β-nicotinamide mononucleotide(β-NMN)is a bioactive nucleotide that indirectly affects various cellular metabolic pathways,and has tremendous potential in the food,pharmaceutical,and cosmetics industries.In the prese...β-nicotinamide mononucleotide(β-NMN)is a bioactive nucleotide that indirectly affects various cellular metabolic pathways,and has tremendous potential in the food,pharmaceutical,and cosmetics industries.In the present study,a multi-enzymatic cascade biocatalyst for the efficient synthesis ofβ-NMN was established.First,the optimal ribokinase gene was selected to construct a biocatalyst in a lower-endotoxin-level Escherichia coli strain to produce NMN from D-ribose.After fine-tuning the gene expression of the three cascade enzymes(RK/PRS/NAMPT),the production of NMN improved to 3.14 g/L.Next,the functions of membrane transporters that import NAM and export NMN were verified for NMN biosynthesis.Through the introduction of an ATP regeneration system,a drastic improvement in NMN biosynthesis at a yield of 8.74 g/L was achieved.The degradation of NMN and NAM in the bioconversion system was further inhibited after systematic modification of the related metabolic pathways.Finally,by incorporating two more copies of the key enzyme NAMPT and one copy of pyrophosphatase involved in inorganic pyrophosphate hydrolysis into the genome of the biocatalyst,the titer of NMN reached 12.2 g/L,with a molar conversion ratio of 98.7%from nicotinamide.This result provides an efficient and economical approach for producing NMN on an industrial scale.展开更多
The rapid development of science and technology calls for an urgent need to systemize the studies in a certain field on the basis of a common target. A case in point was the research and development of atomic bombs th...The rapid development of science and technology calls for an urgent need to systemize the studies in a certain field on the basis of a common target. A case in point was the research and development of atomic bombs that was based on the latest development in many fields.展开更多
Chlorogenic acid(CGA)is a valuable phenolic acid with various pharmaceutical functions.In our previous study,de novo synthesis of CGA in Saccharomyces cerevisiae was achieved.However,its yield required improvement bef...Chlorogenic acid(CGA)is a valuable phenolic acid with various pharmaceutical functions.In our previous study,de novo synthesis of CGA in Saccharomyces cerevisiae was achieved.However,its yield required improvement before large scale production.In this study,systematic metabolic engineering strategy was used to reconstruct chassis cell S.cerevisiae YC0707 to enhance its CGA yield from glucose.To balance the supply of phospho-enolpyruvate(PEP)and erythrose 4-phosphate(E4P),ZWF1(encoding glucose-6-phosphate dehydrogenase)and GND1(encoding 6-phosphogluconate dehydrogenase)were overexpressed by strong promoter PTEF1 swapping,thereby strengthening the pentose phosphate pathway.The mutant of phosphofructokinase(PFK2S718D)was further introduced to weaken the glycolytic pathway.Then,the p-coumaric acid synthesis capacity was enhanced by employing tyrosine ammonia lyase from Rhodotorula glutinis(RgTAL),ΔHAM1,andΔYJL028W.Fusion expression of AtC4H(cinnamate-4-hydroxylase)and At4CL1(4-coumarate CoA ligase 1),together with CsHQT(hydroxycinnamoyl CoA quinate transferase)and AtC3′H(p-coumaroyl shikimate 3-hydroxylase),improved biosynthetic flux to CGA.Subsequently,the microenvironment of P450 enzymes was improved by overexpressing INO2(a transcription factor for lipid biosynthesis)and removal of heme oxygenase gene HMX1.Furthermore,screening potential transporters to facilitate CGA accumulation.Finally,we optimized the fermentation condi-tions.Using these strategies,CGA titers increased from 234.8 mg/L to 837.2 mg/L in shake flasks and reached 1.62 g/L in a 5-L bioreactor,representing the highest report in S.cerevisiae and providing new insights for CGA production.展开更多
基金supported by grant from the National Natural Science Foundation of China(Grant No.22377012).
文摘β-nicotinamide mononucleotide(β-NMN)is a bioactive nucleotide that indirectly affects various cellular metabolic pathways,and has tremendous potential in the food,pharmaceutical,and cosmetics industries.In the present study,a multi-enzymatic cascade biocatalyst for the efficient synthesis ofβ-NMN was established.First,the optimal ribokinase gene was selected to construct a biocatalyst in a lower-endotoxin-level Escherichia coli strain to produce NMN from D-ribose.After fine-tuning the gene expression of the three cascade enzymes(RK/PRS/NAMPT),the production of NMN improved to 3.14 g/L.Next,the functions of membrane transporters that import NAM and export NMN were verified for NMN biosynthesis.Through the introduction of an ATP regeneration system,a drastic improvement in NMN biosynthesis at a yield of 8.74 g/L was achieved.The degradation of NMN and NAM in the bioconversion system was further inhibited after systematic modification of the related metabolic pathways.Finally,by incorporating two more copies of the key enzyme NAMPT and one copy of pyrophosphatase involved in inorganic pyrophosphate hydrolysis into the genome of the biocatalyst,the titer of NMN reached 12.2 g/L,with a molar conversion ratio of 98.7%from nicotinamide.This result provides an efficient and economical approach for producing NMN on an industrial scale.
文摘The rapid development of science and technology calls for an urgent need to systemize the studies in a certain field on the basis of a common target. A case in point was the research and development of atomic bombs that was based on the latest development in many fields.
基金supported by National Key Research and Development Program of China(2021YFA0909500)the National Natural Science Foundation of China(no.31970104).
文摘Chlorogenic acid(CGA)is a valuable phenolic acid with various pharmaceutical functions.In our previous study,de novo synthesis of CGA in Saccharomyces cerevisiae was achieved.However,its yield required improvement before large scale production.In this study,systematic metabolic engineering strategy was used to reconstruct chassis cell S.cerevisiae YC0707 to enhance its CGA yield from glucose.To balance the supply of phospho-enolpyruvate(PEP)and erythrose 4-phosphate(E4P),ZWF1(encoding glucose-6-phosphate dehydrogenase)and GND1(encoding 6-phosphogluconate dehydrogenase)were overexpressed by strong promoter PTEF1 swapping,thereby strengthening the pentose phosphate pathway.The mutant of phosphofructokinase(PFK2S718D)was further introduced to weaken the glycolytic pathway.Then,the p-coumaric acid synthesis capacity was enhanced by employing tyrosine ammonia lyase from Rhodotorula glutinis(RgTAL),ΔHAM1,andΔYJL028W.Fusion expression of AtC4H(cinnamate-4-hydroxylase)and At4CL1(4-coumarate CoA ligase 1),together with CsHQT(hydroxycinnamoyl CoA quinate transferase)and AtC3′H(p-coumaroyl shikimate 3-hydroxylase),improved biosynthetic flux to CGA.Subsequently,the microenvironment of P450 enzymes was improved by overexpressing INO2(a transcription factor for lipid biosynthesis)and removal of heme oxygenase gene HMX1.Furthermore,screening potential transporters to facilitate CGA accumulation.Finally,we optimized the fermentation condi-tions.Using these strategies,CGA titers increased from 234.8 mg/L to 837.2 mg/L in shake flasks and reached 1.62 g/L in a 5-L bioreactor,representing the highest report in S.cerevisiae and providing new insights for CGA production.
