Next Generation Biomanufacturing Technologies

There is a growing interest in replacing chemical catalysts used in several industrial processes with more efficient and environmentally friendly alternatives. To this end, enzymes represent highly efficient biocatalysts that allow for a decrease in overall production costs and environmental pollution. However, one of the biggest challenges to overcome in successfully using biocatalysts instead of chemical catalysts is to find stable and active biocompounds at the various harsh conditions at which industrial processes usually take place. Microorganisms living in extreme environments have proven to be a valuable source of highly efficient and stable biomolecules. These special types of biocatalysts have adapted to withstand extreme conditions, producing low amounts of by-products under a wide range of conditions, and thus are of paramount importance in industrial applications. A good strategy to finding new enzymes as potential biocatalysts is the functional approach. This approach seeks the discovery of extremophiles and their enzymes in extreme environments that display similar conditions to the industrial process in which the biocatalysts are required. After the desired enzyme is obtained, a recombinant version of the enzyme is produced in order to avoid the low biomass and protein yields when working with native enzymes from extremophiles. To obtain enough biomass for protein purifications and downstream analyses, the process must be optimized and scaled up, using quality control points to produce efficient and reproducible enzyme products. This functional approach has been successfully used to develop enzyme products for the market.

Cyanobacteria are photosynthetic prokaryotes. Owing to their efficient photosynthesis ability over other photoautotrophs, simple cell organization, and competency for molecular modification, they have been widely exploited as “green-factories” for production of value-added compounds like carotenoids. Carotenoids are industrially important fine chemicals used in food, pharmaceutical, and cosmetic products. Cyanobacteria, being photosynthetic, can naturally synthesize carotenoids as cellular antioxidants. However, successful demonstrations have been made by the researchers to improve their carotenoid content through genetic engineering. Cyanobacteria are considered low-input, high-output hosts, where such a pathway engineering approach leverages their significance for the commercial markets. This chapter focuses on two important aspects of cyanobacteria as a next-generation resource for biomanufacturing: the significance of carotenoids in cyanobacterial photosynthesis and synthetic biology attributes in the area of cyanobacterial genome editing and engineering them for improved carotenoid yields for commercial exploitation.

Recombinant proteins of therapeutic importance occupy a significant segment of the pharmaceutical industry. Among the type I interferons, interferon alpha 2b (huIFNα2b) is a therapeutic cytokine with the highest commercial footprint possessing antiviral, anti-proliferative, and immunomodulatory functions. It is used in many countries for the treatment of viral diseases such as hepatitis B and C, and some types of cancer such as hairy cell leukemia, melanoma, and AIDS-related Kaposi sarcoma. Different microbial cell factories have been explored for the production of huIFNα2b, and yeasts have gained much attention due to their unique combination of unicellular and eukaryotic assets. The former is displayed in their ease of genetic manipulation and high cell density growth, while the latter is evidence of their capacity to carry out complex post-translational modifications. Even among the yeasts, only three, namely, Saccharomyces cerevisiae, Yarrowia lipolytica, and Pichia pastoris, have been used for the production of huIFNα2b. The present chapter discusses the advantages and disadvantages of different microbial systems for huIFNα2b production with special emphasis on yeasts as expression hosts. Different fermentation strategies of yeast cultivation for efficient production of huIFNα2b are also discussed. Finally, the influence and importance of post-translation modifications on huIFNα2b are emphasized, highlighting the differences in glycosylation pattern among S. cerevisiae, Y. lipolytica, and P. pastoris.

Plants are a potential alternative for the production of commercially important proteins that can be achieved either by stable or transient gene expression. Conventionally, recombinant vaccines are mass produced using mammalian or Escherichia coli-based gene expression systems. However, capital investment warrants a huge cost and requires highly skilled labor, which makes the final product expensive. Being simple, robust, safe, scalable, and cost-effective are the primary reasons the plant expression system is now considered to be a potential platform. Stable transgenics was believed to be the only way to express the transgene in the plants until transient gene expression was introduced. Transient expression in plants is carried out by introducing the recombinant Agrobacterium cells into the intracellular spaces of mature plant leaves by the agroinfiltration technique. The autonomous self-replicating plasmids in Agrobacterium control the plant metabolism and start expressing its cassette and saturate it with the recombinant protein within 4–6 days postinfiltration. However, plants regulate the Agrobacterium and extraneous gene elements by si/miRNA-mediated silencing, posttranslational gene silencing, homology-dependent gene silencing, and so on. In the past two decades, plant high-expression strategies were improved by incorporating viral gene-silencing elements, viral gene regulatory elements, MagnICON technology, and in-plant activation constructs. Another milestone in plant molecular biopharming is the expression of glycoengineered complex mammalian recombinant glycoproteins. Advances in plant molecular farming resulted in several therapeutic products in the pipeline that will be discussed in detail.

Bacterial cell surface display is a widely used technique for expressing heterologous proteins on the surface of Gram-positive and Gram-negative bacterial cells. The chapter describes the various proteins present on the surface of bacteria and their domains which are used for constructing cell surface display systems. The various carrier proteins used and passenger proteins displayed have been described in detail. The applications of bacterial cell surface display system for bioremediation, live vaccine, biosensors, hydro metallurgy, and so forth have been discussed. Since bacterial cell surface display systems are constructed using recombinant vectors, they cannot be used for environmental applications. Bacterial ghosts and Gram-positive enhancer matrix (GEM) particles have been described as attractive alternatives to live cell-based systems for surface display of proteins.

Protein engineering holds immense potential ranging from biocatalyst engineering for improving enzyme stability to drug development and diagnosis. Protein biotherapeutics has gained momentum in recent years due to the enormous progress in the fields of high throughput screening and genetic engineering. This is evident from the rapidly expanding list of protein therapeutics in drugs approved by the U.S. Food and Drug Administration. With the recent innovations in the field of protein engineering and computational tools, we can now design better antibodies with improved efficacy and stability, as well as better pharmacokinetic and pharmacodynamic properties. These so-called “next-generation antibodies” are designed rationally to be more specific and more potent than the traditional monoclonal antibodies and include engineered monoclonal antibodies, antibody drug conjugates, nanobodies, bispecific antibodies, antibody fragments, and antibody-like proteins. The engineered antibody production systems are also advancing with developments in the protein expression and purification process. In this chapter, we focus on the application of protein engineering tools for the development of engineered therapeutic antibodies and discuss the latest developments in the generation of chimeric, humanized, and fragment antibodies, antibody display libraries, and the applications of these next-generation antibodies in therapeutics.

The final yield percentage of product holds the maximum stake in the success of any pharmaceutical process. Similarly, steps to support pharmaceutical biomanufacturing and discovered blocks in progress require immediate corrections and improvements to avoid economical losses related to the lowering of yield and product quality. A robust sensing system for various components of pharmaceutical scavenging can lead the way. Sensing of population abundance, morphological changes, and possible contamination in microorganisms used for pharmaceutical biomanufacturing are some of the important components that require constant attention. Use of bacterial cells as manufacturing units in various pharmaceutical products including antibiotics, antitumor agents, immunomodulators, enzyme inhibitors, coccidiostatic agents, nematicides, and insecticides makes it further urgent to understand it in detail. This chapter is presenting the material and methods that can influence the improvement of bacterial sensing in the process of pharmaceutical biomanufacturing. This chapter includes various methods and the role of material types in the sensing of bacterial population.

Functional oligosaccharides (FOs) are indispensable components in food, feed, pharmaceuticals, cosmetics, and nutraceuticals preparations. They are polysaccharides containing the degree of polymerization of 2–10, not digested (or only partially digested) by gut enzymes, having no or less caloric values, stable at acidic pH, thermostable (>50 °C), and intact in bile salts. Consequently, FOs are neither digested nor degraded in the upper alimentary canal and reach the large intestine of the host. FOs confer several health benefits, such as proliferation of probiotic microbes, elimination of pathogen colonization, enhancement of mineral absorption, evacuation of heavy metal ions by increasing bowel movements, and selective proliferation of specific probiotic microbes in the gut. FOs can be categorized as sucrose based (fructo-oligosaccharides), lactose based (galacto-oligosaccharides and lacto-sucrose), starch related (isomalto/malto-oligosaccharides, trehalose, and cyclodextrins), soy-based oligosaccharides, and non-starch based (xylo/arabino-oligosaccharides, and pectin- and chitosan-based oligosaccharides). FOs are produced by chemical, enzymatic, and chemoenzymatic techniques, but the enzymatic method is preferred because it produces specific FOs under mild, environmentally friendly conditions. FOs are purified and characterized using membrane filtration, chromatography, and spectroscopy. The prebiotic function of FOs in the host’s health has been studied using animal models and a simulated human intestinal microbial ecosystem.

Polyhydroxyalkanoates (PHAs) are polyesters produced in bacterial cells as inclusions and have attracted much scientific and commercial interest as a sustainable alternative to synthetic plastics. Even though PHAs have polymer properties similar to their synthetic counterparts, their commercialization is still in progress. The high cost of production and unstable polymer properties are the main hurdles obstructing the widespread use of these “green polymers.” Efforts to synthesize high-quality PHAs cost effectively have been ongoing for decades, and tremendous progress has been reported. Synthetic biology and metabolic engineering approaches have proved to be a promising strategy for the improvement of PHA-producing microbial systems, and this chapter reviews the significant advancements made in the area of bacterial PHA production through these approaches.

Electrospinning, an electrostatic fiber fabrication method, has emerged as an invincible technique for the production of micro- to nanofibers-based polymeric scaffolds. It has gained significant attention and interest in recent years because of its simplicity, scalability, versatility, and potential application in soft-tissue engineering, biosensors, wound dressings, drug delivery, filtration, and enzyme immobilization. Polymeric solution under the influence of a strong electric field produces fibers of nanoscale dimension. The polymeric scaffold and nonwoven nanofibers produced by electrospinning more closely mimic components of extracellular matrix as compared with conventional methods. Submicron-range electrospun fibers manufactured by the electrospinning process offer unrivaled advantages such as high surface-area-to-volume ratio, tunable porosity, and the ability to sway nanofibers composition to obtain anticipated property and function accompanying tailored morphology and orientation. This chapter aims to briefly discuss the basic concepts of electrospinning and its potential application along with solvent properties, solution parameters, and processing conditions, which considerably affect the morphology and orientation of produced nanofibers. This chapter also intends to highlight advancements, challenges, and various applications along with future prospects of the fabricated natural biopolymer-based electrospun scaffolds produced by this invincible technology.

This chapter deals with current trends in the biomanufacturing of potential, environmentally friendly surface active compounds known as green surfactants. The high manufacturing cost is a major challenge in the commercialization of green surfactants, despite their advantageous properties over synthetic surfactants. This chapter focuses primarily on the green manufacturing of biobased surfactants and biosurfactants. Various strategies and promising developments in the industrial-scale production of green surfactants are also discussed. This discussion includes process-modification technologies and microbial strain improvement methods. Medium optimization techniques, solid-state fermentation using low-cost renewable substrates, and the use of nanotechnology and downstream processing are discussed under the umbrella of process-modification strategies, whereas genetic alteration processes such as various mutagenesis methods and recombinant DNA technology are covered under genetic modification approaches. Finally, the various applications of these commercially important green surfactants in different fields are discussed.

Global warming is one of the major consequences of anthropogenic and industrialization activities, which have caused a substantial increase in the concentration of CO₂ in the atmosphere. This increase in CO₂ concentration has caused measurable global warming and created adverse effects on the ecosystem. This could be resolved by using several technologies—such as chemical catalysis, along with photochemical, electrochemical, biological, bioelectrochemical, and inorganic transformation technologies—by utilizing CO₂ for the production of fuels and value-added products. This chapter discusses the different biological systems, such as photosynthesis, bioelectrosynthesis, and gas fermentation, for CO₂ sequestration. The study also discusses the mechanisms involved in the photosynthesis processes and their metabolic pathways, which regulate the CO₂ in value-added products. Bioelectrosynthesis uses the inorganic compounds through a series of microbially or enzymatically catalyzed reactions in a fuel cell setup for the production of fuels and chemicals by applying external potential. Gas fermentation is an up-and-coming technology, which uses syngas as a substrate by enriching the microorganisms in a moderate temperature and low pressure for the production of low-carbon fuels and synthesis of commodity chemicals.

The potential and applications of biodiesels as the green fuels of the century are summarized. The chapter also discusses choice of raw materials and catalysts for biodiesel synthesis (homogenous and heterogenous catalysts), kinetics of the transesterification process and kinetic models, and design and analysis of reactors for biodiesel manufacture, such as stirred tank, fluidized bed, semifluidized bed, diverging–converging fluidized bed, and inverse fluidized bed reactors. The performance analysis and special characteristics of each of these reactors have been highlighted. Scope and recommendations for future research are also presented.

Biofilms are ubiquitous and the consequence of extracellular polysaccharide secretions by single or multiple microbial populations on an active surface. They are one of the major contributors to biofouling and membrane failure in current membrane-based separation processes. However, biofilm-based systems have also been recognized as aiding in remediation instead of hindering the performance via biosorption and biotransformation of pollutants present in water. This chapter highlights developments in engineered microbial-biofilms-based membrane reactors to bioremediate persistent contaminants like oxyanion species and heavy metal ions.