The Influential Mechanism Of Substrate Supply On Lipid Accumulation In Oleaginous Yeast Yarrowia Lipolytica

Oleaginous microorganisms, which can accumulate significant amount of lipid in the cell,have been studied frequently for production of functional fatty acids and biodiesel because oftheir special growth characteristic and metabolic pathways. Yarrowia lipolytica as one ofoleaginous microorganisms, which produces lipid up to36%of its cell dry weight and growvery effciently on several carbon sources (alkanes, fatty acid, ethanol, acetate, glucose,fructose or glycerol), have been considered as a model organism for studying thebiochemistry and molecular biology of lipid accumulation, and as a cell factory for producingfunctional fatty acids (EPA and CLA) due to its available genome sequence and the existenceof genetic tools for gene manipulation. However the lipid content in Y. lipolytica was lowerthan other oleaginous yeasts and most of wild-type strains accumulate cellular lipid less than20%of their dry cell weight. In this study, enhanced substrate supply (NADPH andacetyl-CoA) for fatty acid synthesis was used to improve the lipid accumulation in Y.lipolytica, we also explored the mechanism of lipid accumulation in oleaginousmicroorganisms and these results of this study may help to develop commercial process forfunctional fatty acid production. The main results are described as follows:(1) Malic enzyme from Y. lipolytica was expressed in E. coli BL21(DE3), andrecombinant protein was purified by Ni-NTA affinity chromatography. The characterstic ofrecombinant malic enzyme was determined and it was found that both NADP+and NAD+were used as cofactor. The affinity for NAD was higher than NADP+, and Km values forNAD+and NADP+were0.63and3.9mM, respectively. Physiologically, NADP+-dependentmalic enzyme activity was too low to be detected and the result of subcellular locatizationprediction showed that malic enzyme was in mitochondrion. These results suggested thatmalic enzyme do not provide the NADPH for lipid accumulation in Y. lipolytica. At themeanwhile, a malic enzyme gene (mce2) from oleaginous fungus, mortierella alpina, wasintegrated into Y. lipolytica genome by vector pINA1312-mce2. The NADP+dependent malicenzyme activity were improved remarkably, however the lipid content in yeast cell wasmaintain the orginal level and was about8%of their dry cell weight. This results suggestedthat malic enzyme in Y. lipolytica is not involved in lipid accumulation.(2) There are three NADPH generated enzymes in the cell, glucose-6-phosphatedehydrogenase,6-phosphogluconate dehydrogenase, and NADP+-dependent isocitratedehydrogenase. These three genes were over-expressed in Y. lipolytica respectively, and theactivities of these NADPH generated enzymes were increased obviously in eachtransformants. The maximal enzyme activities were3104,946and2324U/mg respectively,and had5-fold,3-fold and4-fold increase respectively compared to the control strain. Butover-expression of three NADPH generated enzymes had no significant effect on biomass,lipid content and fatty acid composition. Therefore, NADPH supply is not the bottleneck oflipid accumulation in oleaginous yeast Y. lipolytica strains with low lipid content.(3) When Y. lipolytica was cultivated in H-N and L-N medium, the lipid content was8.7%and14.3%of their dry cell weigth. Based the13C labled metabolic flux analysis in thesecultural conditions, the activities of malic enzyme and glyoxylate cycle were not found in Y.lipolytica, the metabolic flux of pentose phosphate pathway and NADP+dependent isocitrate dehydrogenase activity in H-N medium were lower than L-N medium, these results show thatNADPH supply is not the bottleneck for lipid accumulation in Y. lipolytica. However, the fluxof citrate to acetyl-CoA in H-N medium was higher than L-N medium, it suggested thatATP:citrate lyase provide acetyl-CoA for fatty acid biosynthesis and it may be the key factorfor lipid accumulation in Y. lipolytica.(4) ATP:citrate lyase (ACL) gene from mouse, which had a higher affinity (Km=0.05mM)for citrate than Y. lipolytica, was over-expressed in Y. lipolytica. The results of Westernbloting and enzymatic activity assay in vitro showed that ACL gene was expressed and ACLactivity was improved obviously. The lipid content in yeast cell increased from7.3%to11-23%of their dry cell weight compared to the control strain, and the lipid content in multi-copyintegration transformants was higher than mono-copy integration transformants. The lipidcontent of the transformants was consistent with the copy number of ACL gene intergratedinto genome. The maximal lipid content was found in transformant YL-1292-ACL-6and was2-fold increase compared to the the control strain, meanwhile citrate concentration in themedium decreased from4.0g/L to3.2g/L. These results indicated that acetyl-CoA supplymay be the key factor for lipid accumulation in Y. lipolytica strains with low lipid content.