Next Generation Biomanufacturing Technologies

Next Generation Biomanufacturing Technologies
ACS Symposium Series1329
	Next Generation Biomanufacturing Technologies
		Library of Congress Cataloging-in-Publication Data
Foreword
Preface
Functional Approach for the Development and Production of Novel Extreme Biocatalysts
Cyanobacterial Cell Factories for Improved Carotenoid Biosynthesis through a Synthetic Biology Approach
Yeasts as Microbial Factories for Production of Recombinant Human Interferon Alpha 2b of Therapeutic Importance
Advances in Plant Based Biologics
Bacterial Cell Surface Display
Rational Design of Next-Generation Therapeutic Antibodies Using Protein Engineering Tools
Material and Methods of Bacterial Sensing in the Process of Pharmaceutical Biomanufacturing
Functional Oligosaccharides: Production and Action
Synthetic Biology and Metabolic Engineering Approaches for Improved Production and Recovery of Bacterial Polyhydroxyalkanoates
Electrospinning: An Efficient Biopolymer-Based Micro- and Nanofibers Fabrication Technique
New Trends in the Biomanufacturing of Green Surfactants: Biobased Surfactants and Biosurfactants
Sequestering of CO2 to Value-Added Products through Various Biological Processes
Characteristics and Applications of Biodiesels and Design of Reactors for Their Industrial Manufacture
Microbial Biofilm Membranes for Water Remediation and Photobiocatalysis
Editors’ Biographies
	Indexes
Indexes
Author Index
Subject Index
Preface
	1
Functional Approach for the Development and Production of Novel Extreme Biocatalysts
	Introduction
		Figure 1. Scheme for the development of a new enzyme product. Inside the black rectangle is a summarized scheme of the functional approach. (Photos courtesy of Jenny M. Blamey and Sebastián Muñoz I.)
	Biocatalysts
	Extremophiles and Extremozymes
	Functional Approach
	Environmental Sampling
	Screening and Enzymatic Activity
	Purification and Enzymatic Characterization
	Cloning and Expression of Recombinant Enzymes
	Production of Recombinant Enzyme
	Optimization
	Equation 1. Model of Behavior for DoE
		Figure 2. Response of Surface graphic. 3D Plot of Response of Surface Method. Results for the optimization of pH and temperature values to maximize obtention of biomass.
	Scale-Up
	Equation 2. Mass Balance for Oxygen Accumulation in a Reactor
	Downstream Processing and Purification
	Quality Control Points in the Process
		Figure 3. Quality control points in the enzymatic production process. Proposed quality control points to determine the validity of the process. (Abbreviations: QCP: Quality Control Point; Bio: Biomass; Ct: contamination; Enz: Enzymatic activity; Prot: Protein concentration.)
	Comparison of Native and Recombinant Enzyme Production
	New Trends
	Conclusions
	Acknowledgments
	Author Contributions
	References
		2
Cyanobacterial Cell Factories for Improved Carotenoid Biosynthesis through a Synthetic Biology Approach
	Background
		Figure 1. Schematic representation of the one cell–two wells biorefinery approach, whereby cyanobacterial cells can be engineered for production of carotenoids and leftover or spent biomass could serve as a C source for production of energy molecules.
	Global Carotenoid Market: Scope and Applications
		Figure 2. Applications of carotenoids: Carotenoids are naturally synthesized by the host cells, especially as antioxidants. These properties could be harnessed at the industrial level by supplementing them in food, pharmaceuticals, or cosmetics to improve product quality and shelf life and to exhibit antioxidant functions.
	Cyanobacterial Photosynthesis, Pigments, and Photoprotection
		Figure 3. Cyanobacterial photosynthetic assembly. Illustration of cyanobacterial photosynthetic assembly composed of a phycobilisome complex forming antenna pigments and a thylakoid membrane–associated photophosphorylation machinery composed of PS I and PS II. Subcellular localization and movement of these complexes help in state transition during light stress, increasing concentration of reactive oxygen species and such. During state transition, phycobilisome complex, which is principally associated with PS II operating noncyclic photophosphorylation, apportions to PS I assembly, thereby increasing the rate of cyclic photophosphorylation. This not only drains off the blocked electron carriers but prevents unwanted reactive oxygen species synthesis and degradation of the assembly.
		Figure 4. Cyanobacterial cells perform both photosynthesis as well as respiration in the same cellular compartment, where they efficiently share the electron carriers, namely PQ, plastoquinone; Cyt b6f, cytochrome b6f; PC, plastocyanin; Fd, ferridoxin. The figure also illustrates that a few of the electrons (~30%) transferred during their transition between Fd and NADP, nicotinamide adenine dinucleotide phosphate, are utilized for photosynthetic nitrate assimilation into amino acids. The bidirectional arrow between SDH, succinate dehydrogenase, and PQ indicates reversible reaction. PBS, phycobilisome complex; ChlA, chlorophyll reaction centers; Q, quinone.
	Carotenoids: Biochemistry and Distribution
	Pathways for Carotenoid Production
	MVA Pathway
		Figure 5. Carotenoid biosynthesis pathway. Carotenoid biosynthesis is a three-step process: (A) Ubiquitous glycolysis pathway synthesizes precursors for isoprenoid synthesis: PGAL (phosphoglyceraldehyde), pyruvate, acetyl-CoA, and acetoacetyl CoA. (B) Isoprenoid synthesis occurs through the MVA or MEP pathway, which ultimately culminates into isopentenyl diphosphate and dimethylallyl diphosphate, which fuel terpenoid synthesis and phytoene. (C) Phytoene is the preliminary molecule for carotenoid backbone synthesis. The diagram presents a schematic representation of metabolic pathways involved with biosynthesis of carotenoids. Genes present in cyanobacteria are represented as colored dots ( represents genes from PCC 7002, represents genes from CC 6803, and represents genes from PCC 7942).
	1-Deoxy-D-Xylulose 5-Phosphate /MEP Pathway
	Carotenoid Biosynthesis
	Engineering Cyanobacteria for Carotenoid Production
	Synthetic Biology Approaches in Cyanobacteria
		Figure 6. Schematic representation of cyanobacterial synthetic biology toolbox. (A) Genome editing tools deal with actual cellular modification and analyze the effect of various engineering tactics for improved synthesis of the desired product. (B) Predicting toolbox can be classified as simulative predictions where computer-based software and mathematical models assist in predicting cellular functionality upon genetically engineering, and experimental predictions help in understanding in situ metabolic states when the cell is subjected to different environmental conditions. Combined use of genome editing and predicting tools could give a complete view toward development of efficient cyanobacterial platform factories.
	Conventional Molecular Engineering Approaches To Improve Carotenoid Biosynthesis in Cyanobacteria and Case Studies
	Zeaxanthin
	Astaxanthin
	β-Carotene
	Cyanobacterial Scale-Up, a Major Bottleneck in Valorizing Cyanobacteria
	Conclusion
	References
		3
Yeasts as Microbial Factories for Production of Recombinant Human Interferon Alpha 2b of Therapeutic Importance
	Introduction
		Figure 1. Synthesis and biological actions of human IFNα2b through JAK-STAT pathway.
	Biological Route for the Production of Recombinant huIFNα2b
	Recombinant huIFNα2b Production through Yeast Platform—An Overview
	S. cerevisiae
	P. pastoris
	Y. lipolytica
	Fermentation Strategies for Yeast Expression Systems in huIFNα2b Production
	Batch Fermentation Studies
	Fed-Batch Fermentation Studies
	Significance of Post-translational Modifications on huIFNα2b
		Figure 2. Differences in glycosylation (post-translational modification) of recombinant protein in different yeast platforms.
	Conclusions and Future Prospects
	References
		4
Advances in Plant Based Biologics
	Introduction
	Why Are Plants Considered the Alternate Expression Host?
	Production of Protein in Plants
		Figure 1. The overview of plant biopharming technology (laboratory scale) A. Construction of expression clones—to mobilize into Agrobacterium cells for the agroinfiltration. B. Seedlings of Nicotiana benthamiana. C. Well-grown (3–4 weeks old) N. benthamiana plantlets. D. Syringe infiltration of GFP construct and visualization of GFP under UV after 5–6 days of agroinfiltration. E and F. Vacuum infiltration setup. G. Vacuum-infiltrated N. benthamiana plant. H. Infiltrated plant maintained in the controlled environment for 4–6 days, followed by harvesting of agroinfiltrated leaves. I. Clear plant leaf lysate was prepared with suitable buffer and subjected for the affinity column chromatography for purification. The purified product will be confirmed primarily by SDS-PAGE and western blot, and the protein specific in vitro and in vivo experiments will be conducted to validate the activity. Adapted with permission from reference 13. Copyright 2017 Defence Scientific Information & Documentation Centre.
		Figure 2. Overview of a plant expression system for commercial production. Adapted with permission from reference 13. Copyright 2017 Defence Scientific Information & Documentation Centre.
	Yield-Enhancing Strategies
	The Genetic Engineering Approach To Combat Glycomodifications
	Success Stories of Plants as Expression Hosts
	Plant-Made Veterinary Vaccines
	Influenza Vaccine
	ZMapp
	Status of Plant Based Products on the Market
	Regulatory Challenges To Overcome
	Conclusion
	References
		5
Bacterial Cell Surface Display
	Introduction
	Strategies Used in Bacterial Cell Surface Display
	Carrier Protein
	Passenger Protein
	C-Terminal Fusion
	N-Terminal Fusion
	Sandwich Fusion
	Gram-Negative Bacterial Cell Surface Display
	Outer Membrane Proteins
		Figure 1. Different types of carrier proteins found on the surface of Gram-negative bacteria.
	OmpA
	OmpC
	LamB
	Lipoproteins
	Hybrid Lipoproteins
	Ice Nucleation Protein
	Autotransporters
	S-Layer Protein
	Surface Appendages
	Flagella
	Fimbriae
	Pili
	Secreted Proteins
	Heterologous Proteins
	Systems Based on Virulence Factors
	Gram-Positive Cell Surface Display Systems
		Figure 2. Different types of carrier proteins found on the surface of the Gram-positive bacteria.
	Membrane Associated Proteins
	Proteins Covalently Associated with Cell Wall
	Proteins Noncovalently Associated with Cell Wall
	1) CWBD1 Proteins
	2) CWBD2 Proteins
	3) LysM Proteins
	4) GW Proteins
	5) S-Layer
	Display on Spores
	Non-recombinantly Displayed Proteins on Bacterial Spore Surface
	Display on Gram-Positive Enhancer Matrix Particles
		Figure 3. Schematic diagram showing surface display using GEM particles. (A) Expression vector with protein of our interest (passenger/target protein) translationally fused with the carrier protein containing anchor domain. (B) The plasmid is transformed into a live host and is expressed. The carrier protein helps it to get displayed on the surface, but due to low binding capacity very few proteins bind, and most remain in solution. (C) Live bacteria (nongenetically modified) boiled with the mentioned acids, after the treatment only the PGN matrix will be left. The heat treatment enhances the binding capacity of cell surface. (D) When the heat-treated bacteria are incubated with the purified fused protein secreted by bacteria in (B). Protein gets attached to the entire surface of the treated bacteria, due to high binding affinity.
	Display on Bacterial Ghosts
	Applications
		Figure 4. Applications of bacterial cell surface display.
	Development of Live Bacterial Vaccines
	Biocatalysis
	Bioremediation
	Biosensors
	Screening of the Polypeptides from Their Libraries
	Biohydrometallurgy
	Bioadsorption
	Study of Ligand Receptor Binding Interactions
	Advantages of Bacterial Cell Surface Display
	Conclusions and Future Perspectives
	Acknowledgments
	References
		6
Rational Design of Next-Generation Therapeutic Antibodies Using Protein Engineering Tools
	Introduction
	Methods for Protein Engineering
	Rational Designing: Site-Directed Mutagenesis
	Evolutionary Method: Random Mutagenesis (Error-Prone PCR)
	Computational Methods
	Antibody Engineering
		Figure 1. Schematic representation of the structure of monoclonal antibody and next-generation different engineered antibody formats. Constant regions, part of the fragment crystallization region of the antibody, are shown in blue rectangles. The fragment antigen-binding region consists of a heavy chain (HC) and light chain (LC). Further, each HC and LC consists of a variable domain and constant domains, shown with oval and rectangular boxes. The variable regions are composed of three complementarity-determining regions and four framework regions. These variable regions are not shown on other antibody formats for simplicity. DART: Dual-affinity re-targeting antibody.
	Purposes of Antibody Engineering
		Figure 2. Different formats of engineered antibodies. BiTe: bispecific T cell engager; DART: Dual-affinity re-targeting antibody.
	Reduced Immunogenicity
	Chimeric Antibodies
	Humanized Antibodies
	De-Immunization
	Better Pharmacokinetics
	PEGylation
	Multimerization
	Glycosylation
	Engineering Other Effector Functions of Antibodies
	Optimizing Antigen-Binding Domains for Better Efficacy
	Manufacturability
	Antibody Fragments
	Single-Chain Antibodies and Their Variants
	Single-Domain Antibodies or Nanobodies
	Bispecific Antibodies
	Intrabodies
	Antibody–Drug Conjugates
	Antibody-Like Proteins
	Antibody Display Libraries
	Phage Display
		Figure 3. Overview of antibody library screening using a phage display technique.
	Yeast Display
	Expression Systems for the Development of Engineered Therapeutic Antibodies
	Bacteria
	Escherichia coli
	Lactococcus lactis
	Other Bacteria Expression Systems
	Yeast
	Mammalian Cells
	Plants and Green Algae
	Applications of Engineered Therapeutic Antibodies
	Cancer
	Autoimmunity and Inflammation
	Bacterial, Fungal, and Parasitic Diseases
	Conclusion and Future Prospects
	Acknowledgments
	Conflict of Interest
	Authors’ Contributions
	References
		7
Material and Methods of Bacterial Sensing in the Process of Pharmaceutical Biomanufacturing
	Background
		Scheme 1. Recombinant protein production. Using recombinant DNA techniques, the target human gene can be isolated and ligated to a vector (plasmid). The plasmid containing the human gene is used to transform bacterial cells, which are able to produce high amounts of the recombinant protein. Adapted with permission from reference 8. Copyright 2016 Sociedade Brasileira de Microbiologia.
		Scheme 2. The biopharmaceutical manufacturing technology flowchart exemplifying the upstream and the downstream bioprocess. Adapted with permission from reference 8. Copyright 2016 Sociedade Brasileira de Microbiologia.
	Problems Associated with Lack of Sensing Methods for Bacterial Growth in Biomanufacturing Processes
	Advanced Bacterial Sensing Methods
		Scheme 3. Different types of sensors based on various characteristics used during the process of sensing.
	Role of Material Type and Sensing Efficiency
	Molecular Materials
	Polymers
		Figure 1. Scheme representations of PNIPAm solutions above or below LCST and their structural changes. The function of graphene nanoplatelets in delaying LCST and causing change in the electronics of PNIPAm-GR substrate is also depicted. (A) PNIPAm with chemical structure in sol state (LCST), (B) PNIPAm in sol state with doped GRs and E. coli. (C) Paper-based biosensor chip with PNIPAm-GR prepared and stored at 4°C (