Team:TU-Eindhoven/Industrial

iGEM TU Eindhoven

Co-enzyme Regeneration
Coenzyme regeneration for industry

Modern society is based on a large spectrum of functional and performance chemicals allowing the production of a great variety of products which support our welfare and well-being. Pharmaceuticals are a typical example in this respect.

The challenge for industry is to produce these medicines in an efficient and accurate way. Which often is very difficult, because the synthesis route frequently is a complex one, consisting of multiple reactions, with each reaction potentially increasing the risk of unwanted side reactions. Hence there is a need for specific and efficient chemical conversions. The upcoming modern biotechnology might offer an outcome to this challenge.

All biological processes are based on chemical interactions between molecules. In order to let these processes take place as efficient as possible in nature enzymes are employed. Enzymes are proteins that catalyse biological processes, thus increasing the rate of a chemical reaction. They are substrate specific and only favour an explicit reaction. Enzymes are often affected by other molecules, either inhibitors or activators. Inhibitors are substances that decrease the enzyme’s functionality. Activators increase enzyme functionality and can be divided into two groups, cofactors and coenzymes. Cofactors are inorganic molecules, often metal ions i.e. copper or iron. Coenzymes are not enzymes as such, but a different class of enzyme activators, generally organic compounds.1,2

A first step in order to fill the need for specific, efficient and safer reactions can be taken by the implementation of biocatalysts. A second step could be the use of scaffold proteins, these could be applied for a variety of scenarios: (1) in order to mount several enzymes next to each other to create multi-enzyme synthesis systems; (2) in order to mount an enzyme next to partners to increase stability; (3) in order to create a deteriorated enzyme regeneration system, where the ‘broken’ enzyme is mounted next to a partner which ‘repairs’ it; (4) in order to create cofactor regeneration systems. This last example will be explained more thoroughly hereafter.

The enzymes that are currently used the most are often limited to simple chemical reactions.14 The reason those are used the most, is that the enzymes capable of complex chemical reactions are often coenzyme-dependent. The costs associated with these coenzymes are still too high to be used in an industrial setting. Most coenzymes show behaviour similar to substrates, they are consumed during the enzymatic reaction, efficient regeneration of these coenzymes therefore is a viable way to reduce the costs. Enzymatic reactions are the most practicable choice for coenzyme regeneration, because these reactions require high specificity in order to reach a high enough total turnover number to be economically viable. The total turnover number is the maximal number of conversions per second performed by one active site.3

The TU/e iGEM team developed a scaffold which could be used to provide a viable regeneration system. A coenzyme dependent biocatalyst could be mounted on the scaffold. In order to keep this catalyst functioning there has to be a steady supply of coenzyme. This need could be filled by regenerating the coenzymes. Assembling a coenzyme regeneration biocatalyst on the same scaffold as the enzyme will result in an efficient regeneration system (figure 1). Because around the coenzyme-consuming enzyme will be a high local concentration of consumed coenzymes, which will effectively be regenerated by the second biocatalyst.

Figure 1. shown is a regeneration system mounted on a scaffold. Enzyme 1 (E1) is used to convert substrate A into substrate B, to perform this conversion it uses a coenzyme, which is reduced in the process. A second enzyme (E2) is mounted next to E1 in order to regenerate the consumed coenzyme. E2 regenerates the coenzyme by oxidising a redox mediator (RM), this oxidised mediator (RMox) is able to regenerate the coenzyme by engaging a redox reaction between RMox and the reduced coenzyme (COEred). (note that the mediator and substrate used by E2 are chosen in such a way that they do not interfere with the enzymatic reaction of E1).
Figure 2. show is a regeneration system mounted on a scaffold, this system uses Glucose dehydrogenase (GDH) as E1 and Laccase as E2. GDH converts D-glucose to D-glucos-1,5-lactone and in the process reduces NAD(P)1 to NAD(P)H. Laccase converts oxygen (O2) to water (H2O) and in the process oxidises a redox mediator (RM). The oxidised RM (RMox) engages in a redox reaction in order to oxidise NAD(P)H to NAD(P)1.

In order to create a clearer image a concrete example will be sketched, see figure 2. For this example glucose dehydrogenase (GDH) will be used as E1, NAD(P)+ / NAD(P)H as coenzyme and Laccase as regeneration enzyme E2. GDH uses NAD(P)+ to oxidise D-glucose to D-glucose-1,5-lactone, during this process NAD(P)+ is reduced to NAD(P)H. The Laccase enzyme is employed to regenerate NAD(P)H to NAD(P)+. Laccase converts oxygen (O2) to water (H2O) and in this process a redox mediator is oxidised. This oxidised mediator then is able to perform a redox reaction with NAD(P)H, resulting in NAD(P)+ and the reduced form of the redox mediator.4 So in practice a CT52 peptide will be attached to GDH, a mutated CT52 will be attached to laccase. The scaffold used will consist of two different monomers, each one complementary to the form of CT52 used. This will ensure that each scaffold has an E1 and an E2, and not two E1 or two E2. Other substances present in the medium are D-glucose, NAD(P)+/NAD(P)H, redox mediator, O2/H2O and Fusicoccin to enable the assembly of the enzymes. When all parts are in place a system will occur where GDH consumes NAD(P)+ and Laccase regenerates it.

References
  • [1] Marieb, E. N., & Hoehn, K. (2007).Human anatomy & physiology. Pearson Education.
  • [2] Enzymes Used in Industry. (2016). Boundless. Retrieved from https://www.boundless.com/microbiology/textbooks/boundless-microbiology-textbook/industrial-microbiology-17/microbial-products-in-the-health-industry-199/enzymes-used-in-industry-1004-5469
  • [3] Faber, K., 2000, Biotransformations in organic chemistry. Berlin, Germany, Springer Verlag Science and Media
  • [4] Pham, N. H., Hollmann, F., Kracher, D., Preims, M., Haltrich, D., & Ludwig, R. (2015). Engineering an enzymatic regeneration system for NAD (P) H oxidation. Journal of Molecular Catalysis B: Enzymatic, 120, 38-46.

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