Biomolecular Engineering Solutions for Renewable Specialty Chemicals. Группа авторов
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Название: Biomolecular Engineering Solutions for Renewable Specialty Chemicals
Автор: Группа авторов
Издательство: John Wiley & Sons Limited
Жанр: Биология
isbn: 9781119771944
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1.2 Fundamentals of Genetic Engineering
The advent of genetic engineering, also called rDT, started in 1952 with the discovery of Hershey and Chase, stating DNA as the genetic material (Hershey and Chase, 1952). Cohen and Boyer in the early 1970s were the first to show that the genetic material of one organism can be easily expressed in the other. Genetic engineering (Figure 1.1), in general, is the process in which the DNA is extracted, modified, transformed into a host cell, and a new organism is formed. The DNA from the desired organism is extracted and purified. It is then cleaved using restriction enzymes to get the gene of interest from it. The DNA fragment is then ligated into a vector, which acts as a driving vehicle for the DNA molecule to the host cells. This chimeric DNA molecule is then transformed into the host cells, and selection procedure under suitable stress conditions takes place. Finally, after numerous generations, the organism growing in the stress conditions is said to be recombinant or genetically modified. Genetic engineering has emerged as a crucial step in the development of industrial bioprocesses.
Each and every organism has a different genetic (DNA) makeup, which in turn makes the whole organism different with respect to their carbohydrates, lipids, and proteins. This is due to the fact that DNA transcribes and translates to mRNA and proteins, respectively (central dogma). This makes DNA the choice for manipulation in genetic engineering as manipulating it leads to the generation of a whole new organism. This postulation gives rise to many other disciplines of genetic engineering like recombinant protein production, protein engineering, metabolic engineering, etc.
Every organism being different makes it difficult to use proteins and other biomolecules of one organism to the other. This was the main reason why proteins/enzymes from animals cannot be used by humans. Earlier, insulin was extracted from the pancreas of slaughtered pigs, posing a threat to human health. This leads to the discovery of the first recombinant product, Insulin, approved by the US Food and Drug Administration (FDA) in 1982 (Goeddel et al., 1979). Now synthetic insulin is easily being produced by yeast worldwide as Escherichia coli does not perform post‐translational modifications required to form functional insulin.
Similarly, genetic engineering is now used to produce several other biocommodities. Modifying DNA and getting it expressed inside the host organism requires several steps, as shown in Figure 1.1 and the number of enzymes. These enzymes are explained in further sections with other requirements for genetic engineering.
1.2.1 DNA‐altering Enzymes
The basis of rDT is the manipulation of DNA molecules with the help of molecular biology tools and biocatalysts. The available purified enzymes that can manipulate DNA molecules with specific changes can be categorized in four broad classes: (i) DNA polymerases, (ii) nucleases, (iii) DNA ligases, and (iv) end‐modification enzymes.
Figure 1.1 Basic steps of gene cloning.
1.2.1.1 DNA Polymerases
DNA polymerases is the key enzyme in DNA replication driving the synthesis of new DNA strand from the parent DNA or RNA strand acting as a template. DNA polymerases require an oligo nucleotide (primer) for the initiation of DNA strand synthesis. DNA polymerase‐I (DNA‐dependent DNA polymerase) is widely studied polymerase and has both polymerization and exonuclease activity that can help in synthesizing new strand as well as the degradation for proof reading or repair and primer removal. DNA polymerase I (or Pol I) takes part in the process of prokaryotic DNA replication. It was the first DNA polymerase discovered by Arthur Kornberg in 1956 (Lehman et al., 1958). Pol I has three different enzymatic activities: A 5′ →3′ DNA‐dependent DNA polymerase activity, a 3′ →5′ exonuclease activity that helps in proofreading, and a 5′ → 3′ exonuclease activity mediating nick translation during DNA repair. Pol I having polymerase but lacking nuclease activity is called klenow fragments (Klenow and Henningsen, 1970; Jacobsen et al., 1974).
Taq DNA polymerase, a thermostable DNA polymerase isolated from Thermus aquaticus by Chien et al. (1976). It is frequently used in the polymerase chain reaction (PCR), to amplify small quantities of DNA. It has a functional 5′ → 3′ exonuclease domain at the N‐terminal, and 3′–5′ exonuclease domain was changed so it is not functional. Optimum temperature for Taq pol activity is 75–80 °C, with a half‐life of greater than 2 hours at 92.5 °C and minimum 9 minutes at 97.5 °C, and able to replicate a 1000 bp strand of DNA within 10 seconds at 72 °C.
1.2.1.2 Nucleases
Nucleases can cut or digest the DNA molecules either from one end or in middle by acting on phosphodiester bond, which forms the backbone of DNA (Nishino and Morikawa, 2002). Depending on the position of their digestion nucleases are of two types: exonucleases and endonucleases. Phosphodiester bonds present in the ends of the DNA are digested by exonucleases, removing nucleotides one at a time from either end. Whereas phosphodiester bonds present in the middle of the DNA strand are digested by endonucleases. Specificity of nucleases vary from source to source, Aspergillus oryzae’s endonuclease only cleaves single strands, whereas deoxyribonuclease I (DNase I), extracted from cow pancreas, cuts single as well as double‐stranded DNA molecules. DNase I not being sequence specific cuts DNA at random interior phosphodiester bond, leading to production of mononucleotides and very short oligonucleotides mixture. Some of the examples of nucleases are (i) Mung Bean Nuclease (isolated from mung bean sprouts) – a single‐strand‐specific DNA and RNA endonuclease which can degrade single strand overhangs from the end of DNA and RNA to make blunt ends. (ii) Nuclease S1 (isolated from Aspergillus sp.) – S1 nuclease is a single‐strand‐specific endonuclease that hydrolyzes single‐stranded RNA or DNA into 5′ mononucleotides. The enzyme will hydrolyze single‐stranded extensions in duplex DNA such as loops and gaps. S1 Nuclease is stable at 65 °C (Balabanova et al., 2012). (iii) Exonuclease III (isolated from E. coli) – removes single nucleotides from 3′ termini of the duplex DNA. It is generally used to make a set of nested deletions of the terminal of linear DNA strand. (iv) BAL31 nuclease (isolated from Alteromonas espejiana) – it is a 3′‐exonuclease and removes nucleotides from both 3′‐terminus of the two strands of linear DNA. (v) RNase H – it is an endonuclease that specifically hydrolyzes the phosphodiester bonds of RNA, when the RNA is hybridized to DNA (RNA‐DNA) (Cerritelli and Crouch, 2009). (vi) RNase P – the specificity of this enzyme is to cleave other RNA molecules at the junction of single‐stranded and the 5′ end of double‐stranded regions of RNA (Guerrier‐Takada et al., 1983).
Restriction endonuclease (RE) enzymes recognize and cleave the specific phosphodiester bond present in the DNA molecule (Smith and Welcox, 1970). Restriction enzymes are broadly classified into Type‐I, Type‐II, and Type‐III. For their functioning, they require specific temperature, ATP, and divalent magnesium ions. On digestion of the DNA molecule they can produce both blunt and sticky end. Type I REs interact with unmodified target site in dsDNA. They are bifunctional enzymes having methylase and endonuclease in a single protein molecule. They cleave DNA around 1000 bp away from the recognition site. For their function, both ATP and Mg2+ are required. Type II REs are highly specific and cleave within or very near to the recognition sequence due to this reason type II are used widely in genetic engineering. They do not require ATP for the restriction digestion, only Mg2+ is required. СКАЧАТЬ