propanediol production and screening

1, 3-Propanediol production and screeningSandeep AmetaRoll No.: 06530003Bioschool, IIT BombayBT-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 ProductionGlycerol is a major by-product of industries involved in Biodiesel production

Industrial Aspect of Propanediol ProductionGlycerol is a major by-product of industries involved in Biodiesel productionDihydroxyacetone : Cosmetic Industry

Industrial Aspect of Propanediol ProductionGlycerol is a major by-product of industries involved in Biodiesel productionDihydroxyacetone : Cosmetic Industry1,3-Propanediol : Textile Industry

Chemical Industry

Industrial Aspect of Propanediol ProductionTextile 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 , 52:289glycerol dehydratase oxidoreductase (Microbial route of Propanediol Biosynthesis

Improvement of Production The over expression enzyme: Glycerol dehydratase Limiting enzyme for 1,3-PD production

Alternate Cheap substrate: GlucoseBottleneck: Natural producers dont 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 usinga yeast culture appears not to be very favorable.Mixed Culture ApproachRecombinant E.coli with glycerol producing genes are used with K. pneumoniae asa mixed culture for 1,3-Propanediol production.

Appl Microbiol Biotechnol 2002, 60:60

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 usinga yeast culture appears not to be very favorable.Mixed Culture ApproachAppl Microbiol Biotechnol 2002, 60:60

Mathematical models used for optimization of parameters of 1,3-Propanediol production

For example: Response surface methodology (RSM) is used for optimizing 1,3PD production in recombinant E.coli.

RSM: Response surface methodology (RSM) is a collection of statistical techniques applicable to experimental design, model building, evaluating the effects of factors, and screening optimum conditions of factors for desirable responses.

J Chem Technol Biotechnol 2006, 81:1075Mathematical Approach

Mathematical models used for optimization of parameters of 1,3-Propanediol production

For example: Response surface methodology (RSM) is used for optimizing 1,3-PD production in recombinant E.coli.

Production: 1,3-Propanediol concentration: 43.1 g/L

J Chem Technol Biotechnol 2006, 81:1075Mathematical Approach




Paper:


Novel Redox Potential-Based Screening Strategy for Rapid Isolation of Klebsiella pneumoniae Mutants with Enhanced 1,3-PropanediolProducing CapabilityChenyu Du, Yanping Zhang, Yin Li and Zhuan Cao

Applied and Environmental Microbiology 2007, p- 45154521

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 antibioticsScreening 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 discussionCorrelation of CDW and 1,3-PD production of the mutantsCDW cut off : 1.2g/L is chosen for screening

Results and discussion

Results and discussion40 mutants with CWD is more than 1.2 g/LAverageParent

Results and discussion 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.

Results and discussionOxido-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.

Results and discussionOxido-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 -240mV11 colonies with -280mV

Subjected to 36hr Anaerobic fermentation for 1,3-PD production

Results and discussion-240mV-280mV13 Mutants11 MutantsComparison 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

Results and discussionM5aL: Parent StrainYC1: Mutant screened from CDW correlation methodYF1: Mutant screened from ORP -240mVYMU1: Mutant screened from ORP -280mVYMU2: Aldehyde dehydrogenase Mutant (Previous Work) at ORP -280mV

Results and discussionM5aL: Parent StrainYC1: 5.5 % IncreaseYF1: 22.5 % IncreaseYMU1: 30.5 % IncreaseYMU2: 25.2 % Increase

Results and discussionPreferred 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.

Results and discussionPreferred 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 - 190mVConclusion: -280mV is the preferable ORP for the mutants

Results and discussionChange in the intracellular NAD/NADH ratioTo 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 investigatedNAD/NADH ratio:Parent: 4Muatnt:2ParentMutantThus intracellular environment is more reductive

Results and discussionMetabolic-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

ConclusionA 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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propanediol production and screening

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