Genetic and Metabolic Engineering

Yea-Tyng Yang
Department of Bioengineering and Chemical Engineering
Rice University, P.O. Box 1892, Houston Texas 77251-1892
E-mail: tinayang@rice.edu

George N. Bennett
Department of Biochemistry and Cell Biology
Rice University, P.O. Box 1892, Houston Texas 77251-1892
E-mail: gbennett@bioc.rice.edu

Ka-Yiu San^*Department of Bioengineering and Chemical Engineering
Rice University, P.O. Box 1892, Houston Texas 77251-1892
E-mail: ksan@rice.edu

http://www.rice.edu

Recent advances in molecular biology techniques, analytical methods and mathematical tools have led to a growing interest in using metabolic engineering to redirect the flow through metabolic pathways for industrial and medical purposes. Metabolic engineering is generally referred to as the directed improvement of cellular properties through the modification of specific biochemical reactions or the introduction of new ones, with the use of recombinant DNA technology. This multidisciplinary field draws principles from chemical engineering, biochemistry, molecular and cell biology, and computational sciences. In essence metabolic engineering is the application of engineering principles of design and analysis to the metabolic pathways in order to achieve a particular goal. This goal may be to increase process productivity, such as the production of antibiotics, biosynthetic precursors or polymers, or to extend metabolic capability by the addition of extrinsic activities for chemical production or degradation.

The interest in metabolic engineering is stimulated by potential commercial applications in that improved methods are sought for developing strains which can increase production of useful metabolites. Recent endeavors have focused on the theme of using biologically derived processes as alternatives to chemical processes. Such manufacturing processes pursue goals related to "sustainable development" and "green chemistry" as well as positioning companies to exploit advances in the biotechnology field. Some examples of these new processes include the microbial production of indigo (Genencor) and propylene glycol (DuPont) and others involve improvements in the more traditional areas of antibiotic, and amino acid production by a number of large firms. The extension of metabolic engineering to production of desired compounds in plant tissues and to provide better understanding of genetically determined human metabolic disorders broadens the interest in this field beyond the fermentation industry and bodes well for the future importance of this approach.

Naturally Occurring Genetic Processes

The genetic makeup of an organism is generally quite stable. However, it is not absolutely time invariant. All organisms suffer a certain number of mutations as a result of normal cellular operations. The frequency of these mutation events, known as spontaneous mutations, depends on the type of organism and the exposed environment. Exchange of genetic material also occurs in nature. For example, transfer of genetic material from one microbial cell to another may involve one of the following three mechanisms: transformation, conjugation and transduction. Transformation is a process by which certain bacterial cells import soluble DNA from their surroundings. Bacteria known to be capable of natural transformation include Bacillus subtilis, Streptococcus pneumoniae and Haemophilus influenzae. Cells that are able to carry out this transformation process are said to be competent.

Another mechanism by which direct transfer of genetic material from one bacterial cell to another is called conjugation. This process is mediated by plasmids called conjugative plasmids. Plasmids are self-replicating, double stranded extrachromosomal DNA elements. The first and most studied is the F plasmid (also known as the sex plasmid), which can replicate in Escherichia coli and other related strains. The F plasmid can transfer a replica of the plasmid from a donor cell to a recipient cell.

Consequently, mixing of F⁺ (cells containing an F plasmid) with F^- cells quickly results in a culture full of F⁺ cells. Many R-factors expressing antibiotic resistance are capable of moving to another cell by conjugation. Transduction also may result in transfer of genetic material. In transduction, a small piece of chromosome from a host is incorporated into a maturing phage particle. When this particle infects a new host after its release from the original host, it injects the genetic material from the former host, thereby transferring DNA from one cell to another. Upon entering the cell the DNA can recombine with the host chromosome through various means providing new genes which may help the cell survive in some environment. A variation of the virus infection route is when the virus itself can recombine with the chromosome to give an integrated form which can be carried along with the chromosome during normal replication and cell division. Evidence of unknown integrated viruses has been found from DNA sequencing of microbial genomes.

The consequence of genetic perturbation may be insignificant, however in some cases it may yield desirable traits. Yet in other cases, it may be detrimental to the organism or even have a lethal effect. Historically, mankind has taken advantages of these naturally occurring genetic events. For centuries farmers have practiced selecting and saving of seeds, cuttings, or tubers from superior plants at harvest time for the next planting season. The screening and selection of high yield strains for the production of antibiotics is another example. Other examples are in the isolation and propagation of strains for production of fermented beverages and cheeses which give rise to the traditional specific character of these foods and which are less susceptible to disruptive phage infections or development of off-flavor during the production process.

Metabolic Engineering

Selection of desirable cell lines as a result of natural mutation has proven to be very effective in increasing process productivity. However, the process of natural mutation is slow and random. Exposing organism to mutagenic agents has been used to speed up the mutation process.

Nevertheless, this approach is still random in nature and it is extremely difficult to fully characterize the resulting mutant. Recent advances in molecular and cell biology have provided a unique opportunity to manipulate the genetic makeup in a precise manner.

In summary, genetic and metabolic engineering has the potential to design a process to yield high quality, less expensive and/or completely novel products. In addition, mathematical and systematic treatment of the effect of these genetic perturbations provides more detailed analysis and insight. The combined knowledge is often capable of pointing to a direction for possible genetic changes in order to achieve a desired goal. Metabolic engineering principles have been used to improve biocatalysts either to increase product yield or to synthesize new products in areas such as recombinant protein, amino acid and antibiotic production processes. Examples of application of metabolic engineering to plants include the development of produce that has a longer shelf life, plants that have more resistance to insects and other adverse environments, such as drought or soil with high salt content or that may have improved digestibility or nutritional qualities. Metabolic engineering principles have also been employed to study human diseases such as those associated with the heart and liver. Furthermore, it may also play a role in improving our health care system by pointing to new therapeutic strategies and targets.