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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.
This organization could be a result of the need of a highly precise spatial disposition of the components, as a consequence of one of the major challenges that any cell has to face : channeling large amounts of hydrophobic substances through an aqueous environment to membranes or storage granules. The great majority of microbes are able to synthesize lipid granules but, to our knowledge, these structure were never observed in E. coli before cloning of the genes responsible for the production of poly-hydroxy-butyrate. Considering this cryptic property of E. coli, we hypothesized that any cell possessed the capacity to produce the molecular structures needed to channel lipids to storage granules. Thus, we began to investigate these molecular structures and were particularly interested in understanding how discrete monofunctional enzymes could organize into one single multifunctional enzyme through evolution. In order to try to shed light on the evolution processes responsible for this molecular organization, and maybe find new ways to engineer storage lipid synthesis, we started an enthusiastic exploratory tour of genomic data bases, keeping in mind the famous Carl Woese's sentence "The cell is basically an historical document".
Fatty acid synthesis is an iterative process. At each cycle of four reactions, two atoms of carbon are incorporated into the growing chain. The enzymes catalizing these four reactions are ketoacyl-ACP reductase (KR), hydroxyacyl-ACP dehydrase (DH), enoyl-ACP reductase (ER), and ketoacyl-ACP synthase (KS). During the whole process, the fatty acid chain remains attached to a specific protein, the acyl carrier protein (ACP), which hands the molecule from one enzyme to the next. At the beginning of the process, the first acyl group is transferred from acetyl-CoA to ACP by an acyltransferase (AC). At the end of the process, termination of chain elongation occurs by removing the fatty acid from ACP, often by a transesterification to glycerol catalyzed by a thioesterase (TE).
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.
