Study The Factors Affecting Synthesis Of Trans10, Cis12-CLA In Recombinant Yarrowia Lipolitca

Conjugated linoleic acid (CLA)，the geometric and positional isomer of linoleic acid(LA)，is a collective name for a series of octadeca-fatty acid with conjugated double bonds. Becauseof its promising physiological functions，in the past years CLA has attracted widespreadattention. CLA，obtained by chem-synthesis，is a mixture of various isomers，It is expensivefor separation of the functional isomer. For the biologically safe and high specific isomer，biosynthesis of CLA has become the inevitable technology trends. In this dissertation，therecombinant Yarrowia lipolitca，which can produce high level of t10,c12-CLA，is constructedin our laboratory. With the aim to achieve the high yield of t10，c12-CLA，using biomass，Lip2activity，PAI expression and CLA production as the index. The influence of substrate，different media，culture conditions and fed-batch fermentation on the production of t10，c12-CLA in shaking flasks was investigated. The main results were presented as follows:(1) The effect of different time and concentration of safflower oil (SaO) on CLAproduction in recombinant yeast was studied.When SaO was added during the pre-stationaryphase，the expression level of PAI accumulated to the highest point，the extracellular Lip2production could be effectively increased，and the total CLA yield reached4.3g/L; With theaddition of30g/LSaO，CLA production and conversion rate reached9.1g/L and30.3%.(2) The influence of compounds in culture medium on CLA production by recombinantyeast was investigated. Glucose and organic nitrogen were the optimal carbon and nitrogensource. Tween-80was the optimal emulsifier for the production of CLA，which was enhancedby2.6-fold compared with that of control. Zn2+and Fe2+inhibit the expression of PAI，andCu2+inhibits the growth of cell and Lip2activity，and adding Ca2+at the initial fermentationincreased CLA yield to13.4g/L，which was improved by1.4-fold compared with control.(3) The influence of culture conditions on CLA production by recombinant yeast wasinvestigated. The recombinant strain was found to induce the synthesis of lipase Lip2optimum pH in the6.3-6.5. The biomass was highest in22℃，but it inhibit the Lip2synthesis，28℃is the optimal temperature for induction Lip2synthesis. The temperature transformationstrategy was investigated. The recombinant yeast was incubated at28℃for70h (Lip2synthesis was highest)，then transfer to36℃for another12h. As a result，the CLA yield was16.2g/L，corresponding to a1.7-fold increase compared with28℃incubation.(4) The influence of fed-batch fermentation strategy on CLA production in recombinantyeast.The10g/L glucose was fed during the end of the logarithmic phase could effectivelyimprove the biomass, corresponding to1.2-fold increase compared with control and theexpression levelle of PAI per cell also increased. The CLA production was12.3g/L,1.4-foldcompared with control. When the concentraton of SaO was increased from30to50g/L，CLAproduction was increased to18.9g/L. When glucose was fed to bioreactor twice，combinedwith adding50g/L SaO，the biomass and CLA production increased to19.6g/L and22.3g/L. Comprehensive optimum fermentation conditions，the CLA yield was31.9g/L，correspondingto a1.7-fold increase compared with the highest yield reported.In conclusion，through investigated in shaking flasks experiments，found the factors thatimpact of recombinant yeast synthetic t10,c12-CLA，significantly increased the production ofCLA，proved the recombinant yeast strains has a great potential of synthesis CLA，provide atheoretical and experimental basis for biotransformantion of t10,c12-CLA.