Molecular Mechanism Of Lipid Accumulation In Yarrowia Lipolytica

Objective The aim of this study is to screen and obtain Yarrowia lipolitica strain with high lipid content, and to explore the mechanism of lipid accumulation in Yarrowia lipolitica by analyzing the change of biochemical characteristic,when the yeast was cultivated in N-limited medium.Methods The same N-limited medium was used to screen Yarrowia lipolitica with the highest lipid content, the influence of different carbon sources or nitrogen sources on lipid content and biomass in Yarrowia lipolitica was also evaluated. When the yeast was cultivated in N-limited medium, we need to monitor the carbon residue and nitrogen residue in medium, and determined the biamss, lipid content and fatty acid compositions. The secreted citrate and isocitrate by yeast cells into medium was also determined by HPLC. At the same time, the enzyme assays of malic enzyme, glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase, AMP deaminase, NAD-isocitrate dehydrogenase and ATP citrate lyase were measured in different time using ultraviolet-visible spectrophotometry.A pair of primers with BamHI restriction sites were designed accoding to the malic enzyme gene cDNA sequences in NCBI. The malic enzyme gene was cloned to T-Vector for plasmid pMT-mae by PCR method with the yeast genome as the template. Then the plasmids pMT-mae and pET28a(+) were digested by BamHI, following purified and ligated the fragments. The recombinant pET28a-mae was transformed into E. coli BL21 (DE3). Expression of malic enzyme was induced Induced by IPTG, the fusion protein was expressed under optimized prokaryotic expression conditions. The expressed fusion protein was purified through Ni-NTA affinity chromatography. The enzyme assays of fusion malic enzyme were accomplished in a certain reaction assay and calculated the Km for coenzyme NAD or NADP.Results The lipid content of Yarrowia lipolytica DSMZ 1345 was the highest among all the strains we selected. It was found that glycerol was the best carbon source, the mixture of ammonium tartrate and yeast extract was the best nitrogen source. When the yeast was cultivated in N-limited medium, the nitrogen was exhausted after 7-8 hours, the increase rate of biomass became slow, and the yeast cells started to accumulate lipid, the lipid content was up to 18.1% finally. Besides, a large amount of citrate and isocitrate was secreted by yeast cells, the concertration of citrate and isocitrate in medium was respectively 51.79 g/1 and 14.31 g/1. As the cells ran out of available nitrogen in the medium, AMP deaminase showed a sharp increase in acitivity while the activity of NAD-ICDH was opposite to AMPD, suggesting the onset on lipid accumulation. It was observed that acitivaty of malic enzyme, glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase, used for producing NADPH, were active throughout the fermentation, were not concomitant with the change of lipid accumulation, and thus may not be the regulating enzyme involved in lipid accumulation. However the change of the activity of ACL was in accordance with the change of lipid accumulation, suggesting that ATP citrate lyase might be the key enzyme involved in lipid accumulation in Yarrowia lipolytica.We also cloned the malic enzyme to further investigate the role of malic enzyme in this yeast.The plasmid pET28a-mae expressed the recombinant malic enzyme of Yarrowia lipolytica were constructed and digested by BamHI or Sal I, and to get right bonds in gel. The plasmid pET28a-mae then was transformed into E. coli BL21, The prokaryotic expression condition was optimized as follows:the induced temperature was 20℃; the induced IPTG concentration was 0.2 mM, the induced time was 5 h, and the high level recombinant prorein was obtained. After fusion malic enzyme was purified through Ni-NTA affinity chromatography, the enzyme acivity was determined, the Km(1.57 mM) and Vmax (714.29 nmol/min*mg) of recombinant malic enzyme for NAD were gained, but the affinity for NADP was too low to calculate the Km.Conclusion Glycerol turned out to be the good carbon source for Yarrowia lipolytica DSMZ 1345 to produce both biomass and lipids. The yeast growth fast, it reached exponential phase within a short period of 8-10 hrs in N-limited medium. When the nitrogen in medium was exhausted, the yeast cells started to accumulate lipid. However the lipid content was not high, and cells produced a large amount of citrate and isocitrate,which was secreted into medium. The activity of ATP citrate lyase was correlated to lipid accumulation, and might be the key enzyme in lipid accumulation in Yarrowia lipolytica.The role of malic enzyme was further invisitgated in vitro. The plasmid pET28a-mae expressed the recombinant malic enzyme of Yarrowia lipolytica were constructed successedly, Meanwhile the obtained E coli BL21(DE3) high level recombinant malic enzyme. The recombinant malic enzyme had high affinity for NAD with a Km of 1.57 mM and Vmax of 714.29 nmol/min*mg. However, the recombinant malic enzyme had extremely low affinity for NADP, indicating that it might not be the key enzyme in lipid accumulation in Yarrowia lipolytica.