One-step direct transesterification of wet yeast for biodiesel production catalyzed by magnetic nanoparticle-immobilized lipase

Wang

et al. 2021

Biotechnol Biofuels

Background Liquid lipases are widely used to convert oil into biodiesel. Methanol-resistant lipases with high catalytic activity are the first choice for practical production. Rhizomucor miehei lipase (RML) is a single-chain α/β-type protein that is widely used in biodiesel preparation. Improving the catalytic activity and methanol tolerance of RML is necessary to realise the industrial production of biodiesel. Results In this study, a semi-rational design method was used to optimise the catalytic activity and methanol tolerance of ProRML. After N-glycosylation modification of the α-helix of the mature peptide in ProRML, the resulting mutants N218, N93, N115, N260, and N183 increased enzyme activity by 66.81, 13.54, 10.33, 3.69, and 2.39 times than that of WT, respectively. The residual activities of N218 and N260 were 88.78% and 86.08% after incubation in 50% methanol for 2.5 h, respectively. In addition, the biodiesel yield of all mutants was improved when methanol was added once and reacted for 24 h with colza oil as the raw material. N260 and N218 increased the biodiesel yield from 9.49% to 88.75% and 90.46%, respectively. Conclusions These results indicate that optimising N-glycosylation modification in the α-helix structure is an effective strategy for improving the performance of ProRML. This study provides an effective approach to improve the design of the enzyme and the properties of lipase mutants, thereby rendering them suitable for industrial biomass conversion.

Section: Introductionmentioning

confidence: 99%

Improved methanol tolerance of Rhizomucor miehei lipase based on N‑glycosylation within the α-helix region and its application in biodiesel production

Tian

Wang

et al. 2021

Biotechnol Biofuels

“…Cells were collected after fermentation by centrifugation (12000 rpm, 5 min) and dried to constant weight. Cellular total lipid was extracted by chloroform and methanol with the modified Folch method [ 12 ]. Drying cell pellets were pulverized and shaken for 12 h with 15 mL 4 mol/L .…”

Section: Methodsmentioning

confidence: 99%

“… DSM 27192 could produce lipid and sugar acids from glucose or xylose, and its lipid accumulation is influenced by fermentation factors such as pH, temperature, and carbon sources concentration [ 7 , 9 ]. To increase yields in DSM 27192, many lipid extraction techniques also have been developed [ 10 – 12 ]. However, lipid metabolism mechanisms in still have yet to be investigated.…”

Section: Introductionmentioning

confidence: 99%

GATA-type transcriptional factor SpGAT1 interacts with SpMIG1 and promotes lipid accumulation in the oleaginous yeast $$Saitozyma \ podzolica$$ zwy-2-3

Ran

et al. 2022

Biotechnol Biofuels

Self Cite

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.

“…Cells were collected after fermentation by centrifugation (12000 rpm, 5 min) and dried to constant weight. Cellular total lipid was extracted by chloroform and methanol with the modified Folch method [12]. Drying cell pellets were pulverized and shaken for 12 h with 15 mL 4 mol/L HCl.…”

Section: Determination Of Biomass and Lipidmentioning

confidence: 99%

“…S. podzolica DSM 27192 could produce lipid and sugar acids from glucose or xylose, and its lipid accumulation is influenced by fermentation factors such as pH, temperature, and carbon sources concentration [7,9]. To increase yields in S. podzolica DSM 27192, many lipid extraction techniques also have been developed [10,11,12]. However, lipid metabolism mechanisms in S. podzolica still have yet to be investigated.…”

Section: Introductionmentioning

confidence: 99%

GATA-type Transcriptional Factor SpGAT1 Interacts with SpMIG1 and Promotes Lipid Accumulation in the Oleaginous Yeast Saitozyma podzolica zwy-2-3

Ran

et al. 2022

Preprint

Self Cite

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 zwy-2-3. Results: Compared with WT, ∆gat1, 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 ∆gat1 decreased in the high C/N ratio medium. According to yeast two-hybrid assays, SpGAT1 interacted with SpMIG1, and it's 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.