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Team:INSA-Lyon/Project/Stage3/Theory
From 2010.igem.org
Stage 3 : Design of synthetic multifunctional enzymes
Evolution has been naturally performing synthetic biology for the last thousands years without our knowledge. Evolution combined with mutation and environmental changes has designed and constructed new biological functions and systems not found so far in nature.
This project aims to study how this happened in nature and to use this knowledge to engineer a more complex structure into a chassis that did not possess it.
We were particularly interested in studying how discrete monofuntional enzymes got organised into one single multifunctional enzyme through evolution. Projects 1 and 2 are indeed dealing with the polyhydroxyalkanoate (pha) ABC operon gene which codes for 3 distinct enzymes. It would be the final goal of our global project to increase this lipid production by designing one optimized multifunctional enzyme.
Theory
At the very beginning of our work, we analyzed the literature on storage lipid synthesis and realized that Evolution had performed synthetic biology long before us.
In bacteria, such as E. coli or B. subtilis, and plants, the metabolic reactions leading to fatty acid synthesis are catalized by a collection of separate, "classical", monofunctional enzymes. However, in animals, the different enzymes are integrated into a single multifunctional protein in which substrates are handed from one functional domain to the next. In humans for instance, a 2511-residue polypeptide consisting of 7 domains contains all the catalytic components required to perform the 37 sequential reactions leading to the synthesis of palmitic acid from acetyl- and malonyl-CoA.
FAS I model, extracted from Wikipedia, visualization by Kosi Gramatikoff.
The organization of those FAS, integrated in FAS I, discrete in FAS II, differ from one another but their mechanisms of elongation and reduction are quite alike. Each separated FAS II enzymes can be associated to its equivalent domain in FAS I.
The evolutionary history of fatty acid synthases is therefore a tremendous source of information which will enrich our understanding of why and how FAS I and FAS II have co-evolved.
When the chain is 16 carbon atoms long, a last step is performed where the Acyl Carrier Protein is removed:
FAS I enzyme is therefore a multifunctional protein catalyzing the reactions of Malonyl and Acetyl Transferase, ß Ketoacyl-ACP Synthase, ß Ketoacyl-ACP Reductase, 3 Hydroxyacyl-ACP Dehydrase, Enoyl Reductase and Thioesterase, as refered in the table below with their corresponding EC_numbers.
