Textile Industry Monomer For The Synthesis Of Polyesters

1, 3-Propanediol production and screening

Sandeep AmetaRoll No.: 06530003

Bioschool, IIT Bombay

BT-612 Course Presentation

Scheme of presentation

• Industrial aspect of 1,3-Propanediol production (1,3-PD)

• Strain development to increase production of 1,3-PD

• Paper:

“Novel Redox Potential-Based Screening Strategy for Rapid Isolation ofKlebsiella pneumoniae Mutants with Enhanced 1,3-Propanediol-Producing Capability”

Industrial Aspect of Propanediol Production

Glycerol is a major by-product of industries involved in Biodiesel production



Dihydroxyacetone : Cosmetic Industry



Dihydroxyacetone : Cosmetic Industry

1,3-Propanediol : Textile Industry

Chemical Industry


Textile Industry Monomer for the synthesis of polyesters

PPT: polypropylene terephtalate

PET: polyethylene terephtalate

Better chemical, Mechanical &

biodegradability properties

Chemical Industry Polyurethane, lubricant, solvent and

precursor in the chemical and pharmaceutical

products

Drugs (derivative of 1,3-PD):

Ex: Famvir: Antiviral

Carisoprodol: For acute muscle

pain




• Paper:


Appl Microbiol Biotechnol , 1999 52:289

Appl Microbiol Biotechnol 1999 , 52:289

glycerol dehydratase

oxidoreductas

e (

Mic

robia

l ro

ute

of

Pro

panedio

l B

iosy

nth

esi

s

Improvement of Production

• The over expression enzyme: Glycerol dehydratase Limiting enzyme for 1,3-PD production

• Alternate Cheap substrate: Glucose

Bottleneck: Natural producers don’t have Glucose Propanediol pathway

Recombinant Approach: Heterologous expression of the 1,3-PD pathway genes

Process Approach: Mixed Fermentation

Heterologous expression of the 1,3-PD pathway the genes

Expression of glycerol dehydratase andoxidoreductase genes in S. cerevisiae (Glycerol Producer)

Did not worked: Yield is 0.1g/L

Appl Microbiol Biotechnol 1999, 52:289

Yeast (Glycerol Producer ) and Enterobacteriaceae (Glycerol to Propanediol producer)

in two consecutive stages.

Haynie and Wagner 1996, WO 35799 (E I DuPont de Nemours)

However, because of the repression of microbial 1,3-PD formation by glucose using

a yeast culture appears not to be very favorable.

Mixed Culture Approach

Recombinant E.coli with glycerol producing genes are used with K. pneumoniae as

a mixed culture for 1,3-Propanediol production.


Yeast (Glycerol Producer ) and Enterobacteriaceae (Glycerol to Propanediol producer)

in two consecutive stages.

Haynie and Wagner 1996, WO 35799 (E I DuPont de Nemours)

However, because of the repression of microbial 1,3-PD formation by glucose using

a yeast culture appears not to be very favorable.

Mixed Culture Approach





• Paper:


Novel Redox Potential-Based Screening Strategy for Rapid Isolation

of Klebsiella pneumoniae Mutants with Enhanced 1,3-Propanediol

Producing Capability

Chenyu Du, Yanping Zhang, Yin Li and Zhu’an Cao

Applied and Environmental Microbiology 2007, p- 4515–4521

The approaches used to screen for a metabolite hyper producer can

be generally categorized on the basis:

(i) a change in the absorption peak at a specific wavelength of either

the

target product or a derived compound

(ii) a change in the morphology of mutant colonies

(iii) resistance to extreme conditions such as acid, alkali, or

antibiotics

Screening of a hyper producer

1,3-PD does not have specific absorbance peaks in the visible-UV range.

The chemical functional groups of 1,3-PD are highly similar to those of

the substrate, glycerol, and the by-products, such as ethanol, lactic acid,

and acetic acid.

Moreover, tolerance to 1,3-PD does not directly correlate with 1,3-PD

production.

Problems with 1, 3 PD screening

ORP: (oxidoreduction potential) Screening

Recently, ORP has been used as a parameter to investigate mass and energymetabolic fluxes in several microorganisms

It has been reported that each species, has a preferred redox potential range.

Only within this range is maximum cell growth possible, and the flux may be directed toward the target metabolite.

In K. pneumoniae 1,3-PD production require anaerobic growth on glycerol.

Klebsiella pneumoniae M5aL , the most-preferred ORP levels were -160 to -190 mV.

Higher or lower ORP levels resulted in poor cell growth and poor 1,3-PD production.

J. Appl. Microbiol. 2003 94:280 FEMS Microbiol. Lett. 2002 214:257

Results and discussion

Correlation of CDW and 1,3-PD production of the mutants

CDW cut off : 1.2g/L is chosen for screening



40 mutants with CWD is more than 1.2 g/LAverageParent


• The average 1,3-PD concentration was only 66.1% of that of the parent strain.

• Only 2 of the 67 mutants selected were recognized as positive mutants.

• Cell growth correlation is able to screen but the selection efficiency was not so high.

• The procedure of this method was very laborious.


Oxido-reduction Potential [ORP] Method of Screening

The most-preferred ORP levels of the parent strain: -160 to -190 mV.

Hypothesis: 1,3-PD is synthesized in the bio-reductive branch enhancing

the reductive reactions could improve 1,3-PD production.

ORP tolerance levels of -240 mV and -280 mV were selected to enhance

1,3-PD production.

The mutant colonies cultured in the 5-liter fermentor at an ORP of -240 mV

and -280 mV separately.


Oxido-reduction Potential [ORP] Method of Screening

The most-preferred ORP levels of the parent strain: -160 to -190 mV.

Hypothesis: 1,3-PD is synthesized in the bio-reductive branch enhancing

the reductive reactions could improve 1,3-PD production.

ORP tolerance levels of -240 mV and -280 mV were selected to enhance

1,3-PD production.

The mutant colonies cultured in the 5-liter fermentor at an ORP of -240 mV

and -280 mV separately.

Survived Colonies:

13 colonies with -240mV

11 colonies with -280mV Subjected to 36hr Anaerobic fermentation for

1,3-PD production


-240mV -280mV

13 Mutants 11 Mutants

Comparison of 1,3-PD production by the parent and mutant strains and the average of the isolated mutant strains by the ORP-based screening method


M5aL: Parent Strain

YC1: Mutant screened from CDW correlation method

YF1: Mutant screened from ORP -240mV

YMU1: Mutant screened from ORP -280mV

YMU2: Aldehyde dehydrogenase Mutant (Previous Work) at ORP -280mV


M5aL: Parent Strain

YC1: 5.5 % Increase

YF1: 22.5 % Increase

YMU1: 30.5 % Increase

YMU2: 25.2 % Increase


Preferred ORP range of K. pneumoniae YMU1

As mutant strain K. pneumoniae YMU1 was at -280mV may differ from

that of the parent strain in its preferred ORP range.

To check the preferred ORP of K. pneumoniae YMU1:

Fed-batch fermentations of K. pneumoniae YMU1 were carried out at

constant ORPs of -190 mV, -240 mV, -280 mV, -320 mV

separately.


Preferred ORP range of K. pneumoniae YMU1

As mutant strain K. pneumoniae YMU1 was at -280mV may differ from

that of the parent strain in its preferred ORP range.

To check the preferred ORP of K. pneumoniae YMU1:

Fed-batch fermentations of K. pneumoniae YMU1 were carried out at

constant ORPs of -190 mV, 240 mV, 280 mV, 320 mV

separately.

-280mV

-240mV

-320mVParent

- 190mV

Conclusion: -280mV is the preferable ORP for the mutants


Change in the intracellular NAD/NADH ratio

To verify the change in intracellular redox potential, the time courses of the intracellular NAD/NADH ratio of the parent strain and the mutant strain were investigated

NAD/NADH ratio:

Parent: 4

Muatnt:2

Parent

Mutant

Thus intracellular environment is more reductive


Metabolic-flux analysis of mutant strain K. pneumoniaeYMU1 at-280mV ORP



• Acetate production is decreased Acetate is one of the strongest K. pneumoniae cell growth inhibitors Annu Rev Microbiol 1976, 36:535



• Acetate production is decreased Acetate is one of the strongest K. pneumoniae cell growth inhibitors Annu Rev Microbiol 1976, 36:535

• 2,3 Butanediol Production increased Bio-reductive branch enhanced



• Acetate production is decreased Acetate is one of the strongest K. pneumoniae cell growth inhibitors. Annu Rev Microbiol 1976, 36:535

• 2,3 Butanediol Production increased Bio-reductive branch enhanced

• 1,3 PD production increased

Conclusion

A positive correlation between the CDW of K. pneumoniae and its 1,3-PD biosyn-thesis was observed.

Cell growth correlation screening method was not efficient: 2 out of 67

ORP method shown a increased efficiency: 4 out of 11

The preferred ORP range of the mutant shifted from around -190 mV to -280 mV.

The redistribution of metabolic flux of the mutant strain, with the altered environmental conditions (an ORP of -280 mV), resulted in a decreased intracellular NAD/NADH ratio, which might enhance the activity of 1,3-PD dehydrogenase and consequently accelerate 1,3-PD production.

Thus the bioconversion steps in the reductive branch was improved in isolated mutant strain K. pneumoniae YMU1.

Thank you!!!!

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Textile Industry Monomer For The Synthesis Of Polyesters