Biosynthesis of Ethanol Using E.coli System Group 11 生資所碩士班 924287 呂博凱 資工系博士班 928309 陳炯勳 資工系碩士班 924360 唐宗麟 Jan 14, 2005
Is E. Coli suitable for biosynthesis of ethanol? Pathway1 It is necessary to find some ways that increase the flow of reaction 12. Pathway2 We can overexpress enzyme pyruvate decarboxylase (PDC, EC:22.214.171.124) to produce reaction 15. Pathway 1 Pathway 2 acetaldehyde pyruvate
1.The native alcohol dehydrogenase (ADH) activity of E. coli was not to achieve high ethanol yields. We can transform the adh II gene to E.coli. 2.The plasmid stability in non-selective medium. We can integrate the genes into the chromosome of E.coli, but that reduces the gene dosage, resulting in decreasing the enzyme activity. Pathway 1? or Pathway 2 ? The shortcoming of using pathway1 to produce ethanol. 1. The pathway 1 is unbalanced because one NADH, H+ is generated for each pyruvate made from sugars, and two NADH,H+are required for converting pyruvate into ethanol.E. coli balances it fermentation by also producing acetic and succinic acids 2. E. colicultures are a narrow and neutral pH growth range(pH 6.0–8.0). We choose pathway 2 to produce ethanol in E.coli system and transform the pdc gene into E.coli. The shortcoming of using pathway2 to produce ethanol.
Anaerobic fermentation pathway of the E.coli containing the PDC & ADHII enzymes Glucoseout → Glucosein → G6P → FDP → PEP → Ethanol
VATPase, Y7 Define the relevant variables Metabolites X1 (Glcin, intracellular glucose) X2 (G6P,glucose-6-phosphate) X3 (FDP, fructose diphosphate) X4 (PEP, phosphoenol pyruvate) X5 (ATP) Enzymes Y1 (Vin, sugar transport system) Y2 (VHK, hexokinase) Y3 (VPFK,phosphofructokinase) Y4 (VGADP, glyceraldehyde 3-phosphate dehydrogenase) Y5 (VPK, pyruvate kinase) Y6 (Vcarb, glycogen synthetase) Y7 (VATPase, ATPase)
GMA representation 1.Each rate law can be written as a form of power law functions. 2.a,b means rate constant of the positive and negative reactions. 3.gijk and hijk means kinetic orders of the positives and negative reaction. 4.n means dependent variables and m means independent variables. Define the kinetic orders and rate constants GMA representation of metabolites
VATPase, Y7 Model description
R Xj gij = . Xj R The experiments to determine the metabolic fluxes (Estimation of the kinetic orders and rate constants) Direct estimation from flux data 1.If the effect of Xj on flux Vi+ is to be determined, it would be ideal to keep all variable but Xj at their typical values, and to vary Xj about its normial value.The Flux Vi+ becomes a unvariate function of Xj, and this function is linear when plotted as the logarithm of Vi+ against the logarithm of Xj. 2.The Slope of the function is equivalent to the kinetic order gij(hij) and can estimate the rate constants. Estimation from the literature presentations If the rate law is presented as the function R(X1,…..Xn+m) and the corresponding power-law representation isVi+ , the kinetic order gij is straightforwardly computed form above equation.
The rate law of ethanol biosynthesis pathway Galazzo and Bailey, 1990,1991. In yeast system
GMA representation Estimate the kinetic orders & rate constants Curto, 1995. In yeast system
Objective function & Model constrains Objective function The maximization of production rate and yield of ethanol Model constrains Steady-state constrains Enzyme constrains Metabolite constrains
The ways to maximize yield of ethanol To maximize the ethanol production rate,the enzymes which have to be amplified are six; the amplification ranges between 40 and 50 times the basal enzyme activities. The increase in the rate of ethanol production, VPK/(VPK)basal, is more than 50 times, and the yield of the optimized solution is around 100% of theoretical. The six enzymes: Y1 (Vin, sugar transport system) Y2 (VHK, hexokinase) Y3 (VPFK,phosphofructokinase) Y4 (VGADP, glyceraldehyde 3-phosphate dehydrogenase) Y5 (VPK, pyruvate kinase) Y7 (VATPase, ATPase)