SINGLE CELL OIL PRODUCTION

BYGURUPRASAD VENKATAKRISHNA RAO

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

SO HOW IS RECOMBINANTDNA

TECHNOLOGYUSEFUL HERE ?

BIOSYNTHESIS OF OIL IN MICROBES

GLYCOLISIS

GLYCOLISIS CONTINUED ………..

PRODUCTION OF ACETYL COA

FATE OF ACETYL CoA

ACETYL CoA

LIPOGENESIS CITRIC ACID CYCLE

MY INTEREST

CONVERSION OF ACETYL CoA TO MALONYL CoA

MALONYL CoA TO FATTY ACYL COA

FATTY ACID TO FATTY ACYL COA

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

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.

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.

THE EXPERIMENT

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 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.

CLONING VECTORPMD19-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.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.

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

RESULTS

TIME FACTOR

EFFECT OF SUGAR

EFFECT OF NITROGEN

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…….

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

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 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

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

THANK YOU