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Team:SCUT/Project/System Construction/n-Butanol Prod
From 2014.igem.org
Introduction
n-ButanolSoaring energy costs and increased awareness of global warming have motivated production of renewable, bio-mass-derived fuels and chemicals. Since butanol has a longer chain length than ethanol, it has a higher energy density than ethanol and can be blended up to 85% with gasoline; while ethanol can only be blended up to 10% due to limits set by regulation and requirements of engine modification. The high percentage of butanol-blending renders it an attractive biofuel. (Figure 1) N-Butanol can be produced either chemically from petroleum or fermentatively in a variety of Clostridial species. Clostridia are not ideal because of the relative lack of genetic tools to manipulate their metabolism, their slow growth, their intolerance to n-butanol above 1–2% and oxygen, and their production of butyrate, ace-tone, and ethanol as byproducts.
Figure 1 Butanol is a potential eco fuel in the future.Compared to ethanol, n-butanol is more hydrophobic, has a higher energy density, can be transported through existing pipeline infrastructure and mixed with gasoline at any ratio.
Here we engineered Saccharomyces cerevisiae with an n-butanol biosynthetic pathway. We chose Saccharomyces cerevisiae as a host for n-butanol production because it is a genetically tractable, well-characterized organism, the current industrial strain alcohol (ethanol)producer, and it has been previously manipulated to produce other heterologous metabolites . Recently, S. cerevisiae has been demonstrated to have tolerance to n-butanol.
Mitochondrial matrixThe environment within the mitochondrial matrix differs from the cytoplasm, including '''higher pH, lower oxygen concentration, and a more reducing redox potential''', which may more closely match the optimal for maximal activity of many enzymes. Besides, the smaller volume of mitochondria, could concentrate substrates favoring faster reaction rates and productivity and confine metabolic intermediates avoiding repressive regulatory responses, diversion of intermediates into competing pathways or even toxic effects of intermediates to cytoplasmic or nuclear processes. (Figure 2)
Figure 2 Physicochemical property of mitochondrial matrix: a. powerful coenzyme generation system; b. higher concentration of substrates; c. avoiding competing pathways or toxic effects.
Mitochondrial matrix leading peptide ---- CoxVI
Most mitochondrial proteins are synthesized in the cytosol as larger precursors carrying mitochondria targeting signals. In this pathway, the precursor is bound by cytosolic chaperones and then delivered to a set of receptors on the outer surface of mitochondria. Then, the polypeptide chain is passed through the TOM complex in the outer membrane and the TIM23 complex in the inner membrane. Insertion into the inner membrane is driven electrophoretically by the electrochemical potential across the membrane. Finally, the precursor is pulled completely across the membrane into the matrix by an ATP-powered translocation motor attached to the inner side of the TIM23complex. After that, the protein will be refolded by mitochondrial chaperone, and the mitochondria targeting signal will be cleaved.(Figure 3)
Figure 3 The mechanism of CoxVI signal peptide.
Design
Aimed to take advantage of the potential attributes of the mitochondrial environment, we engineered Saccharomyces cerevisiae mitochondria with a n-butanol biosynthetic pathway original from Clostridium beijerinckii in which isozymes from a number of different organisms (Figure 4) were substituted for the Clostridial enzymes.
Figure 4 The n-butanol biosynthetic pathway. Enzymes are from these organisms: Erg10, S. cerevisiae; Hbd, Crt, AdhE2, Clostridium beijerinckii; Ccr, Streptomyces collinus.
As the pathway showed above, there are three reactions involving NADH (NADPH) as cofactors that are abundant in mitochondrial matrix. Additionally, production of HbCoA by 3-hydroxybutyryl-CoA dehydrogenase is the rate-limiting reaction that determines the n-butanol production in this context. All these suggest the pathway will be more efficient when translocated in mitochondria. To accomplish our proposal, we must first translocate the n-butanol biosynthetic pathway into the mitochondrial matrix. After reading lots of literatures about leading peptide, we chose to use the N-terminal mitochondrial localization signal from subunit VI of the yeast cytochrome c oxidase (CoxVI) (Shown in Figure 3), to target the pathway to the mitochondrial matrix.
Construction
The five genes encoding these six enzymes were cloned into two different plasmids -----YEplac181 and YEp352 (Figure 5), which can coexist in one cell. Considering our multiple-enzyme system, it is necessary to use those compatible plasmids. Most genes were synthesized by Genewiz: Hbd, Crt, AdhE2 (C. beijerinckii), and Ccr ((Streptomyces collinus). While S. cerevisiae gene: Erg10 were cloned from genomic DNA. And all enzymes were under control of GAL1 promoter with CYC1 or ADH1 terminator.
Figure 5 Profile of reconstructed plasmids in n-butanol project.
Besides targeting the n-butanol biosynthesis pathway to mitochondrial matrix with the help of CoxVI, we constructed other two plasmids without CoxVI, which locate the pathway to the cytoplasm as a control group. (Figure 6) The different n-butanol productions of the two parallel assemblies will prove whether the environment of mitochondrial matrix can make the target reactions more efficient.
Figure 6 Construction scheme for the pathwaty targeting to cytoplasm and mitochondrial matrix.
Localization Test
Leading Peptide VerificationTo justify the localization ability of CoxVI, the C-terminus of CoxVI was fused to GFP; while the control group was without CoxVI. (Figure 7) We use MitoTracker Red®CMXRos to stain the mitochondria and do an overlap with GFP. By observing the coefficient value, we can judge that if our leading peptide works.
Figure 7 Construction scheme for leading peptide verification: (a)positive control, (b)negative control.
We adopted fluorescence test to have a visible look at where the fusion protein is located. The following microscope images were obtained. (Figure 8) It was observed that green fluorescence concentrated in mitochondria stained by MitoTracker Red®CMXRos, instead of diffusing in the cytosol. Figure 8(a) proves that CoxVI can locate GFP into the mitochondrial matrix.
Enzyme location confirmationTo justify whether the enzymes can be transported into mitochondrial matrix correctly with the help of CoxVI, we fused GFP to the C- terminus of all five enzymes. And of course, we set up two parallel groups, one with CoxVI targeting to the mitochondrial matrix, another without CoxVI targeting to the cytoplasm. (Figure 9)
Figure 9 Construction scheme for enzyme location verification: (a)positive control, (b)negative control.
Under the confocal microscope, we can observe the location of green fluorescence, thus to confirm the exact subcellular localization of target proteins. (Figure 10)
The result proved that fusion protein with construction strategy could be effectively localized into mitochondrial matrix of yeast.References
[1]KOSTAS TOKATLIDIS, Directing Proteins to Mitochondria by Fusion to Mitochondrial Targeting Signals, METHODS IN ENZYMOLOGY, VOL. 321. [2]José L. Avalos et al. (2013). Compartmentalization of metabolic pathways in yeast mitochondria improves production of branched chain alcohols. Nature Biotechnology 2059. [3]Masayuki Inui, et al. (2008). Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli. Appl Microbiol Biotechnol 77:1305–1316. [4]Fischer CR, et al. (2008). Selection and optimization of microbial hosts for biofuels production. Metabolic Engineering. [5]Eric J Steen, et al. (2008). Metabolic engineering of Saccharomyces cerevisiae for theproduction of n-butanol, Microbial Cell Factories, 7:36. [6] DavidR.Nielsen, et al. (2009). Engineering alternative butanol production platforms in heterologous bacteria. Metabolic Engineering11, 262–273.
