single cell oil production
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SINGLE CELL OIL PRODUCTION
BYGURUPRASAD VENKATAKRISHNA RAO
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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.
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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
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SO HOW IS RECOMBINANTDNA
TECHNOLOGYUSEFUL HERE ?
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BIOSYNTHESIS OF OIL IN MICROBES
GLYCOLISIS
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GLYCOLISIS CONTINUED ………..
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PRODUCTION OF ACETYL COA
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FATE OF ACETYL CoA
ACETYL CoA
LIPOGENESIS CITRIC ACID CYCLE
MY INTEREST
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CONVERSION OF ACETYL CoA TO MALONYL CoA
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MALONYL CoA TO FATTY ACYL COA
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FATTY ACID TO FATTY ACYL COA
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FORMATION OF OIL
Figure 3. Formation of phosphatidic acid from glycerol-3-P or DHAP, and its conversion to triacylglycerol
Lysophosphatidic acid
Phosphatidic acid
Triacylglycerol
NADPH
NADP+
Diacylglycerol
phosphatase
CoA
Acyldihydroxyacetone phosphate
fatty acyl CoA
Dihydroxyacetone phosphate
fatty acyl CoA
CoA
ADP
ATP glycerolkinase
Glycerol-3-P
Glycerol
CoA
fatty acyl CoA
CoA
fatty acyl CoA
Pi
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SO WHAT WE CAN DO NOW ??
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.
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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.
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THE EXPERIMENT
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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.
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Vector : pINA1317
USES A STRONG RECOMBINANT GROWTH PHASE DEPENDET PROMOTER,hp4d.
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INULIN
PRESENT AS RESERVE CARBOHYDRATE IN ROOTS & TUBERS OF PLANT
LOW COST MATERIALWAS 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.
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CLONING VECTORPMD19-T-INU1
CARRIES INULASE GENE CLONED FROM K.maxianus CBS 6556
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CONSTRUCTION OF RECOMBINANT EXPRESSION VECTOR
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CLONING VECTOR WAS PREPARED BY SAMBROOK ET AL.Y LIPOLYTICA WAS TRANSFORMED BY XUAN.ET.ALTO 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.
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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 REFINEDIT WAS CONVERTED INTO FAME & GC ANALYSIS WAS PERFORMED TO CHECK FATTY ACID COMPOSITION
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RESULTS
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TIME FACTOR
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EFFECT OF SUGAR
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EFFECT OF NITROGEN
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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.
IN THE NUTSHELL…….
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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
SUMTHING FOR OIL TECHNOLGIST
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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
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CONCLUSION
SINGLE CELL PRODUCTION IS NOT COMMERCIALIZED METHOD. PROCESS CAN BE COMMERCIALIZED BY1) RESEARCH & DEVELOPMENT IN THIS FIELD .2) INVESTMENTS BY BIG CORPORATES.3) PROMOTION OF THS TECHNOLOGY AT VARIOUS CONFERENCES
WORLDWIDE.
&MOST IMPORTANT
MERGER OF OIL TECHNOLOGIST AND BIOTECHNOLOGIST
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REFERENCES WWW.GOOGLE.COMWWW.WIKIPEDIA.ORGPRINCIPLE OF GENE MANIPULATION BY PRIMROSABIOCHEMISTRY BY LEHNINGER.MODERN TRENDS IN OILS & FATS TECHNOLOGY BY OP NARULA RESEARCH PAPERS ON WWW.SCIENCEDIRECT.COM
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THANK YOU