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
背景:骨代谢紊乱会引起骨相关疾病的发生,而叉头框转录因子O3可以通过调节氧化应激、自噬水平等来影响骨组织细胞增殖、分化与凋亡,调控骨代谢过程。目的:系统性分析叉头框转录因子O3调控骨代谢及其在骨科疾病中作用机制的相关研究文献...背景:骨代谢紊乱会引起骨相关疾病的发生,而叉头框转录因子O3可以通过调节氧化应激、自噬水平等来影响骨组织细胞增殖、分化与凋亡,调控骨代谢过程。目的:系统性分析叉头框转录因子O3调控骨代谢及其在骨科疾病中作用机制的相关研究文献,为后续以叉头框转录因子O3为靶点治疗骨疾病的研究提供参考。方法:以“(SU=FoxO3a OR SU=Foxo3 OR SU=Forkhead box O3 OR SU=叉头框转录因子O3)AND SU=骨”为检索句在中国知网进行检索,以“主题:(“FoxO3a”)OR主题:(“Foxo3”)OR主题:(“Forkhead box O3”)OR主题:(“叉头框转录因子O3”)AND主题:(“骨”)”为检索句在万方医学数据库进行检索;以“((FoxO3a)OR(Foxo3)OR(Forkhead box O3))AND((bone)OR(Skeleton))”为检索句在PubMed数据库进行检索,排除陈旧、重复、质量较差以及不相关的文献,最终纳入56篇文献进行综述分析。结果与结论:①叉头框转录因子O3与骨髓间充质干细胞:叉头框转录因子O3能够促进成骨谱系的形成,还可通过激活自噬促进早期成骨分化。同时,叉头框转录因子O3在骨髓间充质干细胞中体现抗氧化特性,保护细胞免受氧化应激诱导的衰老。②叉头框转录因子O3与成骨细胞:叉头框转录因子O3在成骨细胞中能通过干扰Wnt/β-连环蛋白通路抑制成骨,同时能激活抗氧化酶保护成熟成骨细胞。叉头框转录因子O3能促进成骨祖细胞的增殖,并通过激活自噬促进成骨分化。③叉头框转录因子O3与破骨细胞:叉头框转录因子O3表达可抵抗氧化应激和激活自噬抑制破骨细胞生成。④叉头框转录因子O3与骨细胞:叉头框转录因子O3可通过抗氧化作用保护骨细胞,还可通过抑制p16和p53信号通路和抑制衰老相关分泌表型来减少骨流失。⑤叉头框转录因子O3与软骨细胞:叉头框转录因子O3在骨关节炎中对软骨细胞起到保护作用,抑制软骨细胞分解或凋亡,促进软骨细胞外基质合成,可抑制软骨细胞肥大;然而,叉头框转录因子O3与Runt相关转录因子1在软骨细胞中高度共表达却会促进软骨祖细胞的早期软骨形成和终末肥大。⑥叉头框转录因子O3通过参与氧化应激抵抗与调控自噬等过程影响骨代谢,参与多类骨相关疾病的病理进程。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.
文摘背景:骨代谢紊乱会引起骨相关疾病的发生,而叉头框转录因子O3可以通过调节氧化应激、自噬水平等来影响骨组织细胞增殖、分化与凋亡,调控骨代谢过程。目的:系统性分析叉头框转录因子O3调控骨代谢及其在骨科疾病中作用机制的相关研究文献,为后续以叉头框转录因子O3为靶点治疗骨疾病的研究提供参考。方法:以“(SU=FoxO3a OR SU=Foxo3 OR SU=Forkhead box O3 OR SU=叉头框转录因子O3)AND SU=骨”为检索句在中国知网进行检索,以“主题:(“FoxO3a”)OR主题:(“Foxo3”)OR主题:(“Forkhead box O3”)OR主题:(“叉头框转录因子O3”)AND主题:(“骨”)”为检索句在万方医学数据库进行检索;以“((FoxO3a)OR(Foxo3)OR(Forkhead box O3))AND((bone)OR(Skeleton))”为检索句在PubMed数据库进行检索,排除陈旧、重复、质量较差以及不相关的文献,最终纳入56篇文献进行综述分析。结果与结论:①叉头框转录因子O3与骨髓间充质干细胞:叉头框转录因子O3能够促进成骨谱系的形成,还可通过激活自噬促进早期成骨分化。同时,叉头框转录因子O3在骨髓间充质干细胞中体现抗氧化特性,保护细胞免受氧化应激诱导的衰老。②叉头框转录因子O3与成骨细胞:叉头框转录因子O3在成骨细胞中能通过干扰Wnt/β-连环蛋白通路抑制成骨,同时能激活抗氧化酶保护成熟成骨细胞。叉头框转录因子O3能促进成骨祖细胞的增殖,并通过激活自噬促进成骨分化。③叉头框转录因子O3与破骨细胞:叉头框转录因子O3表达可抵抗氧化应激和激活自噬抑制破骨细胞生成。④叉头框转录因子O3与骨细胞:叉头框转录因子O3可通过抗氧化作用保护骨细胞,还可通过抑制p16和p53信号通路和抑制衰老相关分泌表型来减少骨流失。⑤叉头框转录因子O3与软骨细胞:叉头框转录因子O3在骨关节炎中对软骨细胞起到保护作用,抑制软骨细胞分解或凋亡,促进软骨细胞外基质合成,可抑制软骨细胞肥大;然而,叉头框转录因子O3与Runt相关转录因子1在软骨细胞中高度共表达却会促进软骨祖细胞的早期软骨形成和终末肥大。⑥叉头框转录因子O3通过参与氧化应激抵抗与调控自噬等过程影响骨代谢,参与多类骨相关疾病的病理进程。
