Fungi Bio-prospects in Sustainable Agriculture, Environment and Nano-technology: Volume 3: Fungal Metabolites, Functional Genomics and Nano-technology

By Ajay Kumar

Fungi Bio-prospects in Sustainable Agriculture: Fungal metabolites and Nano-technology is a three-volume series that has been designed to explore the huge potential of the many diverse applications of fungi to human life. The series unveils the latest developments and scientific advances in the study of the biodiversity of fungi, extremophilic fungi, and fungal secondary metabolites and enzymes, while also presenting cutting-edge molecular tools used to study fungi. Readers will learn all about the recent progress and future potential applications of fungi in agriculture, environmental remediation, industry, food safety, medicine, and nanotechnology.Volume 3 provides a comprehensive account of fungal metabolites, including bioactive and host origin compounds, along with other biomolecules, and mycotoxins. This book includes the applications, limitations, and prospects of working with fungal secondary metabolites. The authors explore fungi in the myco-mediated synthesis of nanoparticles along with their biotechnological, industrial, and agricultural uses. This book also discusses advancements in medical mycology for the diagnosis and treatment of fungal infections. Furthermore, this book provides up-to-date and in-depth knowledge about the adoption of advanced CRISPR-Cas9 technology in fungi for gene editing

• Covers the secondary metabolites of fungi including bioactive compounds, mycotoxins and other biomolecules
• Provides insight into the fungal mediated biosynthesis of nanoparticles and its various applications in diverse fields
• Describes advances in diagnosis and treatment of human fungal infections
• Presents the latest information on applications of the CRISPR-Cas9 system in fungi

• Language: English
• Publisher: Academic Press
• Release date: Mar 16, 2021
• ISBN: 9780128217351

Book preview

Fungi Bio-Prospects in Sustainable Agriculture, Environment and Nano-technology

Volume 3: Fungal Metabolites and Nano-technology

Edited by

Vijay Kumar Sharma

Kunming University of Science and Technology, Yunnan Province, P.R. China

Maulin P. Shah

Environmental Microbiology Lab, Bharuch, Gujarat, India

Shobhika Parmar

Kunming University of Science and Technology, Yunnan Province, P.R. China

Ajay Kumar

Agricultural Research Organization - Volcani Centre, Rishon LeZion, Israel

Table of Contents

Cover image

Title page

Copyright

List of contributors

Preface

Chapter 1. Fungal metabolites: a source of bioactive natural products

Abstract

Introduction

Endophytes are lime lights for lovastatin production

Acknowledgment

References

Chapter 2. Endophytic and marine fungi are potential source of antioxidants

Abstract

Introduction

Fungal endophytes from terrestrial plants

Antioxidants from mangrove endophytic fungi

Antioxidants from marine algae-associated fungi

Antioxidants from marine sponges-associated fungi

Antioxidants from marine sediments-derived fungi

Antioxidants from Miscellaneous Marine Fungi

Some of the strategies for cultivation of endophytic and marine fungi

One factor at a time

One strain many compounds

Chemical epigenetic manipulation

Coculture of different strains

Conclusions

References

Chapter 3. Bioactive compounds from marine-derived fungi and their potential applications

Abstract

Introduction

Marine-derived fungi isolated from sponges

Marine-derived fungi isolated from corals

Marine-derived fungi isolated from algae

Marine-derived fungi isolated from deep sea and marine sediment

Marine-derived fungi isolated from mangrove

Marine-derived fungi isolated from other sources

Discussion

Conclusions and future perspective

References

Chapter 4. Marine endophytic fungi isolated from Gulf of Mannar—A source for new generation of pharmaceutical drugs and biosynthesis of silver nanoparticles and its antibacterial efficacy

Abstract

Introduction

Gulf of Mannar

Fungi-mediated synthesis of silver nanoparticles (AGNPs)

Advantages of biologically synthesized silver nanoparticles

Advantages of extracellular synthesis of AGNPs

Procedure for silver nanoparticles from marine fungi

Biotechnological potential of marine fungi

Antimicrobial agents

Anticancer drugs

Antioxidant and biodegradation

Conclusion

References

Chapter 5. Pullulan: a bioactive fungal exopolysaccharide with broad spectrum of applications for human welfare

Abstract

Introduction

Historical background

Structure and properties of pullulan

Microbial sources of pullulan

Pullulan biosynthesis

Derivatives of pullulan

Versatile applications of pullulan in therapeutics

Concluding remarks

Future perspectives

References

Chapter 6. Applications of biomolecules of endophytic fungal origin and its future prospect

Abstract

Introduction

Diversity of the endophytes

Endophytic fungal secondary metabolite and its application

Endophytic fungal enzymes and its application

Limitation and future prospect

References

Chapter 7. Microbial metabolites: as sources of green dye

Abstract

Introduction

Types of color

Effect of artificial dyes

Biocolor

Sources of biocolor in nature

Biotechnological approaches for enhancement of biodyes

Advantage of biocolors

Drawback of biocolors

Conclusion

References

Chapter 8. Fungal endophytes as a potential source of therapeutically important metabolites

Abstract

Introduction

Conclusions

References

Chapter 9. Prevention and control of mycotoxins for food safety and security of human and animal feed

Abstract

Introduction

Major groups of mycotoxins

Metabolism of mycotoxins

Food safety concerns associated with mycotoxin

Factor affecting the mycotoxins in food and agriculture products

Detection of mycotoxins in foods and agriculture products

Prevention and control of mycotoxins

Health and economical impact

Conclusion

References

Chapter 10. Food safety concern related to aflatoxins and control

Abstract

Background

Introduction

Toxicity of aflatoxins

Biosynthesis of aflatoxin

Aflatoxin detection/diagnostics

Management and control strategies

Global regulatory standards for aflatoxin management

Summary

Future perspectives

References

Chapter 11. Role of the endogenous fungal metabolites in the plant growth improvement and stress tolerance

Abstract

Introduction

Ecology of fungi and their metabolites

Fungal–plant interactions

Mechanisms of endophytic fungi–host plant interactions

Future prospective

Conclusion

Acknowledgment

References

Further reading

Chapter 12. Recent trends in fungal biosynthesis of nanoparticles

Abstract

Introduction

Gold nanoparticles

Silver nanoparticles

Gold–silver bimetallic nanoparticles

Copper-based nanoparticles

Cadmium-based nanoparticles

Platinum and palladium nanoparticles

Zinc-based nanoparticles

Other metal and metal oxide nanoparticles

Applications

Antibacterial activity

Antifungal activity

Insecticidal activity

Anticancer activity

Agriculture

Metal removal

Textile

Dye degradation

Future prospects and conclusions

Acknowledgment

References

Chapter 13. Role of fungi in bio-production of nanomaterials at megascale

Abstract

Introduction

Fungi as bionanofactories

Bioproduction of various nanomaterials by fungi

Mycosynthesis of silver nanoparticles

Megascale production of nanoparticles

Conclusion

Acknowledgments

Conflict of interest statement

Author contributions

References

Chapter 14. Mycogenic fabrication of nanoparticles and their applications in modern agricultural practices & food industries

Abstract

Introduction

Isolation and identification of fungus

Application of nanoparticles in agriculture

Application of nanoparticles in food industries

Conclusion

Future trends in application of agriculture and food industries

References

Chapter 15. Role of fungal endophytes in the green synthesis of nanoparticles and the mechanism

Abstract

Introduction

Fungal endophytes

Endophytic fungi as efficient tools for the synthesis of biogenic nanoparticles

Importance and applications of noble metal nanoparticles

Endophytic fungi-mediated synthesis of noble metal nanoparticles

Mechanism of synthesis of nanoparticles by the fungal endophytes

Conclusion

References

Chapter 16. Fungal infections: advances in diagnosis and treatment

Abstract

Fungal infections: advances in diagnosis and treatment

Nucleic acid detection

Polymerase chain reaction

Treatment of fungal infections

References

Chapter 17. CRISPR-Cas9 system for functional genomics of filamentous fungi: applications and challenges

Abstract

Introduction

CRISPR-Cas9 system

Recent developments of CRISPR-Cas9 tools

Current and potential applications of CRISPR-Cas9 system in filamentous fungi

Challenges and ethical concerns of CRISPR-Cas9 tools in filamentous fungi

Conclusion

References

Index

Copyright

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List of contributors

Komal Agrawal,     Bioprocess and Bioenergy Laboratory, Department of Microbiology, Central University of Rajasthan, Ajmer, India

U. Atchayadana,     Department of Microbiology, The American College, Madurai, India

Ashish Bedi,     TERI–Deakin Nano Biotechnology Centre, The Energy and Resources Institute, New Delhi, India

David M. Cahill,     Deakin University School of Life and Environmental Sciences, Waurn Ponds, VIC, Australia

Sonia Chadha

Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai, India

Homi Bhabha National Institute, Mumbai, India

Preeti Chaturvedi,     Department of Biological Science, G.B. Pant University of Agriculture and Technology, Pantnagar, India

Xavier A. Conlan,     Deakin University School of Life and Environmental Sciences, Waurn Ponds, VIC, Australia

Jayashankar Das,     IMS and SUM Hospital, Siksha OAnusandhan (Deemed to be University), Bhubaneswar, India

Shivani Dave,     MBM Engineering College, Jodhpur, India

Sushma Dave,     JIET Group of Institutions, Jodhpur, India

Sunil K. Deshmukh,     TERI–Deakin Nano Biotechnology Centre, The Energy and Resources Institute, New Delhi, India

Sunny Dhiman,     University Institute of Biotechnology, Chandigarh University, Mohali, India

Bharat Z. Dholakiya,     Department of Applied Chemistry, Sardar Vallabhbhai National Institute of Technology (S.V.N.I.T.), Surat, India

Sougata Ghosh

Department of Microbiology, School of Science, RK University, Rajkot, India

Department of Chemical Engineering, Northeastern University, Boston, MA, United States

Manish Kumar Gupta,     SGT College of Pharmacy, SGT University, Gurugram, India

Shubhpriya Gupta,     Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Pietermaritzburg, South Africa

Pratik Jagtap,     Institute of Science, Mumbai, India

Mehul R. Kahimani,     School of Science, P P Savani University, Surat, India

Rishee K. Kalaria,     Aspee Shakilam Biotechnology Institute, Navsari Agricultural University, Surat, India

Ashok Kumar

Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai, India

Homi Bhabha National Institute, Mumbai, India

Suneel Kumar,     Bio-Design Innovation Centre, Rani Durgavati Vishwavidyalaya, Jabalpur, India

Puja Bharti Kumari,     School of Environmental Sciences BBA University, Lucknow, India

J.S. Kushveer,     Department of Biotechnology, School of Life Sciences, Pondicherry University, Kalapet, India

N. Jeny Lydia,     Department of Microbiology, The American College, Madurai, India

Senthamarai Manogaran,     Endophytic Fungal Secondary Metabolite Research Laboratory, Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam, India

Mukesh Meena,     Laboratory of Phytopathology and Microbial Biotechnology, Department of Botany, Mohanlal Sukhadia University, Udaipur, India

Dhruv Mishra,     Department of Biological Science, G.B. Pant University of Agriculture and Technology, Pantnagar, India

Modhurima Misra,     Department of Bio-Engineering, Birla Institute of Technology, Ranchi, India

Padmaja Mohanty,     Department of Botany, Bioinformatics and Climate Change, Gujarat University, Ahmedabad, India

J. Mubina,     Department of Microbiology, The American College, Madurai, India

Gunjan Mukherjee,     University Institute of Biotechnology, Chandigarh University, Mohali, India

Harshajit Nath,     Bodoland University, Kokrajhar, India

Kannan Kilavan Packiam,     Endophytic Fungal Secondary Metabolite Research Laboratory, Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam, India

Sugandha Pant,     Department of Biological Science, G.B. Pant University of Agriculture and Technology, Pantnagar, India

Hiren K. Patel,     School of Science, P P Savani University, Surat, India

PremaLatha Pushpanathan,     Department of Microbiology, ESIC Medical College and PGIMSR, Chennai, India

M. Rashmi,     Department of Biotechnology, School of Life Sciences, Pondicherry University, Kalapet, India

Devashish Rath

Homi Bhabha National Institute, Mumbai, India

Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai, India

Ashish Sachan,     Centre for Life Sciences, Central University of Jharkhand, Ranchi, India

Shashwati Ghosh Sachan,     Department of Bio-Engineering, Birla Institute of Technology, Ranchi, India

Sardul Singh Sandhu,     Bio-Design Innovation Centre, Rani Durgavati Vishwavidyalaya, Jabalpur, India

Roopa Vishwanath Sangvikar,     Department of Botany, N.E.S. Science College, Nanded, India

V.V. Sarma,     Department of Biotechnology, School of Life Sciences, Pondicherry University, Kalapet, India

Gaurav S. Shah,     Department of Biotechnology, Veer Narmad South Gujarat University, Surat, India

Sejal Shah,     Department of Microbiology, School of Science, RK University, Rajkot, India

Mridul Shakya,     Bio-Design Innovation Centre, Rani Durgavati Vishwavidyalaya, Jabalpur, India

Pooja Sharma,     Department of Environmental Microbiology, School for Environmental Sciences, Babasaheb Bhimrao Ambedkar Central University, Lucknow, India

Surendra Pratap Singh,     Plant Molecular Biology Laboratory, Department of Botany, Dayanand Anglo-Vedic (P.G.) College, Chhatrapati Shahu Ji Maharaj University, Kanpur, India

Priyankaraj Sonigra,     Laboratory of Phytopathology and Microbial Biotechnology, Department of Botany, Mohanlal Sukhadia University, Udaipur, India

Immanuel Suresh J,     Department of Microbiology, The American College, Madurai, India

Prashant Swapnil,     Department of Botany, Acharya Narendra Dev College, University of Delhi, India

Poonam Verma,     Bio-Design Innovation Centre, Rani Durgavati Vishwavidyalaya, Jabalpur, India

Pradeep Verma,     Bioprocess and Bioenergy Laboratory, Department of Microbiology, Central University of Rajasthan, Ajmer, India

Rajyoganandh S. Vijayaraman,     Department of Microbiology, Vels Institute of Science, Technology and Advanced Studies, Vels University, Chennai, India

Thomas J. Webster,     Department of Chemical Engineering, Northeastern University, Boston, MA, United States

Garima Yadav,     Laboratory of Phytopathology and Microbial Biotechnology, Department of Botany, Mohanlal Sukhadia University, Udaipur, India

Preface

Vijay Kumar Sharma, Maulin P. Shah, Shobhika Parmar and Ajay Kumar

Fungi play an essential role in a variety of current and future applications in agriculture, the environment, and nanotechnology. They are involved in a range of processes that have significant impacts on the sustainable development of human welfare. While some fungi do produce toxins, others are the major producers of valuable compounds that can be used in the food, agriculture, and pharmaceutical industries. Since fungi are relatively easy to grow, they are well-suited for large-scale production of desired compounds and biotechnological applications. Fungal metabolite profiling has an important role for fungal taxonomy and physiology. Fungus-mediated biosynthesis of nanoparticles is an excellent tool for the fabrication of metal nanoparticles with desired shapes and sizes. The approach is easy, does not involve harmful toxic compounds, and has the future potential for rapid large-scale production and applications. Due to their vast biotechnological potential, filamentous fungi have long been the subject of genome engineering. The advanced genomic tools like the CRISPR-Cas9 system are now available for gene manipulation in filamentous fungi. The uses include the regulation of secondary metabolite pathways, pathogenicity-related genes, and signaling pathways.

Fungi Bio-prospects in Sustainable Agriculture, Environment and Nano-technology is a three-volume series. The book is an attempt to explore the recent advances in fungi from various environments, their diversity, and their diverse applications for the sustainable development of human life. Volume III consists of 17 diverse chapters that provide in-depth accounts of fungal metabolites, including bioactive and host origin compounds, fungal biomolecules, and mycotoxins. The chapters provide information about the extensive applications of fungal secondary metabolites and associated limitations. The book explores role of fungi in the biosynthesis of nanoparticles and their biotechnological, industrial, and agricultural uses. Advances in medical mycology for the diagnosis and treatment of fungal infections were discussed. The book also provide an account of advanced CRISPR-Cas9 technology in fungi for gene editing, and applications in medical mycology, cell biology, multiple signaling cascades, secondary metabolite engineering, waste management, and industries. Chapter 1 discusses fungal metabolites as the source of bioactive natural products. Chapter 2 presents a detailed account of both endophytic and marine fungi as potential sources of antioxidant compounds. Chapter 3 describes the prospects and applications of the compounds isolated from marine-derived fungi and their bioactivity. Chapter 4 gives insights in to the marine endophytic fungi isolated from the Gulf of Mannar, and discusses their prospects for pharmaceutical use and silver nanoparticles biosynthesis. Chapter 5 presents a detailed account of pullulan—a bioactive fungal exopolysaccharide. The chapter further discusses microbial sources of pullulan, structure and properties of pullulan, pullulan biosynthesis, and its therapeutic applications. Chapter 6 describes the biodiversity of the fungal endophytes reported in the literature, their secondary metabolites and other biomolecules, along with their applications, associated limitations, and future prospects. Chapter 7 discusses the biopigments from microbes including those from fungi, their potential applications as chemical dye substitutes, and their advantages and drawbacks. Chapter 8 presents a comprehensive review of therapeutic metabolites produced by endophytic fungi. The chapter also discusses the possible strategies to enhance secondary metabolite production from endophytic fungi. Chapter 9 explains selected groups of mycotoxins, the metabolism of mycotoxins, and associated food safety issues. The chapter also gives a brief account of the factors affecting the mycotoxins in food and agriculture products, along with detection and control measures. Chapter 10 presents the food safety concerns related to aflatoxins; emerging technologies for detection, management, control strategies and global regulatory standards are mentioned. Chapter 11 describes the role of endogenous fungal metabolites in plant growth improvement and stress tolerance. Chapter 12 discusses the recent advances of nanobiotechnological applications of fungi, which include their synthesis, possible mechanisms, and their potential applications. Chapter 13 describes recent progressions regarding the suitability of fungi for large-scale bioproduction of nanoparticles. Chapter 14 describes the bioprospects of fungi in the biofabrication of nanoparticles, their mechanisms, and their potential applications in the agricultural sector and food industries. Chapter 15 discusses fungal endophytes as an alternative tool for the biosynthesis of nanoparticles with a special focus on noble metal nanoparticles. Chapter 16 presents an account of human fungal infections, including advances in diagnosis and treatment. Chapter 17 discusses the CRISPR-Cas9 system for functional genomics of filamentous fungi with applications and challenges.

This volume is the collaborative work of many people. We appreciate and thank all the eminent experts who contributed to various aspects of fungi presented in this book. The views expressed by the authors are their own. We are hopeful that this book will be useful to academics, researchers, postgraduate students, and practitioners in the area of mycology. We express gratitude to Prof. R.N. Kharwar (BHU, India) and Prof. Haiyan Li (KUST, China) for their constant support and encouragement in bringing out this book.

Chapter 1

Fungal metabolites: a source of bioactive natural products

Senthamarai Manogaran and Kannan Kilavan Packiam*,    Endophytic Fungal Secondary Metabolite Research Laboratory, Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam, India*, Corresponding author. Email:[e-mail]

Abstract

Endophytic fungi are novel sources for the production of high-value bioactive compounds which are having huge applications in diverse fields of biotechnology. Euphorbia hirta (L) is one of the weeds which is ignored by people due to lack of awareness but research shows that it can be used for isolating potential endophytic fungi for the production of fungal metabolites at a large scale because of its medicinal properties and rich diversity. Fusarium nectrioides was isolated from this plant and investigated for the production of Lovastatin using an industrial waste called liquid cheese whey due to its lactose content and micronutrients. Growth profile, substrate utilization, and product formation kinetics of this filamentous fungi were studied in detail using unstructured models. From all these models, a suitable model was found with the appropriate fitting of predicted models with an experimental data to analyze the growth behavior of the fungus for the enhanced production of lovastatin. Competition inhibition of HMG-CoA reductase by kit assay revealed that the inhibitory effect of lovastatin on cell proliferation is the consequence of the induction of apoptosis and inhibition of proliferation and lovastatin standards.

Keywords

Endophytic fungi; Euphorbia hirta (L); Fusarium nectrioides; liquid cheese whey and competition inhibition

Introduction

Plants are the major kingdom for the production of a broad spectrum of novel bioactive natural products, but the quantity of these active components is comparatively less than from the microbial community. There is strong evidence for plant–microbe interactions and sharing their potential in order to produce such active metabolites. This is an eye-opener for overcoming the cutting of plants to produce such metabolites for economic benefits. Thus endophytic fungi are the major focus for the production of industrially important secondary metabolites without destroying plants (Rodrigues, 1994). Advanced research studies also reveal that endophytes are good sources for obtaining a vast number of high-value fungal metabolites which have huge applications in various fields.

Endophytes are lime lights for lovastatin production

The isolation and identification of potential fungal isolates to produce such metabolites are the biggest challenges and depend mainly on the growth conditions of the plants, plant health, and the environmental conditions where the plants are growing. This is achieved very effectively using a biotechnological approach through cost-effective production strategies.

Euphorbia hirta (L.) is a pantropical weed (Fig. 1.1) belonging to the Euphorbiaceae family of approximately 2000 species, distributed throughout the hotter parts of India. It is frequently found in wasteland on roadsides.

Figure 1.1 Habitats of Euphorbia hirta (Linn.) collected from Western Ghats of Sathyamangalam.

This plant possesses pharmacological properties, such as, antiseptic, anti-inflammatory, antidiabetic, antispasmodic, antibacterial, antiviral, antifungal, anticonvulsant, antiasthmatic, sedative, antispasmodic, nootropic, antifertile, antifungal, antimalarial, and aphrodisiac properties have already been recorded (Tuhin et al., 2017). The medicinal properties of different parts of the plant are described in Table 1.1.

Table 1.1

Industrial waste as carbon source

Chemical synthesis of lovastatin strictly involves synthetic methods. The cost of production is too high for commercialization purposes and other commercially viable chemical processes only yield poor quality product and involve complicated production steps (Hirama and Iwashita, 1983).

Fusarium sp. is a group of filamentous fungi widely distributed in soil and also found as an endophyte in plants. Fusarium nectrioides was isolated from E. hirta (L) for the production of natural metabolites under controlled environmental conditions (Senthamarai and Kannan, 2019). A phylogenetic analysis of this fungus revealed that there is a strong relationship between this fungus with the pathogenic and nonpathogenic lovastatin-producing fungus F. nectrioides, which is rarely found in the trauma-related eye infections of humans (Schroers et al., 2009), and has a close relationship with other pathogenic Fusarium species such as Fusarium biseptatum, Fusarium penzigii, and Fusarium delphinoides (Bohni et al., 2016).

The genus of this fungus was extensively studied for the production of secondary metabolites. Lovastatin is one of the fungal metabolites produced by F. nectrioides and its production is enhanced using an industrial waste called liquid cheese whey with glucose and histidine. Liquid cheese whey is a yellowish liquid, obtained after the coagulation of milk during cheese production. Due to its rich lactose content and milk nutrients, it has huge commercial applications in industries for the production of whey protein, lactose, and whey protein concentrate. The lovastatin-producing capability was analyzed by UV Spectrophotometry and it was confirmed at the maximum absorbance at 238 nm, as shown in Fig. 1.2.

Figure 1.2 (A) and (B) UV spectrophotometry analysis for the confirmation of lovastatin.

Unstructured models for filamentous fungi

Mathematical models were used to analyze the effects of different media components and to predict the behavior of fungus under the controlled conditions (Chadrashekar et al., 1999) in the reactor. These models are economical as well as simple to implement in complex biological process situations.

Prediction of filamentous fungi behavior using models

Fermentation of F. nectrioides was carried out in the fermenter (bioengineering model: in situ benchtop-type KLF 2000, 3.7 L total volume) using modified media in combination with glucose and lactose as the major carbon sources, as depicted in Fig. 1.3. The fermenter was sterilized in situ prior to the fermentation process

Figure 1.3 Fermentation of media components by Fusarium nectrioides (MH173849) in the reactor.

Growth kinetics of filamentous fungi

Growth kinetics of F. nectrioides inside the reactor were investigated. For the first two days, the cells were in lag phase. They reached an exponential phase at the 3rd day and continued for 8 days, and then entered the stationary phase. It was found that 80% of glucose and liquid cheese whey was utilized at the end of the 8th day of fermentation and biomass concentration was increased to 20.32 g/L, as seen in Fig. 1.4

Figure 1.4 Kinetic profile of cell growth, substrate utilization, and lovastatin production by Fusarium nectrioides.

Malthus law

During the exponential growth phase, growth rate is independent of nutrients due to the high concentration of the nutrients. Hence, the log phase of the filamentous fungus was characterized by the first-order exponential growth equation called Malthus Law. This describes that in a batch fermentation process at ideal conditions, the concentration and biomass increase proportionally with an increase in time. Therefore the probability of multiplication of all the cells will be the same and it is represented as given in Eq. (1.1).

(1.1)

where is the change in growth rate (g/L day), X is the concentration of biomass (g/L), and μnet is the net specific growth rate.

Monod model

The relationship of specific growth rate to substrate concentration is described by saturation kinetics where one of the essential nutrients, which is behaving as a limiting nutrient, will reduce the growth once depleted. Therefore a sigmoidal relationship between specific growth rate μ and the concentration of the limiting substrate S is represented as shown in Eq. (1.2) (Monod, 1949).

(1.2)

where μ is the specific growth rate (per day), S is the concentration of the limiting substrate (g/L), and Ks is the half saturation constant which is equal to concentration of the substrate when μ=μmax/2. The corresponding graphical representations are given in Figs. 1.5 and 1.6.

Figure 1.5 Lineweaver–Burk plot.

Figure 1.6 Comparison of experimental and Monod model predicted biomass.

Contois model

The Contois model is an inverse relationship between microbial concentration and their specific growth, as described in Eq. (1.3). It describes the influence of substrate concentrations on growth kinetics.

(1.3)

where µ is the specific growth rate, µmax is the maximum specific growth rate, S is the substrate concentration, KX is a growth coefficient of the Contois function, and X is the biomass concentration (Contois, 1959).

It also describes that the net specific growth rate of a microbe is not only a function of the limiting substrate concentration but also depends on the microbial cell concentration (Zhi-Wu and Yebo Li, 2014). Hence, as the microbial cell concentration increases, the specific growth rate decreases.

Verhulst model

The Verhulst equation is an unstructured kinetic model developed based on biomass concentration. It states that the growth rate of a population is proportional to the size of the population and to the fraction of the carrying capacity unused by the population (Ardestani, 2012). It is mathematically expressed in Eq. (1.4) and represented in Fig. 1.7

(1.4)

Figure 1.7 Verhulst model.

Tessier model

The Tessier model (Tessier, 1942) is the kinetic growth model developed based on substrate concentration and it is depicted as mentioned in Eq. (1.5). The behavior of the fungus under this model is shown in Fig. 1.8.

(1.5)

Figure 1.8 Tessier model for the substrate utilization and cell growth.

Logistic model

As per the Malthus law, the rate of cell growth is proportional to the concentration of cell mass at the given time. When the cell reaches the stationary phase, the growth rate declines. So a gradual decrease is observed at the end of the exponential phase as given in logistic Eq. (1.6) (Miron et al., 2002), and it is shown in Figs. 1.9 and 1.10.

(1.6)

Figure 1.9 Logistic model for maximum specific growth rate and biomass concentrations.

Figure 1.10 Comparison of experimental biomass with predicted biomass (logistic model).

Luedeking–Piret model for product formation kinetics

The Luedeking–Piret model is such a type of unstructured product kinetic model which is extremely useful in fitting product formation data from different fermentation conditions (Luedeking and Piret, 1959). According to this model, the rate of product formation varies linearly with growth rate and the cell concentration is given in Eq. (1.7), and it is represented in Figs. 1.11–1.13

(1.7)

where dp/dt rate of change of product with change is in time, dX/dt is the change in biomass with change in time, and α (gP/gX) and β (gP/gX/h) are empirical constants.

Figure 1.11 Lovastatin production.

Figure 1.12 Rate of lovastatin production and determination of β.

Figure 1.13 Prediction of lovastation production by comparison of experimental and logistic-incorporated Luedeking-Piret model.

Substrate utilization kinetics

Substrate utilization kinetics is reformation of the Luedeking–Piret model, which describes the conversion of substrate into cell mass, and product and substrate consumption for cell maintenance (Weiss and Ollis, 1980), as given in Eq. (1.8). It is shown in Figs. 1.14–1.16.

(1.8)

where the yield coefficient for biomass with respect to substrate is consumed, is the yield coefficient for product with respect to substrate consumed, and mS is the cellular maintenance.

Figure 1.14 Calculation of δ.

Figure 1.15 Calculation of γ.

Figure 1.16 Comparison of experimental and logistic-incorporated modified Luedeking–Piret model.

Thus various growth models have been used to predict the behavior of filamentous fungi at the reactor level with the combination of modified media and liquid cheese whey. From all these models, a suitable model was found with the appropriate fitting of predicted models with experimental data to analyze the growth behavior of fungus for the enhanced production of lovastatin, as summarized in Table 1.2.

Table 1.2

The Monod, Verhulst, and logistic-incorporated modified Luedeking–Piret models showed good agreement with experimental data. These models also confirmed that lovastatin is a mixed growth associated product since it was produced at the end of an exponential phase and the beginning of the stationary phase. This was achieved by the gradual utilization of lactose by the fungus due to the low metabolic rate of its conversion compared to glucose.

Inhibition of HMG-CoA reductase by kit assay

Transmembrane glycoprotein 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), located on the endoplasmic reticulum, is an enzyme that catalyzes the four-electron reduction of HMG-CoA to coenzyme A (CoA) and mevalonate, which is the rate-limiting step in sterol biosynthesis.

In the case of NADPH-dependent biological reactions, HMG-CoA reductase reduces HMG-CoA to produce mevalonate and CoA. HMGR is the target enzyme of cholesterol-lowering drugs called statins. Inhibition of HMG-CoA reductase by statins induces the expression of low density lipoprotein (LDL) receptors in the liver, which lowers the plasma concentration of cholesterol.

To determine the inhibitory effect of purified lovastatin, HMG-CoA reductase assay kit (Sigma Aldrich, catalog number CS 1090) was used (Koning et al., 1996; Holdgate et al., 2003; Istvan et al., 2000; Kleemann and Kooistra, 2005). The catalytic subunit of HMGR oxidizes NADPH in the presence of the substrate called HMG-Co-A, which was measured using a spectrophotometer at 340 nm, as mentioned in the stoichiometry equation.

1× Assay buffer was diluted fivefold with ultrapure water and NADPH was reconstituted with 1.5 mL of 1× Assay Buffer. After the dilution, the working aliquots of both were stored at –20°C for further use. Since the stability of enzyme and substrate was less in the presence of assay buffer, all the other reaction components were mixed in order to obtain the accurate results.

An aliquot of HMGR and all other reaction components were thawed at room temperature and kept in ice throughout the experiment. Before performing the experiment, spectrophotometer was set at 37°C and 340 nm with the kinetic program. All the reaction components were mixed as mentioned in Table 1.3 to a maximum volume of 1 mL and it was read immediately every 15 s for up to 5 min. From the guidelines of the assay kit, one unit will convert 1.0 µmole of NADPH to NADP+ per 1 min at 37°C.

Table 1.3

Thus the unit specific activity is defined as mmol/min/mg-protein (units/mgP) and calculated using Eq. (1.9). The inhibitor solution pravastatin was provided with the kit and standard lovastatin (LOVs) (Sigma Aldrich) was used as the positive control.

(1.9)

where 12.44=εmM is the extinction coefficient for NADPH at 340 nm, which equals 6.22/mM/cm; 12.44 represents the two NADPH consumed in the reaction; TV is total volume of the reaction in ml (1 mL for cuvettes); V is volume of enzyme used in the assay (mL); 0.6 is enzyme concentration in mg-protein (mgP)/mL; and LP is light path in cm (1 for cuvettes)

Competitive inhibition of HMG-CoA reductase was determined spectrophotometrically (Kleinsek et al., 1997; Sadowitz et al., 2010; Edwards et al., 1979) using the protocol given in the kit. This assay was validated via inhibition studies using three HMG-CoA reductase inhibitors such as standard pravastatin, LOVs and sample lovastatin (LOV). HMG-CoA reductase activity slightly increased initially and then decreased in the presence of inhibitors. All the three inhibitors showed an inhibitory effect against HMG-CoA reductase in a time-dependent manner with respect to concentration (Lachenmeier et al., 2012). Unit specific activity of the inhibitors of HMGR is represented in Table 4.18. In the absence of statins, HMGR converts NADPH into NAD, and thereby the decrease in the concentration of NADPH was confirmed by the decrease in absorbance at 340 nm, whereas in the presence of pravastatin and lovastatin standards, conversion of NADPH into NAD was reduced due to the inhibitory effect of statins (Fig. 1.17). From this assay, the activity of enzyme was determined at different conditions as mentioned in Table 1.4

Figure 1.17 Inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) by three types of statins.

Table 1.4

Activity measured for an enzyme (HMGR), enzyme with standard pravastatin (HMGR+pravastatin), enzyme with sample lovastatin (HMGR+LOV), and enzyme with standard lovastatin (HMGR+LOVs) were 31.48 units/mgP, 15.51 units/mgP, 20.90 units/mgP, and 14.84 units/mgP, respectively. The linear correlations (r values) for standard lovastatin (LOVs), standard parvastatin, and sample lovastatin (LOV) were 0.9899, 0.9944, and 0.9826, respectively (Fig. 1.17).

Thus this assay confirms that lovastatin present in the sample also functions as the inhibitor of the HMGR. Therefore dose-dependent inhibition of HMGR by LOV was observed and it is less than pravastatin and lovastatin standards.

Cell viability assay

To determine the anticancer property of the lovasatin produced, the A549 cells-based cytotoxicity method was used. This method was very simple and easy for interpreting the results. The anticancer effect of the produced lovastatin on cell lines was investigated with and without treatment. This assay was based on the assumption that dead cells or their products do not reduce tetrazolium. In this method, all the cell lines were preincubated on a microtiter plate. This assay was mainly dependent on the cleavage of MTT to a blue formazan derivative by living cells. The numbers of cells were found to be proportional to the extent of formazan production by the cells used.

The cell culture of A-549 was centrifuged and the cell count was adjusted to 1.0×10⁵ cells/mL using Dulbecco’s modified eagle medium containing 10% fetal bovine serum. The diluted (approximately 10,000 cells/well) cell suspension (100 µL) was added in each well and centrifuged after 24 h. After centrifugation, pellets were suspended with 100 µL of different test samples in maintenance media. These were incubated at 37°C for 48 h in 5% CO2 atmosphere, and microscopic examination was carried out and observations recorded every 24 h.

After 48 h, 20 µL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide—2 mg/mL] in MEM-PR (modified eagle medium (MEM) without phenol red) was added. The plates were completely mixed and incubated for 2 h at 37°C in 5% CO2 atmosphere. Further, dimethyl sulfoxide (DMSO, 100 µL) was added to solubilize the formazan with proper agitation.

The absorbance was measured using a microplate reader (ELISA peruser: model: Emax, molecular gadget, USA) at a wavelength of 540 nm. The percentage cell viability was calculated using Eq. (1.10) and the concentrations of purified lovastatin samples needed to inhibit cell growth by 50% values were generated from the dose–response curves for the concentrations of 250, 125, 62.5, 31.25, 15.625 μg/mL (Masahiko et al., 2016).

(1.10)

Percentage viability and the concentration required for a 50% inhibition of viability (IC50) was determined by plotting the graph between the concentration and absorbance (Patel et al., 2009) for both the LOVs and LOV. Control of cell proliferation with and without LOV was compared with the LOVs, as shown in Fig. 1.18. Human lung carcinoma A-549 without lovastatin treatment was kept as the control cell line.

Figure 1.18 Control of cell proliferation by LOVs and LOV.

From this experiment, IC50 values against human lung carcinoma A-549 cells by the LOVs and LOV were 18.45987 and 27.67771 μg/mL, respectively, whereas inhibition of the cholesterol synthesis by simvastatin and pravastatin was 23 and 105 nM, respectively (Cohen et al., 1993). The hydroxyl form of lovastatin showed half maximum inhibition concentration (IC50) of 8 μg/mL in MDAMB468 and 5 μg/mL in MDAMB231 cells, whereas the lactone form of lovastatin showed 9 μg/mL and 7 μg/mL, respectively (Klawitter et al., 2010). Thus these results imply that the inhibitory effect of lovastatin on cell proliferation is the consequence of the induction of apoptosis and inhibition of proliferation and lovastatin standards. The linear correlations (r values) for LOVs, and LOV were 0.859 and 0.970, respectively (Fig. 1.19).

Figure 1.19 Cell viability test for standard and sample lovastatin.

Acknowledgment

We express our indebtedness and sense of gratitude to Chairman, Trustee, and Principal of Bannari Amman Institute of Technology, Sathyamangalam, for providing the necessary facilities to perform the experiments in the Department of Biotechnology.

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Chapter 2

Endophytic and marine fungi are potential source of antioxidants

Ashish Bedi¹, Manish Kumar Gupta², Xavier A. Conlan³, David M. Cahill³ and Sunil K. Deshmukh¹*,    ¹TERI–Deakin Nano Biotechnology Centre, The Energy and Resources Institute, New Delhi, India,    ²SGT College of Pharmacy, SGT University, Gurugram, India,    ³Deakin University School of Life and Environmental Sciences, Waurn Ponds, VIC, Australia*, Corresponding author. Email: [e-mail]

Abstract

Antioxidants are substances that interact both inside and outside of a biological system in protecting against the damaging effects of highly reactive free radicals produced during metabolism. Among the various natural alternative sources of antioxidants, endophytic and marine fungi are good sources of potential antioxidant compounds. Fungi are prolific producers of a broad range of compounds including phenols, perylene derivatives, quinines, coumarins, terpenoids, peptides, polyketides, flavonoids, xanthones, and some of them possess potent scavenging activity. This review compiles the antioxidants reported to have come from endophytic fungi and marine fungi associated with algae, sponges, marine sediments, and other sources discovered during the period from 2013–20 (up to April) using bibliographic databases such as Sci-finder, Scopus, and Google scholar. A total of 224 compounds are reported in this review, out of which 82 present novel antioxidant activity. The scavenging capacity of these compounds against different free radicals is briefly described. Some details, such as host, producing fungi, sources, place of collection, and the antioxidant potentials of these compounds are cataloged in this review. This review aims at displaying some of the novel bioactive compounds isolated recently from endophytic and marine fungi and their potential applications as possible antioxidant candidates.

Keywords

Antioxidant; endophytes; marine fungi

Introduction

Free radicals are primarily produced under stressful conditions and may activate deleterious reactions within the human body. As a result, there are a range of degenerative chronic diseases that can be attributed to oxidative damage, such as atherosclerosis, cancer, asthma, diabetes, and inflammatory joint disease (Florence, 1995). Oxidative damage has also been implicated as the primary factor in suppression of physiological functions that occur during aging. Antioxidants are the molecules that protect call membranes and prevent oxidative stress to the body tissues by neutralizing toxic oxidant molecules and free radicals (Wickens, 2001). Antioxidants are also important for their protective effect against food spoilage and at present several synthetic antioxidants are used in the food industry. Since synthetic compounds are less desirable due to safety concerns, natural antioxidants have been widely investigated for food. The major source of natural antioxidants are fruits, vegetables, mushrooms, spices, and medicinal plants. Filamentous fungi represent an uncommon source of novel entities with diverse bioactivity. The dimension of biosynthetic gene clusters in a single filamentous fungal genome coupled with the historic number of sequenced genomes suggests that the secondary metabolites of filamentous fungi are mostly unexplored (Keller, 2019).

Fungi are multifaceted microorganisms and are the largest group among living organisms after insects. In the year 1990 it was estimated that no less than 1.5 million fungi were in existence and out of these approximately 74,000 species were fully described, that is, c. 5% only (Hawksworth, 1991). By 2017 it was reestimated that the number is around 2.2 to 3.8 million species and out of these approximately 120,000 (i.e., c. 8%) species are now described (Hawksworth and Lücking 2017). Filamentous fungi are well-known producers of a wealth of bioactive metabolites with pharmacological and agricultural applications. In contrast, endophytic fungi are less explored for obtaining novel bioactive metabolites. An endophyte is an endosymbiont, often a bacterium or fungus, that lives within a plant for at least part of its life cycle without causing apparent disease.

Endophytic fungi are prolific producers of biological active compounds belonging to different classes, which include phenols, perylene derivatives, quinines terpenoids cytochalasins, steroids, xanthones, and peptides, (Deshmukh et al., 2015, 2018a, 2019; Gunatilaka, 2006; Gupta et al., 2020; Kharwar et al., 2011). These biologically active compounds possess various pharmacological properties such as anticancer (Verekar et al., 2014, Bedi et al., 2018), antibacterial (Deshmukh et al., 2015), antimycobacterial (Verma et al., 2011), antifungal (Deshmukh et al., 2018b), antiinflammatory (Deshmukh et al., 2009), antidiabetic (Zhang et al., 1999), antiviral (Zhang et al., 2016), antioxidant (Sritharan et al., 2019), enzyme inhibitors (Chen et al., 2013), α-glucosidase inhibitory activity (Xiao et al., 2016), and immunosuppressive agent (Ariefta et al., 2019). The other metabolites include compounds of agricultural importance such as those with antifungal (Wang et al., 2013a; Chapla et al., 2014a) and insecticidal capacity (Kusari et al., 2012).

Marine fungi are another rich source of novel entities or drug molecules from a broad range of chemical classes, such as alkaloids, chromones, coumarins, peptides, polyketides, and terpenes (Deshmukh et al., 2018c, 2020, Sun et al., 2019; Youssef, et al., 2019, Zain ul Arifeen et al., 2020, Carroll et al., 2020), and that have a wide range of biological properties such as antibacterial, antiviral, and anticancer activity. Though many metabolites are reported from marine fungi only a few bioactive metabolites have been analyzed for their radical scavenging potentials, with very limited information available on the fungal diversity, biological targets, or mode of action (Agrawal et al., 2018). Marine fungi are not only important from the perspective of obtaining novel bioactive metabolites but also as a potential source of new molecular scaffolds that can be modified further to obtain the desired action (Fig. 2.1).

Figure 2.1 Percentage of antioxidant compounds reported from different endophytic and marine fungi.

Antioxidants from natural sources such as endophytic and marine fungi can deliver as alternatives to the synthetic antioxidant compounds, usage of which is discouraged due to their harmful side effects and lower bioavailability. A total of 224 compounds are reported in this review, out of which 82 are novel compounds with antioxidant activity from endophytic fungi along with marine fungi associated with algae, sponges, marine sediments, and some other sources discovered during the period 2013–early 2020. The compounds reported in this study are arranged based on their origin, chemical structure, and efficacies.

The total number of compounds, as well as novel compounds obtained from endophytic and marine fungi, in this study is depicted in Fig. 2.2. Antioxidant compounds isolated from fungi associated with terrestrial plant, mangroves, marine algae, marine sediments fungi, and fungi from other marine sources are given in Table 2.1 and the chemical classes of these bioactives are depicted in Table 2.2. An attempt was made to review the recent developments, for increasing the production of bioactive compounds using OFAT, OSMAC epigenetic modification, and the cocultivation method. The bulk of the work in this area has been completed on terrestrial fungi which lends itself well as a starting point for our discussion

Figure 2.2 Novel antoxidents reported from endophytic and marine fungi.

Table 2.1