Biomolecular Engineering Solutions for Renewable Specialty Chemicals E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Fachliteratur
- Sprache: Englisch
Discover biomolecular engineering technologies for the production of biofuels, pharmaceuticals, organic and amino acids, vitamins, biopolymers, 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 discover 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 synthesis, including purification. The articles within cover topics like the rewiring of anaerobic microbial 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 improving microbes and enzymes using protein engineering, metabolic engineering, and systems biology approaches for converting wastes. 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 and future prospects, as well as fermentation strategies in the food and beverage industry * Practical discussions of bioactive oligosaccharides, including their production, characterization, and applications * 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.
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Table of Contents
Cover
Title Page
Copyright Page
Preface
Biocommodity Engineering
List of Contributors
1 Engineered Microorganisms for Production of Biocommodities
1.1 Introduction
1.2 Fundamentals of Genetic Engineering
1.3 Beneficial Biocommodities Produced Through Engineered Microbial Factories
1.4 Photosynthetic Production of Biofuels
1.5 Conclusion
References
2 Microbial Cell Factories for the Biosynthesis of Vanillin and Its Applications
2.1 Introduction
2.2 Natural Sources of Vanilla and Its Production
2.3 Biotechnological Production of Vanillin
2.4 Strain Development for Improved Production of Vanillin
2.5 Bioactive Properties of Vanillin
2.6 Conclusion
Acknowledgments
References
3 Antimicrobials
3.1 Introduction
3.2 Classification of Antibiotics
3.3 Antibacterial Agents
3.4 Antifungal Agents
3.5 Antiviral agents
3.6 Antiparasitic Agents
3.7 Antimicrobial Resistance
3.8 Conclusion
Acknowledgment
References
4 Trends in Antimicrobial Therapy
4.1 Introduction
4.2 Antibiotics: A Brief History
4.3 AMR: A Global Burden
4.4 Antimicrobial Resistance and Virulence
4.5 Alternatives to Antibiotics
4.6 Antibiotics: Global Action Plan on Antimicrobial Resistance
4.7 Conclusion
Acknowledgment
References
5 Fermentation Strategies in the Food and Beverage Industry
5.1 Introduction
5.2 Current Trends in Food Fermentation
5.3 Future Directions
5.4 Conclusions
5.5 Questions for Thought
References
6 Bioactive Oligosaccharides
6.1 Introduction
6.2 Sources, Types, Structure of Oligosaccharides
6.3 Production Methods of Oligosaccharides
6.4 Extraction, Separation, and Purification of Oligosaccharides
6.5 Characterization of Oligosaccharides
6.6 Functional Properties of Oligosaccharides
6.7 Applications of Oligosaccharides
6.8 Market Potential of Oligosaccharides
6.9 Future Prospects
References
7 Biopolymers
7.1 Introduction
7.2 Natures’ Advanced Materials: A Glance at Its Structure and Properties
7.3 Smart Biopolymers
7.4 Fundamental Applications of Biopolymers in Biomedical Engineering
7.5 Processing Techniques for the Contrivance of Biopolymers
7.6 Conclusion
Acknowledgments
References
8 Metabolic Engineering Strategies to Enhance Microbial Production of Biopolymers
8.1 Introduction
8.2 Microbes as Cell Factories for the Production of Speciality Biochemicals
8.3 Microbial Production Pathways for Various Types of Biopolymers
8.4 Tools and Technologies Available for Metabolic Engineering
8.5 Dynamic Metabolic Flux Analysis and its Role in Metabolic Engineering
8.6 Production of Biopolymers from Metabolically Engineered Microbes
8.7 Recovery and Purification of Biopolymers from Fermentation Broth
8.8 Conclusion and Future Challenges
Acknowledgments
References
Web References
9 Bioplastics Production
9.1 Introduction
9.2 Current Trends
9.3 Different Types of Bioplastics
9.4 Challenges Facing the Bioplastics Industry
9.5 Misconceptions and Negative Impacts
9.6 Take Home Message and Future Directions
9.7 Questions for Thought
Acknowledgments
Conflict of Interest
References
10 Conversion of Lignocellulosic Biomass to Ethanol
10.1 Introduction
10.2 LCB: Structure, Composition, and Recalcitrance
10.3 LCB to Ethanol: Bioprocess Strategies
10.4 Pretreatment of LCB
10.5 Enzymatic Hydrolysis
10.6 High Solids Loading Enzymatic Hydrolysis (HSLEH)
10.7 Fermentation
10.8 Genetic Engineering in LCB Bioconversion
10.9 Conclusions
Acknowledgments
References
11 Advancement in Biogas Technology for Sustainable Energy Production
11.1 Introduction
11.2 Biogas Developments Worldwide
11.3 Biogas Development in India
11.4 Recent Issues in Biogas Production
11.5 Current Trends in Biogas Production
11.6 Advanced Anaerobic Digestion Methodologies
11.7 Role of Biotechnology in Enhancing Biogas Production
11.8 Application of Nanotechnology in Biogas and Methane Production
11.9 Biogas Upgrading Technologies
11.10 Conclusion
References
12 Biofertilizers
12.1 Introduction
12.2 Types of Biofertilizers
12.3 Effect on Bioremediation of Environmental Pollutants
12.4 Bioformulations and Its Types
12.5 Preparation of Biofertilizers
12.6 Various Modes of Biofertilizer Application
12.7 Challenges to Commercialization of Biofertilizers
12.8 Future Perspective
References
13 Biofertilizers from Food and Agricultural By‐Products and Wastes
13.1 Introduction
13.2 Biofertilizer
13.3 Agricultural Waste
13.4 Food Waste
13.5 Biofertilizer Production Using Fermentation Technology
13.6 Biofertilizer for Organic Farming
13.7 Conclusion
Conflict of Interest
References
Index
End User License Agreement
List of Tables
Chapter 1
Table 1.1 Genome wide analysis of terpene synthase genes.
Table 1.2 Photoautotrophic production of farnesene.
Chapter 2
Table 2.1 List of microorganisms capable of producing vanillin from various...
Table 2.2 Amount of ferulic acid in different known natural sources.
Table 2.3 Genetically modified (GM) microorganisms for synthesis of biovani...
Chapter 3
Table 3.1 Some bacteriostatic and bactericidal antibiotics.
Table 3.2 Examples of broad‐ and narrow‐spectrum antibiotics.
Table 3.3 Overview of antibacterial agents.
Table 3.4 Overview of antifungal drugs.
Table 3.5 Antiviral drugs and their respective clinical uses with mechanism...
Table 3.6 Overview of antiparasitic drugs.
Chapter 4
Table 4.1 Mechanism action of antibiotics.
Table 4.2 Prioritized antibiotic resistance bacteria.
Chapter 5
Table 5.1 The various fermented products produced by yeast, bacteria, and m...
Chapter 6
Table 6.1 Extraction methods for oligosaccharides.
Table 6.2 Methods for the purification of oligosaccharides.
Chapter 8
Table 8.1 Computational and experimental tools available for metabolic engi...
Chapter 9
Table 9.1 Countries with enacted plastic bag legislations.
Chapter 10
Table 10.1 Composition of crop residues and energy crops.
Table 10.2 Comparison of pretreatment of LCB by different methods.
Table 10.3 Improvement of saccharification yield using enzyme cocktail mixt...
Table 10.4 Ethanol production from lignocellulosic biomass at HSLEH and fer...
Chapter 11
Table 11.1 Biogas upgrading technologies.
Chapter 13
Table 13.1 Elemental compositions of possible waste biomass fertilizers.
Table 13.2 Agro‐industrial wastes and their compositions.
Table 13.3 Recent studies in the area of solid‐state fermentation using var...
Table 13.4 Enzyme production using food waste via solid‐state fermentation ...
List of Illustrations
Chapter 1
Figure 1.1 Basic steps of gene cloning.
Figure 1.2 Representative chemical structures of biopolymers.
Figure 1.3 Different organic acids with their structures.
Figure 1.4 Biosynthetic pathway for production of organic acids.
Chapter 2
Figure 2.1 World map showing the leading countries in the production of vani...
Figure 2.2 Biotransformation pathways of vanillin in microbes using various ...
Chapter 3
Figure 3.1 (a) Basic structure of penicillin; (b) structures of i. natural p...
Figure 3.2 Chemical structures of antibiotics. (a) Cephalexin; (b) i. Erythr...
Figure 3.3 Mechanism of actions of antibacterial agents.
Figure 3.4 (a) Chemical structure of Amphotericin B; (b) i. fluconazole, ii....
Figure 3.5 Mechanisms of action of antifungal agents.
Figure 3.6 Strategies of antibiotic resistance in bacteria.
Chapter 4
Figure 4.1 Evolution of antibiotics.
Figure 4.2 Schematic diagram of mechanism action of antibiotics.
Figure 4.3 Mechanism action of antibiotic resistance in bacteria.
Figure 4.4 Life cycle of
Bdellovibrio
.
Chapter 5
Figure 5.1 Market share of different countries in the food and beverage indu...
Figure 5.2 Biochemical pathways for transformation of fats (blue arrows), ca...
Chapter 6
Figure 6.1 Sources of bioactive oligosaccharides with examples.
Figure 6.2 (a–i) Structures of different oligosaccharides (a)
Malto‐oligosac
...
Figure 6.3 Application areas of bioactive oligosaccharides.
Chapter 7
Figure 7.1 Polypeptide‐based biopolymers: (a) albumin‐based NP‐Abraxane
TM
(B...
Figure 7.2 Cellulose (Richards et al., 2012).
Figure 7.3 Starch (Othman et al., 2018).
Figure 7.4 Cyclodextrin (Baykal et al., 2018).
Figure 7.5 Hyaluronic acid (Sionkowska et al., 2020).
Figure 7.6 Chitin and chitosan (Ruiz & Corrales, 2017).
Figure 7.7 k‐Carrageenan (Solov’eva et al., 2013).
Figure 7.8 Agarose (Garcia et al., 2000).
Figure 7.9 Alginate (Piras & Smith, 2020).
Figure 7.10 (a) Biodistribution of SMID NPs in vivo: i.v. injection of ICG o...
Figure 7.11 (a) In vitro phantom CT contrast images of PFOB@IR825‐HA‐Cy5.5 N...
Figure 7.12 Wound healing process: (a) images of a representative wound site...
Figure 7.13 (a) NICE bioinks use nanosilicates to reinforce an ionic‐covalen...
Chapter 8
Figure 8.1 Overview of different polysaccharide biosynthetic pathways in bac...
Figure 8.2 Metabolic pathways for synthesis of poly hydroxy alkanoates (PHas...
Figure 8.3 Redirection of metabolic flux to the ε‐PL biosynthesis in
S
.
albu
...
Figure 8.4
Xanthomonas campestris pv. campestris
sugar nucleotide metabolism...
Figure 8.5 Steps involved in recovery and purification of xanthan from ferme...
Figure 8.6 Steps involved in ε‐Poly‐
L
‐lysine separation and recovery from fe...
Chapter 9
Figure 9.1 Polymers in market.PE, polyethylene; PET, poly (ethylene tere...
Figure 9.2 Summary of Bio‐PET business and current trend. MEG, mono‐ethylene...
Figure 9.3 Polylactic acid (PLA) polymer in the market.
Figure 9.4 Polyhydroxyalkanoate (PHA) market applications.
Chapter 10
Figure 10.1 (a) Layers in plant cell wall. L – Lumen, PW – Primary wall, S
1
,...
Figure 10.2 Structure of (a) cellulose, (b) xylan polymer, and (c) lignin mo...
Figure 10.3 Biochemical processing of LCB to produce cellulosic ethanol.
Figure 10.5 Structure of the common cations used for ionic liquids: (i) imid...
Figure 10.4 Effect of different pretreatment types on the degradation of lig...
Figure 10.6 Mode of action of (a) cellulases on cellulose and (b) xylanases ...
Figure 10.7 Approaches for construction of genetically engineered ethanologe...
Chapter 11
Figure 11.1 Number of biogas plants in European countries.
Figure 11.2 Number of biogas plants (total) relative to the population of Eu...
Figure 11.3 Development of the number of biomethane plants in Europe.
Figure 11.4 Biogas production in India.
Chapter 12
Figure 12.1 Types of biofertilizers.
Figure 12.2 Types of bioformulations.
Chapter 13
Figure 13.1 Types of biofertilizers.
Figure 13.2 Types of agro‐industrial wastes.
Guide
Cover Page
Title Page
Copyright Page
Preface
List of Contributors
Table of Contents
Begin Reading
Index
Wiley End User License Agreement
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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© 2022 John Wiley and Sons, Inc.
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The right of R. Navanietha Krishnaraj and Rajesh K. Sani to be identified as the authors of the editorial material in this work has been asserted in accordance with law.
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Library of Congress Cataloging‐in‐Publication Data applied for:
ISBN: 9781119771920
Cover design by WileyCover image: © Vetre/Shutterstock
Preface
Biocommodity Engineering
Microorganisms have been realized as promising sources for production of biocommodities 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, stereospecificity 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 potential 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 systems biology approaches for converting the wastes to value‐added products.
Overall, this would be an ideal textbook for bioprocess, biorefinery, biomolecular, and biocommodity engineering courses for chemical, biochemical, and environmental 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 biochemical 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 biochemical synthesis processes.
The editors would like to thank all the authors for their valuable contribution and the Wiley editorial team for their support.
List of Contributors
R. AanandhalakshmiDepartment of Biotechnology School of Bio and Chemical EngineeringKalasalingam Academy of Research and EducationKrishnankoil, TNIndia
Veknesh ArumugamDepartment of Chemical and Environmental EngineeringFaculty of EngineeringUniversiti Putra MalaysiaSerdang, Malaysia
Balasubramaniem AshokkumarDepartment of Genetic EngineeringSchool of BiotechnologyMadurai Kamaraj UniversityMadurai, TNIndia
Saroj BalaDepartment of MicrobiologyPunjab Agricultural UniversityLudhiana, PBIndia
Rathindra Mohan BanikBioprocess Technology LaboratorySchool of Biochemical EngineeringIndian Institute of Technology (Banaras Hindu University)Varanasi, UPIndia
Ramiya BaskaranDepartment of BiotechnologyIndian Institute of Technology MadrasChennai, TNIndia
Mohit BibraZymergen Inc., Emeryville, CAUSA
Rouf Ahmad DarDepartment of MicrobiologyPunjab Agricultural University Ludhiana, PBIndia
Madhuri DuttaBiochemistry and Cell Biology LaboratorySchool of Basic SciencesIndian Institute of Technology Bhubaneswar, ORIndia
Santhalingam GayathriDepartment of Genetic EngineeringSchool of BiotechnologyMadurai Kamaraj UniversityMadurai, TNIndia
Tanvi GovilDepartment of Chemical and Biological EngineeringSouth Dakota MinesRapid City, SDUSA
Composite and Nanocomposite Advanced Manufacturing – Biomaterials CenterRapid City, SDUSA
Muhammad Heikal IsmailDepartment of Chemical and Environmental EngineeringFaculty of EngineeringUniversiti Putra MalaysiaSerdang, Malaysia
Shereena JoyDepartment of BiotechnologyIndian Institute of Technology MadrasChennai, TNIndia
Sukumaran KarthikaDepartment of Genetic EngineeringSchool of BiotechnologyMadurai Kamaraj UniversityMadurai, TNIndia
Chandraraj KrishnanDepartment of BiotechnologyIndian Institute of Technology Madras Chennai, TNIndia
R. Navanietha KrishnarajSouth Dakota School of Mines and TechnologyDepartment of Chemical and Biological EngineeringRapid City, SDUSA
BuG ReMeDEE Consortium, South Dakota School of Mines and TechnologyRapid City, SDUSA
Composite and Nanocomposite Advanced Manufacturing Centre – Biomaterials (CNAM/Bio)Rapid City, SDUSA
Manoj KumarDepartment of Genetic EngineeringSchool of BiotechnologyMadurai Kamaraj UniversityMadurai, TNIndia
Sanjay KumarSchool of Biochemical EngineeringIIT (BHU) VaranasiVaranasi, UPIndia
Satya Sundar MohantyAssistant ProfessorDepartment of BiotechnologySchool of Agriculture and BiosciencesKarunya Institute of Technology and SciencesCoimbatore, TNIndia
Vignesh NatarajanDepartment of BiotechnologyIndian Institute of Technology MadrasChennai, TNIndia
Sinjini PatraBiochemistry and Cell Biology LaboratorySchool of Basic SciencesIndian Institute of Technology Bhubaneswar, ORIndia
Urmila Gupta PhutelaDepartment of MicrobiologyPunjab Agricultural University Ludhiana, PBIndia
Department of Renewable Energy EngineeringPunjab Agricultural UniversityLudhiana, PBIndia
Akhil RautelaSchool of Biochemical EngineeringIIT (BHU) Varanasi, Varanasi UP, India
Gayathri RavichandranDepartment of Biomedical Engineering Indian Institute of Technology, HyderabadKandi, TelanganaUSA
Aravind Kumar RenganDepartment of Biomedical Engineering Indian Institute of Technology, HyderabadKandi, TelanganaIndia
Winny RoutrayDepartment of Food Process EngineeringNational Institute of Technology Rourkela, ORIndia
Anasuya RoychowdhurySchool of Basic SciencesBiochemistry and Cell Biology LaboratoryIndian Institute of Technology Bhubaneswar, ORIndia
David R. SalemDepartment of Chemical and Biological EngineeringSouth Dakota MinesRapid City, SDUSA
Composite and Nanocomposite Advanced Manufacturing – Biomaterials CenterRapid City, SDUSA
Department of Materials and Metallurgical EngineeringSouth Dakota Mines Rapid City, SDUSA
Rajesh K. SaniSouth Dakota School of Mines and TechnologyDepartment of Chemical and Biological EngineeringRapid City, SDUSA
South Dakota School of Mines and TechnologyBuG ReMeDEE ConsortiumRapid City, SDUSA
Composite and Nanocomposite Advanced Manufacturing Centre – Biomaterials (CNAM/Bio)Rapid City, SDUSA
South Dakota School of Mines and TechnologyDepartment of Chemistry and Applied Biological SciencesRapid City, SDUSA
Shivam SaxenaBiochemistry and Cell Biology LaboratorySchool of Basic SciencesIndian Institute of Technology Bhubaneswar, ORIndia
Shailendra Singh SheraDepartment of BiotechnologyFaculty of Engineering & TechnologyRama UniversityKanpur, UPIndia
K. SundarDepartment of BiotechnologySchool of Bio and Chemical EngineeringKalasalingam Academy of Research and EducationKrishnankoil, TNIndia
B. VanavilDepartment of BiotechnologySchool of Bio and Chemical EngineeringKalasalingam Academy of Research and EducationKrishnankoil, TNIndia
Perumal VaralakshmiDepartment of Molecular MicrobiologySchool of BiotechnologyMadurai Kamaraj UniversityMadurai, TNIndia
Mohan Kumar VermaSchool of BiotechnologyDepartment of Molecular MicrobiologyMadurai Kamaraj UniversityMadurai, TNIndia
1Engineered 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 biocommodities 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 biocommodity engineering can be combined to meet these needs. The foundation of biocommodity 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 consumed 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 conventional methods and development of mutant strains for the synthesis of these biocommodities. The last section of the chapter gives an insight about the biofuel production from photoautotrophic organisms such as cyanobacteria and microalgae, which utilizes 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 conditions takes place. Finally, after numerous generations, the organism growing in the stress conditions is said to be recombinant or genetically modified. Genetic engineering 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 biomolecules 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 synthetic 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 biocommodities. 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 molecular biology tools and biocatalysts. The available purified enzymes that can 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.
Figure 1.1 Basic steps of gene cloning.
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 studied 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′ exonuclease activity that helps in proofreading, and a 5′ → 3′ exonuclease activity mediating 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 minutes 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 mononucleotides and very short oligonucleotides mixture. Some of the examples of 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 endonuclease 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 espejiana) – 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 phosphodiester 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 magnesium 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 molecule. 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 segments. 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 fragments 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:
It should have origin of replication, able to replicate autonomously.
It should be easily isolated and purified.
The vector should have suitable selection marker genes that will allow easy selection of the transformed cells from nontransformed cells.
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.
The cells transformed with the vector containing the DNA insert should be easily identifiable and selectable from those transformed cells having unaltered vector.
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 transcription 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 chromosomes (YACs). All the vectors mentioned have different incorporation capacity for foreign DNA. Plasmids are extra chromosomal circular double‐stranded DNA replicating 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 incorporate 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 recombinant 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 transformed 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 initial “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 interest in them inactivates the gene already present in the vector. This leads to insertional 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 religated 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 factors, 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 principles of genetic engineering explained in the earlier section are used to engineer microorganisms to become microbial cell factories to produce essential biocommodities 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 factories 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 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 mesenteriodes synthesize dextran. 40 years after, and PHA reserves were found in B. megaterium, 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 commercially 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 tendon 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 properties 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 cellulose 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, pectin, hemicellulose, and other constituents of lignocellulosic materials (Rahman and Netravali, 2016). There are several bacterial strains that synthesize BC specially the acetic acid bacteria group as they are generally recognized as safe (GRAS) such as Komagataeibacter xylinus, Komagataeibacter hansenii, Komagataeibacter medellinensis, Komagataeibacter nataicola, Komagataeibacter oboediens (Škraban et al., 2018; Castro et al., 2012,2013). Bacterial cellulose synthase (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.
Figure 1.2 Representative chemical structures of biopolymers.
Gluconacetobacter xylinus six genes cmc–ccp–cesAB–cesC–cesD–bgl were overexpressed in Synechococcus sp. PCC 7002 and resulted in very high‐yield production 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 oxygen 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, substrate utilization, BC production, etc. were evaluated. This gives future opportunity 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 combination 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 synthesized 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 aminotransferase 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. licheniformis and B. amyloliquefaciens, capB, C, A, and E, in B. anthraci, while in B. subtilis 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).
