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
Rice straw and sugar cane industrial waste are a plentiful source of lignocellulosic biomass with a high poly-saccharide content,that is hydrolyzed into sugar for microbial growth and their metabolites.3-Hydroxypropio...Rice straw and sugar cane industrial waste are a plentiful source of lignocellulosic biomass with a high poly-saccharide content,that is hydrolyzed into sugar for microbial growth and their metabolites.3-Hydroxypropionic acid(3-HP)is a promising chemical building block that can be produced from renewable resources.The malonyl-CoA pathway is one of the biosynthetic routes for 3-HP production by expressing the malonyl-CoA reductase gene(mcr).However,the problem of the activity imbalance between the C and N-terminal causes a low con-version rate of malonyl-CoA to 3-hydroxypropionic acid.This study aimed to balance the bi-functional MCR enzyme by dissecting MCR into two fragments and enhancing the supply of intermediates to increase the pro-duction of 3-HP.The recombinant strain harboring the dissected mcr gene showed a 21-fold increase in 3-HP titer compared to the strain carrying the full-length mcr gene.The addition of cerulenin and acetate to the fermented medium enhanced 3-HP yield by 8 times,in which recombinant yeast produced 3-HP up to 10 g/L(0.201 gproduct/gsubstrate).The results of using rice straw hydrolysate as a carbon source indicated that Saccharomyces cerevisiae S2 produced 3-HP of 4.02 g/L,which was 0.074 gproduct/gglucose in the diluted hydrolysate.These findings provide an alternative and sustainable strategy for utilizing lignocellulosic biomass for future 3-HP production at an industrial scale.展开更多
Syngas,which contains large amount of CO2 as well as H2 and CO,can be convert to acetic acid chemically or biologically.Nowadays,acetic acid become a cost-effective nonfood-based carbon source for value-added biochemi...Syngas,which contains large amount of CO2 as well as H2 and CO,can be convert to acetic acid chemically or biologically.Nowadays,acetic acid become a cost-effective nonfood-based carbon source for value-added biochemical production.In this study,acetic acid and CO2 were used as substrates for the biosynthesis of 3-hydroxypropionic acid(3-HP)in metabolically engineered Escherichia coli carrying heterogeneous acetyl-CoA carboxylase(Acc)from Corynebacterium glutamicum and codon-optimized malonyl-CoA reductase(MCR)from Chloroflexus aurantiacus.Strategies of metabolic engineering included promoting glyoxylate shunt pathway,inhibiting fatty acid synthesis,dynamic regulating of TCA cycle,and enhancing the assimilation of acetic acid.The engineered strain LNY07(M*DA)accumulated 15.8 g/L of 3-HP with the yield of 0.71 g/g in 48 h by whole-cell biocatalysis.Then,syngas-derived acetic acid was used as substrate instead of pure acetic acid.The concentration of 3-HP reached 11.2 g/L with the yield of 0.55 g/g in LNY07(M*DA).The results could potentially contribute to the future development of an industrial bioprocess of 3-HP production from syngas-derived acetic acid.展开更多
3-Hydroxypropionic Acid(3-HP)is recognized as a high value-added chemical with a broad range of applications.Among the various biosynthetic pathways for 3-HP production,theβ-alanine pathway is particularly noteworthy...3-Hydroxypropionic Acid(3-HP)is recognized as a high value-added chemical with a broad range of applications.Among the various biosynthetic pathways for 3-HP production,theβ-alanine pathway is particularly noteworthy due to its capacity to generate 3-HP from glucose at a high theoretical titer.In this study,theβ-alanine biosynthesis pathway was introduced and optimized in Corynebacterium glutamicum.By strategically regulating the supply of precursors,we successfully engineered a strain capable of efficiently synthesizing 3-HP through theβ-alanine pathway,utilizing glucose as the substrate.The engineered strain CgP36 produced 47.54 g/L 3-HP at a yield of 0.295 g/g glucose during the fed-batch fermentation in a 5 L fermenter,thereby attaining the highest 3-HP titer obtained from glucose via theβ-alanine pathway.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funded by Thailand Science Research and Innova-tion Fund Chulalongkorn University(DIS_FF_68_319_2300_087)Research and Researchers for Industries(RRI)jointly administered by Thailand Research Fund[grant numbers PHD61I0031]Innovation Institute,PTT Public Company Limited provided financial support for this research.
文摘Rice straw and sugar cane industrial waste are a plentiful source of lignocellulosic biomass with a high poly-saccharide content,that is hydrolyzed into sugar for microbial growth and their metabolites.3-Hydroxypropionic acid(3-HP)is a promising chemical building block that can be produced from renewable resources.The malonyl-CoA pathway is one of the biosynthetic routes for 3-HP production by expressing the malonyl-CoA reductase gene(mcr).However,the problem of the activity imbalance between the C and N-terminal causes a low con-version rate of malonyl-CoA to 3-hydroxypropionic acid.This study aimed to balance the bi-functional MCR enzyme by dissecting MCR into two fragments and enhancing the supply of intermediates to increase the pro-duction of 3-HP.The recombinant strain harboring the dissected mcr gene showed a 21-fold increase in 3-HP titer compared to the strain carrying the full-length mcr gene.The addition of cerulenin and acetate to the fermented medium enhanced 3-HP yield by 8 times,in which recombinant yeast produced 3-HP up to 10 g/L(0.201 gproduct/gsubstrate).The results of using rice straw hydrolysate as a carbon source indicated that Saccharomyces cerevisiae S2 produced 3-HP of 4.02 g/L,which was 0.074 gproduct/gglucose in the diluted hydrolysate.These findings provide an alternative and sustainable strategy for utilizing lignocellulosic biomass for future 3-HP production at an industrial scale.
基金supported by the Natural Science Foundation of Shanghai(19ZR1472700)the Fok Ying-Tong Education Foundation,China(Grant No.161017)+2 种基金the National Natural Science Foundation of China(Grant No.21776083)the Fundamental Research Funds for the Central Universities(Grant No.22221818014)supported by Open Funding Project of the CAS Key Laboratory of Synthetic Biology.
文摘Syngas,which contains large amount of CO2 as well as H2 and CO,can be convert to acetic acid chemically or biologically.Nowadays,acetic acid become a cost-effective nonfood-based carbon source for value-added biochemical production.In this study,acetic acid and CO2 were used as substrates for the biosynthesis of 3-hydroxypropionic acid(3-HP)in metabolically engineered Escherichia coli carrying heterogeneous acetyl-CoA carboxylase(Acc)from Corynebacterium glutamicum and codon-optimized malonyl-CoA reductase(MCR)from Chloroflexus aurantiacus.Strategies of metabolic engineering included promoting glyoxylate shunt pathway,inhibiting fatty acid synthesis,dynamic regulating of TCA cycle,and enhancing the assimilation of acetic acid.The engineered strain LNY07(M*DA)accumulated 15.8 g/L of 3-HP with the yield of 0.71 g/g in 48 h by whole-cell biocatalysis.Then,syngas-derived acetic acid was used as substrate instead of pure acetic acid.The concentration of 3-HP reached 11.2 g/L with the yield of 0.55 g/g in LNY07(M*DA).The results could potentially contribute to the future development of an industrial bioprocess of 3-HP production from syngas-derived acetic acid.
基金supported by the National Key Research and Development Program of China(2021YFC2100700)the National Natural Science Foundation of China(NSFC-21776208).
文摘3-Hydroxypropionic Acid(3-HP)is recognized as a high value-added chemical with a broad range of applications.Among the various biosynthetic pathways for 3-HP production,theβ-alanine pathway is particularly noteworthy due to its capacity to generate 3-HP from glucose at a high theoretical titer.In this study,theβ-alanine biosynthesis pathway was introduced and optimized in Corynebacterium glutamicum.By strategically regulating the supply of precursors,we successfully engineered a strain capable of efficiently synthesizing 3-HP through theβ-alanine pathway,utilizing glucose as the substrate.The engineered strain CgP36 produced 47.54 g/L 3-HP at a yield of 0.295 g/g glucose during the fed-batch fermentation in a 5 L fermenter,thereby attaining the highest 3-HP titer obtained from glucose via theβ-alanine pathway.
