Enabling tools are essential for facilitating the methanol bioconversion in Pichia pastoris.However,there is still a relative lack of promoters that can stably express high levels without being affected by the carbon ...Enabling tools are essential for facilitating the methanol bioconversion in Pichia pastoris.However,there is still a relative lack of promoters that can stably express high levels without being affected by the carbon source,which hinders the construction and modification of cell factories containing long metabolic pathways.This study mapped a gene expression intensity library of central metabolic pathways in P.pastoris under methanol and glucose conditions.Through modification of the upstream sequences of promoters,an artificial promoter PS2 was developed with a strong intensity up to 90%of PGAP.By using this promoter,we successfully constructed a hybrid pathway that integrates theβ-alanine and malonyl-CoA pathways for the production of 3-hydroxypro-pionic acid.Further combining rational metabolic engineering strategies,such as optimizing gene copy numbers and blocking byproduct synthesis pathways,the engineered strains CHP9 and CHP20 achieved 3-HP titers of 23 g/L and 22 g/L by using methanol as the sole carbon source.These results indicate that adaptive strength of promoters can facilitate efficient chemical biosynthesis in methanol bioconversion by mitigating glucose repression effects.This work preliminarily explored the expression patterns of genes in the central metabolic pathways of P.pastoris,identified and characterized the intensities of various endogenous promoters,and extended the enabling toolbox for P.pastoris.This result also lays a foundation for the construction of mi-crobial cell factories and the industrial production of 3-HP via methanol bioconversion.展开更多
基金supported by Youth Fund Project of National Natural Science Foundation of China(22308352)China Postdoctoral Science Foundation(2023 M733450)DICP innovation grant(DICP I202335).
文摘Enabling tools are essential for facilitating the methanol bioconversion in Pichia pastoris.However,there is still a relative lack of promoters that can stably express high levels without being affected by the carbon source,which hinders the construction and modification of cell factories containing long metabolic pathways.This study mapped a gene expression intensity library of central metabolic pathways in P.pastoris under methanol and glucose conditions.Through modification of the upstream sequences of promoters,an artificial promoter PS2 was developed with a strong intensity up to 90%of PGAP.By using this promoter,we successfully constructed a hybrid pathway that integrates theβ-alanine and malonyl-CoA pathways for the production of 3-hydroxypro-pionic acid.Further combining rational metabolic engineering strategies,such as optimizing gene copy numbers and blocking byproduct synthesis pathways,the engineered strains CHP9 and CHP20 achieved 3-HP titers of 23 g/L and 22 g/L by using methanol as the sole carbon source.These results indicate that adaptive strength of promoters can facilitate efficient chemical biosynthesis in methanol bioconversion by mitigating glucose repression effects.This work preliminarily explored the expression patterns of genes in the central metabolic pathways of P.pastoris,identified and characterized the intensities of various endogenous promoters,and extended the enabling toolbox for P.pastoris.This result also lays a foundation for the construction of mi-crobial cell factories and the industrial production of 3-HP via methanol bioconversion.
