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The applications of protein engineering can be found everywhere in biology. This field, known as proteomics, is a reliable means of producing novel molecules and has provided scientists a basic roadmap to understanding the essential properties of proteins. Therefore to attempt a complete survey of the land is fruitless. However, it is useful to note some specific chemical compounds proudly offered from A.G. Scientific. In particular, we will be focusing on Polyketide Synthases (PKSs), and their polyketide products in this article.

What are polyketides?

According to the NIH, Polyketides are a broad class of natural products that include a number of products such as antibiotics, anti-fungals, immunosuppressants, and other active pharmacological compounds. Motivated by the value of these natural products, there has been much research focused on developing guidelines for engineering polyketide synthases (PKSs) to generate novel polyketides. Together, these polyketide compounds account for billions of dollars in global annual sales [1,2].

One recent report by The Johns Hopkins University3 provided interesting insights into the enzymatic specificity of the polyketide synthesis pathway, and have demonstrated that it is possible to synthesize the four main chemical types required by PKSs to generate novel polyketides.

Figure 1. Combinatorial Synthesis of the "Four Star" Product Set Required by PKSs.


Image: Pairings of iterative enzymes called non-reducing polyketide synthases, or PKSs by their more familiar acronym. These PKS enzymes catalyze the production of polyketides. It has been a long-standing goal for protein engineers to prep these polyketides for research and possible therapeutic use. The enzymes used in this study produced the four main chemical types of PKSs required to produce novel polyketide molecules (pictured above).

The researchers for the above study remarked, "We found that boundary conditions limit successful chemistry, which are dependent on a set of underlying enzymatic mechanisms. Crucial for successful redirection of catalysis, the rate of productive chemistry must outpace the rate of spontaneous derailment and thioesterase-mediated editing."

To read the full report Click BELOW >>>>>>

Article: Systematic Domain Swaps of Iterative, Nonreducing Polyketide Synthases Provide a Mechanistic Understanding and Rationale For Catalytic Reprogramming (The Johns Hopkins University)

How are polyketides made?

Polyketides are produced naturally from soil dwelling organisms, including fungi. Their synthesis requires the regulated activation of the enzyme cascade for PKSs.

There are less than 10,000 known polyketides, from which an extraordinarily large number of major pharmaceutical products have been derived, according to the journal Current Opinions in Biotechnology [4].

As a class, polyketides are considered one of the richest sources of pharmaceuticals and pharmaceutical revenues, and include numerous antibiotics (e.g. erythromycin, tetracycline), anticancer agents (doxorubicin), immunosuppressants (FK506, rapamycin), antiparasitic agents (avermectin, nemadectin), antifungals (arnphotericin, griseofulvin), cardiovascular agents (lovastatin, compactin), and veterinary prodttcts (monensin) (See Figure 2).

The term "polyketide" is used to refer to a broad class of natural products which includes: aromatic, polyether, and macrolactnne compounds. Although the final structures of polyketide metabolites are diverse, these molecules share common features in their biosynthetic pathways, and intermediates formed in these pathways. For example, all polyketides are formed by the condensation and subsequent modification of acyl units derived from acyl-coenzyme A precursors.

Figure 2. Catalytic Domains within the PKS Complex.


Model displaying artificial "synthetic" domains with having a defined stoichiometry and compatibility based on different recognition specificities (cohesin and dockerin proteins). Cohesin domains are linked together in a defined order into a multimodule scaffolding polypeptide. Catalytic domains to be integrated within the complex are linked to their appropriate dockerin domains in order to interact with their specified extracellular ligand. 

(Image courtesy: Current Opinion Biotechnology [1].) 

How are polyketides made?

Polyketides are made by the contiguous activity of a large number of enzymes and shuttle proteins (usually 5 to > 40) collectively referred to as 'PKSs'. PKSs can exist in two kinetic forms, as either a "modular" (single-use) or "iterative" (conserved) enzyme. In either case, the product that is produced out of its catalytic domain generates the substrate for the next reaction in a neighboring ligand. However, modular polyketides will produce a more complex array of molecules and generally considered by protein engineers to possess greater 'combinatorial potential' [4-5]. As their very nature allows, PKSs can selectively add, delete, and rearrange the order of each catalytic domain. This process generates novel PKSs. Once the PKS is engineered, a new order of catalytic reactions is established which grants the expert protein engineer access to the wealth of new products it produces.

The original coupling reaction experiments were investigated in the late 1980s and early 1990s by Mosbach, Bülow and their co-workers [6, 8]. They tested several fusion proteins, including hybrids of beta-galactosidase and galactose dehydrogenase [6], galactose dehydrogenase and bacterial luciferase [7], and malate dehydrogenase and citrate synthase [8].

The ability to engineer enzymes in order to improve their properties is one of the obvious goals of biotechnology. In addition, it has also become an essential tool of research for basic protein bio- chemistry. The major proof-of-concept work of this technology has been completed, leaving the remaining work to applying the technology to current and new situations.

PKSs are considered essential to the protein chemists toolbelt because it enables them to change the function of a protein from one mode to another. Considering that proteomics has revealed only 60% of proteins known at this time, these tools will continue to be highly prized among the biomedical community for some time to come.


Further Reading

[1] Khosla C1, Zawada RJ. Generation of polyketide libraries via combinatorial biosynthesis. (September, 1996) Current Opinion Biotechnology. 14(9):335-41.

[2] C. Khosla and R.J.X. Zawada, Generation of polyketide libraries via combinatorial biosynthesis. TIBITECH. 1996. 14:335.

[3] Newman, A.G.; Vagstad, A.L.; Storm, P.A.; Townsend, C. A. Systematic Domain Swaps of Iterative, Non-reducing Polyketide Synthases Provide a Mechanistic Understanding and Rationale For Catalytic Reprogramming J. Am. Chem. Soc. 2014, 136, 7348-7362.

[4] Hutchinson CR. "Combinatorial biosynthesis for new drug discovery." Current Opinion in Microbiology. (1998). 1;319-329.

[5] Verdine GL; "The Combinatorial Chemistry of Nature." Nature, 1996. 384:11-13.

[6] Ljungcrantz P, Carlsson H, Mansson M-O, Buckel P, Mosbach K, Bülow L: Construction of an artificial bifunctional enzyme, b-galactosidase/glactose dehydrogenase, exhibiting efficient galactose channeling. Biochemistry 1989, 28:8786-8792.

[7] Lindbladh C, Persson M, Bülow L, Mosbach K: Characterization of a recombinant galactose dehydrogenase/luciferase, displaying an improved bioluminescence in a three-enzyme system. Eur J Biochem 1991, 204:241-247.

[8] Lindbladh C, Rault M, Hagglund C, Small WC, Mosbach K, Bülow L, Evans C, Srere PA: Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase. Biochemistry 1994, 33:11692-11698.


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