Lipid Biosynthesis And Regulation In Marine Yeast Pichia Guilliermondii

Microbial oils have significant value in field of bio-energy, which is a new rawmaterial for biodiesel production. At present the high cost of microbial oils is a majorbarrier to its commercialization. Traditionally, glucose and starch are used as thesubstrates for single cell oils (SCO) production. Therefore, using a low cost rawmaterial is crucial in reducing the cost of microbial oils production. So far, it has beenlittle known about how the oleaginous microorganisms accumulate high level of lipids.Therefore, it is necessary to study the lipid biosynthesis and regulation in oleaginousmicroorganisms.In our previous studies, it is found that inulin had been confirmed to be one ofthe best feedstock for oils fermentation. However, most of the oleaginous yeasts donot synthesize and secrete the inulinase which can catalyze hydrolysis of inulin.Therefore, it is very important to get the oleaginous yeasts which can produce a largeamount of inulinase in order to greatly reduce the cost of raw materials for lipidproduction. The yeast strain Pcla22which was isolated from surface of marine algaecould produce over11.5U/ml of inulinase activity and contained a large amount oflipids. According to the characterizations of colony, cell morphology and thephylogenetic analysis, the yeast strain Pcla22was identified to be Pichiaguilliermondii.In order to directly convert inulin into lipid by the yeast strain Pcla22, theconditions for lipid production by the strain Pcla22were studied. Under the optimalconditions,60.6%(w/w) of lipid based on cell dry weight,20.4g/l of the dry cellmass and SCO yield of0.19g/g were obtained in the culture of the yeast strain Pcla22after96h of the fed-batch fermentation. Over79.8%of the fatty acids from the yeaststrain Pcla22grown in the oil production medium containing inulin was C16:0andC18:1, especially C18:1(57.9%). It was found that over84.3%of the lipids could betransformed into biodiesel and the biodiesel obtained from the produced lipid could beburnt well. The yeast strain Pcla22contained the higher oil content (49.0%w/w) than thestrain TJY22(19.4%w/w) lipid in its cells. In order to know the lipid biosynthesisdifference between the two yeast strains, transcript levels of many genes relevant tolipid biosynthesis in the strains Pcla22and TJY22were investigated. It was found thattranscript level of the PYC gene, one of the genes relevant to oxaloacetate anapleroticreaction, was reduced to7.6%in the strain TJY22compared to that of the same genein the strain Pcla22. Such change in gene expression profile may cause the lower lipidbiosynthesis in the yeast strain of TJY22than that in the yeast strain Pcla22. At thesame time, the roles of calcium carbonate in lipid biosynthesis by the yeast strainTJY22were also investigated. The results indicated that CaCO3not only increased thepyruvate carboxylase activity, but also enhanced the oil production in the yeast strainTJY22.In order to verify the role of pyruvate carboxylase in lipid biosynthesis andregulation, the pyruvate carboxylase gene (PYC) and the ATP-citrate lyase gene(ACL1) were overexpressed in the yeast strain Yarrowia lipolytica ACA-DC50109. Itwas found that the expression of the PYC gene and ACL1gene in ACA-DC50109enhanced citric acid accumulation and single cell oil production. The PYCover-expressing transformant P77and the PYC-ACL1co-over-expressingtransformant PA56contained38.2%(w/w) and45.3%(w/w) of oil, respectively,while the wild-type yeast only contained30.2%(w/w) of oil. The SCO yields in thesestrains (wild-type, P77and PA56) were0.091g/g,0.112g/g and0.136g/g,respectively. However, the citric acid concentrations in the cultures of these strainswere0.5g/l,11.5g/l and6.2g/l, respectively. According to the results mentionedabove, it was found that pyruvate carboxylase could enhance the oxaloacetateanaplerotic reaction, which caused citric acid accumulation, and the ATP-citrate lyasecould convert citrate into lipid.