Plants as Bioreactors for Industrial Molecules E-Book
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PLANTS AS BIOREACTORS FOR INDUSTRIAL MOLECULES An incisive and practical discussion of how to use plants as bioreactors In Plants as Bioreactors for Industrial Molecules, a team of distinguished researchers delivers an insightful and global perspective on the use of plants as bioreactors. In the book, you'll find coverage of the basic, applied, biosynthetic, and translational approaches to the exploitation of plant technology in the production of high-value biomolecules. The authors focus on the yield and quality of amino acids, vitamins, and carbohydrates. The authors explain how high-value biomolecules enable developers to create cost-effective biological systems for the production of biomolecules useful in a variety of sectors. They provide a holistic approach to plant-based biological devices to produce natural molecules of relevance to the health and agriculture industries. Readers will also find: * A thorough overview of plants as bioreactors and discussions of molecular farming for the production of pharmaceutical proteins in plants * Comprehensive explorations of plants as edible vaccines and plant cell culture for biopharmaceuticals * Practical discussions of the production of attenuated viral particles as vaccines in plants and insecticidal protein production in transgenic plants * Extensive treatment of the regulatory challenges involved in using plants as bioreactors Perfect for academics, scientists, and researchers in industrial microbiology and biotechnology, Plants as Bioreactors for Industrial Molecules will also earn a place in the libraries of biotechnology company professionals in applied product development.
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Table of Contents
Cover
Title Page
Copyright Page
About the Editors
List of Contributors
Preface
Acknowledgments
1 Plants as Bioreactors
1.1 Introduction
1.2 Factors Controlling the Production of Recombinant Protein
1.3 Recombinant Proteins in Plants
1.4 Conclusions
References
2 Molecular Farming for the Production of Pharmaceutical Proteins in Plants
2.1 Introduction
2.2 Plant as an Expression Platform
2.3 Plant‐Derived Recombinant Proteins
2.4 Engineering Strategies Utilized for Recombinant Pharmaceutical Protein Production in Plants
2.5 Pharmaceutical Protein Developed Using Plant Expression Platform
2.6 Perspectives
2.7 Conclusion
References
3 Plants as Edible Vaccine
3.1 Introduction
3.2 Mechanism of Action
3.3 Edible Plant Vaccines
3.4 Production of Edible Vaccine (Plant Transformation)
3.5 Plant Species Used as Vaccine Models
3.6 Challenges
3.7 Conclusion
References
4 Plant Cell Culture for Biopharmaceuticals
4.1 Introduction
4.2 Plant Cultures
4.3 Conditions for Plant Cell, Tissue, and Organ Culture
4.4 Types of Plant Cell, Tissue, and Organ Culture
4.5 The Techniques Used in Plant Culture
4.6 Applications of Plant Cultures
4.7 Biopharmaceuticals
4.8 Conclusion
References
5 Microalgal Bioreactors for Pharmaceuticals Production
5.1 Introduction
5.2 Microalgae Strains Selection
5.3 Microalgae Cultivation
5.4 Acquiring Biopharmaceuticals from Microalgae’s
5.5 Microalgal Compounds and their Pharmaceutical Applications
5.6 Conclusions
References
6 Micropropagation for the Improved Production of Secondary Metabolites
6.1 Introduction
6.2 Micropropagation for Production of Secondary Metabolites
6.3 Strategies to Improve Secondary Metabolite Production
6.4 Conclusions
References
7 Metabolic Engineering for Carotenoids Enrichment of Plants
7.1 Background
7.2 Classification of Carotenoid Pigments
7.3 Aspects of the Mechanism of Carotenoid Biosynthesis
7.4 Concluding Remarks and Future Perspectives
References
8 Plant Genome Engineering for Improved Flavonoids Production
8.1 Background
8.2 Structure, Diversity, and Subgroups
8.3 Flavonoid Biosynthesis
8.4 The Mechanism of Action of Flavonoids
8.5 The Role of Flavonoids in Food and Medicine
8.6 Concluding Remarks and Future Perspectives
References
9 Antibody Production in Plants
9.1 Introduction
9.2 How Are Antigens Expressed in Plants?
9.3 Plant‐Derived Antibodies: Are There any Alternative Approaches?
9.4 Antibody Production in Plants: Advantages and Concerns
9.5 Conclusion and Prospects
References
10 Metabolic Engineering of Essential Micronutrients in Plants to Ensure Food Security
10.1 Introduction
10.2 Metabolic Engineering of Crops for Increased Nutritional Value
10.3 Conclusion and Future Perspectives
Acknowledgments
References
11 Plant Hairy Roots as Biofactory for the Production of Industrial Metabolites
11.1 Introduction
11.2 Types of Metabolites and Industrial Metabolites
11.3 Secondary Metabolites
11.4 Importance of Secondary Metabolites
11.5 Enhancement of Secondary Metabolites
11.6 Hairy Roots
11.7 Initiation of Hairy Root Cultures
11.8 Large‐Scale Production of Secondary Metabolites
11.9 Strategies Used
In vitro
11.10 Plants as Bioreactors
11.11 A Case Study
11.12 Conclusion
References
12 Microalgae as Cell Factories for Biofuel and Bioenergetic Precursor Molecules
12.1 Introduction
12.2 Microalgae that Produce Bioenergy and Biofuel Molecules
12.3 Biosynthesis of Molecules for Bioenergy and Biofuels in Microalgae
12.4 Biohydrogen Production
12.5 Starch Biosynthesis
12.6 Lipid Biosynthesis
12.7 Biochemical Regulation of BBPM Associated with Nutritional Conditions
12.8 Physical and Chemical Factors Promote the Accumulation of Molecules for Bioenergy and Biofuels
12.9 Light Intensity
12.10 Salts
12.11 Use of Organic and Inorganic Carbon Sources
12.12 Agitation
12.13 Photobioreactors to Produce Bioenergy and Biofuels
12.14 Open Pond Cultivation Systems
12.15 Closed Systems
12.16 Hybrid Systems
12.17 Conclusions
References
13 Metabolic Engineering for Value Addition in Plant‐Based Lipids/Fatty Acids
13.1 Introduction
13.2 Plant Lipids
13.3 TAG Synthesis in Plants
13.4 Regulatory Factors Involved in TAG Synthesis
13.5 Metabolic Engineering for Lipid/Fatty Acid Synthesis
13.6 Conclusions
References
14 Plants as Bioreactors for the Production of Biopesticides
14.1 Introduction
14.2 Plant Metabolic Engineering for the Production of EOs and their Pure Compounds
14.3 Bioactivity of EOs
14.4
In vitro
Synthesis vs Extraction from Natural Sources: How to Obtain Secondary Metabolites
14.5 Conclusion
References
15 Nutraceuticals Productions from Plants
15.1 Plant‐Derived Nutraceuticals
15.2 Phytochemicals and their Impacts on Human Health
15.3 Engineering Nutraceutical‐Enriched Plants
15.4 Potential Side Effects of Nutraceuticals on Human Health
15.5 Final Considerations
References
16 Green Synthesis of Nanoparticles Using Various Plant Parts and Their Antifungal Activity
16.1 Introduction
16.2 Gold Nanoparticle Synthesis Using Plant Source
16.3 Silver Nanoparticles Synthesis Using Plants Source
16.4 Zinc Oxide Nanoparticles Synthesis Using Plants
16.5 Other Nanoparticles Synthesis Using Plant Source
16.6 Conclusion and Future Perspective
Acknowledgement
Conflicts of Interest
Author Contribution
References
17 Plant‐Based/Herbal Nanobiocatalysts and Their Applications
17.1 Introduction of Nanobiocatalyst
17.2 Nanobiocatalysts from Herbal Alkaloid Plants Are Used in Nanotechnology and Bioengineering
17.3 Why Use Nanobiocatalysts?
17.4 Immobilization of Biocatalyst (Enzymes) and Nanoparticles or Nanomatrix
17.5 Application of the Nanobiocatalyst
17.6 Conclusion
References
18 Potential Plant Bioreactors
18.1 Introduction
18.2 Whole Plants: Stable and Transient Expression Systems
18.3 Unique Features of Using Plant‐based Production Over Microbial and Mammalian Systems
18.4 Strategies to Enhance the Potential of Plant‐based Production Systems
18.5 Concluding Remarks and Future Perspectives
Conflict of Interest
References
19 Production of Nutraceuticals Using Plant Cell and Tissue Culture
19.1 Introduction
19.2 Production of Secondary Metabolites as Nutraceuticals in
In vitro
Cultures
19.3 Conclusions
References
20 Algal Bioreactors for Polysaccharides Production
20.1 Introduction
20.2 Algae
20.3 Biological Activity of Algal Polysaccharides
20.4 Parameters that Iinfluence the Polysaccharides Production by Microalgae
20.5 Algal Bioreactors
20.6 Conclusions and Future Perspectives
Acknowledgments
References
Index
End User License Agreement
List of Tables
Chapter 1
Table 1.1 List of recombinant proteins produced in various plant species.
Chapter 2
Table 2.1 List of various pharmaceutical proteins produced in plants.
Table 2.2 List of various nonpharmaceutical proteins produced in plants....
Chapter 3
Table 3.1 Some popular plant candidates and their desirable traits.
Table 3.2 Plant species used as vaccine models.
Chapter 4
Table 4.1 Composition of synthetic culture media frequently used.
Table 4.2 Some important plant‐derived secondary metabolites of pharmaceuti...
Table 4.3 Some recombinant vaccines expressed in plants.
Table 4.4 Antigens expressed in plants.
Table 4.5 Human pathogen antigens expressed in transgenic plants.
Table 4.6 Antigens of animal pathogens expressed in transgenic plants.
Table 4.7 Plantibodies for therapeutic and diagnostic use.
Table 4.8 Biopharmaceutical proteins produced in transgenic plants.
Chapter 5
Table 5.1 A selected list of cultures collections available worldwide.
Table 5.2 The cultivation methods and their differences.
Table 5.3 Overview of some characteristic differences between open and clos...
Table 5.4 Examples of some high‐value carotenoids and their biological prop...
Chapter 6
Table 6.1 Some examples of secondary metabolites obtained from plants.
Table 6.2 Few examples of elicitors used for secondary metabolite productio...
Table 6.3 Immobilized cell cultures used for secondary metabolite productio...
Table 6.4 Production of secondary metabolites in plant cell cultures using ...
Table 6.5 Use of metabolic engineering in plant secondary metabolite produc...
Chapter 8
Table 8.1 Examples of flavonoids.
Chapter 9
Table 9.1 List of antigenic epitope(s) along with the plant expression syst...
Chapter 10
Table 10.1 Transgenic plants biofortified with different key micronutrients...
Chapter 11
Table 11.1 Classes of secondary metabolites produced by plant tissue cultur...
Table 11.2 Enhancement of secondary metabolites production by using elicito...
Table 11.3 Examples of plant hairy root cultures and secondary metabolites ...
Chapter 12
Table 12.1 Summary of the main microalgae strains producing bioenergy molec...
Chapter 13
Table 13.1 List of transgenic plants with the modified fatty acid or lipid....
Chapter 14
Table 14.1 Antifungal effect of EO against different fungi.
Chapter 15
Table 15.1 Summary of the polyphenols on human health.
Table 15.2 Summary of the terpenoids on human health.
Table 15.3 Summary of the alkaloids on human health.
Table 15.4 Summary of the fatty acids on human health.
Table 15.5 Summary of the dietary fiber on human health.
Chapter 16
Table 16.1 Various plants and their parts used in the synthesis of AuNPs, A...
Table 16.2 Other nanoparticles synthesis using plant source.
Chapter 17
Table 17.1 Immobilization methods with its advantages and disadvantages....
Table 17.2 Plant extracts biocatalyst applied to make nanoparticles and its...
Chapter 18
Table 18.1 Comparison of different plafftorms and representative proteins p...
Table 18.2 Advantages of using plant molecular pharming over mammalian and ...
Chapter 19
Table 19.1
In vitro
anthocyanin production by plant tissue cultures.
Table 19.2 Examples of
in vitro
propagation of bioactive compounds in
Vacci
...
Chapter 20
Table 20.1 Polysaccharide content for different strains of microalgae, cult...
List of Illustrations
Chapter 2
Figure 2.1 An overview of production of pharmaceutical/non‐pharmaceutical pr...
Figure 2.2 Overview of plant‐based expression systems. (a) Advantages and ch...
Figure 2.3 Overview of plant expression platform for pharmaceuticals protein...
Figure 2.4 An overview of plant transformation strategies used to make recom...
Figure 2.5 Overview of production system of plant‐based biopharmaceuticals a...
Chapter 4
Figure 4.1 Flow chart summarizing the tissue culture experiments.
Figure 4.2 Schematic representation of elicitors in plant cells.
Figure 4.3 Overview of plant culture for biopharmaceuticals.
Figure 4.4 Schematic representation of hairy root induction from different e...
Chapter 5
Figure 5.1 Successful selection of microalgae strains for biopharmaceuticals...
Figure 5.2 Some high‐value compounds produced by microalgae.
Chapter 6
Figure 6.1 Plant secondary metabolites classification.
Figure 6.2 Stages of micropropagation.
Figure 6.3 Strategies to enhance the production of secondary metabolites.
Figure 6.4 Classification of plant cell bioreactors.
Chapter 7
Figure 7.1 Schematic illustration of carotenoids classifications.
Figure 7.2 Schematic illustration of carotenoid grouping and metabolic pathw...
Chapter 8
Figure 8.1 Schematic of the structure of the flavone backbone (2‐phenyl‐1,4‐...
Figure 8.2 Schematic illustration of classes of flavonoid compounds.
Chapter 10
Figure 10.1 Approaches to promote iron biofortification in plants.
Figure 10.2 Flow chart depicting the carotenoid biosynthetic pathway in plan...
Chapter 11
Figure 11.1 Steps involved in the production of hairy root culture.
Figure 11.2 Induction of hairy roots and its applications in Biotechnology....
Figure 11.3 Interaction of
Agrobacterium rhizogenes
with plant roots.
Figure 11.4 Complete protocol of hairy root induction and its biotechnologic...
Figure 11.5 Hairy root culture of Artemisia vulgaris. (a) Hairy root initiat...
Chapter 12
Figure 12.1 Representative scheme of the main molecules produced by microalg...
Figure 12.2 Simplified scheme of the metabolic pathways of starch and lipid ...
Figure 12.3 Strategies and factors involved in the design and optimization o...
Chapter 13
Figure 13.1 Overview of Triacylglycerol synthesis pathway. ACCase, acetyl‐Co...
Chapter 14
Figure 14.1 (a) Genera of the Lamiaceae family most commonly used against in...
Figure 14.2 MIC mean values (< 255 μg/ml) of EOs against Gram‐positive and G...
Figure 14.3 MIC mean values (255–4520 μg/ml) of EOs against Gram‐positive an...
Figure 14.4 MIC mean values (> 1790 μg/ml) of EOs against Gram‐positive and ...
Chapter 15
Figure 15.1 Chromones structure.
Figure 15.2 Coumarins structure.
Figure 15.3 Anthocyanidins structure.
Figure 15.4 Flavanols structure.
Figure 15.5 Flavanones structure.
Figure 15.6 Flavones structure.
Figure 15.7 Flavonols structure.
Figure 15.8 Isoflavones structure.
Figure 15.9 Proanthocyanidins structure.
Figure 15.10 Curcumin structure.
Figure 15.11 Resveratrol structure.
Figure 15.12 Xanthones structure.
Figure 15.13 Ginkgolide structure.
Figure 15.14 General phytosterols structure.
Figure 15.15 Structure of Sitosterol, a type of phytosterol.
Figure 15.16 Caffeine structure.
Figure 15.17 Omega‐3 (up) and omega 6 (bottom) structures.
Figure 15.18 Unit of the cellulose, a water‐insoluble polysaccharide formed ...
Chapter 16
Figure 16.1 Conventional methods used for nanoparticle synthesis.
Chapter 17
Figure 17.1 Alkaloid biosynthesis pathway.
Figure 17.2 Graphene‐based biosensor.
Figure 17.3 Diagramatic representation of the hybrid nanoflower.
Chapter 18
Figure 18.1 Overview of production system of plant‐based recombinant protein...
Figure 18.2 Strategies to enhance the potential of plant‐based production sy...
Figure 18.3 Potential of side stream products in plant molecular farming. Ge...
Figure 18.4 Comparative overview of waste/side streams produced during mamma...
Chapter 20
Figure 20.1 Types of bioreactors for microalgae cultivation: (a) vertical tu...
Guide
Cover Page
Title Page
Copyright Page
About the Editors
List of Contributors
Preface
Acknowledgments
Table of Contents
Begin Reading
Index
WILEY END USER LICENSE AGREEMENT
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Plants as Bioreactors for Industrial Molecules
Edited by
Santosh Kumar Upadhyay
Department of Botany, Panjab University
Chandigarh, India
Sudhir P. Singh
Center of Innovative and Applied Bioprocessing (CIAB)
Mohali, Punjab, India
This edition first published 2023© 2023 John Wiley & Sons Ltd
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The right of Santosh Kumar Upadhyay and Sudhir P. Singh 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 DataNames: Upadhyay, Santosh Kumar, editor. | Singh, Sudhir P., editor.Title: Plants as bioreactors for industrial molecules / edited by Santosh Kumar Upadhyay, Punjab University, Chandigarh India, Sudhir P. Singh, Center of Innovative and Applied Bioprocessing, Sector 81, Sahibzada Ajit Singh Nagar, Punjab, India.Description: First edition. | Hoboken, NJ, USA : John Wiley & Sons Inc., 2023. | Includes bibliographical references and index.Identifiers: LCCN 2022041348 (print) | LCCN 2022041349 (ebook) | ISBN 9781119875086 (Hardback) | ISBN 9781119875093 (Adobe PDF) | ISBN 9781119875109 (ePub)Subjects: LCSH: Plant biotechnology. | Bioreactors. | Botanical chemistry.Classification: LCC TP248.27.P55 P584 2023 (print) | LCC TP248.27.P55 (ebook) | DDC 630–dc23/eng/20221006LC record available at https://lccn.loc.gov/2022041348LC ebook record available at https://lccn.loc.gov/20220413499781119875086 (Hardback): 9781119875093 (Adobe PDF): 9781119875109 (ePub)
Cover Design: WileyCover Image: © architect9/Shutterstock
About the Editors
Dr. Santosh Kumar Upadhyay is currently working as an Assistant Professor at the Department of Botany, Panjab University, Chandigarh, India. Prior to this, Dr. Upadhyay was DST‐INSPIRE faculty at the National Agri‐Food Biotechnology Institute, Mohali, Punjab, India. He did his doctoral work at the CSIR‐National Botanical Research Institute, Lucknow and received his Ph.D. in Biotechnology from UP Technical University, Lucknow, India. He has been working in the field of Plant Biotechnology for more than 14 years. His present research focuses in the area of functional genomics. He is involved in the bioprospecting and characterization of various insect toxic proteins from plant biodiversity and defence and stress signaling genes in bread wheat. His research group at PU has characterized numerous important gene families and long noncoding RNAs related to the abiotic and biotic stress tolerance and signaling in bread wheat. He has also established the method for genome editing in bread wheat using CRISPR‐Cas system and developed a tool SSinder for CRISPR target site prediction. His research contribution led to the publication of more than 70 research papers in leading journals of international repute. Further, there are more than five national and international patents, 40 book chapters, and 11 books in his credit. In recognition of his substantial research record, he has been awarded NAAS Young scientist award (2017–2018) and NAAS‐Associate (2018) from the National Academy of Agricultural Sciences, India, INSA Medal for Young Scientist (2013) from the Indian National Science Academy, India, NASI‐Young Scientist Platinum Jubilee Award (2012) from the National Academy of Sciences, India, and Altech Young Scientist Award (2011). He has also been the recipient of the prestigious DST‐INSPIRE Faculty Fellowship (2012), and SERB‐Early Career Research Award (2016) from the Ministry of Science and Technology, Government of India. Dr. Upadhyay also serves as a member of the editorial board and reviewer of several peer‐reviewed international journals.
Dr. Sudhir P. Singh is currently Scientist‐D at the Center of Innovative and Applied Bioprocessing (CIAB), Mohali, India. He has been working in the area of molecular biology and biotechnology for more than a decade. Currently, his primary focus of research is gene mining, biocatalyst engineering, and characterization of novel enzyme variants for the production of high‐value functional biomolecules from low‐cost feedstock. He has generated metagenomic data resources from diverse ecological niches and ethnic food samples and has characterized novel genes encoding enzyme variants with desirable catalytic properties for the biosynthesis of functional sugar molecules such as D‐allulose, turanose, trehalose, and trehalulose. Further, he has achieved enzymatic production of prebiotic molecules, such as fructooligosaccharides, glucooligosaccharides, 4‐galactosyl‐Kojibiose, xylooligosaccharides, levan, dextran, and type III resistant starch. Dr. Singh has published 70 research articles, 07 review articles, and 07 books (edited). Further, he has 08 patents (granted) to his credit as an inventor. He has been conferred the Young Scientist Award (IBA), SBS‐MKU Genomics Award (BRSI), and Professor Hira Lal Chakravarty Award (ISCA, DST). His team was awarded Gandhian Young Technological Innovation Award (SRISTI) in 2019. He is a life member of the National Academy of Sciences, India, and a fellow of the National Academy of Agricultural Sciences (NAAS), India.
List of Contributors
Fernanda AchimónÁrea de Bioplaguicidas, Instituto Multidisciplinario de Biología Vegetal (IMBIV‐CONICET), Córdoba, Argentina; Área Aromas y Pigmentos, Instituto de Ciencia y Tecnología de los Alimentos (ICTA), Universidad Nacional de Córdoba (UNC), Córdoba, Argentina
Deepika AntilDepartment of Botany, Panjab University, Chandigarh, Punjab, India
Vanessa A. ArecoInstituto Multidisciplinario de Investigación y Transferencia Agroalimentaria y Biotecnológica (IMITAB‐CONICET), Universidad Nacional de Villa María (UNVM), Córdoba, Argentina
Gaurav AugustineDepartment of Agricultural Biotechnology, National Agri‐food Biotechnology Institute (An Autonomous Institute of the Department of Biotechnology), Mohali, Punjab, India
J.R. Benavente‐ValdésDepartment of Chemical Engineering, Autonomous University of Coahuila, Saltillo, México
Priscilla Quenia Muniz BezerraLaboratory of Biochemical Engineering, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, Rio Grande do Sul, Brazil
Vanessa D. BritoÁrea de Bioplaguicidas, Instituto Multidisciplinario de Biología Vegetal (IMBIV‐CONICET), Córdoba, Argentina; Área Aromas y Pigmentos, Instituto de Ciencia y Tecnología de los Alimentos (ICTA), Universidad Nacional de Córdoba (UNC), Córdoba, Argentina
Monica ButnariuChemistry & Biochemistry Discipline, University of Life Sciences “King Mihai I” from Timisoara, Timisoara, Timis, Romania
Swarnavo ChakrabortyPost Graduate Department of Biotechnology, St. Xavier’s College (Autonomous), Kolkata, West Bengal, India
Jorge Alberto Vieira CostaLaboratory of Biochemical Engineering, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, Rio Grande do Sul, Brazil
Kricelle Mosquera DeamiciLaboratory of Biochemical Engineering, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, Rio Grande do Sul, Brazil
Zeuko’o Menkem ElisabethDepartment of Biomedical Sciences, University of Buea, Buea, Cameroon; Antimicrobial and Biocontrol Agents Unit, Laboratory for Phytobiochemistry and Medicinal Plant Studies, Department of Biochemistry, University of Yaoundé I, Yaoundé, Cameroon
Dhara GandhiDepartment of Botany, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India
Rajeswaree GohelDepartment of Microbiology, Marwadi University, Rajkot, Gujarat, India
Juliane Karine IshidaBotany Department, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil
Kirthikah KadiresenDivision of Applied Biomedical Sciences and Biotechnology, School of Health Sciences, International Medical University, Kuala Lumpur, Malaysia
Madhu KamleApplied Microbiology Laboratory, Department of Forestry, North Eastern Regional Institute of Science and Technology, Nirjuli,Arunachal Pradesh, India
Elif KarlikDepartment of Molecular Biology and Genetics, University of Istinye, Istanbul, Turkey
Amandeep KaurDepartment of Botany, Panjab University, Chandigarh, Punjab, India
Simardeeep KaurDivision of Biochemistry, ICAR‐Indian Agricultural Research Institute, New Delhi, India
Pradeep KumarApplied Microbiology Laboratory, Department of Forestry, North Eastern Regional Institute of Science and Technology, Nirjuli, Arunachal Pradesh, India; Department of Botany, University of Lucknow, Lucknow, Uttar Pradesh, India
Suelen Goettems KuntzlerLaboratory of Microbiology and Biochemistry, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, Rio Grande do Sul, Brazil
Céline LarocheInstitut Pascal, Université Clermont Auvergne, Clermont‐Ferrand, France
Jia ChooDivision of Applied Biomedical Sciences and Biotechnology, School of Health Sciences, International Medical University, Kuala Lumpur, Malaysia
Anna Pick Kiong LingDivision of Applied Biomedical Sciences and Biotechnology, School of Health Sciences, International Medical University, Kuala Lumpur, Malaysia
MadhuDepartment of Botany, Panjab University, Chandigarh, Punjab, India
A. Méndez‐ZavalaDepartment of Chemical Engineering, Autonomous University of Coahuila, Saltillo, México
Carolina MerloÁrea de Bioplaguicidas, Instituto Multidisciplinario de Biología Vegetal (IMBIV‐CONICET), Córdoba, Argentina; Área Aromas y Pigmentos, Instituto de Ciencia y Tecnología de los Alimentos (ICTA), Universidad Nacional de Córdoba (UNC), Córdoba, Argentina; Departamento de Recursos Naturales, Cátedra de Microbiología Agrícola, Universidad Nacional de Córdoba, Córdoba, Argentina
Amit Kumar MishraDepartment of Botany, School of Life Sciences, Mizoram University, Aizawl, Mizoram, India
Pragati MisraCentre for Tissue Culture Technology, Jacob Institute of Biotechnology and Bioengineering, Sam Higginbottom University of Agriculture Technology and Sciences, Prayagraj, Uttar Pradesh, India
J.C. MontañezDepartment of Chemical Engineering, Autonomous University of Coahuila, Saltillo, México
Michele Greque de MoraisLaboratory of Microbiology and Biochemistry, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, Rio Grande do Sul, Brazil
L. Morales‐OyervidesDepartment of Chemical Engineering, Autonomous University of Coahuila, Saltillo, México
Juliana Botelho MoreiraLaboratory of Microbiology and Biochemistry, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, Rio Grande do Sul, Brazil
Rupasree MukhopadhyayDepartment of Genetics and Biotechnology, University College for Women, Hyderabad, Telangana, India
Alejandra B. OmariniLaboratorio de Biotecnología Fúngica y de los Alimentos, Asociación para el Desarrollo de Villa Elisa y Zona (ADVEZ), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Entre Ríos, Argentina
Elif Aylin OzudogruDepartment of Molecular Biology and Genetics, University of Istinye, Istanbul, Turkey
Ghanshyam PandeyDepartment of Plant Protection and Plant Pathology, Naini Agricultural Institute, Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj, Uttar Pradesh, India
María L. PeschiuttaÁrea de Bioplaguicidas, Instituto Multidisciplinario de Biología Vegetal (IMBIV‐CONICET), Córdoba, Argentina; Área Aromas y Pigmentos, Instituto de Ciencia y Tecnología de los Alimentos (ICTA), Universidad Nacional de Córdoba (UNC), Córdoba, Argentina
Laura Oliveira PiresBotany Department, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil
Romina P. PizzolittoÁrea de Bioplaguicidas, Instituto Multidisciplinario de Biología Vegetal (IMBIV‐CONICET), Córdoba, Argentina; Área Aromas y Pigmentos, Instituto de Ciencia y Tecnología de los Alimentos (ICTA), Universidad Nacional de Córdoba (UNC), Córdoba, Argentina; Departamento de Recursos Naturales, Cátedra de Microbiología Agrícola, Universidad Nacional de Córdoba, Córdoba, Argentina
D. Rodríguez‐ZuñigaDepartment of Chemical Engineering, Autonomous University of Coahuila, Saltillo, México
Isabela Sandy RosaBotany Department, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil
Aryadeep RoychoudhuryPost Graduate Department of Biotechnology, St. Xavier’s College (Autonomous), Kolkata, West Bengal, India
Gaurav SanghviDepartment of Microbiology, Marwadi University, Rajkot, Gujarat, India
Karishma SeemDivision of Biochemistry, ICAR‐Indian Agricultural Research Institute, New Delhi, India
Alok SharmaDepartment of Botany, Panjab University, Chandigarh, Punjab, India
Chikanshi SharmaApplied Microbiology Laboratory, Department of Forestry, North Eastern Regional Institute of Science and Technology, Nirjuli,Arunachal Pradesh, India
Archana ShuklaDepartment of Biological Sciences, Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj,Uttar Pradesh, India
Pradeep Kumar ShuklaDepartment of Biological Sciences, Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj,Uttar Pradesh, India
Prashant Kumar SinghDepartment of Biotechnology, Pachhunga University College Campus, Mizoram University, Aizawl, Mizoram, India
Sudhir P. SinghCenter of Innovative and Applied Bioprocessing (CIAB), Mohali, Punjab, India
Vipin Kumar SinghDepartment of Botany, K.S. Saket P.G. College, Ayodhya, Uttar Pradesh, India
O. Solís‐QuirozDepartment of Chemical Engineering, Autonomous University of Coahuila, Saltillo, México
Nidhi SonkarDepartment of Molecular and Cellular Engineering, Jacob Institute of Biotechnology and Bioengineering, Sam Higginbottom University of Agriculture Technology and Sciences, Prayagraj, Uttar Pradesh, India
Himani ThakkarDepartment of Biochemistry, All India Institute of Medical Sciences, New Delhi, India
Rufin Marie Kouipou ToghueoAntimicrobial & Biocontrol Agents Unit (AmBcAU), Laboratory for Phytobiochemistry and Medicinal Plants Studies, Department of Biochemistry, University of Yaoundé I, Yaoundé, Cameroon
Santosh Kumar UpadhyayDepartment of Botany, Panjab University, Chandigarh, Punjab, India
Vinnyfred VincentDepartment of Biochemistry, All India Institute of Medical Sciences, New Delhi, India
Ying Pei WongDivision of Applied Biomedical Sciences and Biotechnology, School of Health Sciences, International Medical University, Kuala Lumpur, Malaysia
Megan Min Tse YewDivision of Applied Biomedical Sciences and Biotechnology, School of Health Sciences, International Medical University, Kuala Lumpur, Malaysia
Jia Qi YipDivision of Applied Biomedical Sciences and Biotechnology, School of Health Sciences, International Medical University, Kuala Lumpur, Malaysia
María P. ZuninoÁrea de Bioplaguicidas, Instituto Multidisciplinario de Biología Vegetal (IMBIV‐CONICET), Córdoba, Argentina; Área Aromas y Pigmentos, Instituto de Ciencia y Tecnología de los Alimentos (ICTA), Universidad Nacional de Córdoba (UNC), Córdoba, Argentina; Departamento de Química, Cátedra de Química Orgánica, Universidad Nacional de Córdoba, Córdoba, Argentina
Julio A. ZygadloÁrea de Bioplaguicidas, Instituto Multidisciplinario de Biología Vegetal (IMBIV‐CONICET), Córdoba, Argentina; Área Aromas y Pigmentos, Instituto de Ciencia y Tecnología de los Alimentos (ICTA), Universidad Nacional de Córdoba (UNC), Córdoba, Argentina; Departamento de Química, Cátedra de Química Orgánica, Universidad Nacional de Córdoba, Córdoba, Argentina
Preface
In the past two decades, the application of plants as bioreactors for the production of various industrial molecules has evolved into an important research area with numerous new opportunities. Low production cost, easy to scale up, good‐quality produce, and easy downstream processing have attracted the rapid growth of plants as bioreactors in recent years. Genetic and gene engineering methods are helpful in further improving the yield and quality of the product in various plants. Several industrial products have been produced in plant bioreactors, such as pharmaceutical proteins, vaccines, medical diagnostics proteins, industrial proteins, antibodies, attenuated viral particles, and nutritional supplements, including carbohydrates, minerals, vitamins, etc. Since the product quality, concentration, and yield, etc., are essential for commercial products, various strategies such as tissue‐specific expression, enhanced transcript stability, translation optimization, and sub‐cellular accumulation are developed and further improved to increase proteins and other products yields in transgenic plants. Several plant‐derived products have also been reached in the market.
The high‐value biomolecules in the biosphere happen to be an excellent attraction for research and development. The scientific community’s primary objectives are the technological developments to exploit biotic and abiotic components of the ecosystem for societal benefits in a sustainable manner. It is desirable to develop cost‐effective biological systems to produce biomolecules vital in various sectors. Advancement in biotechnological research has enabled the engineering of various plants to produce biomolecules such as proteins, carbohydrates, and lipids, with crucial effects on health and agriculture. These biological systems have been proved to be economical devices for expressing natural molecules of pharmaceutical and nutraceutical significance and, therefore, called bioreactors. Recent biosynthetic technologies have paved the way to develop expression platforms for pilot scale biosynthesis of the metabolites of medical and agricultural importance. The leading advantages of plant bioreactors have emerged the opportunities for the development of edible vaccines and molecular farming of pharmaceutical proteins, insecticidal proteins, antioxidant molecules, secondary metabolites, bioavailable micronutrients, functional food products, etc.
The present book covers the holistic knowledge about plants as bioreactors from a general introduction to the applications in numerous fields, including pharmaceuticals, nutraceuticals, secondary metabolites, carotenoids, flavonoids, biopesticides, biofuels, etc. This book will act as a repository to get comprehensive information on the application of plants as a bioreactor in one place. This information would be significantly valuable for graduate students, academicians, researchers, and the general public.
Santosh Kumar Upadhyay
Department of Botany, Panjab University, Chandigarh, India
Sudhir P. Singh
Center of Innovative and Applied Bioprocessing (CIAB), Mohali, Punjab, India
Acknowledgments
We are thankful to the Panjab University, Chandigarh and the Department of Biotechnology, Government of India, for providing the facility to complete this book. We are grateful to all the esteemed authors for their exceptional contributions and reviewers for their critical evaluation and suggestions for the quality improvement of the book.
We would like to thank Miss Rebecca Ralf (Commissioning Editor), Kelly Labrum (Managing Editor) and Mahalakshmi Pitchai (Production Editor) from John Wiley & Sons Ltd for their excellent management of this project and anonymous reviewers for their positive recommendations about the book.
We also appreciate the support of our friends and research students, whose discussion and comments were beneficial to shaping this book. We thank our numerous colleagues for direct or indirect help in shaping this project.
SKU wish to express gratitude to his parents, wife, and daughter for their endless support, patience, and inspiration. SPS is grateful to his parents and family for consistent moral support. SPS acknowledges the support from CIAB and the Department of Biotechnology.
We would like to warmly thank the department and university faculties and staff for providing a great working environment.
1Plants as Bioreactors: An Overview
Madhu1, Alok Sharma1, Amandeep Kaur1, Deepika Antil1, Sudhir P. Singh2, and Santosh Kumar Upadhyay2
1 Department of Botany, Panjab University, Chandigarh, Punjab, India
2 Center of Innovative and Applied Bioprocessing (CIAB), Mohali, Punjab, India
1.1 Introduction
Plants are the primary producers of the ecosystem on which all other living organisms rely (Malmstrom 2010). They provide essential human diet components, such as carbohydrates, proteins, vitamins, and minerals, and are also a significant source of various phytochemicals (Kumar et al. 2021; Upadhyay and Singh 2021). Phytochemicals are naturally produced by plants as a result of their metabolic pathways and are also referred to as secondary metabolites (Anulika et al. 2016). For a long time, several pharmaceutical companies have used secondary metabolites to treat a variety of diseases. Furthermore, these chemicals are used in various industries, including cosmetics, herbicides, insecticides, flavors, and perfumes (Singh et al. 2017; Dixit et al. 2021a). On the other hand, traditional farming results in variation in the quality and quantity of raw materials due to different environmental conditions in different geographical areas (Seufert et al. 2012). Furthermore, many plants that produce commercially useful metabolites are difficult to cultivate outside their native environments, and as a result, they are overexploited, leading to their extinction (D’Amelia et al. 2021). A survey revealed surprising results that one‐fifth of the 50 000 medicinal plants used today belong to threatened species (Pan et al. 2014). To safeguard these plants, several technologies such as plant‐cell bioreactors and tissue culture have been used (Hussain et al. 2012). But with the discovery of recombinant‐DNA (rDNA) technology, the entire research attention shifted to the development of transgenic crops. Now rDNA technology is mainly focused on improving and modifying proteins of commercial importance (Khan et al. 2016). Transgenic crops are considered bioreactors for the development of commercially important proteins. To develop transgenic crops, we can employ a variety of strategies. The most frequent and widely used strategy is Agrobacterium‐mediated transformation, which includes the transformation of the desired gene into Agrobacterium cells, and then these cells are used to infect plants to transfer the essential genes to the plant genome. Electroporation, particle bombardment, and polyethylene glycol‐mediated gene absorption are some more ways of direct gene transfer (Basso et al. 2020). The recombinant proteins produced in transgenic plants are then directed to different organelles for regular eukaryotic post‐translational modifications (Hofbauer and Stoger 2013). A strong promoter sequence that boosts the expression of the desired product is necessary for excessive production of foreign protein (Gopal and Kumar 2013). Transgenic plants used as a bioreactor to create necessary plant products are a simpler, more appealing, and less expensive technique than microbiological systems (Yao et al. 2015). One big advantage is that plants frequently bloom throughout the year, making it simple to deliver the product to market on time. Plants that produce more biomass have the potential to produce more genetically modified commodities. We can also keep modified proteins in seeds for an extended period without losing their biological capabilities (Gunn et al. 2012). Furthermore, due to their natural and higher biochemical potential, plants are regarded as the best source of alkaloids, carbohydrates, fatty acids, proteins, phenolic compounds, and steroids (Sharma and Sharma 2009). However, for transgenics to be successful, contamination of food crops and their products as a result of gene integration and expression must be addressed (Bawa and Anilakumar 2013). As a result, strict regulatory issues must be addressed to establish transgenics for the development of recombinant proteins (Bawa and Anilakumar 2013). Plant bioreactors have recently achieved major advances in several fields. There is a myriad of products that are produced by plant bioreactors, such as medical, nutritional, industrial, and biodegradable plastics (Sharma and Sharma 2009). The main focus, however, is on the development of therapeutic proteins that can be taken orally or applied topically following extraction and purification (Daniell et al. 2009). The fermentors and bioreactors can be replaced with greenhouses with a proper plant growth chamber to reduce the upstream facility. In addition, plant tissues can be processed for oral delivery of food proteins, which would also reduce downstream processing (Fischer and Buyel 2020). Despite more than 20 years of research and reports about the efficacy of plant‐produced products, very few products have gone all the way through the production and regulatory hurdles (Whitelam et al. 1993). In this chapter, we will discuss the factors that are essential for the optimization of the production of recombinant proteins in plants and different products developed by this approach.
1.2 Factors Controlling the Production of Recombinant Protein
1.2.1 Choice of the Host Species
The host plant species must be carefully chosen to ensure the efficient production of recombinant protein. Before selecting the host, we must consider several factors such as host species, biomass yield, and cost of the scale‐up process (Sharma and Sharma 2009). To achieve great success, we must first investigate the species‐ or tissue‐specific factors that influence recombinant protein accumulation and quality (Whitelam et al. 1993). There are several model plant species, such as alfa–alfa, Arabidopsis, banana, lettuce, pea, potato, purple false brome, maize, rice, soybean, tobacco, and tomato, which we can choose for recombinant protein production (Richter et al. 2000). Plants can produce a variety of products, but each has its own set of requirements for production. As a result, no single species can be ideal for producing all these products (Sharma and Sharma 2009). Plants from the wild are ideal for molecular farming because they do not pose a risk of cross‐contamination (Bawa and Anilakumar 2013). On the other hand, scientists favor domesticated species due to their commercialization and ability to adapt to a wide range of environmental conditions (Sharma and Sharma 2009). Furthermore, self‐pollinating crops should be considered over cross‐pollinating crops to reduce the risk of transgene spread (Daniell 2002). The production sites of recombinant proteins in different plants are different, it may be leaves, cereal grains, oil, etc. (Xu et al. 2018). Major leafy crops used as bioreactors are alfalfa, tobacco, clover, and lettuce. However, leafy crops have a limited shelf life and need immediate processing (Sharma and Sharma 2009). On the other hand, in cereal crops like barley, maize, rice, and wheat, the storage site of protein is grain, where the protein may remain stable for more extended periods without any loss of its proteolytic activity (Garg et al. 2018). Moreover, avidin, which is the first plant‐derived product, also came from a cereal crop (Hood et al. 1997). The recombinant proteins are also produced from oil crops due to their low‐priced downstream processing (Zhu et al. 2018). Some proteins are targeted to the oil bodies, for instance, the protein oleosin. SemBioSys Genetics Inc. has developed this recombinant oleosin protein in safflower seeds (http://www.sembiosys.com) (Fischer et al. 2004). By using the same strategy, thrombin inhibitor hirudin was also produced in transgenic seeds of Brassica napus. It is estimated that more than 1 kg of protein is recovered from 1 ton of seeds (Parmenter et al. 1995). In the field of biopharmaceuticals, bryophytes are considered the best option due to their cost‐effective purposes and also there are no risks associated with the release of the transgenic (Reski et al. 2015). A company called Greenovation has adopted moss as a bioreactor system for the manufacturing of various medicines (http://www.greenovation.com/). Other aquatic plants and green algae can be used to produce recombinant proteins (Fabris et al. 2020). Another essential factor that should be considered before selecting the plant is that the plant genome should be sequenced, annotated, and available on public databases.
1.2.2 Optimization of Expression of Recombinant Protein
In the era of biotechnology, protein production is now accomplished by expression systems derived from different lineages (Ullrich et al. 2015). In recent years, we have had great success in increasing higher protein production in transgenic plants. Transgenic plants are now a viable alternative to traditional expression systems for the production of recombinant proteins (Ullrich et al. 2015). The critical question that arises here is how we can optimize the expression of recombinant protein to commercialize it. If we have to maximize the expression of heterologous protein, we have to optimize it. For optimization, we should consider the adjustable parameters like the structure of mRNA and protein, vector system, and culture conditions (Hayat et al. 2018; Upadhyay et al. 2010a,b, 2011). Each of the parameters should be evaluated individually for the experiments. Moreover, to control the expression of the transgene, other factors like transcription, translation, and posttranslational modifications (PTMs) should also be considered. Here we will discuss how these factors are responsible for the optimization of recombinant protein expression.
1.2.2.1 Transcription
Transcription is a fundamental process that transcribes DNA into RNA. It is a significant determinant of the recombinant protein expression level in transgenic plants. The majority of strategies aimed at increasing recombinant protein expression are based on increasing transgene transcription. Therefore, we should consider several factors to improve transcription levels.
1.2.2.1.1 Promoters
Promoters are the most important regulatory components in the transcription process. Promoters are the nucleotide sequences and crucial binding sites for RNA polymerase to initiate the transcription. In addition, the promoter sequences also act as regulatory sequences (Haberle and Stark 2018). They are usually present adjacent to the gene that is about to be transcribed. Moreover, understanding how a promoter works have opened up a whole new world of possibilities for gene expression regulation. A strong promoter is required to enhance the gene expression in transgenic plants. The most commonly used promoter is the cauliflower mosaic virus 35S (CaMV) promoter, which works constitutively and can induce the expression regardless of the environment or development factors. The CaMV promoter is mainly more effective for dicot plant species than monocot plant species (Bak and Emerson 2020). To improve and strengthen the CaMV 35S promoter, some elements of the promoter have been duplicated, which has ultimately been found to enhance the expression of the reporter transgene in the tobacco plant (Kay et al. 1987). Moreover, when the upstream region of the promoter was repeated, it had no significant impact on the expression of the gusA reporter gene in both tobacco and tomato transformants (Comai et al. 1990). There are several antigenic proteins, which have been produced in plants by using CaMV 35S promoter, for instance, protective antigen, hemagglutinin, cholera toxin subunit B (CTB), severe acute respiratory syndrome coronavirus (SARS‐CoV) vaccine, and rabies virus glycoprotein (Aziz et al. 2005; Huang et al. 2001; Jani et al. 2002; Li et al. 2006; McGarvey et al. 1995).
Another important type of promoter is inducible promoters, which are required for inducible expression vectors (Corrado and Karali 2009). These inducible systems are mainly designed for fundamental and applied research and now they are becoming popular in plant molecular biology. These promoters are usually triggered by wounding and pathogen attacks. For example, the expression of some proteins is only required during any pathogen attack, and their constitutive expression may cause a detrimental effect on the growth and development of the plant. These inducible promoters are mainly regulated by chemical compounds like alcohol, steroids, and tetracycline. Moreover, to make the inducible promoters to be highly efficient, these chemical compounds are known as inducers, should be highly specific to the promoter, have a fast response to induction, and immediately turn off upon withdrawal, nonpoisonous to host, and should be easily available. These inducers provide high inducibility and specificity for the regulation of gene expression. There are many examples where inducible promoters are used to increase the expression of the different heterologous proteins. Human interferon‐gamma and α1‐antitrypsin are expressed by a sucrose starvation‐inducible promoter of the alpha‐amylase gene in rice plants (Chen et al. 2004; Terashima et al. 1999). The Ramy3D promoter, which is induced to express recombinant human granulocyte‐macrophage colony‐stimulating factor (hGM‐CSF) by sucrose deprivation, is another important example of an inducible promoter. Moreover, when compared to tobacco cell‐suspension cultures, the production of hGM‐CSF in transgenic rice seedlings was found to be higher (Shin et al. 2003). Although inducible systems are widely accepted in plant biology, their application is still very limited. As a result, more research is needed to increase the number of inducers that are useful in the field of plant biotechnology.
Some recombinant proteins are expressed in specific tissue or organ, so to control the expression of such proteins, there is a need for tissue‐specific promoters. These promoters are very useful to restrict the final product in specific organs like seeds, tubers, or fruit. For instance, fruits are used to store vaccine antigens, a fruit‐specific promoter E8, is primarily used for various vaccine antigens, and this promoter was identified in the tomato (Deikman et al. 1992; Jiang et al. 2007; Ramirez et al. 2007; Sandhu et al. 2000). There are some other promoters that concentrate the expression of foreign bio‐molecules in seeds, such as porcelain promoter, maize globulin‐1, maize zein, 7S globulin, rice glutelin, soybean P‐conglycinin a′‐subunit (Belanger and Kriz 1991; Chen et al. 1986; Fogher 2000; Lau and Sun 2009; Marks et al. 1985; Osborn et al. 1988; Russell and Fromm 1997; Wu et al. 2005). Some promoters are chloroplast specific, for instance, the 16S ribosomal RNA promoter, which is used for the transgenic products like CTB and labile toxin B (LTB) (Daniell et al. 2001; Kang et al. 2004a; Koya et al. 2005; Ruhlman et al. 2007; Staub et al. 2000). Moreover, a plant‐based pharmaceutical company “Chlorogen Inc.” has patented a technology named chloroplast transformation technology (CTT™) to produce recombinant proteins in the chloroplast. Some recombinant proteins produced by this technology are interferon, cholera vaccine, insulin, and polymers. Another group of researchers has developed a smallpox vaccine and xylanase or E1 endoglucanase using a leaf‐specific rbcS gene promoter in the plant leaves (Dai et al. 2000; Golovkin et al. 2007; Hyunjong et al. 2006).
1.2.2.1.2 DNA Methylation
DNA methylation is an epigenetic modification of the DNA sequence primarily caused by DNA methyltransferases (DNMTs). DNA methylation is found to be involved in the regulation of the expression of various endogenous genes. It has been found that in both plants and mammals, DNA methylation is conserved and critical for growth and development (Zhang et al. 2018). Similarly, DNA methylation also causes suppression of transgenes in transgenic plants, which reduces the expression of chimeric transgenes. For example, in the tobacco plant, two transfer DNA (T‐DNAs) were transformed, and after transformation, it has been found that the expression of T‐DNAs was reduced due to the methylation in the promoters of these genes (Matzke et al. 1989). Moreover, it has been found that in Agrobacterium tumefaciens itself, 0.5% of cytosines are methylated. Thus, it may be possible that transgenes are methylated and get inactivated before transformation (Palmgren et al. 1993). Therefore, to prevent the transgene from methylation, some demethylating agents can be used before and after the transformation. For instance, Palmgren and colleagues used demethylating agent 5‐azacytidine to increase the expression of the β‐glucuronidase gene, which was transferred to tobacco leaf disks by A. tumefaciens (Palmgren et al. 1993).
1.2.2.1.3 Transacting Factors
Transacting factors are the proteins that bind to the cis‐regulatory elements for the regulation of gene expression. These factors are the positive regulators of gene expression. So, these factors are considered important in controlling transgene expression in the plant system (Carrier et al. 2020). To regulate transgene expression, these factors either directly bind to the promoter or interact with other factors and promote their recruitment to the promoter (Carrier et al. 2020). Yang and colleagues have engineered a transcription factor rice endosperm bZIP (REB) in rice plants. It has been found that the engineered REB binds with the rice globulin promoter and enhances the expression level of the lysozyme gene (Yang et al. 2001). In an experiment, the function of a transcription factor on transgene expression was studied by fusing the steroid‐binding domain of the glucocorticoid receptor to a plant transcription factor. This glucocorticoid‐responsive GAL4‐VP16 fusion protein mainly acts as an inducer for the activation of a luciferase reporter gene in transgenic Arabidopsis and tobacco plants. In this study, it has been found that in the absence of a ligand, transcription factor functioning is suppressed. But after induction, the suppression is relieved and the transcription factor resumes its functioning in transgenic plants (Aoyama and Chua 1997). In another study, Lloyd and colleagues have seen that transparent testa glabra (ttg) mutant Arabidopsis plants were not able to produce trichomes, anthocyanins, and seed coat pigments; however, they were generating excess root hairs. So, they have experimented and found that the trichomes and anthocyanin production can be restored by overexpressing the transcription factor R of maize in the constitutive and inducible manner (Lloyd et al. 1994). In one more study, which was conducted by Kermode et al. in 2007, it is mentioned that the recombinant human α‐L‐iduronidase protein production in transgenic tobacco plants could be enhanced by the expression of a conifer abscisic acid insensitive 3 (ABI3) transcription factor (Kermode et al. 2007).
1.2.2.2 Post‐Transcription Modifications
Following the transcription process, the pre‐mRNA undergoes certain modifications known as post‐transcriptional modifications and after these modifications, pre‐mRNA becomes mature mRNA. These modifications include splicing, capping, and polyadenylation. All these modifications occur in the nucleus; mature transcripts are then translocated into the cytoplasm for protein synthesis. These posttranscriptional modifications play a critical role in the gene expression and thus influence the yield of recombinant proteins in transgenic plants (Bandopadhyay et al. 2010). Splicing is the most important modification of pre‐mRNA involving the removal of introns and joining of exons. Introns are the noncoding intragenic sequences containing acceptor and donor sites required for splicing (Wang et al. 2015). The presence of introns has been found to affect transcription levels, but the mechanism is not fully understood. Enhancer sequences in introns are thought to increase expression levels, but the efficiency of splicing and other mRNA stability processing events could also be involved. In a number of cases, introns have been discovered to aid transgene expression in transgenic plants. For example, in Arabidopsis, a profilins (PRF2) promoter and complete expression of β‐glucuronidase require an intron PRF2 (Jeong et al. 2006). In addition, the Arabidopsis agamous gene has an intron with enhancer sequences that are found to be involved in the expression of a reporter from a minimal promoter. Other Arabidopsis genes, such as STK and FLC, and wheat VRN‐1 have enhancer sequences in their introns (Rose 2008). Likewise, in maize plants, the expression of the alcohol dehydrogenase 1 (ADH1) gene is dependent upon the introns (Callis et al. 1987). Moreover, the introns of the ADH1 gene are also responsible for the expression of heterologous gene chloramphenicol acetyltransferase of bacteria in maize protoplasts (Mascarenhas et al. 1990). There are some other examples where introns can influence the expression of genes, for instance, introns of OsTua2, OsTua3, OsTub4, and OsTub6 (Gianì et al. 2009).
1.2.2.3 Translation
The translation is the process where the genetic codons are translated from mRNA to protein by ribosome translocation. To enhance the yield of recombinant protein in a heterologous system, the translation of the transgenes should be regularized. There are several important parameters to increase the translational efficiency and some are discussed later.
1.2.2.3.1 Position and Context of the Initiation Codon
In eukaryotes, thousands of proteins are encoded on which a substantial amount of cellular energy is utilized. If there is any error occurs in the process of translation, it will lead to the production of inactive proteins that will disrupt cellular fitness (Harper and Bennett 2016). So, mRNA must be rightly translated into protein. For this, the ribosome must initiate the translation process with an appropriate codon, incorporate the suitable amino acids, and terminate only at the stop codon (Lutcke et al. 1987). The translation in eukaryotes is mainly initiated with a start codon, i.e. AUG. Furthermore, it has been noticed that the sequences near the initiation codon have an important role in initiating the translation process. In plants, sequences such as AACAAUGGC, GCCAUGGCG, and UAAACAAUGGCU have been identified near the initiation codon (Lutcke et al. 1987). Furthermore, Guerineau et al. 1992 studied the effect of these sequences near the initiation codon on the expression of a GUS reporter gene in tobacco mesophyll protoplast (Guerineau et al. 1992). They have formed an expression cassette by fusing these sequences with a duplicated CaMV 35S promoter. After that, the GUS activity was found to be three times more in translational fusions as compared to transcriptional fusions (Guerineau et al. 1992). Furthermore, Sawant and colleagues studied the effect of sequences downstream of the initiator codon on the gene expression and protein stability in tobacco leaves. They have inserted a sequence GCT TCC TCC after the initiator codon of two reporter genes, uidA and gfp, which further augment the expression of both the genes (Sawant et al. 2001). It has been found that to optimize transgene expression in plants, nucleotide sequence ACC or ACA insertion near the initiation codon will be preferred. In tobacco plants, increasing the expression of the ctxB gene as a C‐terminal fusion protein with native plant protein “ubiquitinin” fragment has been achieved by codon optimization (Mishra et al. 2006).
1.2.2.3.2 Control of Gene Expression by 5′ and 3′ Untranslated Region
To optimize the expression of the transgene, translation efficiency and RNA stability should be there. In some reports, it has been established that 5′ and 3′ untranslated regions (UTRs) of mRNAs play an important role in enhancing translation efficiency (Zeyenko et al. 1994
