1, 3-Propanediol production and screening Sandeep Ameta Roll No.: 06530003 Bioschool, IIT Bombay BT-612 Course Presentation
<ul><li>Scheme of presentation </li></ul><ul><li>Industrial aspect of 1,3-Propanediol production (1,3-PD) </li></ul><ul><l...
Industrial Aspect of Propanediol Production Glycerol is a major by-product of industries involved in Biodiesel production
Industrial Aspect of Propanediol Production Glycerol is a major by-product of industries involved in Biodiesel production ...
Industrial Aspect of Propanediol Production Glycerol is a major by-product of industries involved in Biodiesel production ...
Industrial Aspect of Propanediol Production Textile Industry  Monomer for the synthesis of polyesters  PPT: polypropylene ...
<ul><li>Scheme of presentation </li></ul><ul><li>Industrial aspect of 1,3-Propanediol production (1,3-PD) </li></ul><ul><l...
Appl Microbiol Biotechnol , 1999  52:289
Appl Microbiol Biotechnol  1999 , 52:289 glycerol dehydratase oxidoreductase  ( Microbial route of Propanediol Biosynthesis
Improvement of  Production <ul><li>The over expression enzyme: Glycerol dehydratase </li></ul><ul><li>Limiting enzyme for ...
Heterologous expression of the 1,3-PD pathway the genes  Expression of glycerol dehydratase and oxidoreductase genes in  S...
Yeast (Glycerol Producer ) and Enterobacteriaceae (Glycerol to Propanediol producer) in two consecutive stages. Haynie and...
Yeast (Glycerol Producer ) and Enterobacteriaceae (Glycerol to Propanediol producer) in two consecutive stages. Haynie and...
Mathematical models used for optimization of parameters of 1,3-Propanediol  production  For example: Response surface meth...
Mathematical models used for optimization of parameters of 1,3-Propanediol  production  For example: Response surface meth...
<ul><li>Scheme of presentation </li></ul><ul><li>Industrial aspect of 1,3-Propanediol production (1,3-PD) </li></ul><ul><l...
Novel Redox Potential-Based Screening Strategy for Rapid Isolation of  Klebsiella pneumoniae  Mutants with Enhanced 1,3-Pr...
The approaches used to screen for a metabolite hyper producer can be generally categorized on the basis: (i) a change in t...
1,3-PD does not have specific absorbance peaks in the visible-UV range. The chemical functional groups of 1,3-PD are highl...
ORP: (oxidoreduction potential)  Screening Recently, ORP has been used as a parameter to investigate mass and energy metab...
Results and discussion Correlation of CDW and 1,3-PD production of the mutants CDW cut off : 1.2g/L is chosen for screening
Results and discussion
Results and discussion 40 mutants with CWD is more than 1.2 g/L Average Parent
Results and discussion <ul><li>The average 1,3-PD concentration was only 66.1% of that of the parent strain. </li></ul><ul...
Results and discussion Oxido-reduction Potential [ORP] Method of Screening The most-preferred ORP levels of the parent str...
Results and discussion Oxido-reduction Potential [ORP] Method of Screening The most-preferred ORP levels of the parent str...
Results and discussion -240mV -280mV 13 Mutants 11 Mutants Comparison of 1,3-PD production by the parent and mutant strain...
Results and discussion M5aL: Parent Strain YC1: Mutant screened from CDW correlation method YF1: Mutant screened from ORP ...
Results and discussion M5aL: Parent Strain YC1:  5.5 % Increase YF1:  22.5 % Increase YMU1:  30.5 % Increase YMU2:  25.2 %...
Results and discussion Preferred ORP range of  K. pneumoniae  YMU1  As mutant strain  K. pneumoniae  YMU1 was at -280mV ma...
Results and discussion Preferred ORP range of  K. pneumoniae  YMU1  As mutant strain K. pneumoniae YMU1 was at -280mV may ...
Results and discussion Change in the intracellular NAD/NADH ratio To verify the change in intracellular redox potential, t...
Results and discussion Metabolic-flux analysis of mutant strain  K. pneumoniae YMU1 at-280mV ORP
Results and discussion Metabolic-flux analysis of mutant strain  K. pneumoniae YMU1 at-280mV ORP <ul><li>Acetate productio...
Results and discussion Metabolic-flux analysis of mutant strain  K. pneumoniae YMU1 at-280mV ORP <ul><li>Acetate productio...
Results and discussion Metabolic-flux analysis of mutant strain  K. pneumoniae YMU1 at-280mV ORP <ul><li>Acetate productio...
Conclusion A positive correlation between the CDW of  K. pneumoniae  and its 1,3-PD biosyn- thesis was observed. Cell grow...
Thank you!!!!
Upcoming SlideShare
Loading in...5
×

Textile Industry Monomer For The Synthesis Of Polyesters

639

Published on

Published in: Business, Technology
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total Views
639
On Slideshare
0
From Embeds
0
Number of Embeds
0
Actions
Shares
0
Downloads
21
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Textile Industry Monomer For The Synthesis Of Polyesters

  1. 1. 1, 3-Propanediol production and screening Sandeep Ameta Roll No.: 06530003 Bioschool, IIT Bombay BT-612 Course Presentation
  2. 2. <ul><li>Scheme of presentation </li></ul><ul><li>Industrial aspect of 1,3-Propanediol production (1,3-PD) </li></ul><ul><li>Strain development to increase production of 1,3-PD </li></ul><ul><li>Paper: </li></ul><ul><li>“ Novel Redox Potential-Based Screening Strategy for Rapid Isolation of </li></ul><ul><li>Klebsiella pneumoniae Mutants with Enhanced 1,3-Propanediol-Producing Capability” </li></ul>
  3. 3. Industrial Aspect of Propanediol Production Glycerol is a major by-product of industries involved in Biodiesel production
  4. 4. Industrial Aspect of Propanediol Production Glycerol is a major by-product of industries involved in Biodiesel production Dihydroxyacetone : Cosmetic Industry
  5. 5. Industrial Aspect of Propanediol Production Glycerol is a major by-product of industries involved in Biodiesel production Dihydroxyacetone : Cosmetic Industry 1,3-Propanediol : Textile Industry Chemical Industry
  6. 6. Industrial Aspect of Propanediol Production 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
  7. 7. <ul><li>Scheme of presentation </li></ul><ul><li>Industrial aspect of 1,3-Propanediol production (1,3-PD) </li></ul><ul><li>Strain development to increase production of 1,3-PD </li></ul><ul><li>Paper: </li></ul><ul><li>“ Novel Redox Potential-Based Screening Strategy for Rapid Isolation of </li></ul><ul><li>Klebsiella pneumoniae Mutants with Enhanced 1,3-Propanediol-Producing Capability” </li></ul>
  8. 8. Appl Microbiol Biotechnol , 1999 52:289
  9. 9. Appl Microbiol Biotechnol 1999 , 52:289 glycerol dehydratase oxidoreductase ( Microbial route of Propanediol Biosynthesis
  10. 10. Improvement of Production <ul><li>The over expression enzyme: Glycerol dehydratase </li></ul><ul><li>Limiting enzyme for 1,3-PD production </li></ul><ul><li>Alternate Cheap substrate: Glucose </li></ul>Bottleneck: Natural producers don’t have Glucose Propanediol pathway Recombinant Approach: Heterologous expression of the 1,3-PD pathway genes Process Approach: Mixed Fermentation
  11. 11. Heterologous expression of the 1,3-PD pathway the genes Expression of glycerol dehydratase and oxidoreductase genes in S. cerevisiae (Glycerol Producer) Did not worked: Yield is 0.1g/L Appl Microbiol Biotechnol 1999, 52:289
  12. 12. 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. Appl Microbiol Biotechnol 2002, 60:60
  13. 13. 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 Appl Microbiol Biotechnol 2002, 60:60
  14. 14. 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. 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, 81:1075 Mathematical Approach
  15. 15. 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, 81:1075 Mathematical Approach
  16. 16. <ul><li>Scheme of presentation </li></ul><ul><li>Industrial aspect of 1,3-Propanediol production (1,3-PD) </li></ul><ul><li>Strain development to increase production of 1,3-PD </li></ul><ul><li>Paper: </li></ul><ul><li>“ Novel Redox Potential-Based Screening Strategy for Rapid Isolation of </li></ul><ul><li>Klebsiella pneumoniae Mutants with Enhanced 1,3-Propanediol-Producing Capability” </li></ul>
  17. 17. 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
  18. 18. 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
  19. 19. 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
  20. 20. ORP: (oxidoreduction potential) Screening Recently, ORP has been used as a parameter to investigate mass and energy metabolic 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
  21. 21. Results and discussion Correlation of CDW and 1,3-PD production of the mutants CDW cut off : 1.2g/L is chosen for screening
  22. 22. Results and discussion
  23. 23. Results and discussion 40 mutants with CWD is more than 1.2 g/L Average Parent
  24. 24. Results and discussion <ul><li>The average 1,3-PD concentration was only 66.1% of that of the parent strain. </li></ul><ul><li>Only 2 of the 67 mutants selected were recognized as positive mutants. </li></ul><ul><li>Cell growth correlation is able to screen but the selection efficiency was not so high. </li></ul><ul><li>The procedure of this method was very laborious. </li></ul>
  25. 25. Results and discussion 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.
  26. 26. Results and discussion 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
  27. 27. Results and discussion -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
  28. 28. Results and discussion 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
  29. 29. Results and discussion M5aL: Parent Strain YC1: 5.5 % Increase YF1: 22.5 % Increase YMU1: 30.5 % Increase YMU2: 25.2 % Increase
  30. 30. Results and discussion 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.
  31. 31. Results and discussion 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 - 320mV Parent - 190mV Conclusion: -280mV is the preferable ORP for the mutants
  32. 32. Results and discussion 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
  33. 33. Results and discussion Metabolic-flux analysis of mutant strain K. pneumoniae YMU1 at-280mV ORP
  34. 34. Results and discussion Metabolic-flux analysis of mutant strain K. pneumoniae YMU1 at-280mV ORP <ul><li>Acetate production is decreased </li></ul><ul><li>Acetate is one of the strongest K. pneumoniae </li></ul><ul><li>cell growth inhibitors </li></ul><ul><li>Annu Rev Microbiol 1976, 36:535 </li></ul>
  35. 35. Results and discussion Metabolic-flux analysis of mutant strain K. pneumoniae YMU1 at-280mV ORP <ul><li>Acetate production is decreased </li></ul><ul><li>Acetate is one of the strongest K. pneumoniae </li></ul><ul><li>cell growth inhibitors </li></ul><ul><li>Annu Rev Microbiol 1976, 36:535 </li></ul><ul><li>2,3 Butanediol Production increased </li></ul><ul><li>Bio-reductive branch enhanced </li></ul>
  36. 36. Results and discussion Metabolic-flux analysis of mutant strain K. pneumoniae YMU1 at-280mV ORP <ul><li>Acetate production is decreased </li></ul><ul><li>Acetate is one of the strongest K. pneumoniae </li></ul><ul><li>cell growth inhibitors. </li></ul><ul><li>Annu Rev Microbiol 1976, 36:535 </li></ul><ul><li>2,3 Butanediol Production increased </li></ul><ul><li>Bio-reductive branch enhanced </li></ul><ul><li>1,3 PD production increased </li></ul>
  37. 37. 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 .
  38. 38. Thank you!!!!
  1. A particular slide catching your eye?

    Clipping is a handy way to collect important slides you want to go back to later.

×