Enhancement On Lipid Production By Trichosporon Cutaneum Through Morphological Control And Underlying Mechanism

Biodiesel produced with microbial oils is an alternative to diesel fuel derived from crude oil that is not sustainable and environmentally friendly due to its contribution to greenhouse gas emissions.Trichosporon cutaneum is oleaginous yeast with basidiomycete,which is characterized by broad substrate spectrum for simultaneous utilization of xylose and glucose and tolerant to inhibitors,and thus a candidate for lipid production from lignocellulosic biomass.However,much less has been devoted so far to fundamentals with T.cutaneum such as morphology,physiology,metabolism and genetics,which consequently hinders the development of robust strains and process engineering strategies for produing microbial oils by the species through submerged culture and fermentation.Two significantly different morphologies were observed in T.cutaneum B3,which experienced transition from unicellular yeast-form cells to hyphae under submerged culture and fermentation conditions,and the yeast-form cells synthesized oils more efficiently.While nitrogen limitation,neutral p H and high dissolved oxygen(DO)induced the yeast-form morphology,hyphae were developed under rich nitrogen,acidic p H and low DO conditions.When 5 g/L yeast extract(YE)was supplemented,yeast-form cells inoculated as the seed grew with the hyphae morphology,which compromised utilization of glucose and nitrogen as well as yeast growth due to poor oxygen supply associated with the non-Newtonian fluid properties,and lipid production characterized by intracellular lipid content and lipid productivity was drastically decreased.When YE was supplemented at 3.0 g/L,an obvious dimorphic transition yeast?hyphae?yeast was observed,and hyphae were observed only at the early stage when nitrogen was rich,but all hyphae were transformed to yeast-form morphology near the end of the culture with high lipid productivity and lipid content.When YE supplementation was further decreased to 1.0 g/L,T.cutaneum B3 was grown predominately in the yeast-form morphology during the whole process with much higher intracellular lipid content,but lipid productivity was lower since yeast growth was poor and biomass density was much lower.We performed comparative transcriptomics analysis to identify differently expressed genes,and found genes encoding key enzymes for central carbon metabolism associated with lipid biosynthesis in unicellular yeast-form cells were up-regulated,and on the other hand genes encoding key enzymes related to the degradation of fatty acids via β-oxidation were down-regulated for lipid accumulation.For example,the activities of ATP-dependent citrate synthase(ACL),malic enzyme(MAE),isocitrate dehydrogenase(IDH)and isocitrate lyase(ICL)were up-regulated two folds in the yeast-form cells compared to their activities detected in hyphae to enhance intracellular lipid biosynthesis and accumulation.Comparative transcriptome analysis also indicates that T.cutaneum B3 may mediate its response to nitrogen starvation,p H stimuli and dimorphic transition through cyclic adenosine monophosphate dependent protein kinase A(c AMP-PKA)pathway,RIM101 pathway and mitogen-activated protein kinase(MAPK)cascade signaling pathway.DO was experimentally validated to be a key factor for triggering T.cutaneum B3 dimorphic transition,and consequently affected its lipid production.Computational fluid dynamics analysis showed that when a microporous ceramic membrane sparger was employed with the bioreactor,oxygen mass transfer was enhanced due to high surface area created by the large amoumt of microbubbles for more efficient gas-liquid contact to make DO increased,even at low aeration rate and stirring speed,which benefited for the development of the unicellular yeast-form morphology by T.cutaneum B3 for more efficient lipid production.Based on the DO-control strategy,rice straw hydrolysate without additional supplementation of carbon and nitrogen sources was used for lipid production by T.cutaneum B3 through fed-batch fermentation,and lipid productivity and lipid yield of 0.201 g/L/h and0.250 g/g were achieved,respectively,which are the highest so to our knowledge,and significant for lipid production from lignocellulosic biomass as a feedstock for biodiesel production.Our results provide insights on the morphogenesis and lipogenesis of T.cutaneum B3 for developing robust strains and process engineering strategies to produce lipid more efficiantly,which also laid scientific and technical foundation for morphology engineering strategy to be applied in the production of other fermentation products.