Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Using Recombinant Microorganisms to Produce Commercial Products
Restriction Endonucleases

Until recently, the primary goal of research in molecular biotechnology was The production of various Proteins. However, Recombinant DNA technology can also be used for the large-scale production of many valuable low-molecular-weight compounds, such as Vitamins, Amino Acids, Antibiotics, etc.

With an efficient expression system, obtaining a protein—the product of a specific Gene—is relatively straightforward. The protein can be either the desired end product itself (for example, a restriction endonuclease) or an enzyme that catalyzes a specific chemical reaction (for example, one of the steps in antibiotic Biosynthesis). Sometimes, As a result of genetic manipulation, a microorganism acquires The ability to synthesize a new enzyme and can be used for the in vivo production of low-molecular-weight compounds, such as vitamins, amino acids, Dyes, antibiotics, precursors of various Biopolymers, etc. Such a microorganism becomes a "factory" for the production of useful metabolites.

The Development of recombinant DNA technology would have been impossible without the availability of appropriate restriction endonucleases (restriction Enzymes). Currently, more than 300 different restriction enzymes are commercially available. These enzymes are synthesized by A wide variety of microorganisms: aerobic, anaerobic, photosynthetic, diazotrophic, mesophilic, thermophilic, psychrophilic, slow-growing, and fast-growing. Cultivating each of these requires optimizing Fermentation conditions—Temperature, pH, medium composition, and oxygen concentration—to maximize the yield of the desired enzyme. To avoid growing A large number of different microorganisms, preparing complex media, designing various fermenters, and spending time optimizing growth conditions for numerous organisms, the genes for restriction endonucleases are often cloned into Escherichia coli. This allows for the standardization of production conditions for the required products. Furthermore, E. coli Cell cultures rapidly reach high density and can be adapted for the overproduction of the desired enzyme.

Although the technology for isolating and expressing foreign genes in E. coli and some other microorganisms is well established, It is important to remember that the synthesis of a heterologous protein can have a negative impact on the host Organism. For example, overproduction of such a protein can deplete the host's metabolic resources and adversely affect its growth. The presence of a heterologous protein can even be lethal to the host cell. For instance, restriction sites are present in all DNA molecules, and if the product of the cloned gene is a restriction endonuclease, the host DNA will be cleaved by it in the absence of specific protective mechanisms.

Microorganisms that synthesize restriction endonucleases have developed a self-defense system: they methylate one or more bases of the restriction site, thereby blocking DNA Cleavage at this site by the homologous restriction endonuclease. Gram-negative microorganisms possess an additional defense mechanism: their restriction endonucleases are localized in the periplasmic space. Due to this compartmentalization, There is a physical Separation between the restriction enzymes and the DNA, while still ensuring free access of the methylating (modifying) enzyme to the chromosomal DNA. Furthermore, this protects The Cell from The entry of any foreign DNA, such as viral DNA.

One approach to solving Structure/149.html">The problem of host DNA degradation by heterologous restriction endonucleases is to clone and express both the restriction enzyme gene and the corresponding modifying enzyme gene in the recipient organism. However, cloning both of these genes into a single microorganism is technically challenging if they are located far apart on the donor organism's chromosome. Moreover, to prevent the cleavage of host DNA by restriction endonucleases, the methylating enzyme must be synthesized after transformation before the Synthesis of the restriction enzyme begins.

Fig. 12.1 shows the strategy for isolating and cloning the PstI restriction enzyme gene from the Gram-negative bacterium Providencia stuartii into E. coli.

1. P. stuartii DNA is digested with HindIII, and the fragments are inserted into the HindIII site of the plasmid pBR322.

2. E. coli HB101 Cells are transformed with the recombinant Plasmids, grown in liquid medium, and then infected with bacteriophage λ. If the restriction enzyme gene is expressed in the host cell, it becomes resistant to the lytic action of λ-type phages, whose DNA is actively cleaved by the synthesized restriction enzyme.

3. Transformed cells resistant to phage λ are subjected to osmotic Shock to release periplasmic proteins. The activity of the PstI restriction enzyme in the protein extract is then determined.

4. Positive clones are tested for PstI methylase activity.

One positive clone identified in this experiment contained a 4-kb insert of DNA with the intact PstI restriction-modification Operon and the P. stuartii promoter. In the clone carrying this genetic construct, the natural temporal order of synthesis was maintained: first, the methylating enzyme was synthesized, followed by the restriction endonuclease. The expression level of the PstI restriction enzyme gene in E. coli was approximately 10-fold higher than in P. stuartii. As expected, the restriction enzyme was located in the periplasmic space, while the methylase was in the Cytoplasm. Obtaining PstI by cloning the corresponding gene in E. coli is far more efficient than isolating this enzyme from P. stuartii.

Another approach can also be used to isolate genes encoding Restriction and modification (methylating) enzymes, which consists of the following steps.

1. A genomic library is constructed from the donor organism that produces the known restriction endonuclease. The plasmid vector used for this must contain at least one recognition site for this restriction enzyme.

2. E. coli is transformed with the recombinant plasmids.

3. Plasmid DNA is isolated from the transformed cells grown in a liquid selective medium (i.e., from cells containing the plasmid).

4. The plasmid DNA is digested with the restriction endonuclease of interest.

5. E. coli is transformed with the restriction endonuclease-digested plasmid DNA.

The key to this method is that the plasmid DNA from clones carrying and expressing the modifying enzyme gene is resistant to cleavage by the corresponding restriction endonuclease because its recognition sites are methylated.

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Fig. 12.1. Cloning of the PstI restriction enzyme gene and Selection of transformed bacterial cells carrying it. Chromosomal DNA of P. stuartii is digested with HindIII, and the fragments are inserted into the plasmid pBR322. E. coli is transformed with the recombinant plasmid, the cells are grown in liquid medium, and infected with phage λ. Phage-resistant transformants are selected; these are the ones that carry and express the cloned PstI gene.

Consider the following example. HindIII fragments of Desulfovibrio desulfuricans DNA were inserted into the plasmid pBR322, which was then used to transform E. coli cells. Plasmid DNA isolated from the transformed cells was digested with the restriction enzyme DdeI. Plasmids carrying and expressing the methylating enzyme gene were not cleaved because all eight DdeI recognition sites in pBR322 were methylated. The mixture of DdeI-digested plasmids was used to transform E. coli. Only intact circular plasmid DNA molecules yielded transformants carrying the functional DdeI modifying enzyme gene. The remaining plasmids were cleaved by the restriction endonuclease. To determine which clones contained both the modifying enzyme gene and the restriction endonuclease gene, the transformants were tested for the presence of active DdeI restriction enzyme. This approach can be successfully used to isolate the gene for any restriction enzyme, provided it is located close enough to the gene of the corresponding modifying enzyme and is cloned into a plasmid vector containing at least one recognition site for that enzyme.