Effects Of Vitreoscilla Hemoglobin Expression On Lipid Production In Yarrowia Lipolytica

Yarrowia lipolytica as an oleaginous microorganism can convert excess carbon source into lipids and secrete large amounts of citric acid.Y.lipolytica is an obligate aerobic yeast.Thus,oxygen availability in the fermentation medium is one of most critical parameters for lipid production in Y.lipolytica.To alleviate oxygen limitation and enhance the yield of lipid in the process of Y.lipolytica fermentation,the main researches of this work were carried out as follows:Dissolved oxygen(DO)concentration in the culture medium during fermentation process was controlled by controlling the stirring speed and aeration rate and monitored by dissolved oxygen electrode.DO concentration was selected for five levels i.e.:5%,10%,20%,30%and uncontrolled,to study the influence of DO concentration on lipid accumulation and citric acid secretion in Y.lipolytica.The results showed that the increase of DO concentration caused the increased production of lipid and citric acid in Y.lipolytica.And the maximum yield of lipid and citric acid was achieved by normal fermentation with controlling of stirring speed at 300 rpm and aeration rate at 2.0 vvm.The obtained lipid content and citric acid concentration were 11.62%(w/w)of its dry cell weight and 21.0 g/L,respectively.Furthermore,the different DO concentration also has an influence on the fatty acid composition.High DO concentration could promote the increase of oleic acid content,and the highest content of oleic acid was 48.62%which was obtained in the uncontrolled oxygen level.Therefore,high DO concentration has a positive influence on cell growth and metabolite accumulations of Y.lipolytica.To alleviate oxygen limitation and increase cellular lipid content in fermentation process,Vitreoscilla hemoglobin gene were transformed into Y.lipolytica genome to improve the oxygen availability during fermentation.First of all,hemoglobin gene(vhb)from Vitreoscilla was cloned into the plasmid p INA1312 to construct expression vector p INA1312-vhb.The second,the plasmid p INA1312-vhb containing vhb was introduced into Y.lipolytica using the lithium acetate method.The transformants were verified by PCR with genomic DNA as the template,and then were purified by several cycles of single-colony screening.The expression of VHb was confirmed by CO-difference spectrum analysis.At last,it's the effects of VHb on lipid accumulation and citric acid secretion in the yeast were analyzed.During fermentation,DO concentration was controlled under five different levels(5%,10%,20%,30%and uncontrolled)by associating with stirring speed and aeration.The results showed that the VHb gene expression in Y.lipolytica have no significant influence on cell density.The transformants(VHb~+strain)and control(VHb~-strain)strains exhibited similar biomass.But cell morphology of Y.lipolytica recombinant harboring the VHb gene were changed in different DO concentrations,displaying higher percentage of pseudomycelium than the control strain.The expression of VHb could obviously enhance the lipid accumulation and led to the increase of C18:1 under five DO levels.When DO concentration was controlled at 30%,the TFA content in VHb~+strain was 14.5%and increased by 47.96%compared with VHb~-strain,and the C18:1 content in VHb~+strain increased to 47.8%.Therefore,the expression of VHb can resolve oxygen limitation in fermentation process and enhance metabolites production of Y.lipolytica.This study will provide an important basis for the application of vhb in oleaginous microorganisms.