Omics Technologies and Bio-engineering: Volume 1: Towards Improving Quality of Life

Records.

Part I

Emerging Fields

Outline

Chapter 1 Overview and Principles of Bioengineering

Chapter 2 Omics Approaches Towards Transforming Personalized Medicine

Chapter 3 Omics Approaches in Marine Biotechnology

Chapter 4 Synthetic Biology

Chapter 5 Reverse Engineering and Its Applications

Chapter 6 Omics Approaches in Forensic Biotechnology

Chapter 7 Biotechnology and Bioengineering in Astrobiology

Chapter 8 Lab-on-a-Chip Technology and Its Applications

Chapter 9 Robotics and High-Throughput Techniques

Chapter 10 3D Printing Technologies and Their Applications in Biomedical Science

Chapter 11 Next-Generation Sequencing and Data Analysis

Chapter 12 Computational Techniques in Data Integration and Big Data Handling in Omics

Chapter 13 Bioinformatics and Systems Biology in Bioengineering

Chapter 1

Overview and Principles of Bioengineering

The Drivers of Omics Technologies

Dinesh Yadav, Aiman Tanveer, Neha Malviya and Sangeeta Yadav,    D.D.U Gorakhpur University, Gorakhpur, India

Abstract

The science of omics reflects the innovations in diverse technologies leading to studies of life processes in totality. With the initial emergence of genomics, proteomics, and metabolomics associated with the comprehensive studies of genes, proteins, and metabolites of an organism, respectively, there has been a substantial growth in other branches of omics such as lipidomics, cytomics, phenomics, and transcriptomics. The omics-driven science has a potential application in crop-improvement program, human health sector, environmental sector, and industrial sector. The recent developments in genome sequencing have further expanded the scope of the omics technologies for elucidating the complexity of life processes and understanding the gene expression and regulation in diverse systems. The concept of bioengineering is more relevant to the human health and is often referred to as biomedical engineering. In general, it covers tools and technologies associated with the manipulation of life processes for the benefit of the humankind. In biological system, the central dogma of molecular biology, i.e., replication, transcription, and translation, is the main center of attraction for manipulation using appropriate tools of omics. This chapter highlights the basics of science of omics along with relevant tools and diverse applications.

Keywords

Omics; genomics; transcriptomics; proteomics; metabolomics; glycomics; bioinformatics; bioengineering

1.1 Introduction

1.1.1 Science of Omics

Innovations in technologies have led to the better understanding of the principles of bioengineering by providing potential tools for comprehensive studies. Omics in general reflects totality and, when added as suffix to any specific scientific field, provides an insight with a broader spectrum (Malviya et al., 2016). Genomics is one of the earlier established sciences of omics for comprehensive analysis of the genome, once the whole-genome sequence of an organism is deciphered. Though several omics exists (as mentioned in Table 1.1), genomics, proteomics, metabolomics, transcriptomics, and glycomics are the major branches of omics (Fig. 1.1).

Table 1.1

Brief Description of Different Branches of Science of Omics

Figure 1.1 Bacterial cell showing interplay between the different branches of omics along with the relevant tools and their involvement in the bacterial gene flow.

The growth and by-products productivity, as accomplished by bioengineering, can be modulated by intervention of omics technologies. The tools of omics technologies have paved the way to bioengineering in in vivo as well as in cell cultures (Alyass et al., 2015).

1.2 Genomics

Genomics is a branch of science associated with a comprehensive study of genome of a particular organism. The innovations in sequencing technologies in the recent years have led to the sequencing of several genomes providing substantial importance to the genomics for better understanding of life processes. The genomics aims for

• Decoding the biological information from the whole-genome sequences

• Assessing the total number of genes present in an organism

• Elucidating organization of genes on the chromosomes

• Revealing complexity of genome with reference to distribution of different types of sequences

• Providing an insight into the regulatory sequences present in the genome

• Predicting putative functions of the genes

• Analyzing the genome-wide comparative studies of sequenced genome from evolutionary point of view

• Providing accessibility for genetic manipulation for desired features

• Better understanding of the central dogma of life

• Better understanding of gene expression and regulation

Bioengineering provides a means through which the entire genotype of the biological entity can be altered. Since genes carry all the genetic information stored in them, the genes can be easily moved from one organism to the other, to modulate the target organism depending on our need. Engineering a gene encompasses the basic theme of fragmenting the desired gene and reassembling into the organism of interest. This has proven to be of immense interest ranging from cloning of the gene in unicellular organism to gene therapy in humans. If we are interested in bioengineering of a gene, it can be manipulated inside a living bacterial host or other feasible cell lines. Bacterial cells are the most common host for genetic manipulation as they are easy to culture, relatively cheap to maintain, have faster rate of growth, and have a large number of mutant strains available for multidimensional study of the desired gene (Neus and Villaverde, 2013; Weeks Donald et al., 2016).

The most conventional method for bioengineering is genetic engineering. The basic strategies for production of recombinant proteins are as follows:

• Identification and isolation of the gene of interest

• Selection and designing of appropriate vector

• Ligation of gene of interest into suitable vector

• Transformation/transfection of recombinant clones in suitable host cells

• Selection of recombinants and optimization of transgene integration in host cell

There have been substantial developments at each step of cloning processes and polymerase chain reaction (PCR) is one major breakthrough technologies applied extensively for in vitro amplification of desired genes, provided the sequence information is available (Demain and Vaishnav, 2009). The important microorganisms explored as cell factories for the production of recombinant proteins using the genetic engineering approach along with their unique features and some products are summarized in Table 1.2 (Fisher et al., 2014).

Table 1.2

List of Microorganisms Used for Recombinant Protein Production

Innovations in sequencing technologies leading to deciphering of several genome sequences have expedited the science of genomics. The traditional method of DNA sequencing popularly known as first generation sequencing technique, proposed by Sanger (enzymatic, dideoxy, or chain termination method) and Maxam and Gilbert (chemical method), has undergone several modifications over the years increasing the speed, precision, and read length of the sequences. The automation of Sanger’s method was further upgraded as automated DNA sequencing method with a major change from gel-based to capillary-based analysis. The next-generation sequencing (NGS), which reflects the second, third, and fourth generation sequencing technologies, is currently very popular and highly efficient for genome sequencing. Some of the NGS techniques are pyrosequencing (Roche 454), ABI SOLiD, HeliScope, Ion Torrent PGM, Pacific Biosciences’ real-time single molecule sequencing (PacBioRS), combinatorial probe-anchor ligation (cPAL), Oxford Nanopore, etc. The benefits of advanced sequencing technologies include enhanced speed, accuracy, reduction in cost, increase read length, reduced amount of initial DNA required, less chance of contamination, automation, etc.

Microarray is a high-throughput technique for determination of expression level of large number of genes at a time or for whole-genome typing. Numerous DNA sequences known as probe or spots are attached to the solid surface. The DNA samples to be analyzed are labeled with fluorescent probes and washed against the DNA microarray chip. The complementary DNA sequences of the target bind to the DNA of the chip and are subsequently detected with the help of fluorescent detectors and other imaging techniques. In addition to this core method, there are several modifications of the microarray technique, depending upon its uses in different applications. Microarray can also be used for comparative analysis of different genomes by comparative hybridization. Specific microbes, such as pathogens or microbes in food, can be detected by GeneID microarray. Microarray followed by chromatin immunoprecipitation assay can be used in identifying the DNA sequence bound to any given protein. Single nucleotide polymorphism can also be typed with microarray. Most recent modifications are tiling array, double-stranded B-DNA microarrays, double-stranded Z-DNA microarrays, and multistranded DNA microarrays.

The tools of genomics can be applied in medical field for diseases diagnostics, gene therapy, and somatic cell therapy. In agriculture identification of potential genes associated with desirable agronomic traits can be done from the sequenced genomes and utilized in transgenic technology for developing biotic and abiotic stress-tolerant crops.

1.3 Transcriptomics

Transcriptomics is a comprehensive analysis of whole sets of transcripts for a particular cell, tissue, organ, or whole organism corresponding to a particular time or developmental stages or may be under some specific physiological conditions. Transcriptomics aims for the following:

• Functional annotation of the genome

• Providing an insight into transcriptional structure of the genes

• Deciphering transcriptional start site

• Elucidating splicing

• Understanding posttranslational modifications

• Cataloguing all types of transcripts (mRNA, tRNA, rRNA, siRNA, noncoding RNAs, etc.)

• Providing an insight into the complexity of development processes or to understand diseases

• Elucidating differentially expressed genes

It provides an overview regarding the genetic content and the regulatory system in the cell. The wild-type cells, whether bacterial cell or cell line, possess the same genome content. Based on different applications and conditions, different genes are expressed, resulting in different patterns of gene expression in different organisms. There are cellular regulatory machineries through which the gene expression can be switched on and off. Great deal of work has been performed on the eukaryotes as it contains both introns and exons which through splicing provides better spectrum of the total transcriptome. Trancriptome study of prokaryotes is lagging due to the absence of the 3′-end poly(A) tail, which is considered to be a signature of mature mRNA in eukaryotes. Data related to the expression level of the genes in the given genome, genome profiling, comparative expression levels between different experimental data sets, and effect of different parameters on gene expression can be assessed by the help of transcriptomics tools.

Commonly used techniques for transcriptome study are expressed sequence tag (EST)-based methods, SAGE, hybridization-based microarray, real-time PCR, NGS-based RNA-sequencing (RNA-seq) methods, RNA interference, and bioinformatics tools for transcriptomes analysis. The methodology basically involves RNA isolation, purification, quantification, cDNA library construction, and high-throughput sequencing. The important considerations for selection of tools of transcriptomics are cost effectiveness, sensitivity, high throughput, and minimal concentration of starting RNA.

Sequencing of the ESTs gives an overview regarding the expression level of the gene. Large-scale EST sequencing projects have been applied on the model organisms such as Arabidopsis, Drosophila, and human. EST databases are also available which provide reference for the expression profile. Although it is helpful in studying gene abundance and novel gene discovery, it is highly expensive and time-consuming technique. The cDNA or EST microarrays are available whereby cDNA sequences or ESTs are attached on the microchip. The technique is similar to DNA microarray, but the sample here are the fluorescently labeled cDNA molecules. Being a cost-effective and rapid technique, it helps in the study of relative abundance between the genes. SAGE is another method, which is similar to EST sequencing. It is advantageous over EST because only short tags of about only 15 bases are sequenced. The short fragments generated are then joined together and sequenced. A pool of cDNA can be subjected to high-throughput NGS known as RNA-seq for quantification, discovery of novel ESTs, and profiling of RNAs.

DNA chip, gene chip, biochip, or microarray is a collection of DNA, cDNA, oligonucleotides spots attached to a solid support such as glass, silicon chip forming an array for the purpose of expression profiling; monitoring expression levels of thousands of genes simultaneously and is a hybridization-based method. Several innovations regarding the fabrication of chips, controlling hybridization conditions, and computational analysis of data generated have been achieved over the years. The high-density chip allows relatively low-cost gene-expression profiling, once the fabrication of chip has been achieved. The major limitation of this technology is that genome sequence information is a prerequisite and also higher background inherent of hybridization technique.

The quantitative real-time PCR (qRT-PCR) is a type of PCR preferred for reliable quantification of low-abundance mRNA or low-copy transcripts for transcriptomics studies. The major advantages of this technique are its high sensitivity, better reproducibility, and wide dynamic quantification range. It facilitates gene expressions and regulation studies even in a single cell based on its exponential amplification ability. The availability of diverse types of fluorescence monitoring system attached with the PCR resulted in its popularity for gene-expression studies. The problems of nonspecific amplification, formation of primer-dimers are some of the limitations of qRT-PCR.

RNA-seq is one of the advanced high-throughput technology for transcriptomics. RNA-seq, also known as whole-transcriptome shotgun sequencing, utilizes NGS tools. The advantages of RNA-seq are that it does not rely on the availability of the genome sequence, has no upper quantification limits, shows high reproducibility, and possesses a large dynamic detection range.

The stem cells and cancer cells transcriptomes are emerging field of interest which can provide an insight into the processes of cellular differentiation and carcinogenesis. Transcriptomics can be used in in vitro fertilization for proper embryo selection. Similarly, in biomarker discovery, it can be used in assessing the safety of drugs or chemical risk.

1.4 Proteomics

The study and characterization of entire set of proteins present in cell, organ, or organism at a particular time is referred to as proteomics. Due to alternative splicing and posttranslational modifications, there are vast arrays of protein content in all living cells. Therefore, this diverse and complex proteome needs to be carefully evaluated to get an insight into the cell physiology and other metabolic processes. To overcome this complexity, many proteomic techniques have come up facilitating large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. The basic procedures involved in proteomic study are the isolation of total protein from the cell, its separation, and quantification (Kellner, 2000). The proteomics aims for the following:

• Comprehensive and global study of expression of genetic information at protein level

• Providing an insight into three dimensional structures of proteins

• Assessing protein–protein or DNA–protein interactions

• Elucidating protein structure and function relationship

• Assessing distribution of proteins in different cellular and subcellular components of the cell

• Elucidating the molecular basis of patho(physiological) processes

• Quantitative protein expression profile of a cell, an organism, or a tissue under defined conditions

• Facilitating genome annotation

• Providing an insight into alternative splicing

• Elucidating posttranslational modifications

The tools of proteomics can be applied for diverse applications as listed in Table 1.3.

Table 1.3

Applications of Proteomics

Liquid chromatography (LC) followed by MS is a reliable and powerful tool for proteomics study. It helps in characterization of both native and recombinant proteins. However, the requirement of very concentrated samples and difficulty in handling problematic class of proteins, such as membrane proteins, large-sized and complex folded proteins, and the disulfide rich entities, are the major disadvantages of this technique. To overcome these shortcomings, sample has to be enriched, instrumentation must be improved, and the resulting data should be carefully processed. In addition, vast arrays of other proteomic techniques such as gel-based and gel-free techniques are also available. Gel-based techniques are the commonly used one-dimensional and two-dimensional polyacrylamide gel electrophoresis. Fluorescence two-dimensional difference gel electrophoresis is the modification of two-dimensional polyacrylamide gel electrophoresis wherein the protein samples are fluorescently labeled with Cy2, Cy3, or Cy5 prior to second dimensional electrophoresis. This makes it highly sensitive and quantitative data can also be inferred.

Among the gel-free techniques are the isotope-coded affinity tag (ICAT). As the name indicates, the protein is labeled with the ICAT reagent. The labeled protein after digestion with trypsin and separation by chromatographic procedures is detected with tandem MS and quantitative estimation is done by LC peak areas. But this technique is difficult to work with acidic proteins and works selectively for proteins with high cysteine content. To overcome the limitation of ICAT a newer technique called stable isotope labeling with amino acids in cell culture has emerged. Here the cells are directly labeled with the radiolabeled isotopes which makes the study of differential expression highly convenient. Isobaric tag for relative and absolute quantitation is a high-throughput technique which allows relative quantification of the protein samples. Samples are multiplexed and need to be separated before MS analysis. Multidimensional protein identification technology is an integration of three different techniques. Site-specific tryptic digestion of the protein is performed and the peptides are separated by HPLC. From the HPLC the peptides enter MS column. The MS data is analyzed for the constituent proteins. Similar to DNA and cDNA microarrays there are proteins and antibody microarrays available for the proteomic-based studies.

Mass spectrometry is the key technique in all the proteomic analysis. In a spectrophotometer, first the protein samples are ionized and gas phase ions are produced, these ions are then separated according to their mass to charge ratio and finally the ions are detected for generation of mass spectrophotometry data. The three main parts of a mass spectrophotometer are ion source, mass analyzer, and ion detector. Major ionization sources are MALDI and electrospray ionization, and four major mass analyzers are TOF, ion trap, quadrupole, and Fourier transform ion cyclotron which are mainly applied for protein identification and characterization.

1.5 Metabolomics

It is a comprehensive systematics quantitative assessment of all metabolites in a metabolome under specified conditions. In general, metabolites are substances produced in or by biological processes. It can be either primary metabolite or secondary metabolites. It includes peptides, oligonucleotides, sugars, nucleosides, organic acid, ketones, aldehydes, amines, amino acids, lipids, steroids, alkaloids, drugs, xenobiotics, etc. (Fiehn, 2002). Metabolomics aims for:

• Deciphering the molecular network regulation

• Assessing pathway discovery

• Understanding functional genomics

• Analyzing material samples to gain an understanding of their biochemical composition and structure

• Providing mechanistic insight into diverse type of biological processes

• Unraveling connecting link between genotypes and phenotypes

• Assisting increased metabolic fluxes into valuable biochemical pathways using metabolic engineering

• Investigating the cause of biological effects such as plant–pathogen interactions, plant–environment interactions

The metabolomics tools find significant applications in the study of human diseases and oncobiology. These metabolites are used as biomarkers in several diseases such as male infertility, lung cancer, Alzheimer’s disease, respiratory disease, Huntington’s disease, kidney cancer, and renal cell carcinoma.

The basic methodology of metabolomics involves extraction from the targeted biological tissues/samples, separation of metabolite, and detection followed by identification and quantification (Liao et al., 1996). The basic tools of metabolomics associated with separation are various types of chromatographies such as gas chromatography (GC), HPLC, and capillary electrophoresis (CE), while metabolites can be detected based on different physical properties such as mass, rotation/vibration, absorbance and emittance of light (hv), spin, volatility, size, charge, and hydrophobicity. The methods applicable for the detection of the metabolites are MS, infrared spectrometry, UV-visible spectrometry, fluorescence spectrometry, nuclear magnetic resonance (NMR), GC, LC, CE, and size exclusion chromatography.

1.6 Glycomics

Glycomics deals with a comprehensive study of structural and functional aspects of carbohydrates, which play a significant role in the storage and transmission of biological information associated with diverse types of physiological and pathophysiological processes (Furukawa et al., 2013). It is also considered as a branch of metabolomics and often referred to as glycobiology. Glycans are oligosaccharides or polysaccharides present either in free form or attached to other biomolecules. Contrary to DNA, RNA, and protein, glycans are not encoded through template biosynthetic pathway and also lack proofreading mechanism. Glycosyltransferase is involved in glycan biosynthesis while epimerase, sulfotransferase, etc., are glycan-modifying enzymes. These enzymes are required for site-specific attachment to proteins and lipids to form glycoproteins, proteoglycans, and glycolipids. Glycans attached to proteins are responsible for folding, stability, regulating protein structure and function. Glycoproteins, proteoglycans and glycolipids are diverse and each cell has its own type. According to the data, 70% of therapeutic proteins approved by European and US regulatory agencies are glycoproteins. A large variety of glycans located on the cell surface is associated with vital functions and influences the cell–cell adhesion, immune response, pathogenesis events, cellular process such as development, differentiation, and cellular alteration leading to cancer. The glycomics aims for the following:

• Glycan expression analysis

• Elucidating the diversity of glycan structures

• Elucidating the physiological and pathophysiological roles of glycans located on the surface

• Determining the complexity of glycan with reference to composition, branching, linkages, and anomericity

• Deciphering the potential of glycans as biomarkers for diseases associated with cellular alterations

• Elucidating the N- or O-linked glycans derived from glycoproteins, proteoglycans, and glycosphinolipids, which are important cell surface glycoconjugates

One of the basic techniques to study glycans is high-resolution MS in which even small quantities of glycans can be characterized. The N-glycans of the glycoproteins can be separated and sequenced separately, while the glycolipids can be directly sequenced without separation of the lipid component. Another glycan, glycosaminoglycans, is difficult to analyze through MS due to its large size. Through MS, multiple glycans of any subtype can be profiled. However, mass spectrophotometer cannot perform analysis of complex samples containing multiple glycans of different subtypes. With the help of MS, the mode of binding between the protein and glycans in the glycoproteins can also be studied.

Although MS provides detailed information of glycan, it cannot be used to screen large number of samples at a time. For high-throughput studies, lectin and antibody arrays are available. This is similar to DNA microarray, where lectins (or glycan-specific antibodies) are attached on the array chips instead of DNA spots. The distribution of glycans in different cells and tissues can be routinely analyzed with the help of lectins and glycan-specific antibodies. These act as probe to profile the expression pattern of different glycans. The glycans can also be metabolically labeled with the help of imaging reagents and visualized under the fluorescent microscope. It helps in the real-time imaging of the total glycome content in any cell or tissue.

Many publications are focused on N- and O-glycans as disease markers (de Leoz et al., 2008). Alterations in the degree of branching and levels of sialylation and fucosylation in N-glycans have been reported as a consequence of many diseases such as cancer of prostate, breast, liver, ovary, pancreas, and gastric. Cancer is signified through changes in the branching of N-glycans, truncation of O-glycans, linkage, and acetylation (Varki et al., 2009). The tools of glycomics can be utilized for several clinical applications for disease diagnosis, detection of cancer, and personalized medicine. As therapeutic agents, glycan-integrated nanomaterials can be used as vaccine against viral and bacterial infections and cancer cells.

1.7 Omics-Driven Bioengineering

1.7.1 Cellular Engineering

The cellular engineering is mainly associated with the modifications and alterations of host cells for enhancing the yield and quality of the expressed protein. There are several approaches for altering the cellular processes such as silencing or overexpressing of targeted genes and altering expression of genes using microRNA (miRNA). Gene silencing is the major approach for cellular engineering. RNA silencing is a novel gene regulatory mechanism that limits the transcript level by either suppressing transcription or by activating a sequence-specific RNA degradation process (RNAi). miRNAs are noncoding RNAs which regulate gene expression and further cell physiology. RNA interference is being used to silence multiple genes of different cellular pathways for optimum productivity of bioengineered proteins. RNA interference can be used to target several features such as apoptosis, glycosylation, and enzymes (e.g., dihydrofolatereductase). Once miRNA sequences are identified through complete genome sequence, it can be either overexpressed or silenced to achieve desired regulation of gene expression. New advanced tools that are currently used for gene editing are zinc finger nucleases, modified meganucleases, hybrid DNA/RNA oligonucleotides, Transcription Activator like (TAL) effector nucleases, and modified Clustered Regularly Interspaced Short Palindrome Repeats (CRISPR)/Cas9. These tools have the ability to target one specific DNA sequence in genome, which leads to double-stranded DNA break. DNA repair to these breaks leads to gene knockouts or gene replacement by homologous recombination.

1.7.2 Enzyme Engineering

Enzymes are considered to be one of the major products of biotechnology with immense industrial applications and are known to expedite the rates of a wide range of biochemical reactions along with exquisite specificity. There exist several enzymes especially produced from the microbial sources having diverse application in all sphere of life. The growing need of industrially important enzymes has led to the development of innovations mainly protein engineering–based technologies for developing novel biocatalysts with desired features. The advent of recombinant DNA technology and omics-based technologies, such as genomics and proteomics, has expedited the development of novel biocatalysts in the recent years. The microbial enzymes have been commercially produced by fermentation technology utilizing either submerged fermentation or solid-state fermentation. There is a need for intervention of omics-based technologies at different steps of enzyme technology from production and manipulation of enzymes for desired features to search for novel sources by utilizing the untapped microbial diversity. Protein engineering techniques aim at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity toward nonnatural substrates (Bloom et al., 2005). For past few decades, biocatalysts have been successfully exploited for the synthesis of various complex drug intermediates, speciality chemicals, and even commodity chemicals in the pharmaceutical, chemical, and food industries due to their inherent ability to catalyze reactions with high velocity and unmet specificity under a variety of conditions, as well as their potential as a greener alternative to chemical catalysts. The role of protein engineering is to overcome the limitations of natural enzymes as biocatalysts and engineer process–specific biocatalysts. Some of the tools of protein engineering applied for enzyme manipulation are discussed as follows.

Site-directed mutagenesis is an important tool in enzyme engineering targeting for specific and desired changes in the gene sequences coding for the concerned enzyme. The basic mechanism involves synthesis of an oligonucleotide primer. The desired mutation sites are incorporated into the primer sequence. This may be single-point mutation, multiple-point mutation, or insertion or deletion of the targeted DNA. Next, the DNA polymerase is used to amplify the rest of the DNA sequence. The final DNA sequence after amplification contains the incorporated mutant site which is then transformed in the host cell and cloned. The mutants are confirmed and selected by DNA sequencing.

Directed evolution of enzymes is a well-established high-throughput technology with potential for desired manipulation of enzymes. It is a process which mimics natural evolution carried out in laboratory and involves iterative strategy. The procedure begins by determining a target biomolecule, metabolic pathway, or organism, and a desired phenotypic goal. A diverse library of mutants is generated in vivo or in vitro through methods that mirror the strategies of traditional evolution: introduction of random mutations in the genetic material and/or sexual gene recombination. A high-throughput screening or selection method is used to identify improved progeny among the library, which are subsequently used as parents in a second round of the cycle. The process is repeated until the phenotypic goal is achieved, or when no further improvement of the phenotype is observed despite repeated iterations. Using tools of directed evolution, attempts have been made to enhance catalytic activity of enzyme, improve the stability over a wide range of temperature and pH, alter the specificity, expand or limit substrate specificity, etc.

The search for potential microbial sources for enzymes has been always a limitation in enzyme research as the prerequisite is to develop suitable media for culturing of the microbes in lab conditions. Since only less than 1% of the microbial diversity has been explored based on ability to culture in lab conditions, the remaining 99% is still to be explored. The relatively newer concept of metagenomics approach seems to have immense potential for searching novel sources for potential enzymes. In metagenomics approach, random environmental samples are picked and the DNA is directly isolated. The genomic library of isolated metagenomic DNA is prepared and screened by either structure-based or function-based screening procedures. Since then large number of enzymes have been discovered through this approach.

The deciphering of whole-genome sequences of microbes, known to be potential sources of industrially important enzymes, resulted in genome-wide identification and bioinformatics-based characterization of putative genes coding for the targeted enzymes. Cloning and expression of genes of targeted enzymes could be in-silico analyzed for three-dimensional structural predictions and validation along with deciphering potential catalytic sites prior to subjecting it to wet-lab experimentation (Damborsky and Brezovsky, 2014). The tools of genomics and proteomics can be applied for characterization of enzymes and appropriate enzyme engineering can be targeted based on it.

1.7.3 Metabolic Engineering

Metabolic engineering is basically meant for the production of chemicals, fuels, pharmaceuticals, and medicine by altering the metabolic pathways. This method involves useful alteration of metabolic pathways to better understand and utilize the cellular pathways. Metabolic engineering is motivated by commercial applications by which we can improve the developing strains for production of useful metabolites. This method requires overexpression or downregulation of certain proteins in a metabolic pathway, such that the cell produces a new product. First step for successful engineering requires the complete understanding of host cell for genetic modifications. The effect of genetic manipulation on growth should also be examined. The genetic manipulation may negatively effect on metabolic burden. For example, genetic manipulation for overexpression of phosphoenolpyruvate inhibits heat-shock response and nitrogen regulation. To achieve the product several methods, such as elimination of competitive pathway and toxic byproduct and expression of a heterologous enzyme to improve the synthesis of products, are used for the modification of the host cell. In classical metabolic engineering approach, genetic manipulation is based on the prior knowledge of enzyme network pathway and its kinetics; on the other hand, in inverse metabolic engineering the environmental or genetic conditions are considered for the desired phenotype for genetic manipulation. A number of approaches are used for secondary metabolites improvement using bacteria in metabolic engineering, such as (1) heterologous expression of gene clusters, (2) regulatory networks pathway engineering, (3) gene insertion or deletion, and (4) stimulation using certain substrates. Therefore, metabolic engineering is currently used as cell factories for production of amino acids, biofuels, pharmaceuticals, bioplastics, platform chemicals, silk proteins, etc.

1.8 Bioinformatics Intervention in Omics

The outcome of sequencing projects resulted in substantial increase in the sequence databases globally, demanding the intervention of web-based bioinformatics for proper storage, retrieval, and analysis of sequences. The huge data generated in the field of genomics, proteomics, transcriptomics, metagenomics, and metabolomics needs bioinformatics tools, browsers, or softwares for quick, reliable, and efficient analysis. Several bioinformatics tools have been developed for omics as listed in Table 1.4.

Table 1.4

List of the Important Bioinformatics Tools/Softwares/Browsers for Science of Omics

1.9 Applications of Omics

1.9.1 Food and Agriculture Sector

There have been several innovations for improvement of crop over the years mainly targeting for enhancing the yield or improving the quality. The classical breeding, molecular breeding, and genomics-assisted breeding reflect the developments of technologies. The omics-driven agriculture has great potential for identifying the genes showing potential agronomics traits, developing appropriate molecular markers, applying tools of forward and reverse genetics and also for developing genetically modified or biotech crops with desired features. The genomics provides information regarding the genetic blueprint for making changes at the genetic level. Molecular markers can be identified with the help of genomics, and their association with the different genes can be exploited for genetic improvement. The genome-wide association studies can be made to evaluate the correlation between a specific genetic trait and a marker allele, the selected markers are then used for prediction of the breeding values. Once the breeding values of the genome-wide markers are estimated, they can be substantially applied in crop-improvement programs. Contrary to this strategy, instead of targeting markers, whole-genome sequence can be explored for genetic improvement using traditional breeding or using transgenic approaches.

Genomics-driven biomarkers are also useful in the study of wild habitats and their conservation. These can also be used for assessment of genetic diversity. Similarly, using metagenomics approach one can utilize the unexplored microbial diversity available in different environmental samples such as soil, air, and water for fishing out novel sources for enzymes of metabolites. Intervention of omics is also feasible for identification of contamination in food and relevant molecular markers can be typed for easy identification of pathogens.

1.9.2 Health Sector

The genomics-based information of pathogens and human can facilitate the development of suitable drugs. The effect of drugs on individual may vary, based on the variation of the sequences, and needs to be addressed based on the individual genome sequences. This has led to the concept of personalized medicine. The omics study encompasses all the aspects of information driven from a body related to the genome, transcriptome, proteome, and metabolome. All these parameters can be studied as personalized omics for an individual. In addition, a relatively newer microbiomics providing detailed parameter of all the microorganisms, both good and bad in our body, can be utilized for developing appropriate healthcare technology. Using omics-based technology one can have better understanding about the causative agent for the targeted diseases, can facilitate typing of disease symptoms, can assist in developing relevant biomarkers, etc. There has been substantial improvement in the disease diagnosis, pathogen detection, understanding the physiological and pathological conditions of patients, getting an insight into the cause of genetic or hereditary diseases, and developing appropriate strategies for enhancing life expectancy of human. There have been efforts for omics-based studies for cancer and other diseases over the years.

1.9.3 Environmental Sector

The effect of chemicals on the environment can be monitored by omics technologies. Through the integrated approach of different omics-based methods, the ill-effect of these agents can be monitored. The biomarker for toxicity of specific chemical reagent can be typed to study its mechanism of action and also the risk associated with each hazard-causing chemical. Environmental omics is still in preliminary stage and improvement must be made for characterization of mixture of toxic samples and development of technologies and assays to monitor its impact (Yue Ge et al., 2013). This will help in better understanding of environmental toxicity and sustainable recovery of the environment from health hazards. Omics-based approaches can be used for understanding the interactions of environmental and genetic factors, toxicity mechanism, and short- or long-term effects of environmental chemicals on human health. Using meta-omics, there is a possibility of assessment of microbial communities for identifying novel enzymes associated with degradation of environmental pollutants, developing novel biomarkers for environmental risk monitoring, etc.

1.10 Conclusion

Integrating the omics technologies will assist in better understanding of life processes. Over the years, there have been substantial improvements in technological innovations relevant to different branches of omics. The availability of whole-genome sequences has expedited the growth of genomics, proteomics, and metabolomics along with bioinformatics intervention, and has led to the development of appropriate tools for analysis of genome, proteome, and metabolome efficiently. The extrapolation of the omics information is for the development of strategies for improvement in different sector of life such as agriculture, health, industry, and environment. Efforts have been made to upgrade the tools of omics by enhancing the speed and accuracy, making it cost-effective and easy to operate and analyze the results. In the recent years, based on the success of genomics, proteomics, and metabolomics there have been reports of repertoire of omics dealing exclusively with specific areas. There is an immediate need for developing appropriate links between diverse omics based on the emergence of huge data generated by omics technologies.

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Chapter 2

Omics Approaches Towards Transforming Personalized Medicine

Dipali Dhawan,    PanGenomics International Pvt. Ltd., Ahmedabad, Gujarat, India

Abstract

Pharmacogenomics uses the individual’s genetic profile to determine the response to prescribed drugs. This information enables timely treatment and prevents adverse drug reactions. Genotype–phenotype associations have been well studied in cardiovascular diseases, cancer, infectious diseases, and immune disorders in a number of different populations. Personalized medicine involves particular drug at a tailored dose to an individual based on their genetic makeup. Many of the genetic tests developed have been translated to the clinic and are being recommended by the US Food and Drug Administration (FDA) as well for a better outcome of the patients when prescribed these medicines. In this chapter we provide an overview of pharmacogenomics and also discuss the evolution of the field of companion diagnostics.

Keywords

Personalized medicine; pharmacogenomics; companion diagnostics; genotype; phenotype; drug

2.1 Introduction

The increasing knowledge on genetic variations and their effect on the therapeutic regime have enabled various researchers and clinicians to utilize this information for better patient management. A number of monogenic polymorphisms in gene coding for drug metabolizing enzymes and drug targets have been reported in the literature (Evans and Relling, 1999; Vesell, 1984). A large portion of interindividual variability in drug response has been attributed to genetic variations. However, the applicability of pharmacogenetics has been more prominent in research and academics as compared to clinics (Crews, et al., 2011; Nelson et al., 2011; Pulley et al., 2012; O’Donnell et al., 2012; Cavallari and Nutescu, 2012). There are a number of applications of pharmacogenetics that are summarized in Fig. 2.1. The idea of tailored therapeutics will take a few more years to be completely applied to the clinics and enable efficacious treatment to the patients.

Figure 2.1 Applications of pharmacogenomics.

2.2 Personalized Medicine

Personalized medicine is a very old concept that was coined almost hundred years ago. With newer technologies and availability of genome data, it has been possible to screen individuals to enable the identification of genetic variants affecting diagnosis, prognosis, and theranosis of any disorder. There are a number of variants that affect the drug metabolism and response which serves as a basis for pharmacogenetics to enable personalized medicine. The sequencing of the human genome has enabled the translation of precision medicine from the research phase to the clinical phase. There are a number of tools that have been developed to help in the smooth application of this science to personalize treatment. The Genome-wide Association Studies have enabled the identification of genetic variants for different traits of various populations. The knowledge from these studies has eased the path towards tailored dosing for efficacious treatment. There are several terms being used interchangeably with personalized medicine, including pharmacogenetics, precision medicine, theranosis, and tailored medicine.

2.3 Pharmacogenomics of Various Disorders

2.3.1 Cancer

The applicability of pharmacogenomics in cancer is the highest because the therapeutic window for the cancer drug is narrow, the time of treatment for the patient is critical, and the cost of treatment is very high.

6-Mercaptopurine (6-MP) and Thiopurine methyltransferase (TPMT): Acute lymphoblastic leukemia (ALL) is the most commonly observed childhood malignancy (Pui and Evans, 2006). 6-MP is the most well prescribed therapy for ALL; however, it leads to myelosuppression as a side effect (Lennard et al., 1987). The genetic testing of TPMT has a dual advantage as it is useful in determining the risk of toxicity and the response in terms of minimal residual disease posttreatment (Stanulla, et al., 2005). It has been recommended by the FDA to identify individuals who are homozygous for nonfunctional TPMT alleles or have low or intermediate TPMT activity, and dose determination is done on the basis of this test. Dosing recommendations of mercaptopurine based on the genotype and phenotype have been suggested by the Clinical Pharmacokinetics Implementation Consortium (Relling et al., 2011). About 3%–14% individuals who are heterozygous for nonfunctional TPMT alleles are at a significantly higher risk of toxicity due to mercaptopurine as compared to individuals with two functional alleles (Relling et al., 2011). TPMT*2, TPMT*3A, TPMT*3B, and TPMT*3C are the nonfunctional alleles associated with reduced levels of TPMT activity (McLeod and Siva, 2002).

Irinotecan and UDP glucuronosyltransferase 1 (UGT1A1): Irinotecan is prescribed to patients suffering from rhabdomyosarcoma and other solid tumors. The enzyme UGT1A1 metabolizing irinotecan is highly polymorphic and leads to irinotecan-induced neutropenia (Innocenti et al., 2009; Hoskins et al., 2007). There is a common insertion/deletion in the UGT1A1 promoter TATA box, which reduces the transcriptional activity of UGT1A1. The efficiency of the translation is inversely correlated to the number of tandem TA repeats present in the individual (5, 6, 7, 8 alleles) (Beutler et al., 1998). The most commonly observed are the 6 and 7 repeats. 6 repeats has been classified as UGT1A1*1, whereas 7 repeats has been classified as UGT1A1*28 (Hall et al., 1999). There is a strong evidence for the role of UGT1A1*28 for severe toxicity (Innocenti et al., 2004; Iyer et al., 2002; Rouits et al., 2004; Marcuello et al., 2004; Toffoli et al., 2006; Carlini et al., 2005; Font et al., 2003; Han et al., 2006; Massacesi et al., 2006). The importance of this genetic testing has resulted in the label change of this drug by the FDA, making genetic testing mandatory for irinotecan (Innocenti and Ratain, 2006).

5-Fluorouracil (5-FU) and dihydropyrimidine dehydrogenase (DPYD): 5-FU, used in the treatment of most of the solid tumors, is metabolized by dihydropyrimidine dehydrogenase (DPD). (van Kuilenburg 2004; Heggie et al., 1987). DPD activity is variable among different individuals, and it is reported that 3%–5% of individuals are deficient or show low DPD activity (Etienne et al., 1994; Lu et al., 1993). The gene DPYD is highly polymorphic, and one of the variant allele DPYD*2A (c.1905+1G>A, also called as IVS14+1G>A) has a significant effect on toxicities, including leucopenia and severe mucositis, observed due to 5-FU (Schwab et al., 2008). This variant results in skipping of exon 14 and results in a nonfunctional enzyme leading to toxicities (Wei et al., 1996; Vreken et al., 1996). Various other studies have also reported lethal 5-FU toxicities in patients with DPD deficiency (van Kuilenburg et al. 2003; van Kuilenburg et al. 2001; Johnson and Diasio, 2001; Milano et al., 1999). Furthermore, other concomitant drugs may also interact with 5-FU metabolism and hence affect the risk profile for DPYD variants. The label for 5-FU mentions a warning for DPD-deficient patients not to use this drug; however, genetic testing is not mandatory for it (Yen and McLeod, 2007; Ciccolini et al., 2010).

2.3.2 Cardiovascular Diseases

The ability to diagnose and treat various cardiovascular disorders has improved owing to the better understanding of individual characteristics like genetic variations. Examples of some of these cardiovascular applications have been mentioned below:

Warfarin: Warfarin is widely prescribed for the prevention and treatment of thromboembolic diseases. However, it has varied responses in different individuals due to genetic polymorphisms. The two most well-studied genes for understanding this variable response are CYP2C9 (Cytochrome P450 2C9) and VKORC1 (vitamin K epoxide reductase complex subunit 1), The single nucleotide polymorphisms (SNPs) in these two genes are responsible for more than one-third of the variation observed in warfarin therapeutic outcome (Wadelius et al., 2007; Gage et al., 2008; Klein et al., 2009; Caldwell et al., 2008; Wadelius et al., 2009). To understand whether genotype information could enable the reduction in the incidence rates of hospitalizations occurring due to adverse effects of warfarin, the Medco-Mayo Warfarin Effectiveness Study was conducted with almost 4000 individuals (Epstein et al., 2010). About 900 patients who were starting warfarin therapy were genotyped for CYP2C9 and VKORC1. Their genotype data were sent to their doctors and outcomes were measured in comparison with 2700 controls who were not genotyped. Two external control groups from a different set were also followed during the study, one synchronous with the genotyped group and the other synchronous with the control group. A 31% reduction of hospitalization was observed after a 6-month follow-up in the genotyped patients in comparison to the controls (Epstein et al., 2010). The results of such studies suggest the importance of pharmacogenomic applications in cardiovascular