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WHAT IS THAT EXACTLY ???


Single cell oils (SCO) are the edible
oils extracted from micro-organisms-
the single-celled entities that are at
the bottom of the food chain. Single
celled entities are algae, bacteria,
yeast . Lipids are produced by the
organism from basic units like
carbohydrates for their cell
metabolism & survival.
WHY IT IS SO SPECIAL TO
    OIL TECHNOLOGIST ( LIKE ME ) ???

 THEY CAN BE SOURCE OF OILS & FATS
WHICH CAN SERVE AS RAW MATERIAL
FOR OLEOCHEMICALS INDUSTRIES
 THEY CAN BE SERVE AS MODIFIED
EDIBLE OILS
 THEYARE SOURCES OF PUFA AND
OTHER ESSENTIAL FATTY ACID.
 THEY CAN BE USED TO PRODUCE
MODERN GENERATION BIOFUEL
BIOSYNTHESIS OF OIL IN MICROBES


GLYCOLISIS
GLYCOLISIS CONTINUED ………..
PRODUCTION OF ACETYL COA
FATE OF ACETYL CoA


              ACETYL
               CoA




LIPOGENESIS            CITRIC ACID
                          CYCLE
CONVERSION OF ACETYL CoA TO MALONYL CoA
MALONYL CoA TO FATTY ACYL COA
FATTY ACID TO FATTY ACYL COA
Glycerol
  ATP              phosphatase Dihydroxyacetone phosphate
              glycerol                   fatty acyl CoA
              kinase
  ADP
        Glycerol-3-P                          CoA
               fatty acyl CoA        Acyldihydroxyacetone phosphate
                                                 NADPH
             CoA
 Lysophosphatidic                              NADP+
 acid         fatty acyl CoA
                                Pi
              CoA
   Phosphatidic                  Diacylglycerol
   acid                                       fatty acyl CoA

FORMATION OF OIL                               CoA
                                        Triacylglycerol
  Figure 3. Formation of phosphatidic acid from glycerol-3-P or
  DHAP, and its conversion to triacylglycerol
WE CAN CHANGE THE ACTIVITY OF ENZYME
IN PATHWAY TO PRODUCE LIPID.
   WE CAN IMPROVE THE YIELD OF OIL BY
MAKING APPROPRIATE CHANGE IN
MICROORGANISM .
   WE CAN FORCE MICROBES TO PRODUCE
OIL FROM THOSE ORGANIC MATTER WHICH IS
NOT USEFUL TO US.
  TO EXPLOIT NEWER TACTICS TO ACHIEVE
THE SAME.
SO MY CURRENT DISCUSSIO N WILL BE BASED ON RESEARCH
WORK CONDUCTED BY CHUN-HAI ZHAO & WEI CUI AT UNESCO
CHINESE CENTER OF MARINE BIOTECHNOLOGY AND INSTITUTE
OF MARINE BIODIVERSITY AND EVOLUTION, CHINA.




THE PAPER FOCUS ON HOW RDT
HELPED IN IMPROVING OIL
PRODUCTION FROM YEAST BY
EXPRESSION OF INULASE GENE INTO
IT.
Purpose


To express inulase gene in oleogeinous yeast yarrowcia
lipolytica and thus obtain oil from inuln containing material.



 Host : yarrowia lipolytica

 Reason : 1) fast growth rates
          2) high oil content
          3) Resemblance of their triacyglycerol in plants.
Vector : pINA1317


USES A STRONG
RECOMBINANT
GROWTH PHASE
DEPENDET
PROMOTER,hp4d.
INULIN

  PRESENT AS RESERVE
CARBOHYDRATE IN ROOTS &
TUBERS OF PLANT
  LOW COST MATERIAL
  WAS NOT USED BECAUSE
BECOZ OLEOGINOUS
MAICROBES WERE NOT ABLE
TO SECRETE INULASE.
  SO EXPRESSION OF INULASE
GENE PROMOTES SECRETION
OF INULINASE AND THIS
SUGAR MOIETY WAS
CONVERTED TO OILS.
CLONING VECTOR
 PMD19-T-INU1



CARRIES INULASE GENE
CLONED FROM
K.maxianus CBS 6556
CONSTRUCTION OF RECOMBINANT EXPRESSION VECTOR
CLONING VECTOR WAS PREPARED BY
SAMBROOK ET AL.
Y LIPOLYTICA WAS TRANSFORMED BY
XUAN.ET.AL
TO AMPLIFY INU1 GENE BY PCR,
FORWARD PRIMER WAS PPU & REVERSE
PRIMER WAS PPD.
 CONFIRMATION OF INTEGRATION OF
INULASE GENE WAS DONE BY PCR
FOLLOWED BY CHECKING AT
AUTOMATED GEL DOCUMENTATION &
ANALYSIS SYSTEM.
 DETERMINATION  OF INULASE ACTIVITY WAS
DETERMINED ONYERMS OF INULASE UNIT ( U)
WHICH IS AMOUNT OF ENZYME THAT PRODUCES 1
MICROMOLE OF REDUCING SUGAR PER MINUTE
UNDER THE ASSAY CONDITION.
 EXPERIMENT WAS CONDUCTED IN 250 mL
CONICAL FLASK CONTAINIG 50 ml OF SINGLE
CELL OIL PRODUCTION MEDIUM & 5 ml CELL
MEDIA.
 LIPIDS WAS EXTRACTED BY SOLVENT
EXTRACTION .
IT WAS REFINED
IT WAS CONVERTED INTO FAME & GC ANALYSIS
WAS PERFORMED TO CHECK FATTY ACID
COMPOSITION
TIME FACTOR
EFFECT OF SUGAR
EFFECT OF NITROGEN
IN THE NUTSHELL…….
 TRANSFORMANT Z 31
 ACCUMULATED 46.3 %
 (WT/WT) OF OIL IN ITS CELL
 AND CELL DRY WEIGHT WAS
 11.6 g PER LITRE& ABOUT
 10.7% OF THE SUGAR WAS
 CONVERTED INTO ITS CELL
 OIL.
FATTY ACID   % PERCENTAGE

C14:0        1.68

C16:0        28.32

C16:1        2.12

C18:0        4.65

C18:1        58.48

C18:2        2.14
SCOPE

 THE MENTIONED EXPERIMENT WAS TERMED A
“ HIT “ AT AOCS 2010.
RESEARCH IS NOW BEING DONE TO
IMPROVE FURTHER PRODUCTION BY OVER
EXPRESSION OF GENE ENCODINGATP CITRATE
LYASE, FATTY ACID SYNTHASE AND ACYL COA
CARBOXYLASE AND DISRUPTION OF GENES
ENCODING GLYCEROL-3-PHOSPHATE
DEHYDROGENASE AND ACYL CoENZYME A
OXIDASE
CONCLUSION

 SINGLE CELL PRODUCTION IS NOT COMMERCIALIZED METHOD.
 PROCESS CAN BE COMMERCIALIZED BY
1) RESEARCH & DEVELOPMENT IN THIS FIELD .
2) INVESTMENTS BY BIG CORPORATES.
3) PROMOTION OF THS TECHNOLOGY AT VARIOUS CONFERENCES
   WORLDWIDE.

                            &
                     MOST IMPORTANT
 WWW.GOOGLE.COM
WWW.WIKIPEDIA.ORG
PRINCIPLE OF GENE MANIPULATION BY
PRIMROSA
BIOCHEMISTRY BY LEHNINGER.
MODERN TRENDS IN OILS & FATS
TECHNOLOGY BY OP NARULA
 RESEARCH PAPERS ON
WWW.SCIENCEDIRECT.COM
Single cell oil production

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Single cell oil production

  • 1.
  • 2. WHAT IS THAT EXACTLY ??? Single cell oils (SCO) are the edible oils extracted from micro-organisms- the single-celled entities that are at the bottom of the food chain. Single celled entities are algae, bacteria, yeast . Lipids are produced by the organism from basic units like carbohydrates for their cell metabolism & survival.
  • 3. WHY IT IS SO SPECIAL TO OIL TECHNOLOGIST ( LIKE ME ) ???  THEY CAN BE SOURCE OF OILS & FATS WHICH CAN SERVE AS RAW MATERIAL FOR OLEOCHEMICALS INDUSTRIES  THEY CAN BE SERVE AS MODIFIED EDIBLE OILS  THEYARE SOURCES OF PUFA AND OTHER ESSENTIAL FATTY ACID.  THEY CAN BE USED TO PRODUCE MODERN GENERATION BIOFUEL
  • 4.
  • 5. BIOSYNTHESIS OF OIL IN MICROBES GLYCOLISIS
  • 8. FATE OF ACETYL CoA ACETYL CoA LIPOGENESIS CITRIC ACID CYCLE
  • 9. CONVERSION OF ACETYL CoA TO MALONYL CoA
  • 10. MALONYL CoA TO FATTY ACYL COA
  • 11. FATTY ACID TO FATTY ACYL COA
  • 12. Glycerol ATP phosphatase Dihydroxyacetone phosphate glycerol fatty acyl CoA kinase ADP Glycerol-3-P CoA fatty acyl CoA Acyldihydroxyacetone phosphate NADPH CoA Lysophosphatidic NADP+ acid fatty acyl CoA Pi CoA Phosphatidic Diacylglycerol acid fatty acyl CoA FORMATION OF OIL CoA Triacylglycerol Figure 3. Formation of phosphatidic acid from glycerol-3-P or DHAP, and its conversion to triacylglycerol
  • 13. WE CAN CHANGE THE ACTIVITY OF ENZYME IN PATHWAY TO PRODUCE LIPID. WE CAN IMPROVE THE YIELD OF OIL BY MAKING APPROPRIATE CHANGE IN MICROORGANISM . WE CAN FORCE MICROBES TO PRODUCE OIL FROM THOSE ORGANIC MATTER WHICH IS NOT USEFUL TO US. TO EXPLOIT NEWER TACTICS TO ACHIEVE THE SAME.
  • 14. SO MY CURRENT DISCUSSIO N WILL BE BASED ON RESEARCH WORK CONDUCTED BY CHUN-HAI ZHAO & WEI CUI AT UNESCO CHINESE CENTER OF MARINE BIOTECHNOLOGY AND INSTITUTE OF MARINE BIODIVERSITY AND EVOLUTION, CHINA. THE PAPER FOCUS ON HOW RDT HELPED IN IMPROVING OIL PRODUCTION FROM YEAST BY EXPRESSION OF INULASE GENE INTO IT.
  • 15.
  • 16. Purpose To express inulase gene in oleogeinous yeast yarrowcia lipolytica and thus obtain oil from inuln containing material. Host : yarrowia lipolytica Reason : 1) fast growth rates 2) high oil content 3) Resemblance of their triacyglycerol in plants.
  • 17. Vector : pINA1317 USES A STRONG RECOMBINANT GROWTH PHASE DEPENDET PROMOTER,hp4d.
  • 18. INULIN PRESENT AS RESERVE CARBOHYDRATE IN ROOTS & TUBERS OF PLANT LOW COST MATERIAL WAS NOT USED BECAUSE BECOZ OLEOGINOUS MAICROBES WERE NOT ABLE TO SECRETE INULASE. SO EXPRESSION OF INULASE GENE PROMOTES SECRETION OF INULINASE AND THIS SUGAR MOIETY WAS CONVERTED TO OILS.
  • 19. CLONING VECTOR PMD19-T-INU1 CARRIES INULASE GENE CLONED FROM K.maxianus CBS 6556
  • 20. CONSTRUCTION OF RECOMBINANT EXPRESSION VECTOR
  • 21. CLONING VECTOR WAS PREPARED BY SAMBROOK ET AL. Y LIPOLYTICA WAS TRANSFORMED BY XUAN.ET.AL TO AMPLIFY INU1 GENE BY PCR, FORWARD PRIMER WAS PPU & REVERSE PRIMER WAS PPD.  CONFIRMATION OF INTEGRATION OF INULASE GENE WAS DONE BY PCR FOLLOWED BY CHECKING AT AUTOMATED GEL DOCUMENTATION & ANALYSIS SYSTEM.
  • 22.  DETERMINATION OF INULASE ACTIVITY WAS DETERMINED ONYERMS OF INULASE UNIT ( U) WHICH IS AMOUNT OF ENZYME THAT PRODUCES 1 MICROMOLE OF REDUCING SUGAR PER MINUTE UNDER THE ASSAY CONDITION.  EXPERIMENT WAS CONDUCTED IN 250 mL CONICAL FLASK CONTAINIG 50 ml OF SINGLE CELL OIL PRODUCTION MEDIUM & 5 ml CELL MEDIA.  LIPIDS WAS EXTRACTED BY SOLVENT EXTRACTION . IT WAS REFINED IT WAS CONVERTED INTO FAME & GC ANALYSIS WAS PERFORMED TO CHECK FATTY ACID COMPOSITION
  • 23.
  • 24.
  • 28. IN THE NUTSHELL……. TRANSFORMANT Z 31 ACCUMULATED 46.3 % (WT/WT) OF OIL IN ITS CELL AND CELL DRY WEIGHT WAS 11.6 g PER LITRE& ABOUT 10.7% OF THE SUGAR WAS CONVERTED INTO ITS CELL OIL.
  • 29. FATTY ACID % PERCENTAGE C14:0 1.68 C16:0 28.32 C16:1 2.12 C18:0 4.65 C18:1 58.48 C18:2 2.14
  • 30. SCOPE  THE MENTIONED EXPERIMENT WAS TERMED A “ HIT “ AT AOCS 2010. RESEARCH IS NOW BEING DONE TO IMPROVE FURTHER PRODUCTION BY OVER EXPRESSION OF GENE ENCODINGATP CITRATE LYASE, FATTY ACID SYNTHASE AND ACYL COA CARBOXYLASE AND DISRUPTION OF GENES ENCODING GLYCEROL-3-PHOSPHATE DEHYDROGENASE AND ACYL CoENZYME A OXIDASE
  • 31. CONCLUSION  SINGLE CELL PRODUCTION IS NOT COMMERCIALIZED METHOD.  PROCESS CAN BE COMMERCIALIZED BY 1) RESEARCH & DEVELOPMENT IN THIS FIELD . 2) INVESTMENTS BY BIG CORPORATES. 3) PROMOTION OF THS TECHNOLOGY AT VARIOUS CONFERENCES WORLDWIDE. & MOST IMPORTANT
  • 32.  WWW.GOOGLE.COM WWW.WIKIPEDIA.ORG PRINCIPLE OF GENE MANIPULATION BY PRIMROSA BIOCHEMISTRY BY LEHNINGER. MODERN TRENDS IN OILS & FATS TECHNOLOGY BY OP NARULA  RESEARCH PAPERS ON WWW.SCIENCEDIRECT.COM