Genetic engineering to alter enzyme expression

Genetic engineering allows the in vitro manipulation of DNA with the ability to reintroduce the modified DNA back into host cells. The genetic elements of genes as well as their regulating elements can be altered to achieve changes in enzyme expression. The typical bacteria or fungi used in enzyme production can have between two and 10,000 genes. The bacterium B. subtilis encodes its genes in 4.2 million bases of DNA (Kunst et al., 1997) on one chromosome (one continuous length of DNA). The fungus Aspergillus niger uses approximately 35 million bases of DNA to encode its genes (Debets et al., 1990) on eight chromosomes. Each gene specifies the amino acid composition of a protein. One gene confers the sequence for

Fig. 13.5. Comparison of a-amylase production from Bacillus sp. strains growing on starch agar. The plate on the left shows an industrial overproducing a-amylase strain generated using classical mutagenesis; the plate on the right shows a wild isolate strain. Haloes surrounding the colonies result from a-amylase hydrolysis of the insoluble starch.

Fig. 13.5. Comparison of a-amylase production from Bacillus sp. strains growing on starch agar. The plate on the left shows an industrial overproducing a-amylase strain generated using classical mutagenesis; the plate on the right shows a wild isolate strain. Haloes surrounding the colonies result from a-amylase hydrolysis of the insoluble starch.

one protein. The length of a typical bacterial enzyme gene, encoding a protein of 300 amino acids, is in the order of 1000 nucleotides. In addition to segments of DNA that encode genes, there are also segments of DNA called promoters that control the function of genes. Only a fraction of the genes of an organism are 'on' at any given time. Coordination of the expression of genes is controlled by promoter elements. These regulatory segments of DNA can be thought of as light switches for genes, able to turn a gene on, off or to a state in between.

A key breakthrough in the 1970s that made genetic engineering possible was the discovery of specialized enzymes called restriction endonucleases. These enzymes are capable of cutting DNA in precise locations, in effect acting as molecular knives. Another group of enzymes, called DNA ligases, can join cut ends of DNA together, in effect acting as molecular splicers. Segments of DNA can thus be excised from chromosomes and recombined.

Genetic engineering requires the ability to transfer recombined DNA into a host organism, a process referred to as transformation. The bacteria E. coli and B. subtilis can be transformed fairly easily: mixing cells with calcium chloride renders the cells competent (susceptible) to take up DNA. When genetically engineered DNA is added to such competent cells, the DNA adheres to the cell walls and is transported inside the cell. Though transformation of fungi is more complex, a method was found in the early 1980s to transform the fungus Aspergillus nidulans (Balance et al., 1983). Minor variations soon followed to allow transformation (and hence genetic engineering) of the fungi A. niger and Trichoderma reesei, both of which are frequently used for enzyme manufacturing.

To enable high expression levels of enzymes, DNA can be recombined into an overexpression vector. An overexpression vector is a specialized assembly of DNA that when placed into a host cell causes a specific enzyme to be expressed (produced) above normal levels. Several DNA elements are essential to create a suitable vector. An example of an overexpression vector is one that has been used for production of ferulic acid esterase A from A. niger (the enzyme is abbreviated as FAEA and the gene encoding this enzyme is abbreviated as faeA). FAEA (de Vries et al., 1997) can cleave the ester link between ferulic acid and arabinose present in the cell walls of many feed substrates. For this overexpression vector (Fig. 13.6), recombination of four elements was required: a strong promoter glaA, the faeA gene, the glaA terminator and the pyrG selectable marker. The glaA promoter regulates the faeA gene. The glaA promoter is known to result in high-level expression of genes that it regulates, and for this reason was chosen for the vector. When the overexpression vector depicted in Fig. 13.6 was transferred into an A. niger host, production of the FAEA enzyme increased significantly.

The selectable marker gene allows for a way to determine if a host organism has been transformed with the vector DNA. Before transforming the vector into the A. niger host strain, the A. niger cells are initially mutated to make the native pyrG gene non-functional. The pyrG gene produces an enzyme required to synthesize the DNA building block, pyrimidine. If pyrimidine cannot be made, the cell cannot synthesize DNA, which abolishes any possibility of cell growth. After transformation with the vector, the cells are placed on selective nutrient media that lacks any pyrimidine. In

A. niger glaA terminator

Xba I

Xba I

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