Synthetic non-oxidative glycolysis (NOG) enables complete carbon conservation

Transcription

1 Synthetic non-oxidative glycolysis (NOG) enables complete carbon conservation Igor W. Bogorad, Tzu-Shyang Lin, James C. Liao Nature, vol 502, 2013 Presented by Elise Jyränoja, Kellen Leskinen and Tia Korhonen

2 Motivation: Improving on natural glycolysis Glycolysis involves the partial oxidation and splitting of sugars to pyruvate, which in turn is decarboxylated to produce acetyl-coenzyme A (CoA) for various biosynthetic purposes. The theoretical carbon yield is limited to two moles of two-carbon metabolites (C2) per mole of hexose. So one third of carbon is lost. Image by Thomas Shafee

3 Motivation: Improving on natural glycolysis Authors constructed a non-oxidative, cyclic pathway that allows the production of stoichiometric amounts of C2 metabolites from sugar and sugar phosphates without carbon loss. NOG or non-oxidative glycolysis enables complete carbon conservation in sugar catabolism to acetyl-coenzyme A. CBB - Calvin-Benson-Bassham pathway RuMP - Ribulose monophosphate pathway EMP - Embden-Meyerhof-Parnas pathway F6P = Fructose 6-phosphate NOG was demonstrated both in vitro and in engineered Escherichia coli strains. Industrially this new approach could be used to produce bio-alcohols, fatty acids, biodiesel and isoprenoids from sugars.

4 Non-oxidative Glycolysis Pathway The metabolic logic of the cyclic NOG pathway is broken down into three sections: First, fructose 6-phosphate (F6P) is the input molecule, and the pathway requires an additional investment of two F6P molecules. Second, the three F6P molecules are broken down to three acetyl phosphate (AcP) and three erythorse 4-phosphate (E4P) molecules by the phosphoketolases. Third, these three E4P molecules then undergo carbon rearrangement to regenerate the two initially invested F6P molecules. The net reaction results in the irreversible formation of three AcP molecules. F6P - fructose 6-phosphate AcP - acetyl phosphate E4P - erythorse 4-phosphate

5 Phosphoketolase activity Two different possible phosphoketolase activities Fructose 6-phosphate (F6P) activity termed Fpk Xyloluse 5-phosphate (X5P) activity termed Xpk

6 Example of Carbon Rearrangement Three E4P molecules undergo carbon rearrangement to regenerate the two initially invested F6P molecules. There is two variations of carbon rearrangement networks: FBP-dependent network SBP-dependent network (a) FBP-dependent (b) SBP- dependent FBP - fructose 1,6-bisphosphate SBP - sedoheptulose 1,7-bisphosphate

7 More Examples of NOG networks Because there are two different possible phosphoketolase activities and two variations of the carbon rearrangement networks, many combinations can be devised Three FBP-dependent NOG networks For each of the carbon rearrangement networks these three configurations form the basis to all other combinations. a: NOG using Fpk only b: NOG using Xpk c: NOG using one Fpk with two Xpk

8 In Vitro Tests The core system consisted of eight enzymes * which convert one mole of F6P (fructose 6-phosphate) to three moles of AcP (acetyl phosphate). F6P was completely converted to stoichiometric amounts of AcP. The addition of Ack (acetate kinase) and Pfk (phosphofructokinase) allowed further complete conversion of acetyl phosphate to acetate. * Eight core enzymes were: phosphoketolase with Fpk and Xpk activities (F/Xpk), transaldolase (Tal), transketolase (Tkt), fructose 1,6-bisphosphatase (Fbp), ribulose-5-phosphate epimerase (Rpe), ribose-5-phosphate isomerase (Rpi), FBP aldolase (Fba), and triose phosphate isomerase (Tpi).

9 In Vitro Tests Similar in vitro NOG systems were tested on R5P and G3P, which produced nearly theoretical amounts of acetyl phosphate at a ratio of 2.3 and 1.6, respectively. These in vitro results demonstrated the feasibility of NOG and paved the way for in vivo testing. Conversion of three sugar phosphates to near stoichiometric amounts of acetyl phosphate F6P = fructose 6-phosphate R5P = ribose 5-phosphate G3P = glyceraldehyde 3-phosphate

10 Tests with Engineered E. coli Pathway in E.coli strain NOG: xylose acetate and side products Overexpression enzymes F/Xpk and Fbp (pib4) High copy number plasmid (pib4) Controlled by P L laco 1 and IPTG inducible promoter Grown: Aerobic conditions (growth) and anaerobic conditions Luria-Bertani (LB) with 5% xylose, induced IPTG 0,1µm for testing. Detection Method: His-tag column purification, coupled colorimetric assay to test AcP production

11 Tests with Engineered E. coli Figure B: JCL 118 (control) no AcP Figure C: E. coli strains: Ratio xylose/acetate JCL16 (wild type) (1:0.4) JCL166 ( ldha adhe frdbc) (1:1.1) JCL118 ( ldha adhe frdbc pflb). (1:2.2) Near to theoretical ratio of acetate/xylose were obtained by strain JCL118/pIB4 (1:2.5)

12 Conclusion NOG was demonstrated both in vitro and in engineered Escherichia coli strains. Industrially this new approach could be used to produce bio-alcohols, fatty acids, biodiesel and isoprenoids from sugars. When a sugar (such as xylose or glucose) is used as the initial substrate to generate the input to NOG, acetate can be produced without carbon loss and without additional input of reducing equivalents. Once additional reducing power is provided in the form of hydrogen or formic acid, NOG can be used to produce compounds that are more reduced than acetate, such as ethanol, 1-butanol, isoprenoids and fatty acids.