Team:SCUT/Project/Overview

From 2014.igem.org

Overview

A cellular compartment is an intracellular space enclosed by a single or double lipid layer membrane that separates specific molecules and molecular machineries from the cytosol. Different intracellular pH, different enzyme systems and other substances are isolated within membrane-bound compartments. Due to compartmentalization and positional assembly, a cell can perform different metabolic activities at the same time. This generates specific micro-environments that regulate multistep processes. This project aimed to develop methodologies that allow a better control over complex synthetic reactions.

Mitochondrion is a membrane-enclosed organelle that generates the energy currency of the cell, i.e. ATP, through cellular respiration. In the tricarboxylic acid (TCA) cycle, each pyruvate molecule produced by glycolysis is actively transported across the inner mitochondrial membrane into the matrix where it is oxidized and combined with coenzyme A to form CO2, acetyl-CoA, and NADH. As a consequence, the environment in the mitochondrial matrix has a lower oxygen concentration, a higher pH and a more reducing redox potential than the cytoplasm. This is beneficial to the activity of several enzymes that need reduced cofactors which are synthesized exclusively in mitochondria.

In order to take advantage of the particular features of the mitochondrial environment and increase the effective concentration of each component of an n-butanol pathway, we transferred the complete n-butanol production pathway into the yeast mitochondrial matrix using several leading peptides. Furthermore, a parallel assembly of three carbon sequestration enzymes were organized onto the outer membrane of mitochondria with a scaffold protein. With this machinery, the waste byproduct CO2 released by the TCA cycle was recycled to produce pyruvate, thereby increasing the substrate supply for the production of n-butanol (Fig. 3).

Fig. 1 The engineered pathway. The complete n-butanol pathway was targeted into the     mitochondrial matrix. The carbon sequestration enzymes were anchored onto the outer membrane of mitochondria to recycle the CO2 released through TCA. The darkness of the green indicates concentration of CO2: the darkest green marks the highest value.

Compartmentalization of metabolic pathways into a controlled organelle has been proved to increase the target production effectively. Here, the new concept of PAN-compartmentalization is introduced, which considers both the inner side and the outer side of the compartment as the target. The core principle of pan-compartmentalization is to achieve the higher enzymes concentration, to recycle byproducts and to increase the availability of substrates, in order to maximize the carbon recovery and the efficient use of coenzymes. Simple word is good understanding and make the best of the environment in/out of the specific compartment.