Construction Of Metabolic Model Of Oleaginous Yeast And Lipid Fermentation Performance Prediction From Low-cost Carbon Source And Process Exploration

In the context of the depletion of fossil energy,microbial lipid,as an alternative raw material for biodiesel,is attracting more and more attention from researchers.With the development of multi-omic analysis,the understanding of researchers about the genetic background and biochemical details of oleaginous yeast is gradually deepening.On the other hand,the application of low-cost carbon sources and the development of process routes with potential of industrial production are becoming breakthroughs in the commercial production of microbial lipid.In this paper,focused on the two excellent oleaginous yeasts,Cutaneotri-chosporon oleaginosum and Lipomyces starkeyi,the theoretical simulation and process exploration of microbial lipid production from low-cost carbon sources were carried out.The first small-scale metabolic model of L.starkeyi was constructed in this research.Based on this model,combined with the published small-scale metabolic model of C.oleaginosum,theoretical simulation about production of lipid from various low-cost carbon sources building blocks（glucose,xylose,cellobiose,acetic acid,glycerol,N-acetylglucosamine（Glc NAc）,and glucosamine）was executed.At the same time,for some modification strategies that may promote lipid accumulation,such as replacing or introducing some NADP-dependent oxidoreductases,the simulation verification at the model level and the exploration of related mechanisms are carried out.Furthermore,the law of the lipid production from combined carbon source at the model level was analyzed and simulated.And the combination of acetic acid and other carbon sources could promote the theoretical lipid yield of combined carbon source was found.Based on the toxicity of free acetic acid,the chitin monomer Glc NAc,which contains glucose groups and acetyl groups and with background of low-cost substrate,was as the substrate for the process exploration about fed-batch high-density lipid fermentation in this project.Under the condition of non-nutrient limitation,the molar ratio of carbon to nitrogen（C/N）is 8,and the molar ratio of carbon to phosphorus（C/P）is 253,after 74 hours of fermentation,the biomass,lipid concentration,lipid content,lipid yield and lipid productivity reached 108.79 g/L,59.11 g/L,54.34%,0.171 g/g and 0.797 g/L/h.When the C/P ratio was further increased to 1033,although the lipid productivity decreased to 0.685 g/L/h,the lipid concentration,lipid content,and lipid yield all increased significantly,reaching 74.17 g/L,72.19%and 0.214 g/g.The high concentration of NH₄⁺（26.948 g/L）in the fermentation wastewater was directly removed by the chemical precipitation,and the slow-release nitrogen fertilizer magnesium ammonium phosphate was co-produced,the removal rate can reach the level of 87%-88%.In this study,the foundation for reconstruction of L.starkeyi NRRL Y-11557genome-scale metabolic model was laid,and the potential for high-efficiency lipid production form Glc NAc combined with slow-release fertilizer production was demonstrated.The research provided a new technical route for the high-value utilization of low-cost chitin raw materials.