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

Fundamentals of Molecular Biotechnology
Optimization of Gene Expression Cloned in Prokaryotic Systems
Integration of Foreign DNA into the Host Chromosome

When a Cell contains a plasmid, a portion of its energy resources is diverted to plasmid Replication, METABOLISM/31.html">Transcription, and the Synthesis of the Proteins it encodes. Generally, multicopy Plasmids demand more energy than low-copy ones, causing some Cells to lose their plasmids as the population grows. Cells that have lost their plasmids usually grow faster than those that retain them, eventually becoming the dominant population in the culture. After several generations, this decline in plasmid-bearing cells impacts the yield of the cloned Gene product. At least two approaches have been developed to address this issue. In the laboratory, to maintain plasmids, cells are grown in the presence of Antibiotics or specific metabolites that ensure the survival only of cells harboring the plasmid. However, adding antibiotics or other supplements to large-scale cultures or industrial fermenters significantly drives up the cost of the final product. Ensuring that cloned genes are stably retained—without being lost or transferred to other microorganisms—is especially critical when engineered microorganisms are intended for release outside the laboratory. Such strains must remain not only effective but also environmentally safe. Integrating cloned DNA directly into the host chromosome eliminates The Need for plasmids altogether, thereby preventing the loss of plasmid-borne genes. When inserting a gene of interest into the host chromosomal DNA, care must be taken to ensure that the integration site does not disrupt a gene essential for vital cellular Functions. To avoid this, the foreign gene is inserted into a purposefully non-essential site. Furthermore, to ensure efficient expression, the gene is placed under the control of a regulated promoter. For successful site-specific integration, the introduced gene must be flanked by a nucleotide sequence at least 50 Base Pairs long that is homologous to a region in the host chromosome, within which a physical exchange (recombination) between the two DNA molecules can take place. Briefly, the integration process involves the following steps.

1. Identification of a suitable integration site—that is, a segment of host DNA whose sequence can be interrupted without impairing cellular function.

2. Isolation and cloning of the target chromosomal integration site or a portion of it.

3. Insertion of the gene of interest, together with a regulated promoter, into or near the cloned integration site (Fig. 6.15, A and B, respectively).

4. Transfer of the resulting genetic construct ("chromosomal integration site/cloned gene") into the host cell via a plasmid that is incapable of autonomous replication in that host.

5. Selection and maintenance of host cells that successfully express the cloned gene. Stable Inheritance of the cloned gene is possible only if it integrates into the host chromosome.

When a cell is transformed with a non-replicating plasmid carrying a cloned gene embedded within a cloned fragment of the chromosomal integration site, pairing may occur between the homologous nucleotide sequences of the plasmid and the host DNA (Fig. 6.15, A), followed by integration via a double crossover mediated by host cell Enzymes. Alternatively, the entire plasmid DNA may integrate into the host chromosome through a single crossover event (not shown). Integration of the entire plasmid can also occur when the cloned gene is inserted adjacent to the cloned chromosomal integration site.

Class="center">

Fig. 6.15. Two mechanisms for integrating a plasmid-cloned gene into the chromosome. A. The gene is inserted into the middle of the plasmid-cloned segment ab, which is homologous to segment a' b' in the chromosomal DNA. Through a double crossover (X–X), the cloned gene becomes part of the chromosome. B. The gene is inserted near the plasmid-cloned segment c, which is homologous to segment c' in the chromosomal DNA. A single crossover (X) results in the integration of the entire plasmid, including the inserted gene, into the chromosome.

To test the efficiency of cloned gene integration, B. subtilis was used as a model Organism. An E. coli plasmid was constructed containing the gene for a-amylase (an enzyme involved in starch Hydrolysis) from Bacillus amyloliquefaciens inserted into the middle of a DNA fragment from B. subtilis. Although this plasmid was unable to replicate in B. subtilis, it could be used to transform B. subtilis cells. The resulting transformants synthesized a-amylase, demonstrating that the gene encoding this enzyme had successfully integrated into the B. subtilis chromosome and was actively expressed. The selected recombinants were resistant to ampicillin and chloramphenicol. Because both resistance genes resided on the plasmid, it was evident that a single recombination event had occurred, resulting in the incorporation of the entire plasmid into the B. subtilis chromosomal DNA.

To increase the copy number of the a-amylase gene integrated into the B. subtilis chromosome, the initial transformants were cultivated in the presence of a high concentration of chloramphenicol. Under these selective conditions, survival was restricted to cells that underwent spontaneous duplication of the integrated plasmid. Cells selected for chloramphenicol resistance were subsequently screened for a-amylase activity (Table 6.5). This Procedure yielded cells containing up to 9 copies of the a-amylase gene. The level of enzymatic activity in cells harboring chromosomal copies of the a-amylase genes was significantly higher than that observed when these genes were carried on a multicopy B. subtilis plasmid (20 to 40 copies per cell). In one study, multiple copies of a foreign gene were integrated into distinct, predetermined sites within the B. subtilis chromosome using a two-step procedure for each copy (Fig. 6.16). In the first step, a selectable marker gene (such as an Antibiotic Resistance gene) was inserted into the middle of a well-defined, non-essential region of the B. subtilis chromosomal DNA using a plasmid vector that cannot replicate in this bacterium. Cells expressing the marker gene—indicating successful incorporation of the gene into the chromosomal DNA—were selected. In the second step, the target gene, complete with appropriate transcription and Translation initiation signals, was inserted into the middle of an identical non-essential chromosomal DNA fragment within a plasmid and introduced into the chromosomal DNA via recombination, thereby replacing the marker gene. Cells that no longer expressed the marker gene, but instead carried the integrated target gene in its place, were isolated. This procedure was repeated using different non-essential DNA regions to integrate additional copies of the target gene into the host chromosome.

Table 6.5. Relationship between a-amylase gene copy number and enzyme activity levels in B. subtilis cells¹⁾

¹⁾ Based on Kallio et al., 1987. Appl. Microbiol. Biotechnol. 27: 64–71.

Fig. 6.16. Insertion of a foreign gene into a predetermined site in the B. subtilis chromosome. In Step 1, a marker gene is integrated into the host chromosomal DNA via Homologous Recombination. In Step 2, the marker gene is replaced with the target gene. This process can be repeated for other chromosomal sites.