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To develop a method for direct transesterification of wet yeast using immobilized lipase, the oleaginous yeast Saitozyma podzolica Zwy-2-3 and the lipase producing Burkholderia pyrrolica WZ10-3 were used as materials for production of biodiesel. Fe3O4@SiO2-CHO prepared by modifying Fe3O4 with TEOS, APTES and glutaraldehyde. The biocatalysts covalently cross-linked with WZ10-3 lipase by Fe3O4@SiO2-CHO were characterized by FTIR, XRD and TEM. When the enzyme dosage, glutaraldehyde concentration, temperature and time were 30.22 mL, 2.0%, 40 °C and 4 h, the immobilized lipase activity and immobilization rate reached 10038.0 U/g and 96.9%, respectively. The optimum temperatures for immobilized and free lipase were 60 °C and 40 °C. The immobilized enzyme still had 80% enzymatic activity after 48 d storage at 4 °C. The optimized conditions for the direct conversion of immobilized lipase to esterified wet yeast (one-step) were: enzyme dosage 2.5 g, reaction temperature 35 °C; water content 15%; and molar ratio of n-hexane to methanol 3: 1. The transesterification rates of one-step method for oil and biomass were 98.12% and 56.11%, respectively. In contrast, the two-step method was only 88.75% and 51.21%. The immobilized enzyme had 90% enzyme activity after 10 times of reuse.

Background In oleaginous yeast, nitrogen limitation is a critical parameter for lipid synthesis. GATA-family transcriptional factor GAT1, a member of the target of rapamycin (TOR) pathway and nitrogen catabolite repression (NCR), regulates nitrogen uptake and utilization. Therefore, it is significant to study the SpGAT1 regulatory mechanism of lipid metabolism for conversion of biomass to microbial oil in $$Saitozyma \ podzolica$$ S a i t o z y m a p o d z o l i c a zwy-2-3. Results Compared with WT, $$\Delta gat1$$ Δ g a t 1 , and OE::gat1, the lipid yield of OE::gat1 increased markedly in the low carbon and nitrogen ratio (C/N ratio) mediums, while the lipid yield and residual sugar of $$\Delta gat1$$ Δ g a t 1 decreased in the high C/N ratio medium. According to yeast two-hybrid assays, SpGAT1 interacted with SpMIG1, and its deletion drastically lowered SpMIG1 expression on the high C/N ratio medium. MIG1 deletion has been found in earlier research to affect glucose metabolic capacity, resulting in a prolonged lag period. Therefore, we speculated that SpGAT1 influenced glucose consumption rate across SpMIG1. Based on yeast one-hybrid assays and qRT-PCR analyses, SpGAT1 regulated the glyoxylate cycle genes ICL1, ICL2, and pyruvate bypass pathway gene ACS, irrespective of the C/N ratio. SpGAT1 also could bind to the ACAT2 promoter in the low C/N medium and induce sterol ester (SE) accumulation. Conclusion Our findings indicated that SpGAT1 positively regulated lipid metabolism in S.podzolica zwy-2-3, but that its regulatory patterns varied depending on the C/N ratio. When the C/N ratio was high, SpGAT1 interacted with SpMIG1 to affect carbon absorption and utilization. SpGAT1 also stimulated lipid accumulation by regulating essential lipid anabolism genes. Our insights might spur more research into how nitrogen and carbon metabolism interact to regulate lipid metabolism.

In oleaginous yeast, nitrogen limitation is a critical parameter for lipid synthesis. GATA-family transcriptional factor GAT1, a member of the target of rapamycin (TOR) pathway and nitrogen catabolite repression (NCR), regulates nitrogen uptake and utilization. Therefore, it is significant to study the SpGAT1 regulatory mechanism of lipid metabolism for conversion of biomass to microbial oil in [Formula: see text] zwy-2-3.Compared with WT, [Formula: see text], and OE::gat1, the lipid yield of OE::gat1 increased markedly in the low carbon and nitrogen ratio (C/N ratio) mediums, while the lipid yield and residual sugar of [Formula: see text] decreased in the high C/N ratio medium. According to yeast two-hybrid assays, SpGAT1 interacted with SpMIG1, and its deletion drastically lowered SpMIG1 expression on the high C/N ratio medium. MIG1 deletion has been found in earlier research to affect glucose metabolic capacity, resulting in a prolonged lag period. Therefore, we speculated that SpGAT1 influenced glucose consumption rate across SpMIG1. Based on yeast one-hybrid assays and qRT-PCR analyses, SpGAT1 regulated the glyoxylate cycle genes ICL1, ICL2, and pyruvate bypass pathway gene ACS, irrespective of the C/N ratio. SpGAT1 also could bind to the ACAT2 promoter in the low C/N medium and induce sterol ester (SE) accumulation.Our findings indicated that SpGAT1 positively regulated lipid metabolism in S.podzolica zwy-2-3, but that its regulatory patterns varied depending on the C/N ratio. When the C/N ratio was high, SpGAT1 interacted with SpMIG1 to affect carbon absorption and utilization. SpGAT1 also stimulated lipid accumulation by regulating essential lipid anabolism genes. Our insights might spur more research into how nitrogen and carbon metabolism interact to regulate lipid metabolism.

A cost-efficient system was developed for the production of single cell oil from lignocellulosic biomass. The pretreatment of corn stover by ammonium carbonate-steam explosion was performed. A maximum lignin removal rate of 47.26% was observed and confirmed by SEM, XRD and FTIR. In the process of enzymatic hydrolysis, higher initial enzyme loadings were used to reduce the reaction time to 24 h. Under these conditions, the sugar conversion, sugar conversion rate and sugar yield of the pretreated corn stover were 74.84%, 2.00 g L-1 h-1 and 479.4 g kg-1 corn stover, respectively. The biomass production, lipid concentration and lipid content of Cryptococcus podzolicus SCTCC300292 were 10.56 g L-1, 5.03 g L-1 and 47.60%, respectively, in the fermentation of enzymatic hydrolysate. An improved cycling process of enzymatic hydrolysis was employed to reduce the enzyme loading by 40%. The sugar conversion increased to 80.17% and the sugar yield to 513.45 g kg-1 corn stover with a higher sugar release rate of 2.14 g L-1 h-1. Furthermore, the lipid yield reached 54.6 g kg-1 corn stover for the whole biorefinery process. These results indicated that the process of ammonium carbonate-steam explosion pretreatment and recirculated enzymatic hydrolysis would be a highly efficient path for converting lignocellulosic feedstocks into lipids.