotinamide adenine dinucleotide (reduced form); Pta, phosphotransacetylase; Ptb, phosphotransbutyrylase; Thl, thiolase. The single reactions do not represent stoichiometric fermentation balances.
Figure 9.2 Genes involved in butanol production. (a) sol operon and (b) bcs operon. adhE, aldehyde/alcohol dehydrogenases; ctfA/B, CoA transferase; adc, acetoacetate decarboxylase; ald, aldehyde dehydrogenase; crt, crotonase; bcd, butyryl-CoA dehydrogenase; etfA/B, electron-transferring flavoproteins; hbd, 3-hydroxybutyryl-CoA dehydrogenase. Numbers indicate length in base pairs.
Figure 9.3 Phylogenetic tree of solventogenic clostridia based on 16S rRNA gene sequences. Sequences were aligned using ClustalW 1.6 [96]. The phylogenetic tree was obtained by using the neighbor-joining method within MEGA 5.05 software [97]. Numbers at nodes are bootstrap values calculated from 1000 resamplings to generate a majority consensus tree. C. sporogenes DSM 795T was used as outgroup. The scale bar indicates the nucleotide sequence divergence. The length of the tree branches was scaled according to the number of substitutions per site (see size bar).
Figure 9.4 Simplified Venn diagram representing the pan and core genomes and the number of genome-specific orthologous groups of solventogenic clostridia. Ortholog detection was done with the Proteinortho software (blastp) with an identity cutoff of 50% and an E-value of 1e−10 [98]. The genome of C. beijerinckii AAU1_buffalo was excluded from this analysis owing to an extraordinarily high number of genes annotated (10 315) compared to a genome size of 6.5 Mb.

Chapter 10: Advances in Consolidated Bioprocessing Using Clostridium thermocellum and Thermoanaerobacter saccharolyticum

Figure 10.1 Comparison of biomass solubilization by C. thermocellum relative to fungal cellulases. Open symbols, percent glucan solubilization by C. thermocellum (left axis) as a function of the percent glucan solubilization by fungal enzymes during SSF. The dashed line represents values where solubilization would be equal between C. thermocellum and fungal cellulases. Closed symbols, the same data plotted as the ratio between C. thermocellum and fungal enzyme solubilization (right axis).
Figure 10.2 Typical pathways that can convert cellodextrins (CDs) to pyruvate in C. thermocellum. (a) Transport and conversion of CD to glucose-6-phosphate (G6P), (b) coupling of the membrane gradient to ATP and pyrophosphate (PP_i) formation, (c) catabolism of fructose-6-phosphate (F6P) to phosphoenolpyruvate (PEP), and (d) conversion of PEP to pyruvate (Pyr). ATP yields per hexose equivalent are provided. Glc, glucose; FbP, fructose-1,6-bisphosphate; OAA, oxaloacetate; HK, hexokinase; CD Pase, cellodextrin phosphorylase; PGM, phosphoglucomutase; PPase, pyrophosphatase; PFK, phosphofructokinase; PEPCK, phosphoenolpyruvate carboxykinase; MDH/ME, malate dehydrogenase/malic enzyme; OAADC, oxaloacetate decarboxylase; and PPDK, pyruvate phosphate dikinase.
Figure 10.3 Possible pathways involved in conversion of pyruvate to end products. (a) Homoacetate fementation, (b) mixed acetate/ethanol fermentation, and (c) homoethanol fermentation C. thermocellum. ATP yields per hexose equivalent are provided. Pyr, pyruvate; Ac-CoA, acetyl-Coenzyme A; Fd, ferredoxin; PFL, pyruvate:formate lyase; PTA/ACK, phosphotransacetylase/acetate kinase; ALDH/ADH, aldehyde dehydrogenase/alcohol dehydrogenase; PFOR, pyruvate:ferredoxin oxidoreductase; Ech, energy converting hydrogenase; Rnf, ferredoxin:NAD⁺ oxidoreductase; and NfnAB, NADH-dependent reduced ferredoxin:NADP⁺ oxidoreductase.
Figure 10.4 Ethanol production pathways in T. saccharolyticum. (a) The NADPH-linked pathway. (b) The NADH-linked pathway. Carbon fluxes are shown in black, electron fluxes are shown in gray. For simplicity, only the reduced form of electron carriers are shown (i.e., Fd_red, NADH, and NADPH are shown, but Fd_ox, NAD⁺, and NADP⁺ are not shown).

Chapter 11: Lactic Acid Bacteria

Figure 11.1 Proposed pathway for hexose metabolism of homofermentative LAB (1) and (2) phosphoenolpyruvate (PEP)-dependent sugar phosphotransferase system (PTS); (3) mannitol-specific PTS; (4) phosphoglucose isomerase; (5) mannitol-1-phosphate dehydrogenase; (6) mannitol-1-phosphatase; (7) 6-phosphofructokinase; (8) fructose-diphosphatase; (9) fructose-1,6-diphosphate aldolase; (10) triosephosphate isomerase; (11) glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase; (12) phosphoglyceromutase and enolase; (13) pyruvate kinase; (14) lactate dehydrogenase; (15) pyruvate-formate lyase; (16) acetaldehyde dehydrogenase and alcohol dehydrogenase; (17) pyruvate dehydrogenase; (18) acetate kinase; (19) α-acetolactate synthase; (20) α-acetolactate decarboxylase; and (21) 2,3-butanediol dehydrogenase.
Figure 11.2 Proposed pathway for hexose metabolism of heterofermentative LAB. (1) glucose permease; (2) fructose permease; (3) glucokinase; (4) glucose 6-phosphate dehydrogenase; (5) 6-phosphogluconate dehydrogenase; (6) epimerase; (7) phosphoketolase; (8) glucose phosphate isomerase; (9) fructokinase; (10) mannitol 2-dehydrogenase; (11) glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase; (12) phosphoglyceromutase and enolase; (13) pyruvate kinase; (14) lactate dehydrogenase; (15) phosphate acetyltransferase; (16) acetaldehyde dehydrogenase and alcohol dehydrogenase; (17) acetate kinase; and ? Unknown mannitol transport system.

Chapter 12: Host Organisms: Myxobacterium

Figure 12.1 Cooperative morphogenesis in myxobacteria. The organism shown in the diagram is Chondromyces crocatus. Symbols: A, myxospores released from a sporangiole; B, myxospores' germination and initial stage of swarming; C, advancing and radiating swarm; D, vegetative-type cells in the swarm; E, aggregation of vegetative cells; F–J, fruiting body development; F, knob-/mound-stage; G, development of the slime stalk and cell mass differentiation; H, formation of slime branches; I, clustering of undifferentiated sporangioles; J, matured fruiting body borne with a stalk and bearing multiple branches with clusters of dense ovoid sporangioles; and K, myxospores inside the sporangiole.
Figure 12.2 Classification of myxobacteria. Recently identified novel families/genera are highlighted in bold: Aggregicoccus [12], Vulgatibacteraceae/Vulgatibacter [13], Aetherobacter [14], Racemicystis [15], Minicystis [16], Phasilicystidaceae/Phasilicystis [17], Sandaracinaceae/Sandaracinus [18], Labilithrichaceae/Labilithrix [13], Pseudenhygromyxa [19]. In addition, the family of Haliangiaceae was recently proposed [20] and the genus Anaeromyxobacter was reclassified into Anaeromyxobacteraceae family [13]. The three suborders are distinct phenotypically by growth-stage characteristics. For each suborder, pictures of a fruiting body (left) and vegetative cells (right) are shown for a selected species. (Pictures: Ronald Garcia, ©HZI.) Cystobacter fruiting bodies are composed of clusters of sporangioles held by transparent slime with typical long and needle-shaped rod cells. The fruiting stage of Chondromyces shows a complex treelike structure with cylindrical rod vegetative cells. In case of Nannocystis, several fruiting bodies are visible in agar plate as simple, spherical, and tiny sporangioles with almost cuboidal vegetative rod cells.
Figure 12.3 Stepwise biosynthesis of epothilones by a PKS/NRPS megasynthetase consisting of 6 protein subunits (EpoA, P, B–E) and 50 catalytic domains (shown as circles), which are arranged into 10 modules, each incorporating one precursor molecule into the growing linear epothilone chain [105, 106]. Besides the main products epothilones A–D, 33 additional epothilone derivatives could be identified in culture extracts of the epothilone producer S. cellulosum So ce90 [107] (not all derivatives shown).
Figure 12.4 Biosynthetic potential of the myxobacterial model strain Myxococcus xanthus DK1622. (a) To date, 8 out of 18 identified biosynthetic gene clusters encoding PKS, NRPS, and PKS/NRPS hybrid systems could be assigned to products as indicated in the genome circle. (b) HPLC-MS chromatogram of a M. xanthus DK1622 culture extract showing the presence/detection of five natural product families among various other metabolites and media components (myxoprincomides and compounds c329 and c844 could only be detected in trace amounts, which is not indicated here).
Figure 12.5 Structures of epothilone B in comparison to its semisynthetic derivative ixabepilone and the effect of both compounds on eukaryotic cells (nuclei were stained blue, microtubules were labeled with green).
Figure 12.6 Examples for bioactive compounds from Sorangium cellulosum species.

Chapter 13: Host Organism: Streptomyces

Figure 13.1 Schematic approach of activation of cryptic biosynthetic gene clusters.
Figure 13.2 Natural products produced by Streptomyces albus J1074.

Chapter 14: Extreme Thermophiles as Metabolic Engineering Platforms: Strategies and Current Perspective

Figure 14.1 Current and future targets for metabolic engineering of extreme thermophiles. Boxes with dotted outlines represent potential targets that have not yet been demonstrated.
Figure 14.2 General strategies for chromosomal deletion or insertion. DNA architecture and transformation steps shown for markerless insertion or deletion using circular or linear DNA (a), (b), (e), (f); and marked insertion or deletion using circular or linear DNA (c), (d), (g), (h). Note that pyrF is used as both a marker for selection (uracil prototrophy) and counterselection (5FOA resistance); variations on these strategies may use an alternate marker for selection and a second marker for counterselection, with appropriate chemicals replacing uracil and 5FOA. Abbreviations: 5′ HR and 3′ HR, homology regions 5′ or 3′ to the targeted chromosomal region; pyrF, uracil prototrophy/5FOA resistance marker; kat, kanamycin resistance marker; GOI, gene of interest; PO, “pop-out” region homologous to portion of 5′ HR; ura, uracil; 5FOA, 5-fluoroorotic acid; and kan, kanamycin.
Figure 14.3 Equipment required for plating anaerobic thermophiles. Anaerobic glove box (a) for manipulating cells and paint canister (b) adapted for incubating plates anaerobically at high temperatures.
Figure 14.4 Modeling and optimization of a thermophilic n-butanol pathway in vitro. Enzymes from multiple thermophilic bacteria were assembled in vitro to measure conversion of carbon in acetyl-CoA to products. Reaction kinetic modeling was used to optimize the relative amount of each enzyme (as mol%) to maximize selectivity for n-butanol production.

Chapter 15: Cyanobacteria as a Host Organism

Figure 15.1 Overview of pathway for biofuels and chemicals produced from cyanobacteria.
Figure 15.2 1-Butanol production via the CoA-dependent pathway implemented in S. elongatus PCC7942. Gene symbols are accABCD, acetyl-CoA carboxylase; nphT7, acetoacetyl-CoA synthase; phaB, acetoacetyl-CoA reductase; phaJ, R-specific enoyl-CoA hydratase; ter, trans enoyl-CoA reductase; pduP, CoA-acylating propionaldehyde dehydrogenase; bldh, butyraldehyde dehydrogenase; and yqhD, NADPH-dependent alcohol dehydrogenase.
Figure 15.3 3-Hydroxypropionate (3HP) synthesis via glycerol-dependent, malonyl-CoA-dependent, and β-alanine-dependent pathways. Gene symbols are ppc, phosphoenolpyruvate carboxylase; aspC, aspartate transaminase; panD or adc: aspartate decarboxylase; skpyD4, β-alanine aminotransferase; mcr, malonyl-CoA reductase; msr, malonate semialdehyde reductase; gdh, glycerol dehydratase; and adh, alcohol dehydrogenase.
Figure 15.4 Isobutanol and 2-methyl butanol (2MB) production via keto acid pathway in cyanobacteria. Gene/protein symbols are cimA, citramalate synthase; leuCD, isopropylmalate isomerase; leuB, 3-isopropylmalate dehydrogenase; AHAS, acetohydroxyacid synthase; ilvC, acetohydroxy acid isomeroreductase; ilvD, dihydroxy acid dehydratase; kivd, ketoisovalerate decarboxylase; yqhD, alcohol dehydrogenase.

Chapter 16: Host Organisms: Algae

Figure 16.1 Schematic of two typical expression vectors for Chlamydomonas nuclear transformation. (a) A standard nuclear expression vector, including a selectable marker cassette and a gene of interest cassette, each with their own promoters, 5′ UTRs and 3′ UTRs. (b) Vector in which the gene of interest has been transcriptionally fused to zeocin resistance by the 2A self-cleaving peptide to drive high gene of interest expression. UTR, untranslated region.
Figure 16.2 Schematic of a typical Chlamydomonas chloroplast transformation vector. The gene of interest and the selectable marker are expressed in two separate cassettes. Two sites are commonly targeted: the 3HB site is a neutral site in the chloroplast genome that tolerates transgene insertion, and the psbA site is replaced by a transgene for high expression in psbA knockouts. The flanking homology regions contain sequence homologous to the chloroplast genome at the intended site of integration. UTR, untranslated region.

Chapter 17: Host Organisms: Mammalian Cells

Figure 17.1 Scheme of the compartmentalization of mammalian cells. The nucleus, host of genetic information, is the cell organelle where DNA replication and transcription of DNA occur. A small genome is as well found in mitochondria where important metabolic pathways, for example, TCA cycle, oxidative phosphorylation, and β-oxidation, take place. A variety of acid hydrolases enables the degradation of all kinds of macromolecules in the cell's lysosomes. The degradation of hydrogen peroxide and β-oxidation of fatty acids occurs in the peroxisomes. The rough endoplasmic reticulum (studded with ribosomes) represents the site for protein synthesis, processing, and translocation, while the smooth endoplasmic reticulum is often involved in assembly of vesicles for transport to the Golgi apparatus. Glycosyltransferases, kinases, and proteases modify proteins or lipids on their way through the Golgi apparatus until they are finally delivered to their cellular destinations.
Figure 17.2 Exemplified time series of some substrates, products, and by-products of a CHO fed-batch culture feeding glucose (and glutamine) and producing the therapeutic protein. For the sake of brevity, dynamics of uptake and release of other amino acids were excluded. Roughly two phases can be identified (I and II) that distinguish by lactate, glutamine, and ammonia kinetics. Dotted lines outline putative variations of the said components. It is assumed that a CHO DHFR production system is used, which explains the slight accumulation of glutamate. If CHO producers possess the GS genotype, glutamine would be synthesized internally via glutamine synthetase using glutamate as external substrate. HPLC, high-performance liquid chromatography; HPLC-MS/MS, high-performance liquid chromatography-tandem mass spectrometry; GC-MS, gas chromatography-mass spectrometry; NMR, nuclear magnetic resonance.
Figure 17.3 Overview of sample processing protocols to access intracellular metabolome patterns. Metabolism of suspended cells is rapidly halted by cooling using either quenching fluids or micro heat exchanger. Alternatively, fast filtration that prevents additional centrifugation for cell/liquid separation may be used. The growth medium of adherent cells (grown, e.g., in six-well plates) can be easily sucked (also in combination with liquid nitrogen addition for metabolism stop). Typically, cells are frozen prior to extraction which is followed by metabolite analytics.
Figure 17.4 Schematic view of citrate availability in mitochondria and in the cytosol. Citrate, which is produced in the TCA cycle inside mitochondria without ATP consumption, is exported into the cytosol using the citrate/malate antiport shuttle. Next, citrate is further catabolized via ATP-dependent citrate lyase in the cytosol to produce acetyl-CoA and oxaloacetate. Hence, cellular compartmentalization finally yields at net ATP need for citrate production in the cytosol, while simplifying models, only concentrating on TCA, ignore this existing ATP demand.

Chapter 18: Industrial Microorganisms: Saccharomyces cerevisiae and other Yeasts

Figure 18.1 Substrates and products of yeast bioprocesses.
Figure 18.2 Candida curvata grown with limiting nitrogen. Total lipid content approximately 40%. M, mitochondrion and L, lipid droplets.

Chapter 19: Industrial Microorganisms: Pichia pastoris

Figure 19.1 Metabolic pathways of methanol utilization in P. pastoris. (a) Overview of assimilation and dissimilation pathways. (b) Xylulose monophosphate (XuMP) cycle for methanol assimilation. Metabolites entering and leaving the peroxisomes are marked in gray boxes. GSH: reduced glutathione; GS-CH₂OH: S-(hydroxymethyl)glutathione; GS-CHO: S-formylglutathione; Cat1: catalase; Fld1: formaldehyde dehydrogenase; Fgh1: S-formylglutathione hydrolase; Fdh1: formate dehydrogenase; Aox1/2: alcohol oxidase 1 and 2; Das1/2: dihydroxyacetone synthase 1 and 2; Dak2: dihydroxyacetone kinase; Tpi1: triosephosphate isomerase; Fba1-2: fructose 1,6-bisphosphate aldolase; Fbp1: fructose 1,6-bisphosphatase; Shb17: sedoheptulose 1,7-bisphosphatase; Rki1-2: ribose 5-phosphate ketol-isomerase; and Rpe1-2: d-ribulose 5-phosphate 3-epimerase.

List of Tables

Chapter 1: History of Industrial Biotechnology

Table 1.1 (a) The largest pharmaceutical companies, with sales in 2014 and (b) the largest biotechnology companies with sales listed [72, 89a,b]
Table 1.2 Selected products made by fermentation (worldwide, 2003–2005) [72, 96, 111–114]
Table 1.3 Examples of rDNA products for medical use compiled and extended after [72, 87, 131, 133]
Table 1.4 Laboratory procedures on which the growth of recombinant DNA was also dependent
Table 1.5 Genetic engineering and sequencing required pure well-characterized enzymes
Table 1.6 Methods for enriching mRNAs/detecting specific clones or gene products

Chapter 3: Toward Genome-Scale Metabolic Pathway Analysis

Table 3.1 Main topological properties of the six metabolic models used in this study

Chapter 4: Cell-Free Synthetic Systems for Metabolic Engineering and Biosynthetic Pathway Prototyping

Table 4.1 Examples of industrial biotechnology (time and cost)
Table 4.2 Productivities, yields, and scales of CFME

Chapter 5: Industrial Biotechnology: Escherichia coli as a Host

Table 5.1 Some of the economically successful industrial E. coli bioprocesses

Chapter 6: Industrial Microorganisms: Corynebacterium glutamicum

Table 6.1 Enzymes and corresponding genes of the central metabolism of Corynebacterium glutamicum including known effectors and transcriptional regulation
Table 6.2 Precursor demand for biomass synthesis in C. glutamicum with additional consideration of the ATP demand for polymerization and assembly [66–68]
Table 6.3 General features of the publicly available genome sequences of C. glutamicum ATCC 13032 obtained in different whole genome sequencing project by Kalinowski and co-workers (Accession No. BX927147) [18], and Ikeda and co-workers (Accession No. BA000036/NC003450) [160], respectively
Table 6.4 Overview on the industrial amino acid market [96] and the production performance of Corynebacterium glutamicum [223, 230, 237]

Chapter 8: Host Organism: Pseudomonas putida

Table 8.1 Available plasmids for genetic engineering of P. putida.
Table 8.2 Modules available among the pSEVA vectors (http://wwwuser.cnb.csic.es/∼seva/)

Chapter 9: Host Organisms: Clostridium acetobutylicum/Clostridium beijerinckii and Related Organisms

Table 9.1 General GenBank features of genomes of solventogenic clostridia
Table 9.2 General features of genomes of solventogenic clostridia
Table 9.3 Main directed genome editing systems relevant to Clostridium acetobutylicum and C. beijerinckii

Chapter 10: Advances in Consolidated Bioprocessing Using Clostridium thermocellum and Thermoanaerobacter saccharolyticum

Table 10.1 Comparative solubilization by C. thermocellum and fungal cellulase
Table 10.2 Genes associated with reactions in the T. saccharolyticum pyruvate-to-ethanol pathway
Table 10.3 Current state of strain development of C. thermocellum and T. saccharolyticum.

Chapter 11: Lactic Acid Bacteria

Table 11.1 Fermented foods and beverages, their applications, and the lactic acid bacteria involved in each fermentative process
Table 11.2 Industrially relevant compounds produced by lactic acid bacteria and their applications
Table 11.3 Examples of production values of industrially relevant metabolites by lactic acid bacteria

Chapter 12: Host Organisms: Myxobacterium

Table 12.1 Characteristics of 13 myxobacteria representatives with completely sequenced genomes
Table 12.2 Myxobacteria as multi-producers of bioactive compounds exemplified with two species
Table 12.3 Heterologous production of secondary metabolites in Myxococcus xanthus

Chapter 14: Extreme Thermophiles as Metabolic Engineering Platforms: Strategies and Current Perspective

Table 14.1 Methods for genetic manipulation of extreme thermophiles (T_opt ≥ 70 °C)
Table 14.2 Extremely thermophilic metabolic engineering hosts.^a
Table 14.3 Metabolic engineering applications of extreme thermophiles
Table 14.4 Genetic parts available for engineering of extreme thermophiles

Chapter 15: Cyanobacteria as a Host Organism

Table 15.1 Titers for biofuel and chemical productions from cyanobacteria

Chapter 16: Host Organisms: Algae

Table 16.1 Reporter genes for nuclear expression
Table 16.2 Reporter genes for chloroplast expression

Chapter 18: Industrial Microorganisms: Saccharomyces cerevisiae and other Yeasts

Table 18.1 Yeasts used in biotechnology

Chapter 19: Industrial Microorganisms: Pichia pastoris

Table 19.1 First generation of P. pastoris promoters used for recombinant protein production
Table 19.2 New generation of P. pastoris promoters
Table 19.3 Recessive selection markers used for P. pastoris.
Table 19.4 Dominant selection markers used for P. pastoris

Related Titles

Mozzi, F., Raya, R.R., Vignolo, G.M. (eds.)

Biotechnology of Lactic Acid Bacteria - Novel Applications 2e

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Print ISBN: 978-1-118-86840-9

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(Further Volumes of the “Advanved Biotechnology” Series:)

Published:

Villadsen, J. (ed.)

Fundamental Bioengineering

2016

Print ISBN: 978-3-527-33674-6

Love, J. Ch. (ed.)

Micro- and Nanosystems for Biotechnology

2016

Print ISBN: 978-3-527-33281-6

Coming soon:

Yoshida, T. (ed.)

Applied Bioengineering

2017

Print ISBN: 978-3-527-34075-0

Wittmann, Ch., and Liao, J.C. (eds.)

Industrial Biotechnology}{Products and Processes

2017

Print ISBN: 978-3-527-34181-8

Planned:

Systems Biology

J. Nielsen & S. Hohmann (Chalmers University, Sweden)

2017

Print ISBN: 9783527335589

Emerging Areas in Bioengineering

H. N. Chang (KAIST, South Korea)

2017

Print ISBN: 9783527340880

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To Heike, Isabelle, Felix, and Florian, with thanks from Christoph for their love, support, and inspiration.