Team:SCUT/Project/System Construction/Co2 Fixation

Introduction

Having utilizing the high-concentration ATP and CO2 around mitochodria as the starting point, we did research on both endogenous metabolic pathways of S.cerevisiae and exogenous pathways that consuming CO2 and, finally set employing PRK ( phosphoribulokinase ), RuBisCo ( Ribulose-1,5-bisphosphate carboxylase ) and CA ( carbonic anhydrase ) to fix CO2 to improve the C sequestration of yeast，in other words, to increase the yield of pyruvate for butanol production as the standing point. To make full use of ATP and CO2, we tried to find out a leading peptide for locating and exogenous scaffold proteins to fix the above-mentioned enzymes to a limited space. Finally we employed Tom22, a leading peptide of outer mitochondrial membrane, and GBD, SH3 and PDZ and their ligands, to reach that goal.

Enzymes

PRK

A phosphoribulokinase is an enzyme that catalyzes the chemical reaction:
ATP + D-ribulose 5-phosphate → ADP + D-ribulose 1,5-bisphosphate

This enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing groups (phosphotransferases) with an alcohol group as acceptor. This enzyme participates in carbon fixation. As is stated in previous research, increased expression levels of PKR in yeast result in an overall positive effect on the ethanol yield. In the meantime, though, these would bring a small metabolic burden to the host cell. Therefore, its expression level should be controlled.

The PRK employed in this pathway is from Spinacia oleracea, catalyting the first reaction of this pathway.

RuBisCo

Ribulose-1,5-bisphosphate carboxylase/oxygenase, commonly known by the abbreviation RuBisCO, is an enzyme involved in the first major step of carbon fixation, a process by which atmospheric carbon dioxide is converted by plants to energy-rich molecules such as glucose.
Ribulose 1,5-bisphosphate + CO2 → 2x 3-phosphoglycerate

In cyanobacteria and many chemolithoauto-trophic bacteria, most if not all of RuBisCo is packaged in protein microcompartments called carboxysomes. It is probably the most abundant protein on Earth. However, RuBisCO also catalyses a reaction between ribulose-1,5-bisphosphate and molecular oxygen (O2) instead of carbon dioxide (CO2). Thus, something must be taken to increase concentration of the substrate of interest (CO2)to bring a high yield.

The RuBisCo we use in this pathway, cbbs, is a prokaryotic form-Ⅱ RuBisCo from Thiobacillus denitrificans and used for catalyting the second reaction of this pathway

CA

Carbonic anhydrases (or carbonate dehydratases) form a family of enzymes that catalyze the rapid interconversion of carbon dioxide and water to bicarbonate and protons (or vice versa), a reversible reaction that occurs relatively slowly in the absence of a catalyst. The active site of most carbonic anhydrases contains a zincion; they are therefore classified as metalloenzymes. The reaction catalyzed by carbonic anhydrase is:
H2CO3 → CO2 + H2O

The reaction rate of carbonic anhydrase is one of the fastest of all enzymes, and its rate is typically limited by the diffusion rate of its substrates. The epsilon(ε) class of CAs occurs exclusively in bacteria in a few chemolithotrophs and marine cyanobacteria that contain cso-carboxysomes.

The CA we use is a component of the carboxysome shell of Halothiobacillus neapolitanus c2.

Chaperons

It is stated that Functional expression of RuBisCo would be strongly stimulated in the presence of its chaperons. Compared with  the endogenous chaperon couple of S.cerevisiae, Hsp60/Hsp10, existing in mitochondrial matrix, EroGL/EroGS, two E.coli protein-folding chaperones, are much more efficient since RuBisCo expression requires their activity in the cytosol.

Scaffold Protein

Scaffold protein, without enzymatic activity, can combine with two or more proteins which increases the efficiency of interaction between individual partner molecules by the simple tethering mechanism. Beyond that, these proteins can also exert complex allosteric control over their partners as well as themselves.

Obviously, scaffold proteins offer a simple, flexible strategy for regulating selectivity in pathways, shaping output behaviors, and achieving new responses from preexisting signaling components. Scaffold proteins hence have been exploited by evolution, pathogens and cellular engineers to reshape cellular behavior.

Leading Peptide

In Saccharomyces cerevisiae, Tom protein complex is positioned on the outer mitochondria membrane. Almost all precursor proteins transported into mitochondria must cross the Tom protein complex. Tom family, including Tom20, Tom22, Tom40, Tom5, Tom6 and Tom7. Tom20 and Tom70, are the initial receptors of Tom complex. Tom22 is anchored to the outer membrane by its hydrophobic segment in the middle of the sequence and it can carry the precursor protein into the channel formed by Tom40, the β-barrier protein which precursor protein can cross, while Tom 5, Tom 6, Tom7 subunits play an important role in stabilize the Tom complex.