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

Flow diagram

Pathway1It is necessary to find some ways that increase the flow of reaction 12.

Pathway2We can overexpress enzyme pyruvate decarboxylase (PDC, EC:4.1.1.1) to produce reaction 15.

Pathway 2

Pathway 1

pyruvate acetaldehyde

Is E. Coli suitable for biosynthesis of ethanol?

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. coli cultures 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.

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.

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 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

Model description

VATPase, Y7

The experiments to determine the metabolic fluxes (Estimation of the kinetic orders and rate constants)

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.

Direct estimation from flux data

Estimation from the literature presentations

gij = . R

Xj

Xj

R

If the rate law is presented as the function R(X1,…..Xn+m) and the corresponding power-la

w representation is Vi+ , 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

Model constrainsSteady-state constrains

Enzyme constrains

Metabolite constrains

The maximization of production rate and yield of ethanol

The optimization

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)