In the present study,reference is made to a pigsty with an estimated total animal weight of 500,000 kg.The quantities and quality(typical characteristics)of the generated waste are calculated.The total waste occurs af...In the present study,reference is made to a pigsty with an estimated total animal weight of 500,000 kg.The quantities and quality(typical characteristics)of the generated waste are calculated.The total waste occurs after the cleaning washes in the premises of the breeding unit.Semi-liquid waste is treated in solid-liquid phase mechanical separation plants.The resulting solids,waste(reduced humidity),go to storage areas,manure.These areas are shaped concrete basins,in which there is aerobic digestion“composting”.This process results in odorless organochumic material which is further used as a soil conditioner.展开更多
5-Methyltetrahydrofolate(5-MTHF)is the essential methyl donor for S-adenosylmethionine(SAM)-dependent methylation,yet its chemical instability restricts intracellular availability and limits biosynthetic efficiency.In...5-Methyltetrahydrofolate(5-MTHF)is the essential methyl donor for S-adenosylmethionine(SAM)-dependent methylation,yet its chemical instability restricts intracellular availability and limits biosynthetic efficiency.In this study,heterologous expression of the corrinoid-dependent methyltransferase AcsE from Moorella thermoa-cetica in Escherichia coli was found to substantially enhance intracellular 5-MTHF accumulation.To elucidate the molecular mechanism underlying this phenotype,we conducted a comprehensive biochemical characterization.In vitro stability tests revealed that 5-MTHF undergoes rapid degradation in the presence of methylenetetrahy-drofolate reductase(MetF),whereas the co-presence of AcsE effectively mitigates this loss.Although glutathione S-transferase pull-down assays detected no direct AcsE-MetF interaction,isothermal titration calorimetry demonstrated that AcsE binds 5-MTHF with high affinity,which is 25.6-fold stronger than that of MetF.These findings identify a non-canonical mechanism wherein AcsE stabilizes the 5-MTHF pool via competitive sequestration,thereby shielding it from MetF-associated instability.Guided by this insight,we engineered an AcsE-based 5-MTHF enhancement module to boost SAM-dependent methylation.This module resulted in a 54%increase in ferulic acid production relative to the control,achieving a titer of 2.6 g/L in fed-batch fermentation.Collectively,this work uncovers a previously unrecognized role of AcsE in stabilizing 5-MTHF and provides a generalizable biostabilization strategy to improve 5-MTHF availability and SAM-dependent methylation efficiency.展开更多
文摘In the present study,reference is made to a pigsty with an estimated total animal weight of 500,000 kg.The quantities and quality(typical characteristics)of the generated waste are calculated.The total waste occurs after the cleaning washes in the premises of the breeding unit.Semi-liquid waste is treated in solid-liquid phase mechanical separation plants.The resulting solids,waste(reduced humidity),go to storage areas,manure.These areas are shaped concrete basins,in which there is aerobic digestion“composting”.This process results in odorless organochumic material which is further used as a soil conditioner.
基金supported by the Taishan Scholars Program(No.tsqn202312271)the Key R&D Program of Shandong Province,China(No.2023JMRH0201)the Key Science and Technology Program of Jiangsu Tobacco Industry Co.,Ltd(No.H202534).
文摘5-Methyltetrahydrofolate(5-MTHF)is the essential methyl donor for S-adenosylmethionine(SAM)-dependent methylation,yet its chemical instability restricts intracellular availability and limits biosynthetic efficiency.In this study,heterologous expression of the corrinoid-dependent methyltransferase AcsE from Moorella thermoa-cetica in Escherichia coli was found to substantially enhance intracellular 5-MTHF accumulation.To elucidate the molecular mechanism underlying this phenotype,we conducted a comprehensive biochemical characterization.In vitro stability tests revealed that 5-MTHF undergoes rapid degradation in the presence of methylenetetrahy-drofolate reductase(MetF),whereas the co-presence of AcsE effectively mitigates this loss.Although glutathione S-transferase pull-down assays detected no direct AcsE-MetF interaction,isothermal titration calorimetry demonstrated that AcsE binds 5-MTHF with high affinity,which is 25.6-fold stronger than that of MetF.These findings identify a non-canonical mechanism wherein AcsE stabilizes the 5-MTHF pool via competitive sequestration,thereby shielding it from MetF-associated instability.Guided by this insight,we engineered an AcsE-based 5-MTHF enhancement module to boost SAM-dependent methylation.This module resulted in a 54%increase in ferulic acid production relative to the control,achieving a titer of 2.6 g/L in fed-batch fermentation.Collectively,this work uncovers a previously unrecognized role of AcsE in stabilizing 5-MTHF and provides a generalizable biostabilization strategy to improve 5-MTHF availability and SAM-dependent methylation efficiency.
