Cofactor engineering

Summary of Cofactor Engineering
Cofactor engineering is a subset of metabolic engineering where engineers alter an organism’s metabolic pathways. Cofactor engineering utilizes computational analysis of metabolic pathways and recombinant DNA technology. Cofactor engineering has applications in industrial manufacturing, pharmaceuticals, and medicine.
History of Cofactor Engineering
The concept of a cofactor was first conceived by Arthur Harden and William Youndin in 1906. Harden and Youndin performed an experiment where they added boiled yeast extracts to unboiled yeast extracts. They noticed that the rate of alcoholic fermentation increased when the boiled yeast extract was added. A few years after, Hans von Euler-Chelpin identified the cofactor that Harden and Youndin found as NAD+. (Wiki 63). Other cofactors, such as ATP and coenzyme A, were discovered during the 1900’s. Cofactors throughout the nineteenth century were not understood well. In 1936, Otto Heinrich Warburg determined that NAD+ functioned as an electron acceptor. Eventually in the early 1990’s recombinant DNA technology emerged. This created the fields of metabolic engineering and cofactor engineering. Cofactor engineering, which has been studied less than metabolic engineering, is still young. Cofactors and the metabolic pathways that utilize them need to be studied further.

Tools and Processes of Cofactor Engineering

Cofactor engineering requires a medium to occur in. The medium is often supplemented with glucose and will contain various molecules needed by the specific experiments. Pre-cultures are then grown on these mediums in shake flasks. The flasks are left on a Shaker machine while they grow. This agitates the culture resulting in its aeration. An aerated culture is important because if it wasn’t the growing organisms wouldn’t be able to breathe. The Shaker machines usually run around a few hundred rpm’s. Once the pre-cultures are ready they are fermented in bio-reactor. The bio-reactors are systems in which one can allow a culture to grow in a controlled environment. The needed temperature, pH, and various other environmental factors can be controlled by the bio-reactor to accomplish this. Once samples are finished growing in the reactor they can be removed and studied to determine whether the alterations to the organism where beneficial or not. Metabolic Engineering Jul2006, Vol. 8 Issue 4, p303-314 Metabolic Engineering Apr2004, Vol. 6 Issue 2, p133
Cofactor Engineering as a Subset of Metabolic Engineering
Metabolic engineering is considered the manipulation of the reactions that take place within cells. Cofactor engineering represents a more recently explored approach within the field of metabolic engineering, which involves more specifically the manipulation of the way cell’s metabolic pathways utilize cofactors. Like metabolic engineering, cofactor engineering requires the use of recombinant DNA technology, but metabolic engineering involves changes in the expression of all sorts of enzymes and other proteins involved in metabolic pathways. Cofactor engineering is confined to the manipulation and study of the proteins, which directly bind to, or other wise effect cofactors.

Cofactors
Cofactors are described as simple compounds, which bind to proteins, and are necessary for at least one of a protein’s functions within a cell. The proteins considered here are generally enzymes, which are considered either apoenzymes (not bound to cofactor) or holoenzymes (bound to cofactor), when there are in their active and inactive states respectively. However, some enzymes require the presence of several cofactors in order to function properly. Cofactors may be either organic or inorganic, but some sources consider cofactors to be solely inorganic. Some examples of cofactors include metallic ions, vitamins, and their derivates, such as NAD⁺, which are important dietary components of many organisms. This is indicative of their importance to the overall metabolism of such organisms.

Potential Applications

A. Changing an enzymes cofactor from NADPH to NADH

Both NADPH and NADH are important cofactors used by many enzymes to catalyze reactions. In the manufacturing world, NADPH is more expense and less stable than NADH. For these reasons, manufacturing plants prefer enzymes to use NADH over NADPH. Cofactor engineering has been successful in altering an enzyme to prefer NADH as a cofactor instead of NADPH.

In 2010, a group of scientists performed cofactor engineering on Saccharomyces cerevisiae, a yeast able to create enantiopure chiral compounds commonly used in pharmaceutical production. Saccharomyces cerevisiae has a natural preference to use NADPH as a cofactor instead of NADH. The scientists used site-directed mutagenesis to alter the cofactor site of the catalyst involved in producing enantiopure chiral compounds. The results showed that the new catalyst was capable of using NADH as a cofactor instead of NADPH. This allows chemical manufacturing plants to take advantage of the benefits of NADH over NADPH.

B. Wine and Beer Industry
Yeast is commonly used in the beer and wine industry since they are capable of producing ethanol. Ethanol is produced from an enzyme that uses NADH as a cofactor. Scientists engineered a Saccharomyces cerevisiae to contain a water forming NADH oxidase and tested how this modification would impact the ethanol yield. The results showed that yeast with NADH oxidase produced 15 percent less ethanol. This occurred because the NADH oxidase lowered the NADH concentration in the cell, which prevented enzymes from producing ethanol. This is applicable to the beer and wine industry who want to adjust their ethanol levels in their beverages. The advancements in the wine industry have caused a steady increase in ethanol content of their beverages, and winemakers want to be able to reduce the ethanol levels.

C. Citric Acid Cycle

Cofactors are essential in the Citric Acid Cycle. The manipulation of cofactors CoA/acetyl-CoA during the pyruvate dehydrogenase reaction has shown to lead to an increase in succinate and lycopene production. (http://scholarship.rice.edu/handle/1911/18718) Both succinate and lycopene have beneficial effects on the human body. An increase in succinate, which is used as a catalyst during the process, may lead to an increase in the speed of the Citric Acid Cycle. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1314946/) An increase in lycopene, a compound that has been shown to decrease the risk of prostate cancer, may help reduce the risk of prostate cancer.

D. Biofuels
The idea of using biomass for fuel has been a possibility for the last three decades although not until recent advancements in cofactor engineering has allowed for these fuels to produced more cost effectively and possibly vault biofuels into a larger percentage of energy generation in America. Although the specific technology to reach this goal has not been created yet there is a strong possibility that as cofactor engineering advance the technology can be applied to the conversion of biomass to biofuel. By adjusting the process of ethanol creation in cells ethanol could be produced 10 to 30 percent cheaper than it currently is today.

E. Manufacturing

Many industrial useful enzymes utilize cofactors to catalyze reactions. Cofactor engineering is young and has not been studied to its fullest extent. However, cofactor engineering’s other potential applications are similar to metabolic engineering it involves the manipulation of metabolic pathways. Some of these applications include: lowering manufacturing costs, improving manufacturing efficiency, and lowering the pollution from chemical manufacturing plants. (http://www.springerlink.com/content/9ndfbb37pvch6f6b/) One case that shows all three manufacturing benefits involves the genetic engineering of aspen trees. In the tree to paper process, manufacturing plants have to remove lignin, a biochemical compound that gives the tree trunk its stiffness, from the wood. The removal process requires the manufacturing plant to use expensive and toxic chemicals. A group of genetic engineers created a particular aspen tree that produced less lignin. These genetically engineered trees allowed for paper mills to reduce their costs, pollution, and manufacturing time.