High Value Fermentation Products, Volume 1
Human Health

Green technologies are no longer the ?future? of science, but the present.  With more and more mature industries, such as the process industries, making large strides seemingly every single day, and more consumers demanding products created from green technologies, it is essential for any business in any industry to be familiar with the latest processes and technologies.  It is all part of a global effort to ?go greener,? and this is nowhere more apparent than in fermentation technology. 

This book describes relevant aspects of industrial-scale fermentation, an expanding area of activity, which already generates commercial values of over one third of a trillion US dollars annually, and which will most likely radically change the way we produce chemicals in the long-term future. From biofuels and bulk amino acids to monoclonal antibodies and stem cells, they all rely on mass suspension cultivation of cells in stirred bioreactors, which is the most widely used and versatile way to produce. Today, a wide array of cells can be cultivated in this way, and for most of them genetic engineering tools are also available. Examples of products, operating procedures, engineering and design aspects, economic drivers and cost, and regulatory issues are addressed. In addition, there will be a discussion of how we got to where we are today, and of the real world in industrial fermentation. This chapter is exclusively dedicated to large-scale production used in industrial settings.

Foreword xvii

About the Editors xix

List of Contributors xxi

Preface xxv

Acknowledgement xxvii

1 Introduction, Scope and Significance of Fermentation Technology 1
Saurabh Saran, Alok Malaviya and Asha Chaubey

1.1 Introduction 1

1.2 Background of Fermentation Technology 2

1.3 Market of Fermentation Products 3

1.4 Types of Fermentation 4

1.4.1 Solid State Fermentation (SSF) 4

1.4.2 Submerged Fermentation (SmF) 7

1.4.3 Solid State (SSF) vs. Submerged (SmF) Fermentation 9

1.5 Classification of Fermentation 9

1.6 Design and Parts of Fermentors 10

1.7 Types of Fermentor 15

1.7.1 Stirred Tank Fermentor 15

1.7.2 Airlift Fermentor 16

1.7.3 Bubble Column Fermentor 17

1.7.4 Fluidized Bed Fermentor 18

1.7.5 Packed Bed Fermentor 19

1.7.6 Photo Bioreactor 19

1.8 Industrial Applications of Fermentation Technology 21

1.9 Scope and Global Market of Fermentation Technology 22

1.10 Conclusions 23

References 24

2 Extraction of Bioactive Molecules through Fermentation and Enzymatic Assisted Technologies 27
Ramón Larios-Cruz, Liliana Londoño-Hernández, Ricardo Gómez-García, Ivanoe García, Leonardo Sepulveda, Raúl Rodríguez-Herrera and Cristóbal N. Aguilar

2.1 Introduction 27

2.2 Definition of Bioactives Compounds 29

2.2.1 Polyphenols and Polypeptides 29

2.2.2 Importance and Applications of Bioactive Compounds 29

2.2.3 Bioactive Peptides 31

2.3 Traditional Processes for Obtaining Bioactive Compounds 33

2.3.1 Soxhlet Extraction 33

2.3.2 Liquid-Liquid and Solid-Liquid Extraction 34

2.3.3 Maceration Extraction 35

2.4 Fermentation and Enzymatic Technologies for Obtaining Bioactive Compounds 35

2.4.1 Soft Chemistry in Bioactive Compounds 35

2.4.2 Biotransformation of Bioactive Compounds 36

2.4.3 Enzymatic and Fermentation Technologies 39

2.5 Use of Agroindustrial Waste in the Fermentation Process 45

2.5.1 Cereal Wastes 46

2.5.2 Fruit and Plant Waste 46

2.6 General Parameters in the Optimization of Fermentation Processes 49

2.6.1 Response Surface Methodology 49

2.6.2 First-Order Model 49

2.6.3 Second-Order Model 49

2.7 Final Comments 52

Acknowledgements 52

References 52

3 Antibiotics Against Gram Positive Bacteria 61
Rahul Vikram Singh, Hitesh Sharma, Anshela Koul and Vikash Babu

3.1 Introduction 61

3.2 Target of Antibiotics Against Gram Positive Bacteria 64

3.2.1 Cell Wall Synthesis Inhibition 65

3.2.2 Protein Synthesis Inhibition 70

3.2.3 DNA Synthesis Inhibition 72

3.3 Antibiotics Production Processes 72

3.4 Conclusion 75

References 76

4 Antibiotic Against Gram-Negative Bacteria 79
Maryam Faiyaz, Shikha Gupta and Divya Gupta

4.1 Introduction 79

4.2 Gram-Negative Bacteria and Antibiotics 80

4.2.1 β-Lactam Drugs 81

4.2.2 Macrolide 82

4.2.3 Aminoglycosides 84

4.2.4 Fluoroquinolones 84

4.3 Production of Antibiotics 85

4.3.1 Strain Development 85

4.3.2 Media Formulation and Optimization 88

4.3.3 Fermentation 90

4.3.4 Downstream Processing and Purification 92

4.3.5 Quality Control 95

4.4 Conclusion 95

References 96

5 Role of Antifungal Drugs in Combating Invasive Fungal Diseases 103
Kakoli Dutt

5.1 Introduction 103

5.2 Antifungal Agents 105

5.2.1 Azoles 114

5.2.2 Polyenes 115

5.2.3 Allylamine/Thiocarbonates 116

5.2.4 Other Antifungal Agents 117

5.3 Targets of Antifungal Agents 120

5.3.1 Cell Wall Biosynthesis Inhibitors 120

5.3.2 Sphingolipid Synthesis Inhibitors 123

5.3.3 Ergosterol Synthesis Inhibitors 125

5.3.4 Protein Synthesis Inhibitors 126

5.3.5 Novel Targets 128

5.4 Development of Resistance towards Antifungal Agents 130

5.4.1 Minimum Inhibitory Concentration 130

5.4.2 Antifungal-Drug-Resistance Mechanisms 131

5.5 Market and Drug Development 134

5.6 Conclusions 136

Acknowledgement 137

References 137

6 Current Update on Rapamycin Production and Its Potential Clinical Implications 145
Girijesh K. Patel, Ruchika Goyal1 and Syed M. Waheed

6.1 Introduction 145

6.2 Biosynthesis of Rapamycin 146

6.2.1 Microbial Strain 147

6.2.2 Optimization of Carbon, Nitrogen Sources and Salts 147

6.2.3 Strain Manipulation to Improve Rapamycin Production 148

6.3 Organic Synthesis of Rapamycin 152

6.4 Extraction and Quantification of Rapamycin 152

6.5 Physiological Factors Affecting Rapamycin Biosynthesis 153

6.5.1 Effect of Media Components 153

6.5.2 Effect of pH on Rapamycin Production 153

6.5.3 Effect of Physical Gravity 154

6.5.4 Effect of Morphological Changes 154

6.5.5 Effect of Dissolved Oxygen (DO) and Carbon Dioxide (DCO₂) 154

6.6 Production of Rapamycin Analogs 154

6.7 Mechanism of Action of Rapamycin 155

6.8 Use of Rapamycin in Medicine 157

6.8.1 Anti-Fungal Agent 157

6.8.2 Immunosuppression 158

6.8.3 Anti-Cancer Agent 158

6.8.4 Anti-Aging Agent 158

6.8.5 Role in HIV Treatment 158

6.8.6 Rheumatoid Arthritis 159

6.9 Side Effects of Long-term Use of Rapamycin 159

6.10 Conclusions 159

Acknowledgements 160

References 160

7 Advances in Production of Therapeutic Monoclonal Antibodies 165
Richi V Mahajan, Subhash Chand, Mahendra Pal Singh, Apurwa Kestwal and Surinder Singh

7.1 Introduction 165

7.2 Discovery and Clinical Development 166

7.3 Structure and Classification 167

7.4 Nomenclature of Monoclonal Antibodies 168

7.5 Production of Monoclonal Antibodies 170

7.5.1 Hybridoma Technology 170

7.5.2 Epstein-Barr Virus Technology 172

7.5.3 Phage Display Technology 172

7.5.4 Cell Line Based Production Techniques 173

7.5.5 Chemical Modifications of Monoclonal Antibodies 183

7.5.6 Advances in Antibody Technology 183

7.6 Conclusions 185

References 186

8 Antimicrobial Peptides from Bacterial Origin: Potential Alternative to Conventional Antibiotics 193
Lipsy Chopra, Gurdeep Singh, Ramita Taggar, Akanksha Dwivedi, Jitender Nandal, Pradeep Kumar and Debendra K. Sahoo

8.1 Introduction 193

8.2 Classification of Bacteriocins 194

8.2.1 Bacteriocins from Gram-Negative Bacteria 194

8.2.2 Bacteriocins from Gram-Positive Bacteria 194

8.3 Mode of Action 196

8.3.1 Pore-Forming Bacteriocins 196

8.3.2 Non-Pore-Forming Bacteriocins: Intracellular Targets 198

8.4 Applications 198

8.4.1 Food Bio Preservative 198

8.4.2 Food Packaging (In Packaging Films) 198

8.4.3 Hurdle Technology to Enhance Food Safety 199

8.4.4 Therapeutic Potential 200

8.4.5 Effect of Bacteriocins on Biofilms 200

8.5 Conclusions 202

Acknowledgments 202

Abbreviations 202

References 202

9 Non-Ribosomal Peptide Synthetases: Nature’s Indispensable Drug Factories 205
Richa Sharma, Ravi S. Manhas and Asha Chaubey

9.1 Introduction 205

9.1.1 Non-Ribosomal Peptides as Natural Products 205

9.1.2 Non-Ribosomal Peptides as Drugs 206

9.2 NRPS Machinery 208

9.3 Catalytic Domains of NRPSs 208

9.3.1 Adenylation (A) Domains 208

9.3.2 Thiolation (T) or PCP Domains 209

9.3.3 Condensation (C) Domains 209

9.3.4 Thioesterase (Te) Domains 209

9.4 Types of NRPS 210

9.4.1 Type A (Linear NRPS) 210

9.4.2 Type B (Iterative NRPS) 210

9.4.3 Type C (Non-linear NRPS) 210

9.5 Working of NRPSs 210

9.5.1 Priming Thiolation Domain of NRPS 211

9.5.2 Substrate Recognition and Activation 211

9.5.3 Peptide Bond Formation between NRP Monomers 211

9.5.4 Chain Termination of NRP Synthesis 212

9.5.5 NRP Tailoring 212

9.6 Sources of NRPs 213

9.7 Production of Non-Ribosomal Peptides 216

9.8 Future Scope 218

Acknowledgements 219

References 219

10 Enzymes as Therapeutic Agents in Human Disease Management 225
Babbal, Adivitiya, Shilpa Mohanty and Yogender Pal Khasa

10.1 Introduction 225

10.2 Pancreatic Enzymes 230

10.2.1 Trypsin (EC 3.4.21.4) 230

10.2.2 Pancreatic Lipase (EC 3.1.1.3) 231

10.2.3 Amylases (EC 3.2.1.1) 231

10.3 Oncolytic Enzymes 232

10.3.1 L-Asparaginase (EC 3.5.1.1) 232

10.3.2 L-Glutaminase (EC 3.5.1.2) 233

10.3.3 Arginine Deiminase (ADI) (EC 3.5.3.6) 233

10.4 Antidiabetic Enzymes 234

10.4.1 Glucokinase (EC2.7.1.1)

10.5 Liver Enzymes 235

10.5.1 Superoxide Dismutase (SOD) (EC 1.15.1.1) 235

10.5.2 Alkaline Phosphatase (ALP) (EC 3.1.3.1) 236

10.6 Kidney Disorder 237

10.6.1 Uricase (EC 1.7.3.3) 237

10.6.2 Urease (EC 3.5.1.5) 238

10.7 DNA- and RNA-Based Enzymes 238

10.7.1 Dornase 239

10.7.2 Adenosine Deaminase 240

10.7.3 Ribonuclease 240

10.8 Enzymes for the Treatment of Cardiovascular Disorders 241

10.8.1 The Hemostatic System 242

10.8.2 Enzymes of the Hemostatic System 244

10.9 Lysosomal Storage Disorders 251

10.9.1 α-Galactosidase A (EC 3.2.1.22) 251

10.9.2 Glucocerebrosidase (EC 3.2.1.45) 252

10.9.3 Acid Alpha-Glucosidase (GAA) (EC 3.2.1.20) 253

10.9.4 α-L-iduronidase (Laronidase) (EC 3.2.1.76) 253

10.10 Miscellaneous Enzymes 254

10.10.1 Phenylalanine Hydroxylase (EC 1.14.16.1) 254

10.10.2 Collagenase (EC 3.4.24.3) 255

10.10.3 Hyaluronidase 256

10.10.4 Bromelain 256

10.11 Conclusions 256

References 257

11 Erythritol: A Sugar Substitute 265
Kanti N. Mihooliya, Jitender Nandal, Himanshu Verma and Debendra K. Sahoo

11.1 Introduction 265

11.1.1 Background of Erythritol 265

11.1.2 History of Erythritol 268

11.1.3 Occurrence of Erythritol 268

11.1.4 General Characteristics 268

11.2 Chemical and Physical Properties of Erythritol 271

11.3 Estimation of Erythritol 271

11.3.1 Thin Layer Chromatography (TLC) 273

11.3.2 Colorimetric Assay for Detection of Polyols 273

11.3.3 High-Performance Liquid Chromatography (HPLC) 273

11.3.4 Capillary Electrophoresis (CE) 273

11.4 Production Methods for Erythritol 274

11.4.1 Chemical Methods for Erythritol Production 274

11.4.2 Fermentative Methods for Erythritol Production 274

11.5 Optimization of Erythritol Production 275

11.5.1 One Factor at a Time 276

11.5.2 Statistical Design Approaches 277

11.6 Toxicology of Erythritol 277

11.7 Applications of Erythritol 277

11.7.1 Confectioneries 278

11.7.2 Bakery 279

11.7.3 Pharmaceuticals 279

11.7.4 Cosmetics 279

11.7.5 Beverages 279

11.8 Precautions for Erythritol Usage 279

11.9 Global Market for Erythritol 280

11.10 Conclusions 280

References 281

12 Sugar and Sugar Alcohols: Xylitol 285
Bhumica Agarwal and Lalit Kumar Singh

12.1 Introduction 285

12.1.1 Lignocellulosic Biomass 286

12.1.2 Properties of Xylitol 287

12.1.3 Occurrence and Production of Xylitol 289

12.2 Biomass Conversion Process 289

12.2.1 Pretreatment Methodologies 289

12.2.2 Enzymatic Hydrolysis 292

12.2.3 Detoxification Techniques 293

12.3 Utilization of Xylose 296

12.3.1 Microorganisms Utilizing Xylose 296

12.3.2 Metabolism of Xylose 297

12.4 Process Variables 299

12.4.1 Temperature and pH 299

12.4.2 Substrate Concentration 300

12.4.3 Aeration 301

References 303

13 Trehalose: An Anonymity Turns Into Necessity 309
Manali Datta and Dignya Desai

13.1 Introduction 309

13.2 Trehalose Metabolism Pathways 310

13.3 Physicochemical Properties and its Biological Significance 311

13.4 Trehalose Production 312

13.4.1 Enzymatic Conversion to Trehalose 312

13.4.2 Microbe Mediated Fermentation 314

13.4.3 Purification and Detection of Trehalose in Fermentation Process 316

13.5 Application of Trehalose 317

13.5.1 Role of Trehalose in Food Industries 317

13.5.2 Role of Trehalose in Cosmetics and Pharmaceutics 318

13.6 Conclusions 319

References 320

14 Production of Yeast Derived Microsomal Human CYP450 Enzymes (Sacchrosomes) in High Yields, and Activities Superior to Commercially Available Microsomal Enzymes 323
Ibidapo Stephen Williams and Bhabatosh Chaudhuri

14.1 Introduction 323

14.1.1 Cytochrome P450 (CYP) Enzymes in Humans 323

14.1.2 Human Cytochrome P450 Enzymes and their Role in Drug Metabolism 324

14.1.3 Requirement of Activating Proteins to Form Functional Human CYP Enzymes 325

14.1.4 Use of Yeast Biased Codons for the Syntheses of Human Cytochrome P450 Genes 325

14.1.5 Expression of Human CYP Genes in Baker’s Yeast from an Episomal Plasmid 325

14.1.6 Expression of Human CYP Genes in Baker’s Yeast from Integrative Plasmids 327

14.1.7 The ADH2 Promoter for Production of Human CYP Enzymes in Baker’s Yeast 327

14.1.8 Growth of Yeast Cells Containing Integrated Copies of CYP Gene Expression Cassettes, Driven by the ADH2 Promoter, for Production of CYP Enzymes 328

14.2 Amounts of Microsomal CYP Enzyme Isolated from Yeast Strains Containing Chromosomally Integrated CYP Gene Expression Cassettes are far Higher than Strains Harbouring an Episomal Expression Plasmid Encoding a CYP Gene 328

14.2.1 Preparation of Microsomal CYP Enzymes 328

14.2.2 Measurement of the Amounts of Functional CYPs in Microsomes Isolated from Baker’s Yeast 329

14.2.3 Production of Functional Human CYP1A2 Microsomal Enzyme from Baker’s Yeast 330

14.2.4 Production of Functional Human CYP3A4 Microsomal Enzyme from Baker’s Yeast 330

14.2.5 Production of Functional Human CYP2D6 Microsomal Enzyme from Baker’s Yeast 331

14.2.6 Production of Functional Human CYP2C19 Microsomal Enzyme from Baker’s Yeast 332

14.2.7 Production of Functional Human CYP2C9 Microsomal Enzyme from Baker’s Yeast 333

14.2.8 Production of Functional Human CYP2E1 Microsomal Enzyme from Baker’s Yeast 333

14.2.9 Comments on the Production of Human CYP Enzymes from Baker’s Yeast 334

14.3 Comparison of CYP Enzyme Activity of Yeast-Derived Microsomes (Sacchrosomes) with Commercially Available Microsomes Isolated from Insect and Bacterial Cells 336

14.3.1 Fluorescence-based Assays for Determining CYP Enzyme Activities in Isolated Microsomes 336

14.3.2 Comparison of Enzyme Activity of CYP1A2 Sacchrosomes with Commercially Available CYP1A2 Microsomes Isolated from Insect and Bacterial Cells 336

14.3.3 Comparison of Enzyme Activity of CYP2C9 Sacchrosomes with Those of Commercially Available CYP2C9 Microsomes from Insect and Bacterial Cells 337

14.3.4 Comparison of Enzyme Activity of CYP2C19 Sacchrosomes with Those of Commercially Available CYP2C19 Microsomes from Insect and Bacterial Cells 337

14.3.5 Comparison of Enzyme Activity of CYP2D6 Sacchrosomes with Those of Commercially Available CYP2D6 Microsomes from Insect and Bacterial Cells 338

14.3.6 Comparison of Enzyme Activity of CYP3A4 Sacchrosomes with Those of Commercially Available CYP3A4 Microsomes from Insect and Bacterial Cells 338

14.3.7 Comparison of Enzyme Activity of CYP2E1 Sacchrosomes with One of the Commercial CYP2E1 Microsomes Available from Insect Cells 339

14.4 IC₅₀ Values of Known CYP Inhibitors Using Sacchrosomes, Commercial Enzymes and HLMs 339

14.5 Stabilisation of Sacchrosomes through Freeze-drying 340

14.6 Conclusions 342

References 345

15 Artemisinin: A Potent Antimalarial Drug 347
Alok Malaviya, Karan Malhotra, Anil Agarwal and Katherine Saikia

15.1 Introduction 347

15.2 Biosynthesis of Artemisinin in Artemisia annua and Pathways Involved 348

15.3 Yield Enhancement Strategies in A. annua 351

15.4 Artemisinin Production Using Heterologous Hosts 352

15.4.1 Microbial Engineering 352

15.4.2 Plant Metabolic Engineering 353

15.5 Spread of Artemisinin Resistance 357

15.6 Challenges in Large-Scale Production 358

15.7 Future Prospects 360

References 360

16 Microbial Production of Flavonoids: Engineering Strategies for Improved Production 365
Aravind Madhavan, Raveendran Sindhu, KB Arun, Ashok Pandey, Parameswaran Binod and Edgard Gnansounou

16.1 Introduction 365

16.2 Flavonoids 366

16.3 Flavonoid Chemistry and Classes 366

16.4 Health Benefits of Flavonoids 367

16.5 Flavonoid Biosynthesis in Microorganism 368

16.6 Engineering of Flavonoid Biosynthesis Pathway 370

16.7 Metabolic Engineering Strategies 370

16.8 Applications of Synthetic Biology in Flavonoid Production 371

16.9 Post-modification of Flavonoids 374

16.10 Purification of Flavonoids 374

16.11 Conclusion 375

Acknowledgements 375

References 376

17 Astaxanthin: Current Advances in Metabolic Engineering of the Carotenoid 381
Manmeet Ahuja, Jayesh Varavadekar, Mansi Vora, Piyush Sethia, Harikrishna Reddy and Vidhya Rangaswamy

17.1 Introduction 381

17.1.1 Structure of Astaxanthin 382

17.1.2 Natural vs. Synthetic Astaxanthin 382

17.1.3 Uses and Market of Astaxanthin 383

17.2 Pathway of Astaxanthin 384

17.2.1 Bacteria 384

17.2.2 Algae 384

17.2.3 Yeast 385

17.2.4 Plants 386

17.3 Challenges/Current State of the Art in Fermentation/Commercial Production 386

17.4 Metabolic Engineering for Astaxanthin 388

17.4.1 Bacteria 388

17.4.2 Plants 390

17.4.3 Synechocystis 391

17.4.4 Algae 391

17.4.5 Yeast 392

17.5 Future Prospects 393

References 395

18 Exploitation of Fungal Endophytes as Bio-factories for Production of Functional Metabolites through Metabolic Engineering; Emphasizing on Taxol Production 401
Sanjog Garyali, Puja Tandon, M. Sudhakara Reddy and Yong Wang

18.1 Introduction 401

18.2 Taxol: History and Clinical Impact 403

18.3 Endophytes 403

18.3.1 Biodiversity of Endophytes 405

18.3.2 Endophyte vs. Host Plant: the Relationship 405

18.4 The Plausibility of Horizontal Gene Transfer (HGT) Hypothesis 407

18.5 Endophytes as Biological Factories of Functional Metabolites 409

18.6 Taxol Producing Endophytic Fungi 410

18.7 Molecular Basis of Taxol Production by Taxus Plants (Taxol Biosynthetic Pathway) 412

18.8 Metabolic Engineering for Synthesis of Taxol: Next Generation Tool 416

18.8.1 Plant Cell Culture 417

18.8.2 Microbial Metabolic Engineering for Synthesis of Taxol and Its Precursors 418

18.8.3 Metabolic Engineering in Heterologous Plant for Synthesis of Taxol and Its Precursors 420

18.9 Future Perspectives 421

Acknowledgements 423

References 423

Index 431

Saurabh Saran, PhD, is a microbiologist and fermentation scientist with over ten years of experience in industrial microbiology, biotechnology and fermentation technology. He received his doctorate from Delhi University, and he has extensive experience in both the academic and industrial worlds, in multiple countries. He is currently Senior Scientist in the Fermentation Technology Division at the Indian Institute of Integrative Medicine, Jammu. He has three patents and more than 25 international publications in peer reviewed international journals on fermentation technology to his credit.

Vikash Babu, PhD, has a doctorate from the Indian Institute of Technology and has over ten years of experience in graduate and postgraduate work and teaching. After working at Mangalayatan University and Graphic Era University, he joined the Indian Institute of Integrative Medicine as a scientist. He has been the editor on one book, also available from Wiley-Scrivener.

Asha Chaubey, PhD, is Senior Scientist at the Fermentation Technology Division at the Indian Institute of Integrative Medicine, Jammu, India. Her research interests include exploration and exploitation of microbes for bioactives & enzymes production, immobilization of enzymes, biotransformation, kinetic resolution of racemic drug intermediates, development of biosensors for health care and environmental monitoring.Cover Design: Kris Hackerott