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
The methylotrophic yeast Pichia pastoris(syn.Komagataella phaffii)has been extensively engineered for protein production,and is attracting attention as a chassis cell for methanol biotransformation toward production o...The methylotrophic yeast Pichia pastoris(syn.Komagataella phaffii)has been extensively engineered for protein production,and is attracting attention as a chassis cell for methanol biotransformation toward production of small molecules.However,the relatively unclear methanol metabolism hampers the metabolic rewiring to improve the biosynthetic efficiency.We here performed a label-free quantitative proteomic analysis of Pichia pastoris when cultivated in minimal media containing methanol and glucose,respectively.There were 243,158 up-regulated proteins and 244,304 down-regulated proteins in log and stationary phase,respectively,when cultivated in methanol medium compared with that of glucose medium.Peroxisome enrichment further improved the characterization of more differentially expressed proteins(481 proteins in log phase and 524 proteins in stationary phase).We demonstrated the transaldolase isoenzyme(Tal2,Protein ID:C4R244)was highly up-regulated in methanol medium cultivation,which plays an important role in methanol utilization.Our work provides important information for understanding methanol metabolism in methyltrophic yeast and will help to engineer methanol biotransformation in P.pastoris.展开更多
The blue-emitting YPO4 phosphors doped with Yb3＋ were prepared by a simple hydrothermal method. All the products were characterized by XRD and TEM, which revealed that they were zircon structure with leaf-like morpho...The blue-emitting YPO4 phosphors doped with Yb3＋ were prepared by a simple hydrothermal method. All the products were characterized by XRD and TEM, which revealed that they were zircon structure with leaf-like morphology. According to the analysis of photoluminescence spectra, upon ultraviolet （275 nm） excitation, the Yb3＋ doped YPO4 phosphor showed an intense blue emission composed of two main bands at 420 and 620 nm assigned to charge transfer state （CTS） → 2Fs/2 and CTS →ZF7/2, respectively. Moreover, the optimum doping concentration of Yb3＋ in YPO4 phosphor was 1%, which exhibited the maximum emission intensity. The possible physical mechanism of concentration quenching was discussed, and the critical transfer distance determined to be 23.889 A. In particular, the color purity of the as-synthesized Yb3＋ doped YPO4 phosphor was as high as 83%, which made it an excellent candidate for blue-emitting materials.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.
基金supported by National Natural Science Foundation of China(22161142008,M-0246)DMTO research grant(grant no.DICP DMTO_(2)01701)from Dalian institute of Chemical Physics,CAS.
文摘The methylotrophic yeast Pichia pastoris(syn.Komagataella phaffii)has been extensively engineered for protein production,and is attracting attention as a chassis cell for methanol biotransformation toward production of small molecules.However,the relatively unclear methanol metabolism hampers the metabolic rewiring to improve the biosynthetic efficiency.We here performed a label-free quantitative proteomic analysis of Pichia pastoris when cultivated in minimal media containing methanol and glucose,respectively.There were 243,158 up-regulated proteins and 244,304 down-regulated proteins in log and stationary phase,respectively,when cultivated in methanol medium compared with that of glucose medium.Peroxisome enrichment further improved the characterization of more differentially expressed proteins(481 proteins in log phase and 524 proteins in stationary phase).We demonstrated the transaldolase isoenzyme(Tal2,Protein ID:C4R244)was highly up-regulated in methanol medium cultivation,which plays an important role in methanol utilization.Our work provides important information for understanding methanol metabolism in methyltrophic yeast and will help to engineer methanol biotransformation in P.pastoris.
文摘The blue-emitting YPO4 phosphors doped with Yb3＋ were prepared by a simple hydrothermal method. All the products were characterized by XRD and TEM, which revealed that they were zircon structure with leaf-like morphology. According to the analysis of photoluminescence spectra, upon ultraviolet （275 nm） excitation, the Yb3＋ doped YPO4 phosphor showed an intense blue emission composed of two main bands at 420 and 620 nm assigned to charge transfer state （CTS） → 2Fs/2 and CTS →ZF7/2, respectively. Moreover, the optimum doping concentration of Yb3＋ in YPO4 phosphor was 1%, which exhibited the maximum emission intensity. The possible physical mechanism of concentration quenching was discussed, and the critical transfer distance determined to be 23.889 A. In particular, the color purity of the as-synthesized Yb3＋ doped YPO4 phosphor was as high as 83%, which made it an excellent candidate for blue-emitting materials.
