Pseudouridine(Ψ),a crucial modified nucleoside in RNA,holds significant application value in biomedical fields.However,its industrial-scale production still faces challenges because of the low efficiency and the high...Pseudouridine(Ψ),a crucial modified nucleoside in RNA,holds significant application value in biomedical fields.However,its industrial-scale production still faces challenges because of the low efficiency and the high costs.In this study,a Ψ high-producing strain was constructed from the E.coli BL21(DE3)through metabolic engineering and dynamic regulation strategies.To do this,the key enzymes,i.e.,pseudouridine 5′-monophosphate glycosidase(Ψ MP glycosidase)and phosphatase,were firstly optimized to obtain the best co-expression system for Ψ biosynthesis,and the results indicated that co-expression of Ψ MP glycosidase(i.e.,EcPsuG)and phosphatase(EcYjjG)from E.coli showed the highest Ψ production and the resultant strain Tran-B4 produced 1.09±0.035 g/L of Ψ.Subsequently,the supply of R5P was enhanced by overexpressing the rate-limiting enzymes in the PP pathway and dynamically regulating the expression of pgi gene using a growth-phase-dependent promoter(i.e.,P_(fliC)),and the resultant strain Tran-D4 produced 2.35±0.085 g/L of Ψ.Lastly,the transmembrane transports and the competitive metabolic pathway were rationally modified to increase Ψ production,and the resultant strain Tran-F2 produced 2.81±0.098 g/L of Ψ in shake-flask fermentation.The final strain Tran-F2 could produce 29.5 g/L of Ψ with a productivity of 0.670 g/(L·h)and the molar conversion ratio of uracil was 90.4%in fed-batch culture.As far as we know,this is the best efficiency of Ψ production using glucose and uracil as feedstock.This study provides an efficient and cost-effective biosynthetic strategy for the Ψ production in industry.展开更多
基金supported by the National Natural Science Foundation of China(Grant number 32271534)the Class I project of independent innovation of agricultural science and technology in Jiangsu Province(grant numbers CX(22)1012)+1 种基金the Green Bio-fabrication Program of China(Grant number 2021YFC2100900)the Top-Notch Academic Programs Project of Jiangsu Higher Education Institutions,the 111 project(Grant number 111-2-06).
文摘Pseudouridine(Ψ),a crucial modified nucleoside in RNA,holds significant application value in biomedical fields.However,its industrial-scale production still faces challenges because of the low efficiency and the high costs.In this study,a Ψ high-producing strain was constructed from the E.coli BL21(DE3)through metabolic engineering and dynamic regulation strategies.To do this,the key enzymes,i.e.,pseudouridine 5′-monophosphate glycosidase(Ψ MP glycosidase)and phosphatase,were firstly optimized to obtain the best co-expression system for Ψ biosynthesis,and the results indicated that co-expression of Ψ MP glycosidase(i.e.,EcPsuG)and phosphatase(EcYjjG)from E.coli showed the highest Ψ production and the resultant strain Tran-B4 produced 1.09±0.035 g/L of Ψ.Subsequently,the supply of R5P was enhanced by overexpressing the rate-limiting enzymes in the PP pathway and dynamically regulating the expression of pgi gene using a growth-phase-dependent promoter(i.e.,P_(fliC)),and the resultant strain Tran-D4 produced 2.35±0.085 g/L of Ψ.Lastly,the transmembrane transports and the competitive metabolic pathway were rationally modified to increase Ψ production,and the resultant strain Tran-F2 produced 2.81±0.098 g/L of Ψ in shake-flask fermentation.The final strain Tran-F2 could produce 29.5 g/L of Ψ with a productivity of 0.670 g/(L·h)and the molar conversion ratio of uracil was 90.4%in fed-batch culture.As far as we know,this is the best efficiency of Ψ production using glucose and uracil as feedstock.This study provides an efficient and cost-effective biosynthetic strategy for the Ψ production in industry.
