Chapter 12& 13 & 14 - Metabolic Engineering examples & Ethyl acetate production & Lactic acid production at low pH

degree of reduction

C=4, H=1, O=-2

what is the degree of reduction of glucose (C6H12O6)?

24

what is the degree of reduction of butanethanol (C4H10O2, BDO)?

22

how many glucose (C6H12O6) can be converted into BDO (C4H10O2)?

22 glucose convert into 24 BDO

overall reaction of glucose --\> BDO

𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 1/2 O2+ 𝐴𝐷𝑃 + 𝑃𝑖 --\> 𝐵𝐷𝑂 + 2 𝐶𝑂2+ 𝐴𝑇𝑃

is the overall reaction of glucose --\> BDO equal to maximum yield?

It depends on the degree of freedom

the reaction of maximum yield

5.5 glucose + 8 ATP --\> 6 BDO + 9 CO2 + 3 H2O + 8 ADP + 8Pi, this reaction consumes ATP

pathway yield

24 glucose = 20 ethyl acetate + 4 formate

max theoretical yield

5 glucose = 6 ethyl acetate + 6 CO2 + 6 H2O

anaerobic pathway

glucose + 2 ADP +2Pi = ethylacetate + 2ATP + 2CO2 + 2H2

what happends when the reaction consumes ATP?

it's unfavourable for the m.o., no growth

ATP is produced from glucose during product formation,...

for biomass and maintenance

ATP produced from the breakdown of glucose to CO2 and H2O,...

is used for conversion of glucose to product, and for production of biomass and maintenance

suitable genes for knockout

genes that are not contributing or making byproducts to the production of the product

No energy (ATP) using transport

uses diffusion

proton symport

uses proton motive force

ABC transporters

uses an ATP-binding cassette, directly utilize ATP

Phosphotransferase

uses energy formed during phosphorylation

Redox cofactors

drives of redox reaction

Characteristics of ethyl acetate?

highly volatile, non-toxic, biodegradable

Ethyl acetate reaction

ethyl acetate --\> ethanol acetate --\> CO2

optimization of ethyl acetate production

pathway, gene expression levels, portein production, process

alcoholysis and thiolysis

transfering alcohol group to other alcohol group

exchaning thio

ethyl acetate production in yeast

high (bulk) amounts produced only under iron/oxygen limitation

maximal yield is when

all electrons from the substrate end up in the product

optimization ways for maximum yield

dynamic flux balance analysis, reduce side activities and other esters

lactic acid production at low pH (2.8) reaction

sugar --\> 2 lactic acid (aq) --\> 2 lactic acid

lactic acid production at pH7

proton (H+) is pumped out by ATPase when lactic acid diffuse faster than pumping out. pH decrease and cell dies

lactic acid production at pH3

No ATP production, proton (H+) is pumped out by ATP produced by glucose during lactic acid synthesis

lactic acid production classical (pH 7) reaction

sugar + Ca(OH)2 --\> Ca(lactate)2 + H2SO4--\> 2 lactic acid +CaSO4

prerequisites for efficient lactic acid production - phylogenetic traits during the process

lactic acid production, low pH, oxygen output, mineral mediam, robust

prerequisites for efficient lactic acid production - phylogenetic traits of cell factories

tolerant to high lactic acid concentrations, acidophile, aerobic, prototrophs, tolerant to high sugar concentrations

prerequisites for efficient lactic acid production - monogenetic traits during process

lactic acid production, anly D or L, no by products, low cost sugar

prerequisites for efficient lactic acid production - monogenetic traits of cell factories

making lactic acid, highly specific NLDH, no byproducts, able to use several sugars

isolation and selection of lactic aid tolerant strains

isolate in mineral media with low pH, glucose/xylose and lactic acid. plate it and choose colonies to transfer to liquid media with increasing lactic acid concentrations