Biomolecular Engineering Solutions For Renewable Specialty Chemicals Microorganisms Products and Processes R Navanietha Krishnaraj Full Chapter

Biomolecular Engineering Solutions for
Renewable Specialty Chemicals:

Microorganisms, Products and
Processes R. Navanietha Krishnaraj
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Discover biomolecular engineering technologies for the production of KRISHNARAJ

biofuels, pharmaceuticals, organic and amino acids, vitamins, biopolymers, SANI
surfactants, detergents, and enzymes
In Biomolecular Engineering Solutions for Renewable Specialty Chemicals, distinguished researchers and
editors Drs. R. Navanietha Krishnaraj and Rajesh K. Sani deliver a collection of insightful resources on
advanced technologies in the synthesis and purification of value-added compounds. Readers will dis-
BIOMOLECULAR
cover new technologies that assist in the commercialization of the production of value-added products.
The editors also include resources that offer strategies for overcoming current limitations in biochemical
ENGINEERING SOLUTIONS
FOR RENEWABLE
BIOMOLECULAR ENGINEERING SOLUTIONS

synthesis, including purification. The articles within cover topics like the rewiring of anaerobic microbial
FOR RENEWABLE SPECIALTY CHEMICALS

processes for methane and hythane production, the extremophilic bioprocessing of wastes to biofuels,
reverse methanogenesis of methane to biopolymers and value-added products, and more.
The book presents advanced concepts and biomolecular engineering technologies for the production
of high-value, low-volume products, like therapeutic molecules, and describes methods for improv-
SPECIALTY CHEMICALS
ing microbes and enzymes using protein engineering, metabolic engineering, and systems biology
approaches for converting wastes.
MICROORGANISMS, PRODUCTS, AND PROCESSES
Readers will also discover:
• A thorough introduction to engineered microorganisms for the production of

biocommodities and microbial production of vanillin from ferulic acid
• Explorations of antibiotic trends in microbial therapy, including current approaches
EDITED BY
and future prospects, as well as fermentation strategies in the food and beverage industry
• Practical discussions of bioactive oligosaccharides, including their production,
R. NAVANIETHA KRISHNARAJ
characterization, and applications
RAJESH K. SANI
• In-depth treatments of biopolymers, including a retrospective analysis in the facets
of biomedical engineering
Perfect for researchers and practicing professionals in the areas of environmental and industrial
biotechnology, biomedicine, and the biological sciences, Biomolecular Engineering Solutions for
Renewable Specialty Chemicals is also an invaluable resource for students taking courses involving
biorefineries, biovalorization, industrial biotechnology, and environmental biotechnology.
R. Navanietha Krishnaraj, PhD, is Research Professor in the Composite and Nanocomposite

Advanced Manufacturing-Biomaterials Center in the Department of Chemical and Biological Engi-
neering at the South Dakota School of Mines and Technology. He received the Award for Cutting
Edge Research (Fulbright Faculty Award) in 2016.
Rajesh K. Sani, PhD, is Professor in the Departments of Chemical and Biological Engineering at
South Dakota School of Mines and Technology, South Dakota, USA. He is the Biocatalysis Program
Committee Member for the Society for Industrial Microbiology and Biotechnology.
Cover Design: Wiley

Cover Image: © Vetre/Shutterstock
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Biomolecular Engineering
Solutions for Renewable
Specialty Chemicals
Biomolecular Engineering Solutions
for Renewable Specialty Chemicals
Microorganisms, Products, and Processes
Edited by
R. Navanietha Krishnaraj
South Dakota School of Mines and Technology
Rapid City, SD, USA
Rajesh K. Sani
South Dakota School of Mines and Technology
Rapid City, SD, USA
This edition first published 2022
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Set in 9.5/12.5pt STIXTwoText by Straive Pondicherry, India
10 9 8 7 6 5 4 3 2 1
v
Contents
Preface xvii
List of Contributors xix
1 Engineered Microorganisms for Production of Biocommodities 1

Akhil Rautela and Sanjay Kumar
1.1 Introduction 1
1.2 Fundamentals of Genetic Engineering 2
1.2.1 DNA-altering Enzymes 2
1.2.1.1 DNA Polymerases 4
1.2.1.2 Nucleases 4
1.2.1.3 Ligases 5
1.2.1.4 DNA-modifying Enzymes 6
1.2.2 Vectors 7
1.2.3 Incorporation of Modified DNA into Host 8
1.2.3.1 Introducing Recombinants into Prokaryotes 8
1.2.3.2 Introducing Recombinants into Eukaryotic Hosts 9
1.2.4 Selection of Transformants 10
1.2.4.1 Direct Selection 10
1.2.4.2 Identification of the Clone from a Gene Library 11
1.3 Beneficial Biocommodities Produced Through Engineered
Microbial Factories 12
1.3.1 Biopolymers 13
1.3.1.1 Cellulose 14
1.3.1.2 Poly-ϒ-glutamic Acid 15
1.3.1.3 Hyaluronic Acid 16
1.3.1.4 Polyhydroxyalkoate 18
1.3.2 Organic Acids 20
1.3.2.1 Citric Acid 21
1.3.2.2 Lactic Acid 23
vi Contents
1.3.2.3 Succinic Acid 24

1.3.2.4 Fumaric Acid 26
1.3.3 Therapeutic Proteins 27
1.4 Photosynthetic Production of Biofuels 28
1.4.1 Biohydrogen 29
1.4.2 Biodiesel 30
1.4.3 Bioethanol 31
1.4.4 Terpenoids 32
1.5 Conclusion 34
References 34
2 Microbial Cell Factories for the Biosynthesis of Vanillin and Its

Applications 49
Sukumaran Karthika, Manoj Kumar, Santhalingam Gayathri,
Perumal Varalakshmi, and Balasubramaniem Ashokkumar
2.1 Introduction 49
2.2 Natural Sources of Vanilla and Its Production 51
2.3 Biotechnological Production of Vanillin 52
2.3.1 Enzymatic Synthesis of Vanillin 52
2.3.2 Microbial Biotransformation of Ferulic Acid to Vanillin 54
2.3.3 Agro-wastes as a Source for Biovanillin Production 58
2.4 Strain Development for Improved Production of Vanillin 60
2.4.1 Metabolic and Genetic Engineering 60
2.5 Bioactive Properties of Vanillin 63
2.5.1 Antimicrobial Activity 63
2.5.2 Antioxidant Activity 63
2.5.3 Anticancer Activity 64
2.5.3.1 Apoptosis Pathway 64
2.5.3.2 Tumor Necrosis Factor-induced Apoptosis 64
2.5.3.3 Cell Cycle Arrest 65
2.5.3.4 Nuclear Factor κB (NF-κB) Pathway 65
2.5.4 Anti-sickling Activity 65
2.5.5 Hypolipidemic Activity 66
2.6 Conclusion 66
Acknowledgments 66
References 67
3 Antimicrobials: Targets, Functions, and Resistance 77

Madhuri Dutta, Sinjini Patra, Shivam Saxena, and Anasuya Roychowdhury
3.1 Introduction 77
3.2 Classification of Antibiotics 77
3.2.1 Classification of Antibiotics Based on Mode of Action: Bactericidal
and Bacteriostatic 78
Contents vii
3.2.2 Classification of Antibiotics Based on the Spectrum of Action:

Broad- and Narrow-spectrum Antibiotics 79
3.3 Antibacterial Agents 79
3.3.1 Penicillins 79
3.3.1.1 Mechanism of Action 82
3.3.1.2 Clinical Implications 83
3.3.2 Cephalosporins 83
3.3.2.2 Clinical Indications 85
3.3.3 Macrolides 85
3.3.3.2 Clinical Indications 85
3.3.4 Fluoroquinolones 86
3.3.4.2 Clinical Indication 86
3.3.5 Sulfonamides 87
3.3.6 Tetracyclines 88
3.3.7 Aminoglycosides 89
3.4 Antifungal Agents 89
3.4.1 Polyenes 90
3.4.2 Azoles 90
3.4.3 Echinocandins 93
3.4.4 Flucytosine 95
3.4.4.2 Clinical Implication 95
3.5 Antiviral agents 95
3.6 Antiparasitic Agents 98
3.6.1 Antiprotozoan Agents 98
3.6.2 Antihelminthic Agents 101
3.6.3 Ectoparasiticides 101
viii Contents
3.7 ntimicrobial Resistance 101

A
3.7.1 Genetic Basis of AMR 102
3.7.2 Mechanistic Basis of Antimicrobial Resistance 102
3.8 Conclusion 103
Acknowledgment 104
References 104
4 Trends in Antimicrobial Therapy: Current Approaches and Future

Prospects 111
Mohan Kumar Verma, Santhalingam Gayathri, Balasubramaniem
Ashokkumar, and Perumal Varalakshmi
4.1 Introduction 111
4.2 Antibiotics: A Brief History 112
4.2.1 Classification of Antibiotics 113
4.2.2 Evolution of Antibiotics 113
4.2.3 Mechanism of Action of Antibiotics 113
4.3 AMR: A Global Burden 113
4.3.1 Global Scenario 114
4.3.2 Origin of SUPERBUGS and the “END of Antibiotics” 116
4.4 Antimicrobial Resistance and Virulence 117
4.4.1 Molecular Insights and Mechanism of AMR 117
4.4.2 Antibiotic Resistance in Bacteria 118
4.4.2.1 Horizontal Gene Transfer 118
4.4.2.2 Increased Mutation Rate 118
4.4.2.3 Antibiotic Inactivation 118
4.4.2.4 Alteration of the Antibiotic Targets 119
4.4.2.5 Changes in Cell Permeability and Efflux 119
4.4.2.6 The Major Facilitator Superfamily 119
4.4.2.7 The ATP-Binding Cassette Superfamily 119
4.4.2.8 The Multidrug and Toxic Compound Extrusion Family 120
4.4.2.9 The Resistance–Nodulation–Division (RND) Superfamily 120
4.4.2.10 The Small Multidrug-Resistance Family 120
4.4.3 Development of Antibiotic Resistance 120
4.4.4 Prioritization of Antibiotic Resistant Bacteria 120
4.4.5 Understanding Biofilm Resistance 122
4.5 Alternatives to Antibiotics 122
4.5.1 Peptide Antibiotics 122
4.5.1.1 Cationic Antimicrobial Peptides (CAMPs) 122
4.5.1.2 Marine Antimicrobial Peptides 123
4.5.2 Nano Drugs 124
4.5.3 Probiotics 125
4.5.4 Bacteriocins 126
Contents ix
4.5.5 Bdellovibrio 127

4.5.6 Bdellovibrio as Live Antimicrobial Agent 128
4.6 Antibiotics: Global Action Plan on Antimicrobial Resistance 129
4.7 Conclusion 130
Acknowledgment 130
References 131
5 Fermentation Strategies in the Food and Beverage Industry 141

Mohit Bibra, R. Navanietha Krishnaraj, and Rajesh K. Sani
5.2 Current Trends in Food Fermentation 143
5.2.1 Fermentation Types 144
5.2.1.1 Spontaneous Fermentation 144
5.2.1.2 Back-Slopping Fermentation 144
5.2.1.3 Starter-Culture Fermentation 144
5.2.2 Microbial Cultures 145
5.2.2.1 Starter Cultures 145
5.2.2.2 Adjunct Cultures 155
5.2.2.3 Bio-protective Cultures 155
5.2.2.4 Probiotic Cultures 155
5.3 Future Directions 156
5.3.1 Use of Defined Mixed Cultures 156
5.3.2 Nanotechnology 157
5.3.2.1 Nanosensors 157
5.3.2.2 Nanoparticles 157
5.3.2.3 Nanocomposites 157
5.3.3 Meat Analogues 158
5.4 Conclusions 158
5.5 Questions for Thought 159
References 160
6 Bioactive Oligosaccharides: Production, Characterization,

and Applications 165
R. Aanandhalakshmi, K. Sundar, and B. Vanavil
6.2 Sources, Types, Structure of Oligosaccharides 166
6.2.1 Plant Source 166
6.2.2 Animal Source 167
6.2.3 Insect Source 167
6.2.4 Marine Source 167
6.2.5 Microbial Source 168
6.2.6 Synthetic Oligosaccharides 168
x Contents
6.2.7 Pseudo-oligosaccharides 168

6.3 Production Methods of Oligosaccharides 169
6.3.1 Chemical Methods 169
6.3.2 Physical Methods 169
6.3.3 Enzymatic Hydrolysis 171
6.3.4 Microbial Production of Oligosaccharides 171
6.4 Extraction, Separation, and Purification of Oligosaccharides 172
6.5 Characterization of Oligosaccharides 174
6.6 Functional Properties of Oligosaccharides 174
6.7 Applications of Oligosaccharides 175
6.7.1 Functional Foods, Nutraceuticals, and Prebiotics 176
6.7.2 Pharmaceutical and Medical Applications 176
6.7.2.1 Effects on Intestinal Microflora 176
6.7.2.2 Effects on Urogenital Infections 177
6.7.2.3 Type II Diabetes and Obesity 177
6.7.2.4 Immunomodulatory and Antitumor Activities 178
6.7.2.5 Effect on Cardiovascular Risk 178
6.7.2.6 Lowering of Cholesterol 178
6.7.2.7 Role in Osteoporosis 178
6.7.2.8 Antihypertensive Effects 179
6.7.2.9 Hepatic Protection 179
6.7.2.10 Antioxidant and Neuroprotective Agent 179
6.7.2.11 Antimicrobial Activity 180
6.7.2.12 Antibiotics 180
6.7.2.13 Oligosaccharides as Vaccine Components 181
6.7.3 Environmental Fortification 181
6.7.4 Cosmetics 182
6.7.5 Elicitors and Agriculture 182
6.7.6 Novel Biomaterials 183
6.8 Market Potential of Oligosaccharides 183
6.9 Future Prospects 184
References 184
7 Biopolymers: A Retrospective Analysis in the Facet of Biomedical

Engineering 201
Gayathri Ravichandran and Aravind Kumar Rengan
7.2 Natures’ Advanced Materials: A Glance at Its Structure
and Properties 202
7.2.1 Polypeptides 202
7.2.1.1 Collagen 202
Contents xi
7.2.1.2 Elastin 202

7.2.1.3 Silk Fibroin 204
7.2.1.4 Gelatin 204
7.2.1.5 Albumin 205
7.2.1.6 Casein 205
7.2.2 Polysaccharides 205
7.2.2.1 Cellulose 205
7.2.2.2 Starch 206
7.2.2.3 Cyclodextrin 207
7.2.2.4 Hyaluronic Acid 207
7.2.2.5 Chitosan 208
7.2.2.6 K-carrageenan 208
7.2.2.7 Agarose 209
7.2.2.8 Alginate 209
7.2.3 Polynucleotides-based Biopolymers 210
7.3 Smart Biopolymers 211
7.3.1 Chemical-Responsive Biopolymers 211
7.3.1.1 pH-Sensitive Smart Biopolymers 211
7.3.1.2 Glucose-Responsive Biopolymers 212
7.3.2 Physically Responsive Biopolymers 212
7.3.2.1 Temperature-Sensitive Smart Biopolymers 212
7.3.2.2 Light-Responsive Smart Polymers 213
7.3.2.3 Electric-Responsive Smart Polymers 213
7.3.2.4 Magnetic-Responsive Smart Polymers 214
7.3.2.5 Redox-Responsive Biopolymer 215
7.3.3 Biochemical Stimuli-Responsive Biopolymers 215
7.3.3.1 Enzyme-Responsive Biopolymer 215
7.4 Fundamental Applications of Biopolymers in Biomedical
Engineering 216
7.4.1 Biopolymers in Cancer Theranostics 216
7.4.1.1 Drug Delivery 217
7.4.1.2 Cancer Diagnosis and Molecular Imaging 218
7.4.2 Biopolymeric-based Biosensor 219
7.4.3 Wound Healing 222
7.4.4 Tissue Engineering and Regenerative Medicine 223
7.4.4.1 Biopolymers as Bioink for 3D Scaffolds 225
7.4.4.2 Corneal Regeneration 225
7.4.4.3 Neural Tissue Engineering 226
7.4.4.4 Bone Tissue Engineering 227
7.4.4.5 Cartilage Tissue Regeneration 227
7.4.5 Biopolymers for Biological Implants 229
xii Contents
7.4.6 Biopolymers in Other Applications 230

7.5 Processing Techniques for the Contrivance of Biopolymers 230
7.5.1 3D Bioprinting 230
7.5.2 4D Bioprinting 232
7.5.3 Electrospinning 233
7.6 Conclusion 234
Acknowledgments 234
References 234
8 Metabolic Engineering Strategies to Enhance Microbial Production

of Biopolymers 247
Shailendra Singh Shera and Rathindra Mohan Banik
8.2 Microbes as Cell Factories for the Production of Speciality
Biochemicals 248
8.2.1 Bacteria as Cell Factories for the Production of Biopolymers 249
8.2.1.1 Polysaccharides 249
8.2.1.2 Polyesters 250
8.2.1.3 Polyamides 251
8.2.2 Fungus as Cell Factories for the Production of Biopolymers 252
8.2.2.1 Polysaccharides 253
8.2.2.2 Polyester 253
8.2.3 Microalgae as Cell Factories for the Production of Biopolymers 254
8.2.3.1 Polysaccharides from Microalgae 255
8.2.3.2 Polyester 255
8.3 Microbial Production Pathways for Various Types of
Biopolymers 256
8.3.1 Polysaccharide Production Pathways in Bacteria 256
8.3.2 Mechanism of Fungal Polysaccharides Synthesis 260
8.3.3 Mechanism of Synthesis of Polyester in Bacteria 260
8.3.4 Mechanism of Synthesis of Polyamide in Bacteria 262
8.4 Tools and Technologies Available for Metabolic Engineering 262
8.4.1 Metabolic Pathway Reconstruction 263
8.4.2 Metabolic Flux Analysis 264
8.4.3 Metabolic Control Analysis 266
8.4.4 Omics Analysis 266
8.5 Dynamic Metabolic Flux Analysis and its Role in Metabolic
Engineering 268
8.6 Production of Biopolymers from Metabolically Engineered
Microbes 269
Contents xiii
8.6.1 Metabolic Modification of Pathway for Synthesis

of Polysaccharides 269
8.6.2 Levan 271
8.6.3 Metabolic Modification of Pathway for Synthesis of Polyester 271
8.6.4 Metabolic Modification of Pathway for Synthesis of Polyamides 272
8.6.5 Culture of Metabolically Engineered Microbes in Fermentation or
Bioreactor for Production of Biopolymer 273
8.7 Recovery and Purification of Biopolymers from Fermentation Broth 275
8.7.1 Separation and Purification of Xanthan 275
8.7.2 Separation of Poly-l-lysine 277
8.8 Conclusion and Future Challenges 278
Acknowledgments 278
References 279
Web References 285
9 Bioplastics Production: What Have We Achieved? 287

Tanvi Govil, David R. Salem, and Rajesh K. Sani
9.2 Current Trends 289
9.3 Different Types of Bioplastics 291
9.3.1 Bio-based Polyethylene (Bio-PE) 291
9.3.2 Bio-based PET 292
9.3.3 Polylactic Acid 293
9.3.4 Starch Blends 294
9.3.5 Polyhydroxyalkanoate 295
9.3.6 Polybutylene Succinate 298
9.3.7 Polybutylene Adipate Terephthalate 299
9.3.8 Polycaprolactone 299
9.3.9 Epoxies 300
9.3.10 Cellulose Acetate 300
9.4 Challenges Facing the Bioplastics Industry 301
9.5 Misconceptions and Negative Impacts 301
9.6 Take Home Message and Future Directions 302
9.7 Questions for Thought 303
Acknowledgments 304
Conflict of Interest 304
References 304
10 Conversion of Lignocellulosic Biomass to Ethanol: Recent

Advances 311
Ramiya Baskaran, Vignesh Natarajan, Shereena Joy,
and Chandraraj Krishnan
xiv Contents
10.1 I ntroduction 311

10.2 LCB: Structure, Composition, and Recalcitrance 312
10.3 LCB to Ethanol: Bioprocess Strategies 313
10.4 Pretreatment of LCB 313
10.4.1 Physical Pretreatment 316
10.4.2 Physicochemical Pretreatment 319
10.4.2.1 Steam Explosion 320
10.4.2.2 Liquid Hot Water 320
10.4.2.3 Ammonia Fiber Explosion 321
10.4.3 Chemical Pretreatment 321
10.4.3.1 Dilute Acid Pretreatment (DAP) 322
10.4.3.2 Alkali Pretreatment 322
10.4.3.3 Organosolv 323
10.4.3.4 Ionic Liquid (IL) and Deep Eutectic Solvent (DES) 323
10.4.3.5 Supercritical Fluid Pretreatment 324
10.4.4 Biological Pretreatment 325
10.4.4.1 Bacterial Pretreatment 325
10.4.4.2 Fungal Pretreatment 325
10.4.4.3 Enzymatic Pretreatment 326
10.4.5 Optimization of Pretreatment Process 326
10.5 Enzymatic Hydrolysis 327
10.5.1 Cellulose Hydrolysis 327
10.5.2 Xylan Hydrolysis 328
10.5.3 Accessory Enzymes 328
10.5.4 Auxiliary Activity and Non-Hydrolytic Enzymes 330
10.5.5 Enzyme Cocktail for Biomass Hydrolysis 331
10.5.5.1 Cocktail Development 331
10.6 High Solids Loading Enzymatic Hydrolysis (HSLEH) 335
10.6.1 Enzyme Inhibitors and Detoxification 335
10.6.2 Cellulase Feedback Inhibition 336
10.6.3 Rheology 337
10.6.4 Reactors and Impellers 337
10.7 Fermentation 338
10.8 Genetic Engineering in LCB Bioconversion 343
10.9 Conclusions 344
Acknowledgments 344
References 344
11 Advancement in Biogas Technology for Sustainable Energy

Production 359
Rouf Ahmad Dar, Saroj Bala, and Urmila Gupta Phutela
Contents xv
11.2 iogas Developments Worldwide 360

B
11.3 Biogas Development in India 363
11.4 Recent Issues in Biogas Production 365
11.5 Current Trends in Biogas Production 365
11.6 Advanced Anaerobic Digestion Methodologies 367
11.6.1 Anaerobic Membrane Reactor (AnMBRs) 368
11.6.2 Dry Anaerobic Digestion Technology (DADT) 368
11.6.3 Anaerobic Co-digestion Technology (AcoD) 369
11.7 Role of Biotechnology in Enhancing Biogas Production 370
11.8 Application of Nanotechnology in Biogas and Methane
Production 371
11.9 Biogas Upgrading Technologies 372
11.10 Conclusion 372
References 378
12 Biofertilizers: A Sustainable Approach Towards Enhancing

the Agricultural Productivity 387
Satya Sundar Mohanty
12.2 Types of Biofertilizers 388
12.2.1 Nitrogen-Fixing Biofertilizer 389
12.2.1.1 Free-Living Nitrogen-Fixing Microorganisms 390
12.2.1.2 Photosynthetic Nitrogen-Fixing Microorganisms 390
12.2.2 Phosphorus Biofertilizer 391
12.2.2.1 Phosphate-solubilizing Bacteria (PSB) 392
12.2.2.2 Phosphate-mobilizing Microorganisms 394
12.2.3 Plant-Growth-promoting Biofertilizers 394
12.3 Effect on Bioremediation of Environmental Pollutants 396
12.4 Bioformulations and Its Types 398
12.5 Preparation of Biofertilizers 401
12.6 Various Modes of Biofertilizer Application 402
12.7 Challenges to Commercialization of Biofertilizers 403
12.8 Future Perspective 403
References 404
13 Biofertilizers from Food and Agricultural By-Products

and Wastes 419
Veknesh Arumugam, Muhammad Heikal Ismail, and Winny Routray
13.2 Biofertilizer 420
13.2.1 N2-fixing Biofertilizer 422
13.2.1.1 Free-living N2-fixing Biofertilizer 422
xvi Contents
13.2.1.2 Symbiotic N2-Fixing Biofertilizer 424

13.2.2 Phosphate-solubilizing Biofertilizers 425
13.2.3 Phosphate-mobilizing Biofertilizer 425
13.2.4 Plant-Growth- promoting Biofertilizers 426
13.3 Agricultural Waste 426
13.3.1 Agro-industrial Wastes 428
13.4 Food Waste 430
13.5 Biofertilizer Production Using Fermentation Technology 432
13.5.1 Solid-State Fermentation (SSF) 433
13.5.2 Submerged Fermentation (SmF) 435
13.5.3 Production of N2-fixing Biofertilizer 438
13.5.3.1 Production of Rhizobium Biofertilizer 438
13.5.3.2 Production of Azotobacter Biofertilizer 438
13.5.3.3 Production of Azospirillum Biofertilizer 439
13.5.4 Production of Phosphate-solubilizing Biofertilizer 439
13.5.5 Production of Phosphate-mobilizing Biofertilizer 439
13.6 Biofertilizer for Organic Farming 440
13.7 Conclusion 441
Conflict of Interest 441
References 442
Index 449
xvii
Preface
Biocommodity Engineering
Microorganisms have been realized as promising sources for production of biocom-

modities such as biofuels, pharmaceuticals, organic acids, amino acids, vitamins,
biopolymers, surfactants, detergents, and enzymes. They offer several advantages
over the conventional chemical processes including mild operating conditions, ste-
reospecificity of the products, environmentally benign nature, and ecofriendly.
Translating the bioproducts from laboratory to the industry remains a bottleneck.
Biocommodity and biomolecular engineering approaches help in overcoming
these limitations, developing new products, and improving the processes.
Considering the importance of the field, this book is specifically focused on poten-
tial technologies that can help in commercializing the processes. The objective of
the book is to provide advanced technologies in producing different products using
improved microorganisms/enzymes. This book will also discuss on improving the
microbes or enzymes using protein engineering, metabolic engineering, and sys-
tems biology approaches for converting the wastes to value‐added products.
Overall, this would be an ideal textbook for bioprocess, biorefinery, biomolecu-
lar, and biocommodity engineering courses for chemical, biochemical, and envi-
ronmental engineering students. We have also included glossary and reasoning
type questions at the end of each chapter. This book will also help the scientists to
understand the advanced concepts in biomanufacturing. This book discusses the
promising strategies that will help overcome the current limitations in the bio-
chemical synthesis processes. The book will help the readers working in industry
or research to know about the new ways for improving the efficiency of the bio-
chemical synthesis processes.
The editors would like to thank all the authors for their valuable contribution
and the Wiley editorial team for their support.
xix
List of Contributors
R. Aanandhalakshmi Rathindra Mohan Banik

Department of Biotechnology Bioprocess Technology
School of Bio and Chemical Engineering Laboratory
Kalasalingam Academy of Research School of Biochemical
and Education Engineering
Krishnankoil, TN Indian Institute of Technology
India (Banaras Hindu University)
Varanasi, UP
Veknesh Arumugam India
Department of Chemical and
Environmental Engineering Ramiya Baskaran
Faculty of Engineering Department of Biotechnology
Universiti Putra Malaysia Indian Institute of Technology Madras
Serdang, Malaysia Chennai, TN
India
Balasubramaniem Ashokkumar
Department of Genetic Engineering Mohit Bibra
School of Biotechnology Zymergen Inc.,
Madurai Kamaraj University Emeryville, CA
Madurai, TN USA
India
Rouf Ahmad Dar
Saroj Bala Department of Microbiology
Department of Microbiology Punjab Agricultural University
Punjab Agricultural University Ludhiana, PB
Ludhiana, PB India
India
xx List of Contributors
Madhuri Dutta Sukumaran Karthika

Biochemistry and Cell Biology Department of Genetic
Laboratory Engineering
School of Basic Sciences School of Biotechnology
Indian Institute of Technology Madurai Kamaraj University
Bhubaneswar, OR Madurai, TN
India India
Santhalingam Gayathri Chandraraj Krishnan

Department of Genetic Engineering Department of Biotechnology
School of Biotechnology Indian Institute of Technology Madras
Madurai Kamaraj University Chennai, TN
Madurai, TN India
India
R. Navanietha Krishnaraj
Tanvi Govil South Dakota School of Mines and
Department of Chemical and Technology
Biological Engineering Department of Chemical and
South Dakota Mines Biological Engineering
Rapid City, SD Rapid City, SD
USA USA
Composite and Nanocomposite BuG ReMeDEE Consortium,
Advanced Manufacturing – South Dakota School of Mines and
Biomaterials Center Technology
Rapid City, SD Rapid City, SD
USA USA
Composite and Nanocomposite
Muhammad Heikal Ismail
Advanced Manufacturing Centre –
Department of Chemical and
Biomaterials (CNAM/Bio)
Environmental Engineering
Rapid City, SD
Faculty of Engineering
USA
Universiti Putra Malaysia
Serdang, Malaysia
Manoj Kumar
Shereena Joy Department of Genetic Engineering
Department of Biotechnology School of Biotechnology
Indian Institute of Technology Madras Madurai Kamaraj University
Chennai, TN Madurai, TN
India India
List of Contributors xxi
Sanjay Kumar Akhil Rautela

School of Biochemical Engineering School of Biochemical Engineering
IIT (BHU) Varanasi IIT (BHU) Varanasi, Varanasi
Varanasi, UP UP, India
India
Gayathri Ravichandran
Satya Sundar Mohanty Department of Biomedical
Assistant Professor Engineering Indian Institute of
Department of Biotechnology Technology, Hyderabad
School of Agriculture and Biosciences Kandi, Telangana
Karunya Institute of Technology and India
Sciences
Coimbatore, TN
Aravind Kumar Rengan
India
Department of Biomedical
Engineering Indian Institute of
Vignesh Natarajan
Technology, Hyderabad
Department of Biotechnology
Kandi, Telangana
Indian Institute of Technology
India
Madras
Chennai, TN
India Winny Routray
Department of Food Process
Engineering
Sinjini Patra
National Institute of Technology
Biochemistry and Cell Biology Laboratory
Rourkela, OR
School of Basic Sciences
India
Bhubaneswar, OR
Anasuya Roychowdhury
India
School of Basic Sciences
Biochemistry and Cell Biology
Urmila Gupta Phutela
Laboratory
Department of Microbiology
Punjab Agricultural University
Bhubaneswar, OR
Ludhiana, PB
India
India
Department of Renewable Energy David R. Salem
Engineering Department of Chemical and
Punjab Agricultural University Biological Engineering
Ludhiana, PB South Dakota Mines
India Rapid City, SD
USA
xxii List of Contributors
Composite and Nanocomposite Shailendra Singh Shera

Advanced Manufacturing – Department of Biotechnology
Biomaterials Center Faculty of Engineering & Technology
Rapid City, SD Rama University
USA Kanpur, UP
India
Department of Materials and
Metallurgical Engineering
K. Sundar
South Dakota Mines
Rapid City, SD
School of Bio and Chemical
USA
Engineering
Kalasalingam Academy of Research
Rajesh K. Sani
and Education
South Dakota School of Mines and
Krishnankoil, TN
Technology
India
Department of Chemical
and Biological Engineering
B. Vanavil
Rapid City, SD
USA
School of Bio and Chemical
South Dakota School of Mines Engineering
and Technology Kalasalingam Academy of Research
BuG ReMeDEE Consortium and Education
Rapid City, SD Krishnankoil, TN
USA India
Composite and Nanocomposite
Perumal Varalakshmi
Advanced Manufacturing Centre –
Department of Molecular
Biomaterials (CNAM/Bio)
Microbiology
Rapid City, SD
School of Biotechnology
USA
Madurai Kamaraj University
South Dakota School of Mines and Madurai, TN
Technology India
Department of Chemistry and
Applied Biological Sciences Mohan Kumar Verma
Rapid City, SD School of Biotechnology
USA Department of Molecular
Microbiology
Shivam Saxena Madurai Kamaraj University
Biochemistry and Cell Biology Laboratory Madurai, TN
School of Basic Sciences India
Bhubaneswar, OR
India
1
Engineered Microorganisms for Production

of Biocommodities
Akhil Rautela and Sanjay Kumar
School of Biochemical Engineering, IIT (BHU) Varanasi, Varanasi, UP, India
1.1 Introduction
As we are going toward becoming more developed, we tend to see our transition
toward more sustainable resources and knowing and understanding the life form
more. This leads to the use of the living system and engineer them to produce bio-
commodities such as fuels, polymers, hormones, therapeutic proteins and peptides,
and neurotransmitters, which is termed as biocommodity engineering. It basically
deals with the need of society. Biotechnology, genetic engineering, and biocommod-
ity engineering can be combined to meet these needs. The foundation of biocom-
modity engineering lies in molecular biology, which is also the foundation of genetic
engineering or recombinant DNA technology (rDT). Therefore, it can be said that
these terms are interrelated to each other. The majority of the biocommodities con-
sumed by humans were earlier isolated from plants and animals, posing the threat
of activation of immune reactions in humans. So, the machinery of the synthesis of
these biocommodities can be engineered in microorganisms.
Its main aim is to engineer microorganisms to get a high yield of the product, use
cheap raw material as a substrate so that cost of the product can be minimized, easy
downstream processing, increasing robustness of the microorganism, etc. All this
can be achieved by genetically modifying the organisms using genetic toolkits. This
chapter deals with the basics of genetic engineering, giving details about the enzymes
used, transformation techniques, and how to select a transformant from non-
transformants. Further sections compile the comprehensive data of the problems in
the production of biopolymers, organic acids, and therapeutic proteins from conven-
tional methods and development of mutant strains for the synthesis of these biocom-
modities. The last section of the chapter gives an insight about the biofuel production
Biomolecular Engineering Solutions for Renewable Specialty Chemicals: Microorganisms,

Products, and Processes, First Edition. Edited by R. Navanietha Krishnaraj and Rajesh K. Sani.
© 2022 John Wiley & Sons, Inc. Published 2022 by John Wiley & Sons, Inc.
2 1 Engineered Microorganisms for Production of Biocommodities
from photoautotrophic organisms such as cyanobacteria and microalgae, which uti-

lizes sunlight and carbon dioxide as energy and carbon source, respectively.
1.2 Fundamentals of Genetic Engineering
The advent of genetic engineering, also called rDT, started in 1952 with the discovery
of Hershey and Chase, stating DNA as the genetic material (Hershey and Chase, 1952).
Cohen and Boyer in the early 1970s were the first to show that the genetic material of
one organism can be easily expressed in the other. Genetic engineering (Figure 1.1), in
general, is the process in which the DNA is extracted, modified, transformed into a
host cell, and a new organism is formed. The DNA from the desired organism is
extracted and purified. It is then cleaved using restriction enzymes to get the gene of
interest from it. The DNA fragment is then ligated into a vector, which acts as a driving
vehicle for the DNA molecule to the host cells. This chimeric DNA molecule is then
transformed into the host cells, and selection procedure under suitable stress condi-
tions takes place. Finally, after numerous generations, the organism growing in the
stress conditions is said to be recombinant or genetically modified. Genetic engineer-
ing has emerged as a crucial step in the development of industrial bioprocesses.
Each and every organism has a different genetic (DNA) makeup, which in turn
makes the whole organism different with respect to their carbohydrates, lipids, and
proteins. This is due to the fact that DNA transcribes and translates to mRNA and
proteins, respectively (central dogma). This makes DNA the choice for manipulation
in genetic engineering as manipulating it leads to the generation of a whole new
organism. This postulation gives rise to many other disciplines of genetic engineering
like recombinant protein production, protein engineering, metabolic engineering, etc.
Every organism being different makes it difficult to use proteins and other bio-
molecules of one organism to the other. This was the main reason why proteins/
enzymes from animals cannot be used by humans. Earlier, insulin was extracted
from the pancreas of slaughtered pigs, posing a threat to human health. This leads
to the discovery of the first recombinant product, Insulin, approved by the US
Food and Drug Administration (FDA) in 1982 (Goeddel et al., 1979). Now syn-
thetic insulin is easily being produced by yeast worldwide as Escherichia coli does
not perform post-translational modifications required to form functional insulin.
Similarly, genetic engineering is now used to produce several other biocommodi-
ties. Modifying DNA and getting it expressed inside the host organism requires
several steps, as shown in Figure 1.1 and the number of enzymes. These enzymes
are explained in further sections with other requirements for genetic engineering.
1.2.1 DNA-altering Enzymes

The basis of rDT is the manipulation of DNA molecules with the help of molecu-
lar biology tools and biocatalysts. The available purified enzymes that can
1. Desired organism
selection
Restriction digestion of DNA
Bacterial cell Animal cell
Restriction
2. Extraction of DNA enzyme
dsDNA
3. Restriction digestion of
DNA
Smal EcoRI
Gene of interest CCC GGG G AATT C
GGG CCC CTTAA G
Blunt ends Sticky ends
4. Restriction
digestion of vector
5. Joining of vector and 6. Vector 7. Insertion of 8. Genetically

gene of interest by DNA containing gene of vector into host engineered
ligase interest organism organism
Figure 1.1 Basic steps of gene cloning.

manipulate DNA molecules with specific changes can be categorized in four

broad classes: (i) DNA polymerases, (ii) nucleases, (iii) DNA ligases, and (iv) end-
modification enzymes.
1.2.1.1 DNA Polymerases

DNA polymerases is the key enzyme in DNA replication driving the synthesis of
new DNA strand from the parent DNA or RNA strand acting as a template. DNA
polymerases require an oligo nucleotide (primer) for the initiation of DNA strand
synthesis. DNA polymerase-I (DNA-dependent DNA polymerase) is widely stud-
ied polymerase and has both polymerization and exonuclease activity that can
help in synthesizing new strand as well as the degradation for proof reading or
repair and primer removal. DNA polymerase I (or Pol I) takes part in the process
of prokaryotic DNA replication. It was the first DNA polymerase discovered by
Arthur Kornberg in 1956 (Lehman et al., 1958). Pol I has three different enzymatic
activities: A 5′ →3′ DNA-dependent DNA polymerase activity, a 3′ →5′ exonucle-
ase activity that helps in proofreading, and a 5′ → 3′ exonuclease activity mediat-
ing nick translation during DNA repair. Pol I having polymerase but lacking
nuclease activity is called klenow fragments (Klenow and Henningsen, 1970;
Jacobsen et al., 1974).
Taq DNA polymerase, a thermostable DNA polymerase isolated from Thermus
aquaticus by Chien et al. (1976). It is frequently used in the polymerase chain
reaction (PCR), to amplify small quantities of DNA. It has a functional 5′ → 3′
exonuclease domain at the N-terminal, and 3′–5′ exonuclease domain was
changed so it is not functional. Optimum temperature for Taq pol activity is
75–80 °C, with a half-life of greater than 2 hours at 92.5 °C and minimum 9 min-
utes at 97.5 °C, and able to replicate a 1000 bp strand of DNA within 10 seconds
at 72 °C.
1.2.1.2 Nucleases
Nucleases can cut or digest the DNA molecules either from one end or in middle
by acting on phosphodiester bond, which forms the backbone of DNA (Nishino
and Morikawa, 2002). Depending on the position of their digestion nucleases are
of two types: exonucleases and endonucleases. Phosphodiester bonds present in
the ends of the DNA are digested by exonucleases, removing nucleotides one at a
time from either end. Whereas phosphodiester bonds present in the middle of the
DNA strand are digested by endonucleases. Specificity of nucleases vary from
source to source, Aspergillus oryzae’s endonuclease only cleaves single strands,
whereas deoxyribonuclease I (DNase I), extracted from cow pancreas, cuts single
as well as double-stranded DNA molecules. DNase I not being sequence specific
cuts DNA at random interior phosphodiester bond, leading to production of mon-
onucleotides and very short oligonucleotides mixture. Some of the examples of
1.2 Fundamentals of Genetic Engineerin 5
nucleases are (i) Mung Bean Nuclease (isolated from mung bean sprouts) – a
single-strand-specific DNA and RNA endonuclease which can degrade single
strand overhangs from the end of DNA and RNA to make blunt ends. (ii) Nuclease
S1 (isolated from Aspergillus sp.) – S1 nuclease is a single-strand-specific endonu-
clease that hydrolyzes single-stranded RNA or DNA into 5′ mononucleotides. The
enzyme will hydrolyze single-stranded extensions in duplex DNA such as loops
and gaps. S1 Nuclease is stable at 65 °C (Balabanova et al., 2012). (iii) Exonuclease
III (isolated from E. coli) – removes single nucleotides from 3′ termini of the
duplex DNA. It is generally used to make a set of nested deletions of the terminal
of linear DNA strand. (iv) BAL31 nuclease (isolated from Alteromonas espeji-
ana) – it is a 3′-exonuclease and removes nucleotides from both 3′-terminus of the
two strands of linear DNA. (v) RNase H – it is an endonuclease that specifically
hydrolyzes the phosphodiester bonds of RNA, when the RNA is hybridized to
DNA (RNA-DNA) (Cerritelli and Crouch, 2009). (vi) RNase P – the specificity of
this enzyme is to cleave other RNA molecules at the junction of single-stranded
and the 5′ end of double-stranded regions of RNA (Guerrier-Takada et al., 1983).
Restriction endonuclease (RE) enzymes recognize and cleave the specific phos-
phodiester bond present in the DNA molecule (Smith and Welcox, 1970).
Restriction enzymes are broadly classified into Type-I, Type-II, and Type-III. For
their functioning, they require specific temperature, ATP, and divalent magne-
sium ions. On digestion of the DNA molecule they can produce both blunt and
sticky end. Type I REs interact with unmodified target site in dsDNA. They are
bifunctional enzymes having methylase and endonuclease in a single protein mol-
ecule. They cleave DNA around 1000 bp away from the recognition site. For their
function, both ATP and Mg2+ are required. Type II REs are highly specific and
cleave within or very near to the recognition sequence due to this reason type II
are used widely in genetic engineering. They do not require ATP for the restriction
digestion, only Mg2+ is required. Type III REs cleave dsDNA at defined positions
and need ATP, Mg2+. They cleave the DNA 24–26 bp away from the specific site.
1.2.1.3 Ligases
Ligases are considered to be molecular glue in rDT, used to join two DNA seg-
ments. It also has role in various aspects of molecular biology such as replication,
recombination, and cloning. In the presence of ligase enzyme when two DNA
fragments are mixed under a certain condition, base pairing between two frag-
ments occur which results in sealing of two different DNA fragments to make a
chimera (Pascal et al., 2004). It occurs due to covalent bonds formation between
2′-PO4 group and 3′-OH group of adjustment strands.
1.2.1.3.1 Mechanism of Action The DNA ligation reaction has two main steps.
First, the DNA ends collide with each other by chance and reside together for
enough time for the ligase to join them. For sticky overhangs, there is an additional
reason; lower temperature stabilizes the hydrogen bonding between the
complementary nucleotides, which really helps to join easily. DNA ligase catalyzes
the joining of the 3′-OH to the 5′-phosphate via a two-step process. First, the AMP
nucleotide, which is linked to a lysine residue in the enzyme’s active site, is shifted
to the 5′-phosphate. Then the AMP-phosphate bond is attacked by the 3′-OH,
forming the covalent bond and discharging AMP. To allow the enzyme to conduct
further reactions, the AMP in the enzyme’s active site must be restocked by
ATP. The DNA ligase enzyme has optimal activity at 25 °C so the ligation reaction
is carried out at a temperature range of 16–25 °C. Normally, 1 hour at 16 °C is best
for ligation but since bringing the DNA ends together is the least efficient part of
the reaction favoring this by lowering the temperature to 4 °C can give even more
efficiency. Two types of ligases are there that are used in genetic engineering.
E. coli ligases which is generally used to join two sticky or cohesive ends. It is
mainly used to catalyze a phosphodiester bond between duplex DNA-containing
cohesive ends. It will not efficiently ligate blunt-ended fragments as much as it
efficient for sticky end ligation. NAD+ is required as a cofactor for the ligation
reaction. T4 DNA ligase that is generally used for the ligation of blunt-ended DNA
molecules. This enzyme can join blunt-ended termini as well as ends with cohesive
overhangs (either 3′ or 5′ complementary overhangs). This enzyme will also
function for the repair of single-stranded nicks in duplex DNA, RNA, or DNA/
RNA duplexes. ATP is required as a cofactor for the ligation reaction. All the
reactions are generally carried out at 16 °C. Sometimes PEG (polyethylene glycol)
can be added that helps in fusion and increases the frequency of collision between
two DNA molecules.
1.2.1.4 DNA-modifying Enzymes

1.2.1.4.1 Alkaline Phosphatase Alkaline phosphatase prevents self-ligation of
DNA molecule (vectors and gene of interest) in genetic engineering experiment
by dephosphorylating phosphate group on 3′ end of the DNA molecule. It is
extracted from E. coli or calf intestine. The enzymes catalyze the hydrolysis of
monoesters in phosphoric acid which can moreover catalyze a trans-phosphorylation
reaction with large concentrations of phosphate acceptors. It can be used to
prevent self-ligation of vectors in the cloning experiments because alkaline
phosphate-treated DNA fragments lack the 5′-phosphophate in the terminal,
required for the actions of DNA ligases (Tamás et al., 2002).
1.2.1.4.2 T4 Poly Nucleotide Kinase Polynucleotide kinase enzyme is exact

reverse of alkaline phosphatase. It adds phosphate group to on the 5′ end of the
DNA molecule and is extracted from phage-infected E. coli. The reaction with
polynucleotides can be made reversible, which consents the exchange of the
γ-phosphate of ATP with the 5′-terminal phosphate of a polynucleotide, thus

entangled the need of dephosphorylation the substrate DNA molecule with
alkaline phosphatase (Cameron and Uhlenbeck, 1977).
1.2.1.4.3 Terminal Transferase Terminal deoxy-nucleotidyl transferase [Terminal

transferase (TdT)] has the capability to transfer or add polynucleotide in 3′
terminus of the DNA molecule. It is extracted from calf thymus. TdT is a template
impartial polymerase that catalyzes the addition of deoxynucleotides to the 3′
hydroxyl terminus of DNA molecules with none template strand. In rDT, blunt-
ended DNA strands are required every now and then to make it cohesive via the
addition of nucleotides at 3′ of one strand (Greider and Blackburn, 1985)
1.2.1.4.4 Topoisomerases Topoisomerases change the affirmation of covalently

closed circular DNA molecule, such as plasmids with the aid of removing the
supercoils present in the round DNA molecule and alternate the linking number.
It performs a major role in replication and transcription when the DNA unwinds,
doing away with positive and negative supercoils (Liu, 1989).
1.2.2 Vectors
A vector is a DNA molecule, which act as molecular transporter, that can replicate
autonomously in an appropriate host cell and into which the gene of interest (a
foreign gene) is inserted. Insertion of a foreign gene into the vector is aiming
either to get numerous copies of the gene of interest or obtaining a product from
this gene. Vector is basically of two types: cloning vector and expression vector.
Characteristics of an ideal cloning vector are mentioned below:
1) It should have origin of replication, able to replicate autonomously.
2) It should be easily isolated and purified.
3) The vector should have suitable selection marker genes that will allow easy
selection of the transformed cells from nontransformed cells.
4) For gene transfer, vector should have the ability to integrate either itself or the
DNA insert it carries into the genome of the host cell.
5) The cells transformed with the vector containing the DNA insert should be
easily identifiable and selectable from those transformed cells having unal-
tered vector.
6) Unique restriction digestion sites should be present where gene of interest can
be inserted.
Expression vectors are different from cloning vectors. They are designed in such a
way that they should have a promoter sequence to express the gene. Expression
plasmid has all the information regarding transcription which is followed by
translation to synthesize proteins from mRNA. Expression vectors should have the
followings gene sequences: A strong promoter for the initiation, termination codon,
adaptation of distance between the promoter and cloned gene, incorporated tran-
scription termination sequence, and a compact translation initiation sequence.
Number of cloning vectors are also present which includes plasmid, cosmid,
phage vectors, bacterial artificial chromosomes (BACs), and yeast artificial chromo-
somes (YACs). All the vectors mentioned have different incorporation capacity for
foreign DNA. Plasmids are extra chromosomal circular double-stranded DNA repli-
cating elements present in bacterial cells. Plasmids size is ranging from 5.0 to 400 kb.
Plasmids are inserted into bacterial calls by a transformation. Plasmids can incorpo-
rate an insert size of up to 10 kb DNA fragment. Bacteriophage infects bacteria and
has a very unique mechanism for delivering its genome into bacterial cell. Hence, it
can be used as a cloning vector for larger DNA segments insertion. Phage vectors
can insert DNA fragments of size up to 20 kb. BACs are simple plasmid vectors that
are designed to integrate large DNA fragments of size 75–300 kb (Kim et al., 1996b).
BACs have antibiotic resistance marker genes and a very stable OriC that promotes
the distribution of plasmid after bacterial cell division and maintaining the plasmid
copy number to one or two per cell. YACs are yeast expression vectors. A very large
DNA fragments sizes ranging from 100 to 3000 kb can be cloned using YACs (Riley
et al., 1990). YACs have an extra benefit over BACs in expressing eukaryotic proteins
which need posttranslational modifications.
1.2.3 Incorporation of Modified DNA into Host

After engineering the DNA with the help of restriction enzymes, ligase, and getting
it attached to the vector, it is ready to go inside the host cells. How this vector that is
a recombinant vector now is made to enter inside the host cells is explained in brief.
1.2.3.1 Introducing Recombinants into Prokaryotes

The most frequently used model organism E. coli has the potential for becoming a
host for cloning purposes. A huge number of E. coli vectors are also available.
Transformation is the most used and effortless method used to carry the recombi-
nant DNA inside the host cells. There are several other methods some of which
are discussed.
1.2.3.1.1 Transformation Transformation is the most commonly used method in

cloning. In this simple technique, the DNA from the surrounding is taken up by
the competent cells. Some of the host cells like E. coli, yeast, and mammalian cells
are not naturally competent. These cells are made competent chemically so that
they can easily take external DNA. Calcium and magnesium chloride in specific
concentrations in cold condition can make E. coli cells competent.
1.2.3.1.2 Transduction Using phage particles for transferring foreign DNA into
the host cells is called transduction. Therefore, it is also called as phage-mediated
gene transfer. It can be classified into generalized and specialized transduction.
Generalized transduction involves phage particles attacking host cells and as the
assembly of phage particles takes place host DNA gets packed inside them. When
this phage particle infects another host cell, the DNA gets integrated into the host
genome through recombination. While in specialized transduction, the phage
genes get integrated with host genome and lysogenic cycle takes place. When
some stimulus is given, the phage particles carry more than one genes from the
host. These phage particles can infect a naive host cell by which the gene of
interest can be inserted into the host genome.
1.2.3.1.3 Conjugation Conjugation is the procedure of movement of hereditary

material from a donor cell to a beneficiary cell when they are in close contact. It
was found by Lederberg and Tatum in 1946 who indicated that two distinct strains
of microscopic organisms with various development prerequisites could trade
qualities. It was found by Lederberg and Tatum in 1946 who indicated that two
distinct strains of microscopic organisms with various development prerequisites
could trade qualities. They derived that the bacterial cells must cooperate with one
another so as to move the genetic material and the procedure is currently known
as sexual conjugation by direct contact. A section (not often all) of the donor’s
chromosome recombines with the recipient chromosome through homologous
recombination. For conjugation the key characteristic is the physical contact. The
two cells should be in close proximity. Recipients containing donor DNA are called
transconjugants. Genetic exchange through conjugation is unidirectional. Fertility
plasmid (F plasmid) plays an important role in conjugation. The donor cells are F+
and have F plasmid, while the recipient is not having F plasmid and is F−.
1.2.3.2 Introducing Recombinants into Eukaryotic Hosts

The usage of bacterial hosts for genetic engineering laid the muse of rDT era;
however, researchers have additionally had high-quality avocation in genetically
engineering eukaryotic cells, especially the ones of plant life and animals.
1.2.3.2.1 Transfection Every other technique to transfer rDNA into host cells
includes mixing the foreign DNA with charged substances like calcium phosphate,
cationic liposomes, or diethylaminoethyl (DEAE)-dextran is a polycationic
derivative of the carbohydrate polymer dextran and covering on recipient host
cells. Host cells take in the DNA in a technique called transfection.
1.2.3.2.2 Electroporation An electric current is used to create transient

microscopic pores in the recipient host cell membrane allowing rDNA to enter.
1.2.3.2.3 Microinjection Exogenous DNA can also be brought immediately into

animal and plant cells without using eukaryotic vectors in the system of
microinjection, foreign DNA is directly injected into recipient cells the use of a
quality micro syringe under a section contrast microscope to useful resource
imaginative and prescient.
1.2.3.2.4 Biolistics An excellent technique that has been developed to introduce

foreign DNA into in particular plant cells is by means of using a gene or particle
gun. Microscopic particles of gold or tungsten are covered with the DNA of
interest and bombarded onto cells with a device just like a particle gun.
Subsequently, the time period biolistic is used.
1.2.4 Selection of Transformants

The artwork of cloning is to determine the precise transformed cell that consists
of the cloning vector with the gene of interest (referred to as a recombinant cell).
It is also very important to find the recombinant cells from the culturing trans-
formed petri plate where nonrecombinant (containing the vector without insert
DNA) transformed cells are also present. If the final goal is achieved, that means
our desired gene of interest is inserted into the vector then it will be called as
clone. Direct selection and selection from gene library are the two basic concepts
for selection procedures. Direct selection involves designing of experiment in
such a way that transformation takes place only for the clones containing specific
gene of interest. Selection happens on the plating-out stage of the transformation
where the colonies are grown on agar plates. It is the preferred technique because
of the ease and unambiguous in nature. Selection from gene library involves ini-
tial “shotgun” cloning test, to produce a clone library representing all or most of
the genes present inside the cell, observed through evaluation of each individual
clones to identify the correct one.
1.2.4.1 Direct Selection

Majority of cloning vectors are designed in such a way that inserting gene of inter-
est in them inactivates the gene already present in the vector. This leads to inser-
tional inactivation of gene. Two of the common examples of insertional
inactivation, i.e., direct antibiotic resistance screening and blue white screening
are discussed below.
1.2.4.1.1 Direct Antibiotic Resistance Screening Cloning vectors are planned in

such a way that insertion sites are present in between the genes in the vectors,
often antibiotic resistance gene. Vector carrying ampicillin resistance gene confers
resistance to ampicillin to the cells in which it will be transformed to. Transformed
cells when grown in agar plates containing ampicillin shows resistant to the
antibiotic and are capable to grow. But this does not guarantee the presence of
gene of interest in the vector or the vector is recombinant. Ampicillin resistance
gene become disrupted in vector having gene of interest. Thus, the transformed
cells having self-ligated vector grows on ampicillin agar plate, whereas the cells
having recombinant vector does not show growth. The recombinant cells can be
selected from the non-recombinant one through replica plating method.
1.2.4.1.2 Blue–White Color Screening Blue-white screening is also based on

insertional inactivation of a gene giving blue color compound as a visual
representation of the result. The gene that plays a key role in this screening is lacZ
gene which codes for the enzyme β-galactosidase is under the control of the
inducible lac promoter. lacZ gene is repressed by lac repressor and is induced by
IPTG (isopropyl-β-d-thiogalactopyranoside). The enzyme formed can breakdown
X-gal (five-bromo-4-chlore-3-indolyl-β-d-galactopyranoside) to give a blue color
compound.
Insertional inactivation of lacZ gene due to insertion of desired gene prevents
the formation of the enzyme, breakdown of X-gal, and hence no blue color is
obtained. The cells are plated on agar plates having IPTG, X-gal, and an antibiotic
giving combination of blue and white colonies. The transformed cells with reli-
gated vector having functional lacZ gene shows blue color colonies. Whereas the
cells with gene of interest and disrupted lacZ gene forms colorless colonies.
Further the colorless colonies can be picked and plated again.
1.2.4.2 Identification of the Clone from a Gene Library

Screening a positive clone from gene library involves typical techniques such as
nucleic acid hybridization, functional screening, and chromosome walking.
Nucleic acid hybridization needs preliminary information of both the gene of
interest and area of gene to be cloned. Contrary, functional screening entails the
information about the vector.
1.2.4.2.1 Nucleic Acid Hybridization Nucleic acid hybridization utilises short

synthetic radiolabeled oligonucleotides called as probe. These probes are used for
locating complementary sequences in individual cells or phages containing an
insert. The success of the hybridization experiment relies upon the probe.
Therefore, probe should be designed with care and should have some part
complementary to the sequence of the cloned gene. If there is no information
about the gene, then the sequences from the related protein can be derived and
degenerate probes can be synthesized to be used. So, in this technique first the
colonies are transferred to a nylon membrane and then lysis of cells will be done.
The released DNA will be denatured through an alkali treatment. The denatured
single-stranded DNA will be heat treated so that they could bind to the membrane.
After that the membrane will be submerged into solution containing DNA probes
and incubated for a certain time to get hybridized by complementary base pairing.
Finally, the hybridized radiolabeled probes will be identified by autoradiography.
1.2.4.2.2 Functional Screening If the gene encodes for a product that has a
specific role, then the expression library is by means of cloning DNA (cDNA in
case of eukaryotes). These libraries are prepared using unique cloning vectors for
expression of cloned genes. The prepared library can be used to detect the product
or the clones generated by using antibodies or other ligands. These antibodies or
ligands binds with the encoded product and clones.
1.2.4.2.3 Chromosome Walking Usually a genomic clone may not be includ all of
the sequences for a specific gene so it is required to isolate overlapping clones that
protect the genomic segment of interest. This process is known as chromosome
walking.
1.3 Beneficial Biocommodities Produced Through

Engineered Microbial Factories
Earlier biocommodities (up to some extent now also) were extracted from plants
and animals leading to their overexploitation (Brower, 2008). Plants are the great
source of secondary metabolites that have complex structures and used by
humans, e.g. medicines, terpenes, flavoring and coloring agents, etc. (Facchini
et al., 2012). Their yields are greatly affected by the time, climate, and other fac-
tors, which affect circadian rhythm of the plants (Li and Vederas, 2009). Moreover,
the yields cannot satiate the current demand of the population. Being structurally
complex in nature relying on chemical synthesis of the secondary metabolites is
not feasible. Chemical synthesis also leads to toxicity of the end products and
environmental pollution (Du et al., 2011). To overcome these challenges, the prin-
ciples of genetic engineering explained in the earlier section are used to engineer
microorganisms to become microbial cell factories to produce essential biocom-
modities such as bioplastic, biofuels, and antibiotics important to human welfare.
Advancement in the knowledge of metabolic pathways for synthesis of secondary
metabolites and full genome sequencing made fabrication of microbial cell facto-
ries easier (Keasling, 2012). Fabrication generally involves introduction of the
gene of interest in the host organism so the heterologous pathway begins in it. It
also includes exclusion of native pathways that are not crucial for the growth of
the host organism (Tyo et al., 2007). Apart from the development of genetically
modified strains, optimum conditions according to the product of interest and
1.3 Beneficial Biocommodities Produced Through Engineered Microbial Factorie 13
growth of strains is to be provided in a contained environment (bioreactor). This

is done to observe the full potential of the strain (Kiss and Stephanopoulos, 1992).
1.3.1 Biopolymers
Any substance that is made up of repeating monomeric units is called as polymer.
The polymers that are naturally synthesized by the living organisms are called
biopolymers such as proteins, polysaccharides, polyphenols, lipids, polyamides,
and polyhydroxyalkanoate (PHA) (spanning from liquid solutions to bioplastics).
They are biodegradable hence eco-friendly. Naturally occurring polymers form
the basis for life. Polynucleotides (DNA and RNA) and proteins (amino acids) are
present in every living form. Starch is the reserve form of carbohydrate in which
plants store food and is the main carbohydrate in the human diet. Lipids store
energy and its bilayer acts as a barrier in the living cells. Whereas cellulose is the
primary component in the cell wall of plant kingdom providing structural rigidity.
Microbial synthesis of biopolymers is known from old age. Louis Pasteur and Van
Tieghem, in the mid nineteenth century, discovered that Leuconostoc mesenteri-
odes synthesize dextran. 40 years after, and PHA reserves were found in B. mega-
terium, which serves as a basis for bioplastics to date. As stated earlier,
understanding in the molecular pathways for synthesis of biopolymers leads to
the engineering of microorganism for the production of custom made, altered
biopolymers (Rehm, 2010).
Biopolymers have a wide range of application in biomedical, food industry,
packaging to cosmetics, electronics, etc. Its biomedical applications are in drug
delivery, tissue engineering, wound healing, and medical and dental devices.
Porous gelatin, electrospun poly (lactic-co-glycolic acid), etc. are used as a scaffold
for this purpose (Van Vlierberghe et al., 2007; Zhao et al., 2016). Each biopolymer
has a specific characteristic to be used for particular conditions. Poly (d,l-lactides)
scaffolds are used for bone engineering, whereas poly (trimethylene carbonate)
scaffolds are used for soft tissue engineering. Poly(l-lactide-co-glycolide) scaffolds
provides good adhesion and proliferation (Ulery et al., 2011). Some of the com-
mercially available scaffolds are BioFiber™, an orthopedic scaffold made of a leno
weave of P4HB monofilaments (Tornier) for tendon repair, BioFiber®-CM, an
orthopedic scaffold made of P4HB coated with bovine collagen (Tornier) for ten-
don repair, etc. Use of plastic in packaging industry is growing day by day leading
to their disposal problem. Plastic films production takes one-third portion of the
total plastic production. Cellulose and starch are widely used for this purpose
after some modifications. The material to be used for packaging should have prop-
erties similar to polyethylene terephthalate (PET) i.e. barrier, sealing and thermal
properties. Standard cosmetics have nonbiodegradable polymers in them such as
polyethylene microparticles in face and body scrubs. These microparticles
eventually enter in the ecosystem through waste water stream. Biodegradable

poly lactic acid (PLA) powder can be used in exfoliating agents. Chitin absorbs UV
light and effectively used in sunscreen lotions. Also, PLA mixed with titanium
dioxide and zinc oxide powder shows photocatalytic activity.
Some of the biopolymers having industrial application are highlighted in the
section below with their structures shown in Figure 1.2.
1.3.1.1 Cellulose
Cellulose is the major biopolymer found in nature and commercially extracted
from wood (Klemm et al., 2005). It is homopolysaccharide of glucose linked by
β-1,4 glycosidic bonds (Cannon and Anderson, 1991). In 2015, the value of cellu-
lose market calculated was US$20.61 billion and is estimated to reach US$48.37
billion by 2025 (Cellulose Fiber Market Size and Share Industry Report,
2014–2025 market [Accessed 20 December 2017]). To meet such high demands
overcutting of trees are being done leading to imbalance in nature and global
warming. Bacterial cellulose (BC) is the alternative and green approach toward it
which is biodegradable, nontoxic, and biocompatible. Apart from this, BC can be
used directly in its native form as it is free from contaminants such as lignin, pec-
tin, hemicellulose, and other constituents of lignocellulosic materials (Rahman
and Netravali, 2016). There are several bacterial strains that synthesize BC spe-
cially the acetic acid bacteria group as they are generally recognized as safe
(GRAS) such as Komagataeibacter xylinus, Komagataeibacter hansenii,
Komagataeibacter medellinensis, Komagataeibacter nataicola, Komagataeibacter
HO O
CH2OH CH2OH
H O H O O
H H O H H N
OH OH
H
H
O H
O
H OH H OH
n n
Cellulose Poly-γ-glutamic acid
COO– CH2OH
R O
H O H O O
H
OH H H H
O H
OH
H H O OH
m n
H OH H NHCOCH3
n
Hyaluronic acid Polyhydroxyalkanoate
Figure 1.2 Representative chemical structures of biopolymers.

oboediens (Škraban et al., 2018; Castro et al., 2012,2013). Bacterial cellulose syn-
thase (Bcs) is the primary enzyme for cellulose synthesis which adds glucose
monomers to the growing chain. There are two subunits BcsA and BcsB necessary
for the formation of polysaccharide chain (Römling and Galperin, 2015).
Commercial usage of BC and low yields from the strains producing BC lead to the
development of microbial cell factories. These cell factories overexpress genes
essential for BC like cmc (carboxymethylcellulose), ccp (cellulose complementing
factor protein), cesAB, cesC, cesD, bgl, bcsABCD (BC synthase operon), etc. into
the host organism to increase the yields and crystallinity of BC.
Gluconacetobacter xylinus six genes cmc–ccp–cesAB–cesC–cesD–bgl were over-
expressed in Synechococcus sp. PCC 7002 and resulted in very high-yield produc-
tion of extracellular type-I cellulose (Zhao et al., 2015). BC synthase operon
(bcsABCD) from Gluconacetobacter hansenii was engineered in E. coli with its
upstream operon (cmcax and ccpAx) giving BC of 1000–3000 μm and a diameter of
10–20 μm (Buldum et al., 2018). Cellulose synthase D subunit (bcsD) increases the
crystallinity structure of BC, but not the yield. G. xylinus bcsD when engineered in
E. coli synthesizes BC with high crystallinity. FTIR results showed crystallinity
index of 0.84, which was 17% more than the wild-type strain (Sajadi et al., 2017).
Oxygen plays an important role in the synthesis of BC. G. xylinus was engineered
with Vitreoscilla hemoglobin (VHb)-encoding gene vgb. vgb help cells to grow in
hypoxic conditions. The mutant strains showed significant increase in BC in oxy-
gen tension also (Liu et al., 2018). Recently, Komagataeibacter sp. nov. CGMCC
17276 genome was completely sequenced. BC operon genes were aligned with
other BC producers to find sequence similarity. Apart from this growth rate, sub-
strate utilization, BC production, etc. were evaluated. This gives future opportu-
nity to engineer this strain for better BC synthesis (Jang et al., 2019).
1.3.1.2 Poly-ϒ-glutamic Acid

Poly-ϒ-glutamic acid is a naturally occurring, abundant poly amino acid which is
water-soluble, anionic, biodegradable, and edible biopolymer produced primarily
by Bacillus subtilis. Glutamic acid can be l- and d- in nature linked by ϒ-amide
bond (Cao et al., 2018). Due to the presence of ϒ-amide linkage, it is resistant to
cleavage by proteases (Candela and Fouet, 2006). It is gaining importance because
of being the potential candidate for drug delivery, cryoprotectant, thickening
agent, biopolymer flocculant, bioabsorption of heavy metals, etc. (Bhattacharyya
et al., 1998; Shih and Van, 2001). It is also widely used in skin serums in combina-
tion with vitamin C to increase skin elasticity and making it smooth
(Tanimoto, 2010; BEN-ZUR and GOLDMAN, 2007). Predominantly, Bacillus sp.,
such as Bacillus licheniformis, Bacillus subtilis, B. megaterium, Bacillus pumilis,
Bacillus mojavensis, and Bacillus amyloliquefaciens, are producer of PGA. It is syn-
thesized in ribosome independent manner. Glutamate is the precursor of PGA,
which is derived from glutamic acid biosynthesis or glutamate transportation. In

turn, α-keto glutaric acid serves as a precursor for glutamate in tricarboxylic acid
cycle (TCA) also known as Krebs cycle. The reaction is catalyzed by glutamate
dehydrogenase in absence of glutamic acid, while 2-oxoglutarate aminotrans-
ferase in presence of glutamic acid (Stadtman, 1966; Holzer, 1969). Furthermore,
the glutamate units are joined together to produce PGA through racemization,
polymerization, and anchoring or releasing. This can be studied in detail in review
by Najar and Das (2015). PGA synthetase (Pgs), a membrane associated enzyme
polymerizes glutamate to PGA. Genes encoding Pgs are different in different
Bacillus species. Like Pgs is encoded by four genes (pgsB, C, A, and E) in B. licheni-
formis and B. amyloliquefaciens, capB, C, A, and E, in B. anthraci, while in B. sub-
tilis they are ywsC, ywtABC. Role of pgsBCA operon was shown in B. subtilis for
PGA production. Mutants with disrupted pgsBCA did not showed PGA synthesis.
While the xylose-induced pgsBCA operon started PGA synthesis (Ashiuchi
et al., 2006). Sometimes the overexpression of pgsBCA leads to decrease in PGA
production as seen in mutants of B. amyloliquefaciens (Feng et al., 2015).
Under nutrient deprivation conditions PGA is sometimes hydrolyzed by PGA
hydrolases. Deletion of PGA hydrolases can increase the yield of PGA. Depending
on where the PGA hydrolase will cleave PGA, it can be (i) PgdS (the γ-dl-glutamyl
hydrolase) cleaves the γ-glutamyl bond between d-and l-glutamic acids residues
of γ-PGA; (ii) the d/l-endopeptidase hydrolyzes the peptide bond between the
glutamic acid residues; and (iii) the γ-glutamyltransferase (GGT) possesses a pow-
erful exo-γ-glutamyl hydrolase activity. The GGT is different from PgdS and d/l-
endopeptidase as it hydrolyzes the γ-PGA from the N-terminal to release both
d-and l-glutamic acids (Cao et al., 2018).
1.3.1.3 Hyaluronic Acid

Hyaluronic acid (HA) also called as hyaluronan is a structural biopolymer pro-
duced by extracellular matrix of animal and human epithelial, neural, and con-
nective tissues. HA is copolymer disaccharide between N-acetyl-o-glucosamine
and o-glucuronic acid linked by β1 → 3 and β1 → 4 glycosidic bonds. Due to the
water retention properties of HA, it is widely used in cosmetics as it binds water
to collagen and makes skin look plumper and more hydrated. Hyaluronic acid
synthase (HAS-a) is the integral enzyme present in the plasma membrane of
mammalian cells and bacteria and synthesizes HA (Chen, 2002). It has several
isoforms depending on the organism in which it is forming. Traditionally, it was
extracted from animals that pose threat of infection from the animal source. Its
bacterial production started with using Streptococcus zooepidemicus with a pro-
duction titer value of 5–10 g/l in batch fermentation. Later, S. zooepidemicus was
considered to be a human pathogen and was not GRAS. HA being viscous in
nature pose problems in fermentation leading to lower yields. This leads to opti-
mization of fermentation conditions and improvement in the strains genetically
(Chong et al., 2005).
Hyaluronidase enzyme activity in the strains is not beneficial as it depolymer-
izes HA. Kim et al. (1996a) selected Streptococcus equi chemically derived mutant
strain (after nitroglycerine treatment) which is nonhemolytic, hyaluronidase-
negative in nature, thus producing high molecular weight HA. Controlled expres-
sion of hyaluronidase is beneficial too as it reduces the molecular weight and
viscosity of HA. Expressing tuaD, gtaB, glmU, glmM, and glmS genes plus glyco-
lytic pathway genes in B. subtilis with controlled expression of hyaluronidase by
N-terminal engineering increased production from 5.96 to 19.38 g/l (Jin
et al., 2016). HA produced from this strategy was low in molecular weight in the
range of 2.20 × 103 to 1.42×106 Da. One of the reasons Streptococcus sp. were not
considered as GRAS is that they produce streptolysin which causes haemolysis.
Streptococcus equisimilis CVCC55116 mutated by ultraviolet ray combined with
60
Co-γ ray treatment produces 174.76 mg/L HA without producing streptolysin
(Chen et al., 2012).
In an attempt to find the GRAS producers of HA, B. subtilis, Lactococcus lactis,
E. coli, Pichia pastoris, Corynebacterium glutamicum, etc. were engineered to
express HAS-a, b (UDP-glucose 6-dehygrogenase), c (glucose-1-P uridyltrans-
ferase), d and e gene, which using precursors N-acetyl-o-glucosamine and o-
glucuronic acid forms HA (Jia et al., 2013; Kaur and Jayaraman, 2016; Jeong
et al., 2014). Lactococcus lactis is not the natural producer of HA due to absence of
HAS gene. The attempt to express HAS gene in L. lactis was performed by number
of research groups by using non-integrative plasmids pRKN, pNZ8148, etc.
(Prasad et al., 2010; Chien and Lee, 2007; Prasad et al., 2012). Strains expressing
only HAS-a and b genes give a production of 0.097 g/l, while the one expressing
HAS-c gene also gives the maximum production of 0.234 g/l (Prasad et al., 2010).
This indicates the importance of HAS-c gene expression for HA production. This
is due to the fact that HAS-c diverts the flux of glucose-1-phosphate toward UDP-
glucose synthesis, which is critical for HA synthesis. Genome integrative approach
is also carried in Lactobacillus sp. giving twofold increase in the production and
molecular weight (MW) of HA in comparison to plasmid bearing strains (Hmar
et al., 2014). It was explained by the precursors (N-acetyl-o-glucosamine/o-
glucuronic acid) and HAS-a/HAS-b mRNA ratio. In genome-integrated strains
HAS-b expression was high compared to HAS-a, which gives greater availability
of precursors to bind to HA synthase leading to high MW HA. E. coli the most
common microbe to be engineered has a wide variety of genetic tools available to
be modified. It was the first microorganism in which human HAS was success-
fully expressed (Hoshi et al., 2004).
1.3.1.4 Polyhydroxyalkoate
Poly-3-hydroxyalkanoates (PHA) are polyesters that have gained economic impor-
tance due to their biodegradability and tendency to replace petroleum synthetic
polymers. PHAs are produced in the form of intracellular inclusion bodies under
nutrient deprivation and carbon source excess conditions. These inclusion bodies
or PHA granules serve as carbon reserves for microorganisms during stress condi-
tions (Sudesh et al., 2000). The inclusion bodies have PHA hydrophobic core sur-
rounded by PHA synthase involved in synthesis of PHA (Grage et al., 2009).
Depending on the number of monomeric units PHA can be divided into short
chain length (SCL), medium chain length (MCL), and combination of both (SCL/
MCL). SCL PHA have three to five carbons in the monomeric unit, is thermoplas-
tic nature, brittle, and lacks toughness (3-hydroxypropionate). MCL PHA have
monomers consisting of six to fourteen carbons and have elastomeric property
(3-hydroxytetradecanoate). SCL/MCL PHA are derived from both short-chain-
length and long-chain-length PHA having three to fourteen carbons. It has a wide
range of physical and thermal properties. The generalized structure of PHA is
shown in Figure 1.2. Till date 150 different PHAs composition have been identi-
fied according to the modifications made in existing PHA and genetically engi-
neered organisms to produce PHA (Loos, 2011; Zinn and Hany, 2005). Apart from
its application to produce bioplastics PHA has application in the field of tissue
engineering, drug carrier, biomedical, etc. The widespread use of PHA is still
restricted due to the fact that the cost of production of PHA is 5–10 times the cost
of petrochemical-derived plastic (Chen, 2009).
PHA synthase (PHAc) is the primary enzymes which polymerizes R-3-
hydroxyacyl-CoA precursors. For this all the carbon metabolism is shifted to
form R-3-hydroxyacyl-CoA thioester. PHA synthase is broadly classified into
four classes. Class I consists of one type of PHAc, while class II contains two
type of synthases PHAc1 and PHAc2. Classes II and IV consist of PHAc-PHAe
and PHAc-PHAr units, respectively (Pötter and Steinbüchel, 2005). Classes I,
III, IV favor SCL-PHA, while Class II favors MCL-PHA synthesis. Ralstonia
eutropha is a model organism for PHA production as it accumulates polymer in
nutrient-deficient condition. Its wild type only synthesizes SCL-PHA and has to
be modified for tailor-made PHAs (Pohlmann et al., 2006). R. eutropha is engi-
neered to produce copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)
(PHBHHx) from palm oil. Engineered strain contains PHAc from Rhodococcus
aetherivorans, enoyl-CoA hydratase gene (PHAj) from Pseudomonas aeruginosa,
and acetoacetyl-CoA reducatase gene (PHAb). PHAj accumulates PHA, while
PHAb alters the level of hydroxyhexanoate (Budde et al., 2011). Since R. eutropha
grows slowly and difficult to lyse engineering E. coli is better option. E. coli is not
a natural producer of PHAs, as it does not have PHA synthase gene. The first
heterologous production of polyhydroxybutyrate (PHB) in E. coli was done in

1988 (Schubert et al., 1988). PHB synthase gene from R. eutropha was cloned in
E. coli. The engineered E. coli is able to synthesize PHB but lack the ability to
accumulate it.
Increasing the number of PHA synthase gene copies in an organism only
increases the PHA levels occasionally that too when the cell dry weight increases.
This indicates the importance of cell dry weight over additional copies of PHA
synthase gene and shown by many researchers (Tombolini et al., 1995; Huisman
et al., 1992; Huisman et al., 1991). E. coli binary fission pattern is changed to mul-
tiple fission, by deleting the genes minC and minD (related to binary fission) and
overexpressing the genes ftsQ, ftsL, ftsW, ftsN, and ftsZ (related to cell division)
(Wu et al., 2016a). This results in more dry weight and more PHB accumulation.
Apart from the cell dry weight, cell shape also matters. Along with the deletion of
minC and minD gene, deleting gene envC and nlpD which degrades peptidogly-
can layer leads to filamentous shape and accumulated 70% PHB (Wu et al., 2016b).
All carbon source getting converted to biomass and no product is not beneficial
industrially. Therefore, a glucose-sensing toggle switch is created that automati-
cally senses glucose limitation and activates product formation leading to shorter
growth phases and good titers of PH (Bothfeld et al., 2017).
There is no doubt that genetic engineering increases the production of PHAs in
many aspects. But the substrate used in PHA production is costly, and unusual
substrates (whey, lipid, molasses, glycerol, etc.) are not directly converted to
PHA. Whey contains 4.5% of lactose, engineering Cupriavidus necator which is
the natural producer of PHA so that it can grow on lactose. For this purpose, lac
operon consisting of lac I, lac Z, lac O from E. coli is inserted into C. necator
(Povolo et al., 2010). The lac operon is inserted within the depolymerase gene
(phaZ1) which reduces the depolymerization of the polymer and increase in its
accumulation. E. coli which naturally utilizes lactose but do not have PHA syn-
thase gene is engineered with PHA synthase gene from C. necator. The recombi-
nant strain was grown in cell recycle fed-batch culture giving 87% PHB content
(Ahn et al., 2001). Molasses, which is the predominant waste produced by sugar
industry, contains almost 50% sucrose. Microorganisms should be engineered
with sucrose utilization genes in order to use molasses as the carbon source for
PHA production. One such gene is sucrose permease (csc) of E. coli which has
been engineered in C. necator to produce PHBHHx using sucrose as a sole carbon
source (Loo et al., 2005). Glycerol is the major waste of soap industry by fat
hydrolysis and is now also being generated from biodiesel manufacturing. Aerobic
metabolism gene from E. coli, glycerol kinase, aquaglyceroporin, FAD-dependent
glycerol 3-phosphate dehydrogenase are transformed in C. necator to obtain PHB
accumulation (Fukui et al., 2014).
1.3.2 Organic Acids

Organic acids are widely distributed in nature from ages and have been used by us
in our daily life be it lactic acid (in curd) or citric acid (in fruits like lemon, orange,
etc.), indicating them to be the building block chemicals. Most of the organic acids
can be produced microbially or as an intermediate compound. These microorgan-
isms can be wild type or improved genetically to increase the yield. Products pro-
duced by microorganisms are of high purity, selectivity, and are cost efficient (Singh
et al., 2016). Organic acids have a wide range of applications and its market is
expected to grow at a rate of 60% till 2021. Though the direct demand of the particu-
lar organic acids in the market is low but can be created by chemical industry (Sauer
et al., 2008). For example, maleic anhydride can be replaced by succinic acid if there
is no price limitation. There are number of products that can be derived from suc-
cinic acid which includes butanediol, butyrolactone, tetrahydrofuran, bionolle, etc.
Bionolle is the polymer of succinic acid and has property similar to polyethylene.
Their applications are in food, beverages, pharmaceuticals, textile, detergents,
solvents, petrochemicals, dyes and adhesives, rubber, perfumes, and plastics, etc.
If we talk about gluconic acid then gluconic acid and its component have a variety
of applications. Gluconic acid is used to prevent milk stone formation in dairy
industry and to clean aluminium cans. Various salts of gluconic acid like sodium,
calcium, and iron are used as additive in cement, supplement in calcium and iron
deficiency, respectively (Ramachandran et al., 2006).
Citric acid, succinic acid, lactic acid, and gluconic acid are discussed below with
their structure shown in Figure 1.3.
HO
O
O H O
OH
OH HO OH
HO O
O H
Citric acid
Fumaric acid
Organic
acids
O O
H3C HO
OH OH
OH
O
Lactic acid Succinic acid
Figure 1.3 Different organic acids with their structures.

1.3.2.1 Citric Acid

Citric acid (2-hydroxy-propane-1,2,3-tricarboxylic acid) is colorless, odorless, in
its pure form, and is water soluble. It is widely distributed in plants and animals
and is omnipresent in nature as the intermediate compound in krebs cycle or tri-
carboxylic acid cycle. It is used as flavoring agent in food industry (Soccol
et al., 2006). It has a major role in pharmaceutical industry as a blood preservative,
in chemical industry as antifoam agent, and in soap industry as stabilization of
fats and oils (Max et al., 2010). Its estimated production capacity is approximately
2 million tons per year (Steiger et al., 2017). Citric acid synthesis pathway along
with other organic acids synthesis is shown in Figure 1.4.
It was first discovered in lemon (citrus fruit) and now about 99% of citric acid
is produced from microbial fermentation. Wehemer (1893) discovered the pro-
duction of citric acid by Penicillium glaucum, but could not scale up the process
(Vandenberghe et al., 1999). In 1917, strains of Aspergillus niger were found to
synthesize citric acid and soon became the method of choice for industrial pro-
duction of citric acid (Chen and Nielsen, 2016). Highest producing strains of
Aspergillus can accumulate 200 g/l citric acid under optimum conditions (Steiger
et al., 2017). For improved production rate, metabolic and genetic engineering
of Aspergillus strains are done which can tolerate low pH levels and can secrete
excess citric acid in the medium (Ruijter et al., 1997; Steiger et al., 2019). Carbon
source utilization plays an important role in industrial production of citric acid.
Despite giving higher yields glucose cannot be used industrially as it is not cost
efficient. Therefore, liquified corn starch, glycogen, etc. are used. Using liquified
corn starch leads to the production of isomaltose with the action of enzyme
glucosidase and cannot be used for citric acid production. There is another
enzyme glucoamylase that releases glucose from starch and is beneficial. So,
Wang et al., (2016) deleted the glucosidase encoding gene and overexpressed
glucoamylase gene in A. niger. This leads to the decrease in residual sugar to
about 88.2% and increase in citric acid production to 16.9%, reaching up to
185.7 g/l.
From Figure 1.4, it can be seen that oxaloacetate and Acetyl-CoA act as a pre-
cursor for citric acid production. Oxaloacetate is generated by malate dehydroge-
nase and fumarase while Acetyl-CoA by pyruvate dehydrogenase, acetyl-CoA
synthetase, and ATP-citrate lyase (ACL1, ACL2). ATP-citrate lyase, as the name
suggests consumes citrate to synthesize Acetyl-CoA. There was an increase in cit-
ric acid production when ACL1 was deleted from A. niger (Meijer et al., 2009). But
when both the subunits ACL1 and 2 were deleted, there was a decrease in levels
of citric acid (Chen et al., 2014). Overexpression of fumarase gave increased cit-
rate levels, while malate dehydrogenase only expedites levels initially. Role of cit-
rate exporter (CexA) is also very important. Citric acid produced inside the cell
and not able to get released in the medium could lead to its inhibition. CexA is the
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After reflecting upon what you had been so good as to
inform me off I could not fail to write the enclosed as I have all
the reason in the World to believe you my friend, so I take the
liberty to beg that after perusal in case you find it not too
assuming and in a stile sufficiently respectly you will be so
good as take the trouble to deliver it with an appology.... [Not
legible.]
(Signed) J. Broun.[156]
My Lord,—I heartyly regreted it was not in
my power when so near as London to do
myself the honour of waiting upon your Murray to [157] Earl
Lordship for Reasons I beg Mr. Smith to give Marischal
and at the same time assure your Lordship
that without the honour of being known to you there was none
who had a more hearty and sincere Regard for your Lordship’s
property.... [Not legible.] my Lord, abstracting from your many
private Virtues makes you dear to every true Scotsman and
from what I could easily learn gives your Lordship great weight
with the English. This, my Lord, encourages me to suggest of
what use you may be att this juncture to his Majesty’s affairs by
uniting those people together who to my great concern I found
(so far as I could judge) quite inactive, diffident of one another
and distrustfull of those they employ. Your Lordship’s being so
near them will I hope make the work short as well as Effectual,
and in my poor Judgement were there any unanimity, any
harmony and concord amongst them it would easily be in our
power to shake off the yoke of Bondage and Slavery we now
groan under; this, my Lord, I propose with great submission to
your Lordship’s serious reflection and experience, I shall only
add that as I have no other in any thing I do but to promote the
real Interest of my King and Country so I have all the reason in
the world to believe that healing the wounds and cementing the
differences now subsisting amongst our Neighbours is the
greatest ambition of our countrymen and must render their
gratitude to your Lordship for so great a work unalterable. I
begg your Lordship will pardon my presumption in writing
without being asked and believe I am with the most sincere
regard and esteem, My Lord, etc.[158]
These two preceding letters I gave to my L[ord] T[ra]q[uai]r on the 5th
of Aprile 1743, the day before his Lordship sett out from his own house to
London which he was to show Mr. McGregar who told my Lord that this
was not a time to send such letters and besides that the letter to Lord
Marshall was not such an one he proposed, by which means they never
went. I shall here mention what occurred to me what was the reason for
Mr. McGregars stoping them never having any further reason given than
the above and not knowing even that till L[ord T]raq[uai]r’s return to
Scotland, during the short stay I made att London after my return from
Paris in company with Mr. McGregar as I said before I made it my
business to learn of Coll. Cicel and Mr. Smith all the Complaints they had
against Lord Semple and Mr. McGregar which I generally communicated
to him being then fully perswaded of their Honesty and Abilitys. Upon
telling him what Mr. Smith had said in relation to Lord Marshall he
purposed I should write to his Lordship a Letter in the same Stile Lord
Semple had done, purposely to let him know how little he was in favour
with his Countrymen and show him how much their liking depended upon
his being well with them two, and att the same time to acquaint him that
his Countrymen’s regard went no further than so far as he should act
entirely conformable to the King’s will, he saying that Lord Marshall
pretended to stand entirely upon his own legs from the great Number of
Admirers he had in Scotland, and imagined that unless he was the sole
manager of the King’s affairs his Majesty’s subjects would not be brought
to act for him, in short that he looked upon the King’s interest to depend
upon [him] alone. I suppose he thought by my writing in this stile to draw
something from Lord Marshall undervalueing of his Countrymen whereby
to hurt him with the generality but particularly with those of the Concert
who had first employed and still supported him, whereby he might verify to
them what he had so long advance against his Lordship, but this was a
thing I refused for many reasons; first that it must hurt the King’s affairs to
create differences amongst his friends of which their then subsisted too
many; secondly it was running myself headlong into a party quarrel, a
thing I ever had the worst oppinion off; and thirdly, not to be too tedious in
giving many more, it was a thing I had not the smallest tittle to do. But on
the contrary I thought it was a fair opportunity of uniting people together
and commencing a Correspondence with Lord Marshall whereby if I gain
his friendship and confidence I [should] thereby be enabled to inform the
King of the pleas of both partys without letting either of them know of it,
which is acting a part some people may think odd as it is seemingly
playing with both hands, but in my Eyes not only honourable but my duty,
when for the King’s interest, I receive from time to time letters from L[ord]
T[ra]q[uai]r when att London informing me in his way of his success with
the English; but as there was nothing material in them and that they were
signed by himself I did not care to keep them in case of accidents. Some
time in the month of June I received the following letter from Mr. Edgar
with two enclosed from the King himself in answer to two I had wrote when
at Paris.
Copy Mr. Edgar’s letter, dated March 14th, 1743.
Sir,—You will see by the enclosed I send
you from our friend Mr. Edwards (he means
the King) that he leaves me nothing to say in Edgar to
return to your two letters of the 18th and 25th Murray
of february. This Packet is sent open to Lord Semple to forward
to you, you will see it is write in his Cypher, a Copy of which I
know Mr. McGregor left with L[ord] T[ra]q[uai]r so I reckon you
may easily gett this read, for suppose you may have that
Cypher in your own Custody. You will be glad to know that the
family is well, I heartyly wish you all health and happiness and
longing to have the pleasure of Embracing you, I am with great
Esteem, etc.
Letter from the Chevalier to John Murray.
I received a few days ago yours of the 18th
of February and am far from disaproving your
The Chevalier
comming into France att this Time.[159] The de St. George
Settling of a Correspondence betwixt us on to Murray
this Side of the Sea and our friends in
Scotland may be of consequence in this juncture I hope you
will have concerted some safe method for this effect with Lord
Semple before you leave him and that once determined you
will I think have done very well to return home where you may
be of more use than abroad. I shall say nothing here of what’s
passing in France of which you will have been informed by
Lord Semple, and you may be well assured that depends upon
me to induce the French to assist us as is reasonable to hope
they will if their be a general war. But if they ever undertake
anything in my favour, I shall to be sure have little warning of it
before and by consequence I fear it will be impossible that
General Keith can come in time into Scotland how much
soever both I, and I am perswaded himself also, desires it
because you will easily see that one of his rank and distinction
cannot well quite the Service he is in either abruptly or upon an
uncertainty. I remark all you say to me on that subject and
when the time comes it shall be my care to dispose all such
matters in as much as in me lys for what I may then think for
the real good of my Service and for my friends Satisfaction also
for in such sort of particulars it is scarce possible to take proper
Resolutions before the time of Execution. I had some time ago
a proposal made me in Relation to the Seizing of Stirling
Castle[160] what I then heard and what you now say on that
subject is so general, that I think it is not impossible but that the
two proposals may be found originally one and the same
project; I wish therefore you would enter a little more into
particulars that I may be the better able to determine what
directions to send. As to what is represented about the Vassals
I suppose what you mean is the same with what I have
inserted in a draught of a Declaration for Scotland I have long
had by me, viz., That the Vassals of those who should appear
against my forces on a landing should be freed of their
Vassalidge and hold immediately of the Crown provided such
Vassals should declare for me and join heartyly in my Cause.
As this is my intention I allow my friends to make such prudent
use of it as they may think fitt. Before you gett this you will
probably have received what was wrote to you from hence
about the Scotts Episcopal Clergy so that I need say nothing
on that subject here more than that I hope the steps taken by
me will give satisfaction and promote union in that Body.
It is a great comfort to me to see the Gentlemen of the
Concert so zealous so united and so frank in all that relates to
my service and I desire you will say all that is kind to them in
my name, I remark you have advanced one hundred pounds of
your own money[161] for Sir J[ames] C[ampbe]ll which I take
very well of you but I desire you would not give me any more
proofs of that kind of your goodwill towards me and as for what
is past I look upon it as a personal Debt and shall take care
that it be repayed, I remark what you say about the difficulty
their is of raising money I foresaw that would be no easy matter
and think it should not be insisted upon, I think I have now
taken notice of all that required any answer in what you wrote
to me and Edgar and shall add nothing further here but to
assure you of the continuance of any good opinion of you and
that your prudent and zealous indeavours toward my service
shall never be forgott by me.
(Signed) J. Edwards.[162]
Dated March 11th,

1743
.
At the same time came the following:—
Since I wrote to you on 11th I have seen
what you wrote to Edgar of the same date; I
remark what you say on extending a Concert The Chevalier
to Murray
in the Low Country and the Method[163] it has
been done in the Highlands such a measure may be a great
advantage to the cause and therefore I cannot but much
approve of it, provided it be gone about with great prudence
and Caution, which I earnestly recommend to you not only on
your own account but mine also, for we must in this juncture
carefully avoid anything that may give the Government any
jealousie or pretence to molest our friends. I am well pleased to
observe what you say of L[ochie]l and if you have occasion
make him a kind Compliment from me and the Prince, this is all
I have to add or present in this paper.
Signed as befor and dated 14th, 1743.
In the first letter I wrote to Mr. Edgar after my return home I told him
that the Packet for Bishop Rattrae was still in my Custody being informed
that he intended to be soon in town so thought it fitt to delay doing any
thing in it till he should come, accordingly whenever I heard of his arrival I
went immediately to the Country where the Cypher Mr. Edgar mentioned
there was, and brought it to town with me but to my Surprise found it did
not answer. Yett I nevertheless resolved to deliver the Packet to him att
the same time reading the paragraph in mine concerning it so sent for Mr.
Rae[164] one of the Presbyters in Edinburgh and told him I wished to see
Mr. Rattrae and desired he would go to him and acquaint him with my
intention and make an appointment for me which he agreed to do next
mourning. He came to me on the morrow and told me had missed him but
would indeavour to find him sometime that day upon which he left me and
found him dinning with his daughter Mrs. —— so delayed it till next
morning, but when he went was informed by his daughter Mrs. Clark in
whose house he lived that he had been taken ill the night before of an
Epidimical Distemper that at that time raged almost all of over Europe of
which he died in three or four days illness, by which means that Packet
still remains in my Custody, never since having gott any directions about it.
From that time I have keept my Resolution of never having any more to do
with the Clergy, for when I was asked some few days after by Mr. Rae
what I thought they should do in their present situation, I told him I thought
they should draw up a full and distinct state of their [affairs] without
neglecting the least thing and send it to the King leaving him to determine
without any further representations, but shunned writting or taking any
Commissions about it, nor do I since know any of their procedure none of
them having ever spoke to me on the subject.
About this Time the Duke of P[erth] came from England and as
L[ochie]l and [I] had often Schemed together what we ourselves were able
to do in the present posture of affairs and seemed to agree in Opinion that
should the french disapoint us we were more able to restore the King by
our own strength during the absence of the Army than with the Assistance
of 10,000 men were our Troops once returned, for which reason did the
English fail to give the assurances to the french they required, but we
should gett the people at home to take it in hand by themselves. With this
View I had already wrote Mr. Edgar on that Strain and now we agreed to
sound the Duke of P[erth] on the same Subject which we did and found
him abundantly forward. He was then going to the Country and Sir
A[lexander] Mc[Donal]d[165] was with his brother-in-law A[irl]y so desired
the Duke would try him and some days after had a letter from hime
wherein he said that he had spoke to him as was agreed and found him
very keen that he said the sooner it was done the better and in place of
700 men which his Uncle carried with him in the year 1715 he would now
bring 1200. At the same time when his Grace was talking to us in
Edinburgh on this subject he said the people in that part of the Country
where he had come from very honest and that the Mayer and Aldermen
had spoke to him in the strongest terms, which he then told us, and
desired I might acquaint the King of it, so I desired his Grace would putt it
in writting and that I should not fail to transmit it to his Majesty; upon which
he went to another room and brought me a sheet of paper mostly write
which I did not care to transmitt in his own words but abridged it; however
shall say no more of it here as I will putt down the letter I wrote the King
and another to Mr. Edgar. I must only observe that I read the Duke’s
memorial to my L[ord] T[ra]q[uai]r att his own house upon giving him some
paper he had putt in my custody att his leaving Scotland, at which time his
Lordship and I agreed to put it in the fire as the letter I had wrote and
which I att the same time read to my Lord contained the substance of it,
this I have frequently repented since, as his Grace’s memory did not seem
to serve him exactly to what he had wrote. In answer to his Majesty’s I
wrote the following, Dated July 5th, 1743:—
Sir,—I had the honour of your Majesty’s
commands some weeks ago of the 11th of
March. I communicated the paragraph of your Murray to the
Majesty’s relating to the certainty of General Chevalier
Keith’s coming to Scotland to the Duke of P[erth] and L[ochie]l
who are equally well satisfied with what your Majesty is
pleased to say on that subject. Your Majesty’s Declaration with
regard to the Scotts Vassals and the Liberty you are most
graciously pleased to grant of insinuating so much to them give
L[ochie]l extream pleasure being convinced it will tend greatly
to promote your Majesty’s interest. The Duke of P[erth] desired
me to acquaint your Majesty that the Mayer and Aldermen of
York had freely opened their minds to him on the Subject of the
Restoration and gave him a Commission humble to Represent
their Loyalty and firm attachment to your Majesty’s cause. They
engage that upon hearing of your Majesty’s aproach with a
Sufficient Body to support them they will raise 10,000 men in
that County and have no doubt of an equal Number from the
Countys Adjacent. There are two gentlemen one of them a
present member the other was last parliament but declined it
these have the Sole management of the County and did they
appear would certainly be followed by every one in it. His
Grace had not the good fortune to see them they being at
London but left them his Compliments by some of their friends
who assured him they were ready upon your Majesty’s desire
to enter into any Scheme to promote your Majesty’s interest.
His Grace desired me likewise to inform your Majesty that he is
fully convinced and may almost affirm that upon shewing an
order from your Majesty to treat with them they will sign any
declaration or assurance of whatever Nature shall be thought
most for your Majesty’s interest and to have the Mayer and
Aldermen, at least those of them upon whose Secrecy they can
the most depend to do the like. In the Duke’s Memorandum to
me he neglected the two Gentlemen’s names. As the Election
of their Mayer goes by Rotation it falls next year upon a Whig
which his grace thinks can be of no consequence as none of
that Kidney have any Interest, in the town, but he was told that
notwithstanding the custom, if your Majesty desired it they
would indeavour to have another chosen. I shall incroatch no
further upon your Majesty’s time having wrote to Mr. Edgar but
beg leave to subscribe myself with the greatest Veneration and
Duty your Majesty’s, etc.
Letter to Mr. Edgar, dated July 5th, 1743.
Since I had the pleasure of receiving your
last letter the Duke of P[erth] returned from
York after having gott a very possitive and Murray to
harsh Refusall from the Lady to whom he was Edgar
making his addresses, during his stay there he had some
commissions from the Mayer and Aldermen to the King with
which I had the honour to acquaint the King by the enclosed
amongst others they begged he might offer their humble Duty
and assure him of their unalterable Regard to his Interest. They
intended to send his Highness the Duke[166] the freedom of
their toun in a Gold Box as the highest mark of their regard for
his Highness who does them the honour to bear the Name of
their Town but being afraid least a discovery should be made
by Workmen or others who would render them the less usefull
to his Majesty they must humbly beg his Highness would look
upon the Compliment as real and Honour them with his
Acceptance.
L[ochie]l and I spoke to the Duke upon the supposition we
should obtain no assistance from France how far he thought it
would be prudent at this juncture to undertake the King’s
Restoration ourselves. Upon his Grace’s approving of the
scheme, I proposed when he went to the Country he should
talk to Sir A[lexander] M[acdonal]d upon the same, which he
accordingly did, and sent an answer to L[ochie]l wherin he says
that found him entirely of the same Opinion, that he seemed to
think there was a necessity for it, and that it ought to be done
as soon as possible and that in place of 700 men his Uncle
brought with him in the year 1715 he would engage to bring
1200. I could not fail to acquaint you with his Opinion, he being
the most reserved cautious man I ever knew, and the least apt
to say or do anything rashly, everybody is of Opinion the
Government designs by all methods to Ruin the Highlands
which to be sure makes the Gentlemen fond to have something
done before it be out of their power to be of service, especially
as there are some of them whose Estates are so low such as
G[lengar]ry C[lanranal]d A[p]p[i]n C[ap]p[oc]h that they will be
obliged either to sell their lands or conform to the Government
through necessity; and am very credibly[167] informed that
Ca[p]p[oc]h was this Winter at London on a Scheme of raising
an independent Company. Since Mr. Smith came to this place
he has insisted with Lo[chie]l to go over to see Lord Marshall
who he tells him has had several different accounts of the
State of the Highlands so that he is very anxious to talk to him
upon that head. L[ochie]l excuses himself from going but told
me Mr. Smith supposed his Lordship was informed of
everything by the King and that the State given in by Mr.
Drummond was just he is certain; but now things are much
better and that Mr. Drummond rather erred in making the
number too small[168] being determined to advance nothing but
what he could answer for. I had a letter lately from my L[ord]
T[ra]q[uai]r wherein he seems to hint that his Majesty’s friends
in England are in a way of acting with more vigour and
unanimity than they have hitherto done. I hope they are now
become sensible of their weak and groundless prejudices
against Lord Semple (a Catalogue of which I sent you in my
last) as well as of the necessity there is to act Vigorously and
Resolutely for his Majesty’s Restoration. It would seem L[ord]
T[ra]q[uai]r and Mr. Drummond have not judged it fitt to send
my letter to L[ord] Marshall (a Copy of which I sent you in my
last) for I have never had the smallest hint of it from Mr. Smith
neither has L[ord] T[ra]q[uai]r taken occasion to mention it in
any of his Letters to me. Upon Bishop Rattrae’s coming to town
I went immediately to the Country for the Cypher when I found
it did not in the least correspond to that Coll. Urquhart must
certainly have been mistaken. I nevertheless resolved to
deliver the letters att the same time showing him the paragraph
in mine relative to him and to tell him the cause of the mistake,
but this day I sent one of his Brethern to acquaint him that I
would wait on him in the afternoon, he was abroad and the
next day when I sent again found he was taken ill of a Pluirisy
of which he died two days after[169] which to be sure is a very
great misfortune and not the less so that Mr. Keith is now
Senior Bishop, what they are now doing or intend to do I don’t
know I have sent you the Cypher inclosed but retained the
Letter till further Orders.
We had great rejoicings some days ago on account of a
Battle said to be gained by the Allies in Germany,[170] but by
latter Accounts, the Case seems to have been that the Army of
the Allies had gott too far into the Country and finding a
Scarcity of provisions (especially amongst the English) they
resolved to return from whence they came when they were
attacked by a body of French to the number of 25,000 or
thereabouts, some write they intended to attack their rear and
harrass them on their march, others they proposed intercepting
12,000 Hannoverians and Hessians that were on their march to
join the Army, but whatever was their Intention they attacked
our foot. Whereupon a very smart engagement ensued,
wherein both partys seem to have suffered severely. The
French repassed the river and we continued our march and are
now gott near to frankfort where its said the Emperor is and
talks of a Suspension of Arms I wish too good a peace may not
ensue. You certainly cant help laughing when you consider with
what Vigour and Success we carry on our war with Spain.
Never was a poor Country in so miserable a Condition as we
are in att present, neither Money nor Trade nor Credit, nay nor
so much as the smallest degree of honour or Character left us.
The Duke of P[erth]’s Stay here was so short that I could not
gett him to explain his Scheme of Seizing Stirlling Castle, but I
wont neglect the first opportunity to have it from him. I am Still
obliged to continue the old Cannal of Correspondence not
being able while att London to find any Safe Conveyance from
thence, but I hope Lord T[ra]q[uai]r will have effectuated that
before his return; I have now write everything that occurrs to
me I wish may not already have tired you.—So with hearty
prayers for the familys wellfare and all friends with you and that
we may soon meet on a Solid and happy footing, I beg that you
would believe that I ever am, etc.
Dated July 5th, 1743.
P.S.—I had almost neglected to tell you that while I was att
London I ordered a Countryman of ours to work Six pair of the
finest Silk-Stockens he could possibly make, which as they are
home manufactory and finest I believe were mad in the Island I
have ventured to send them by Mr. Smith to Rome hoping the
Prince will do his Country and me the honour to accept of
them.
My Lord T[ra]q[uai]r having now been three months att London and we
all that time receiving no Material accounts of Success things looked as if
this Summer would be Spent as well as the former without any Resolution
taken of coming to Blows in the Autumn or Winter which made all those of
the concert very uneasy but particularly my Lord L[ova]t, so that he
seemed to give up all hopes of the Schemes ever succeeding and wrote
to L[ochie]l several Letters wherein he resolved to settle his affairs and go
to London where, after he had sollicited an appeal he intended to lodge
against Chisholm, he would then go over to France with his son. L[ochie]l
was so good as show me the most of his Letters and from some things he
said, but particularly a paragraph in one of them we had reason to
conjecture he proposed to end his days in a Religious house; this we were
the more easily induced to believe as his Lordship had now lived to a
great age during the most of which he had acted a part in the world not
looked upon by the Generality of Mankind in a favourable light. We
therefore agreed that L[ochie]l Should write him dissuading him from his
design but at the same time he seemed so positive that it was thought
Necessary to acquaint the King of it as his Majesty’s orders appeared to
us the only mean whereby to prevent his journey, knowing that his leaving
the Country would be of the worst Consequence as there was not a man
in that part of the Country capable to manage it but himself and in general
that his appearing publickly in Arms for the King must be of great Service,
for which Reasons the first Occasion that occurred I wrote to the King
dated September 5th 1743:
Sir,—I had the honour to write to your
majesty the 5th of July last which I hope is
come Safe. I have of late seen several Letters Murray to the
from Lord L[ovat] to L[ochie]l wherein he Chevalier
Express great Anxiety and impatience that things are not like to
come to a conclusion. I take it to be upon that account that he
has settled his affairs att home and is now taking leave of his
friends with a Resolution of going this Winter to London there
to solicite an appeal and from thence to France with his Son.
Your Majesty will see at first View all the inconveniencys that
may attend such a procedure more readily than I can express,
he being the only man in the Country capable to manage that
part of the Country alloted him, and indeed I am afraid from a
paragraph in one of his Letters that he has some thoughts of
Ending his days in a monastry Since he thinks he cannot do it
in your Majestys Service. Affairs abroad seem now to tend
more and more to a War with france which Should it happen
will I hope putt a Stop to his journey, but failing that I am apt to
believe nothing will do save your Majestys orders, etc.
To Mr. Edgar.
September 5, 1743.
Sir,—I gave you the trouble of a pretty
long Letter the fifth of July last, since which I
have not had the pleasure of hearing from Murray to
you. Lord T[ra]q[uai]r is still att London but Edgar
proposes to be soon down here, which I heartily wish, some
folks being vastly anxious for his return expecting upon that
Event to be intirely satisfied as to what may be hoped for from
the Kings friends in England. Upon the Highland deserters
being shott att London,[171] which has greatly disobliged their
Countrymen, I took it upon me to acquaint some of the
Gentlemen that it was his Majestys pleasure they should
endeavour to prevent as much as possible any of their
followers from inlisting in the Service of the present
Government. This I thought the more necessary as a great
many of them have been carried out of the Country for some
years past, the Dutch having gott several hundreds upon their
last Augmentation.[172] My Lord K[e]n[mur]e is returned from
Portugall perfectly recovered. I said some obliging things to
him in his Majestys Name of gaining the Cameronians
(amongst whom he lived) to his Majestys Interest. I am very
sensible what a fickle Sett of people they are and how difficult
an undertaking of this kind may prove. Yett as Sir Th[oma]s
G[ord][o]n of E[arls]t[o]n,[173] a leading man amongst them two
years ago, spoke to the late Lord of the precarious Situation of
the present Government, and in case of a Restoration begged
his protection, this Lord seemed the fitter person to learn his
present Sentiments. Your Friend Sir J[ames] S[tewar]t[174] who
deservedly well liked by all his acquaintances is to be married
to Lord W[emy]ss eldest daughter, a Match made by Lord
E[lcho][175] who left this the beginning of Summer and I
understand, is now at Boulogne, so that I had no opportunity to
deliver the Compliments his Majesty and the Prince honour him
with. I beg you will believe me, etc.
Some time in the month of August the Laird of
Mc[Leo]d[176] came to Edinburgh and told L[ochie]l on his way
here, who desired he might see him, and that he had several
Commissions to us, so desired he would make an appointment
with me. When L[ochie]l spoke to me of it I agreed to ride out
with him on the Saturday to Peggie Vints where he proposed to
dine and see a son of Lord L[ovat]s who was then at the
School of Preston,[177] but we were both afraid from his saying
that he had several Commissions that his Lordship had been
too open with him, contrary to the engagement all these of the
Concert had come under to one another; for which reason we
resolved to be very cautious and determined, in case we found
it as we suspected, to say nothing of it to him. We according
mett, dined in the Country and adjurned to the Tavern in
Edinburgh where we resolved to give him leave to say or ask
as few questions as possible and took occasion to speak a
good deal on the present miserable Situation of the Country,
and tell him that we thought him one of the fittest Persons we
knew to instigate the English to join heartily for promoting the
Kings interest, being both a highland man and one of power in
the Country; and at the same time told him it was his Majestys
pleasure that the Chiefs of the Clans should allow none of their
men to leave the Country. To which he answered that he and
Sir A[lexander] Mc[Donal]d had taken care to let none of theirs
inlist, and said a good deal of his readiness to serve the King
so soon as an occasion should offer, and that he had already
during his being att London made it his business to incite and
encourage the English to every thing that cou’d conduce to his
Majestys interest; and as to Lord L[ova]ts commissions, they
turned out only to inquiring about Lord T[ra]q[uai]r and what
news or good hopes he had. From this time nothing passed
worthy the noticeing, I had some compliments from Lord
L[ova]t in his letters to L[ochie]l wherein he acquainted him with
the success he had in a Circuit he made over the Country and
then gott a Letter or two from himself on these subjects and
desiring his Majesty might be acquainted with it and at the
same time saying he was resolved to continue at Home in
expectation of something satisfactory upon my Lord
T[ra]q[uai]r’s return. Upon this I wrote the following Letter to Mr.
Edgar, dated October 28th, 1743.
Sir,[178]—I has the pleasure of writing to

you the fifth of last moneth with one inclosed
Murray to
to his Majesty, which makes me give you the Edgar
trouble of this to acquaint you that upon
L[ochie]ls repeated Letters to Lord L[ovat] together with Copys
of my Lord T[ra]q[uai]rs from London which I sent him, he is
determined not to stir from home this Winter. I should be
greatly to blame did I neglect to inform you that his Lordship
has been most assiduous this Summer to promote his
Majestys Interest in his district so that I have great reason to
believe that he is sure of all those he engaged for. He seems to
be in great spirits upon account of his success in his Circuit he
lately made over the Country when he gained most of the
Monroes,[179] a people as little to have been expected as any
in the Highlands. He keeps an open table by which means he
is become very popular, and I believe, generally speaking, has
more to say than any in that Country. L[ochie]l is still here
expecting every day Lord T[ra]q[uai]rs arrival etc. His Lordship,
to the best of my Remmembrance, came to Scotland sometime
in the moneth when L[ochie]l and I immediately mett with him.
He acquainted us that Mr. Drummond had left London a great
while before him and promised so soon as he gott to Paris that
the King of France and his ministers should be acquainted with
the favourable accounts he had to give of his Success and that
he would forthwith inform Lord T[ra]q[uai]r of every Resolution
that was taken. His Lordship likewise told us what had passed
during his Stay at London, which I shall not pretend to give a
particular detail of, having no authority to intermeddle with, nor
ever had, any particular concern in what regarded the English;
so shall leave it to his Lordship to give a particular account of
his Negotiations in that Country, if he shall think it necessary. I
shall only mention here that his Lordship informed us that he
had talk’d with the principal people of the Tory party some of
which were very timerous, others such as Lord Bar[rymo]re
very ready to join in any thing that could conduce to forward
the Restoration, and that he had frankly offered, when they
proposed a sum of Money to be ready to the Value of £12,000
which was scrupled at by some, to provide it himself. That Lord
O[rre]ry[180] I had made two several apointments with him and
Mr. Drummond, neither of which he keept, but Stept out of
Town without Seeing of them. But I must observe that from all I
can Remmember of the Story no particular Concert was
formed nor was their any appearance given the french of
meeting with provisions, Carriages and horses att their landing,
as Mr. Amalet proposed at Versails; for to the contrary
when was spoke to who lived in the neighbourhood of
where they proposed to land, concerning the providing of these
several Necessary he said he had no Idea that any thing had
been so suddenly designed for the King, so could make no
promises. Mr. Butler, the Gentleman sent over by the King of
France to enquire into the Situation of the Country, was
introduced by his Lordship to the most of the people. He knew
and was sent to the Country to a meeting at Litchfield Races
there to meet with Lord B[arrymor]e, Sir W[atkin Williams]
W[ynn][181] where he was with about 80 or more gentlemen all
of them but one reckoned honest people, which to be sure
gave a good aspect to the party in general; but nevertheless I
don’t see he went away with such a satisfactory account as Mr.
Amalet seemed to require. He assured my Lord he had several
Instructions from the King himself, but I wish his principall
Errand may not have been to purchase horses with a View to
the Kings equipage for the insuing Campaign, he having
bought to the value of 3 or 4000£. This reflection may seem
harsh but I cannot reconcile their bestowing that sum upon
horses for which he said they had no Occasion only by a way
of blind and not allowing the prince not above one half of it for
a whole years expences, and I don’t think it would be just to
argue in opposition to it that they then knew nothing of the
Campaign his Majesty intended to make. The french are rather
too far sighted not to allow them to design so short a while as
some moneths before hand. In short from his Lordships return
till the moneth of february we had no Letters from france. In the
interim L[ochie]l went to the Highlands when he acquainted Sir
J[ames] C[ampbell] and Lord L[ova]t with all yt had passed and
that we soon expected the french would come to a final
determination one or tother. We spent the time greatly
shagerin’d, vex’d to have no Accounts of any kind considering
that Mr. Drummond promised at his leaving London in
Company with Mr. Butler to write over immediately. Att last we
received two letters, one inclosing another Copy, of which I
shall insert Copy of Mr. Drummonds Letter to the Earl of
T[ra]q[uai]r, dated ...
* * * * *
After I had made this answer with the Consent and advice of the Duke
of P[erth] and was signed by them, the nixt day being Ash Wedensday[182]
his Grace resolved to leave the place being apprehensive that as the
newspapers were there very full of the french preparations he might be
suspected and seized and so not in his power to appear when any thing
came to be done. My Lord T[ra]q[uai]r, att whose Lodgings I had the
Honour to meet his Grace that mourning, was very much against his
leaving the place in such a hurry, and on that day especially. I likewise
took the Liberty to remonstrate to him a little against it but all to no
purpose and he accordingly sett out about Eleven O Clock and went that
night the Length of Dumblain. This was immediately looked upon by the
people of the Government with a very jealous Eye and the more so that
his Brother Lord J[oh]n had come to Scotland some little time before upon
a Scheme of raising a Scots Regiment for the french Service, stayed only
ten days or little more at Edinburgh and went from that to the Highlands to
solicite the Gentlemen there to be assisting to him in making his Levies. It
unluckily hapened for the Duke that upon the back of Lord J[oh]ns going to
the Country the Government began to be alarmed with the Accounts of the
Princes being come to france and the intended invasion in his Majestys
interest which made our little Ministers conjecture that Lord J[oh]n had
been sent over with the accounts of it to the Highlands and consequently
that the Duke had left the town with an intention to foment an insurrection
in the Country. This was made no secret of, being publickly talked of
immediately upon the Duke disappearing two days, so after his leaving the
town a servant of his was dispatched to London with the Letter I had wrote
to Mr. Drummond their being no other means of conveiying one to him but
by express, we not having any settled Cannal of Correspondence from
hence there. This I think was about the 8th or 10th of february [1744]. My
Lord T[ra]q[uai]r stayed some days in town after, designedly to create no
suspicion.
In about a week or so after I went on a Sunday Evening to see Mr.
H[a]y[183][184] who, when I was talking in a ludicrous way of the paragraph
in the papers about the manner in which his Highness had left Italy, he told
me in a very serious way that it was no Joke and said that the sooner I
went to the Country the better as my living quietly at home would give no
suspicion to the Government, but if I stayed any time in town he did not
know what might happen, that upon his honour he had not heard me
mentioned, which perhaps was owing to my near Relation with him, but
that att that same time I was suspected to correspond with Rome. I told
him I intended to stay some days longer in Town and would so soon as I
had formerly proposed, that I had no cause of fear and so would not run
away, and as to their suspecting my corresponding with Rome, that I
laughed at but thanked him for his kind concern. I was not at all sorry to
find they were so alarmed and afraid, which to me appeared a sure sign of
their weakness, and indeed their fear for some days after increased to the
most abject pusilanimity. His saying that he had not heard me mentioned
was telling plainly that they had been consulting of who were the persons
first to be laid hands; and I am apt to believe had the management of
affairs been left to Lord Arniston,[185] Sir John Inglis,[186] Commissioner
Arthburthnet[187] and the rest of the present Ministry there would have
been little lenity shown any Body they had the least reason to suspect. But
affairs were afterwards taken out of their hands and putt into these of
Justice Clerks,[188] who tho’ as Violent a Whig yet not so hott and Violent
a Man. I went next day, being Munday, in the morning to wait of General
Guest, who then Commanded in Chief in Scotland[189] with a View to hear
what a Notion or Idea he had of matters. He told me that the Repeal of the
Habeas Corpus Act was expected that night by the Post, but added that,
was it come, upon his honour he did not know a man he suspected
enough to lay up, which I was exceedingly well pleased with. Also att the
same time I could have marked out a great many and the general seemed
not in the least to be affraid and laughed att the hurry and confusion the
other folks were. I was taken very ill that day after dinner and gave up
thoughts of going to Mr. Hunters of Poolmood’s Burrial[190] which was to
be on the Thursday, and I was desired out by the Widow on the tuesday,
which evening about six O Clock Mr. Mc[Douga]ll[191] brought me a Letter
directed to the Countess of T[ra]q[uai]r. As I was then expecting one every
day from Mr. Drummond, as he had promised in his last, I began to
suspect a little notwithstanding it had come by the Common Post, a very
odd method of Conveyance. In such a critical juncture I opened it when I
found a blank Cover and Still directed as before. This confirmed me in my
suspicion and under that I found a Letter for my Lord which I immediately
opened and tho’ partly in Cypher, could easily understand that things were
directly to be putt in execution. This struck me a good deal as I said I
would not go to the Country. However, I sent Mr. Mc[Douga]ll with
directions immediately to sent the Letter off to Lord T[ra]q[uai]r and to
desire D. C[ ]n[192] to come down as of no design and tell me I might go
to the Country next day if I pleased, which he accordingly did and hyrred a
Chaise, not being able to ride. In the mean while Sir J. S[tewar]t came to
see me, who I acquainted of it and att the same time wrote a Letter to Lord
K[enmu]re who I had spoke to the Munday before, desiring him to meet
me at T[ra]q[uai]r the thursday night as likewise one to Mr. J[oh]n
Mc[leo]d[193] telling him I thought it would be fitt to send Sir J[ames]
C[ampbe]lls son to the Highlands, who his father designed should serve
him therein being assistant to raise the Country. I accordingly sett out next
mourning for the Country and the day following Lord T[ra]q[uai]r mett me
at Polmood and shewed me the Letter when decyphered which I shall
here give a Copy off, and att the same time a Letter he had received the
night before by express from Edinburgh telling him that their was a
Warrant out to apprehend him which determined his Lordship to go
immediately to the D[uke] of P[erth]. Upon which so soon as the Burrial
was over we came to my house, where I wrote a Letter to Lord K[enmu]re
desiring he would follow us next mourning to Hartrie[194] where we
intended to sleep that night and sent it Express to T[ra]q[uai]r with other
Letters of my Lords, expecting Lord K[enmo]re would be there that night,
but he nevertheless continued the whole time in Edinburgh. We set out
next day from Hartrie which was the fryday, that the french fleet was
dispersed and the Transports run a Shore,[195] and the night after gott to
Drummond Castle, from whence his Grace sent immediately an express
with the Copy of the last Letter we received to L[ochie]l. We continued
som weeks there always in Expectation to hear of a landing and in the
mean time heard that several informations was given in against the Duke
of Perth as having numbers of armed men about his house which was
absolutely false. At last a party of 150 foot and 30 Horse were sent from
Stirling to make him Prisoner, but he had intelligence of it and went out of
the way.[196] All this time Lord T[ra]q[uai]r was sculking about the Country
having returned from the jaunt he had made over the Highlands.
After staying in that Country till the beginning of Aprile, without
receiving any Accounts from abroad and giving up all hopes of a Landing,
I left my Lord T[ra]q[uai]r there and came to Stirlingshire where I stayed
about three weeks and so came to Edinburgh, and from that went to the
Country the 11th of May. In the beginning of June, when Lord T[ra]q[uai]r
returned, I went to wait of him, and being very uneasy to think we had
received no accounts from Abroad, I said if I could afford the expence I
would go over on pretence of seeing the Army in flanders and so see the
Prince myself and learn distinctly what situation things were in. This his
Lordship was well pleased with but I did not say anything positively, but
upon Reflexion by the Road, I thought it was hard that people who had
been for so long concerned in the Kings affairs and putt to so great
Charge about it with the hazard of their Lives and fortunes should now be
left in the dark as to every thing. Wherefore I resolved to do it, and next
day wrote a Letter to my Lord telling him that if the Duke of P[erth] would
give me a 100£ I would be att the rest of the expence myself and go over
and in case his Lordship approved of it, he would be so good as meet me
at Peebles on Saturday, which he did, and after talking with him I came
home and sett out the same night about O Clock and gott to Drummond
[Castle] next afternoon. The Duke immediately agreed to the thing and
gave me an order for the money. About this time came a letter from Lord
Semple to Lord T[ra]q[uai]r by way of an account of their precedure in the
Spring, which I went to T[ra]q[uai]r and assisted his Lordship to decypher,
but was so little to our satisfaction that my Lord still thought my going over
more necessary than before. About this time I received a letter from
St[uar]t of Ard[shie]ll,[197] telling me that he would have come to the
Country to wait of me but his dress made him remarkable, being in
Highland Cloaths, but as he had comed to Town purposely to meet with
me, he hoped I would give him a meeting, and yt he had seen L[ochie]l
lately. I went to town in a day or two after and dined with him. His Errand
was to know of me if I had gott any Accounts lately, and what hopes I had.
I did not think it att all proper to let him know any thing of my having seen
such a letter as Lord Semple had wrote, nor indeed that any Accounts had
come; for in that case he would have expected something positive. But I

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Biomolecular Engineering Solutions for

Renewable Specialty Chemicals:

Discover biomolecular engineering technologies for the production of KRISHNARAJ

BIOMOLECULAR ENGINEERING SOLUTIONS

FOR RENEWABLE SPECIALTY CHEMICALS

• A thorough introduction to engineered microorganisms for the production of

R. Navanietha Krishnaraj, PhD, is Research Professor in the Composite and Nanocomposite

Cover Design: Wiley

Microorganisms, Products, and Processes

Limit of Liability/Disclaimer of Warranty

Library of Congress Cataloging-­in-­Publication Data applied for:

Cover design by Wiley

Set in 9.5/12.5pt STIXTwoText by Straive Pondicherry, India

1 Engineered Microorganisms for Production of Biocommodities 1

1.3.2.3 Succinic Acid 24

2 Microbial Cell Factories for the Biosynthesis of Vanillin and Its

3 Antimicrobials: Targets, Functions, and Resistance 77

3.2.2 Classification of Antibiotics Based on the Spectrum of Action:

3.7 ­ ntimicrobial Resistance 101

4 Trends in Antimicrobial Therapy: Current Approaches and Future

4.5.5 Bdellovibrio 127

5 Fermentation Strategies in the Food and Beverage Industry 141

6 Bioactive Oligosaccharides: Production, Characterization,

6.2.7 Pseudo-­oligosaccharides 168

7 Biopolymers: A Retrospective Analysis in the Facet of Biomedical

7.2.1.2 Elastin 202

7.4.6 Biopolymers in Other Applications 230

8 Metabolic Engineering Strategies to Enhance Microbial Production

8.6.1 Metabolic Modification of Pathway for Synthesis

9 Bioplastics Production: What Have We Achieved? 287

10 Conversion of Lignocellulosic Biomass to Ethanol: Recent

10.1 I­ ntroduction 311

11 Advancement in Biogas Technology for Sustainable Energy

11.2 ­ iogas Developments Worldwide 360

12 Biofertilizers: A Sustainable Approach Towards Enhancing

13 Biofertilizers from Food and Agricultural By-­Products

13.2.1.2 Symbiotic N2-­Fixing Biofertilizer 424

Microorganisms have been realized as promising sources for production of biocom-

R. Aanandhalakshmi Rathindra Mohan Banik

Madhuri Dutta Sukumaran Karthika

Santhalingam Gayathri Chandraraj Krishnan

Sanjay Kumar Akhil Rautela

Composite and Nanocomposite Shailendra Singh Shera

Engineered Microorganisms for Production

Biomolecular Engineering Solutions for Renewable Specialty Chemicals: Microorganisms,

from photoautotrophic organisms such as cyanobacteria and microalgae, which uti-

1.2 ­Fundamentals of Genetic Engineering

1.2.1 DNA-­altering Enzymes

Blunt ends Sticky ends

5. Joining of vector and 6. Vector 7. Insertion of 8. Genetically

Figure 1.1 Basic steps of gene cloning.

manipulate DNA molecules with specific changes can be categorized in four

1.2.1.1 DNA Polymerases

1.2.1.4 DNA-­modifying Enzymes

1.2.1.4.2 T4 Poly Nucleotide Kinase Polynucleotide kinase enzyme is exact

γ-­phosphate of ATP with the 5′-­terminal phosphate of a polynucleotide, thus

1.2.1.4.3 Terminal Transferase Terminal deoxy-­nucleotidyl transferase [Terminal

1.2.1.4.4 Topoisomerases Topoisomerases change the affirmation of covalently

1.2.3 Incorporation of Modified DNA into Host

1.2.3.1 Introducing Recombinants into Prokaryotes

1.2.3.1.1 Transformation Transformation is the most commonly used method in

1.2.3.1.3 Conjugation Conjugation is the procedure of movement of hereditary

1.2.3.2 Introducing Recombinants into Eukaryotic Hosts

• A thorough introduction to engineered microorganisms for the production of

Library of Congress Cataloging-in-Publication Data applied for:

3.7 ntimicrobial Resistance 101

6.2.7 Pseudo-oligosaccharides 168

10.1 I ntroduction 311

11.2 iogas Developments Worldwide 360

13 Biofertilizers from Food and Agricultural By-Products

13.2.1.2 Symbiotic N2-Fixing Biofertilizer 424

1.2 Fundamentals of Genetic Engineering

1.2.1 DNA-altering Enzymes

1.2.1.4 DNA-modifying Enzymes

γ-phosphate of ATP with the 5′-terminal phosphate of a polynucleotide, thus

1.2.1.4.3 Terminal Transferase Terminal deoxy-nucleotidyl transferase [Terminal

1.2.4.1.2 Blue–White Color Screening Blue-white screening is also based on

1.3 Beneficial Biocommodities Produced Through

1.3.1.2 Poly-ϒ-glutamic Acid