Although cytochrome P450 enzymes are the most versatile biocatalysts in nature,there is insufficient comprehension of the molecular mechanism underlying their functional innovation process.Here,by combining ancestral ...Although cytochrome P450 enzymes are the most versatile biocatalysts in nature,there is insufficient comprehension of the molecular mechanism underlying their functional innovation process.Here,by combining ancestral sequence reconstruction,reverse mutation assay,and progressive forward accumulation,we identified 5 founder residues in the catalytic pocket of flavone 6-hydroxylase(F6H)and proposed a"3-point fixation"model to elucidate the functional innovation mechanisms of P450s in nature.According to this design principle of catalytic pocket,we further developed a de novo diffusion model(P450Diffusion)to generate artificial P450s.Ultimately,among the 17 non-natural P450s we generated,10 designs exhibited significant F6H activity and 6 exhibited a 1.3-to 3.5-fold increase in catalytic capacity compared to the natural CYP706X1.This work not only explores the design principle of catalytic pockets of P450s,but also provides an insight into the artificial design of P450 enzymes with desired functions.展开更多
Taxus,commonly known as yew,is a well-known gymnosperm with great ornamental and medicinal value.In this study,by assembling a chromosome-level genome of the Himalayan yew(Taxus wallichiana)with 10.9 Gb in 12 chromoso...Taxus,commonly known as yew,is a well-known gymnosperm with great ornamental and medicinal value.In this study,by assembling a chromosome-level genome of the Himalayan yew(Taxus wallichiana)with 10.9 Gb in 12 chromosomes,we revealed that tandem duplication acts as the driving force of gene family evolution in the yew genome,resulting in the main genes for paclitaxel biosynthesis,i.e.those encoding the taxadiene synthase,P450s,and transferases,being clustered on the same chromosome.The tandem duplication may also provide genetic resources for the nature to sculpt the core structure of taxoids at different positions and subsequently establish the complex pathway of paclitaxel by neofunctionalization.Furthermore,we confirmed that there are two genes in the cluster encoding isoenzymes of a known enzyme in the paclitaxel biosynthetic pathway.The reference genome of the Himalayan yew will serve as a platform for decoding the complete biosynthetic pathway of paclitaxel and understanding the chemodi-versity of taxoids in gymnosperms.展开更多
Phloretin is an important skin-lightening and depigmenting agent from the peel of apples. Although de novo production of phloretin has been realized in microbes using the natural pathway from plants, the efficiency of...Phloretin is an important skin-lightening and depigmenting agent from the peel of apples. Although de novo production of phloretin has been realized in microbes using the natural pathway from plants, the efficiency of phloretin production is still not enough for industrial application. Here, we established an artificial pathway in the yeast to produce phloretin via assembling two genes of p-coumaroyl-CoA ligase(4CL) and chalcone synthase(CHS). CHS is a key enzyme which conventionally condenses a CoA-tethered starter with three molecules of malonyl-CoA to form the backbone of flavonoids. However, there was 33% of byproduct generated via CHS by condensing two molecules of malonyl-CoA during the fermentation process. Hence, we introduced a more efficient CHS and improved the supply of malonyl-CoA through two pathways;the by-product ratio was decreased from 33% to 17% and the production of phloretin was improved from 48 to 83.2 mg L^(-1). Finally, a fed-batch fermentation process was optimized and the production of phloretin reached 619.5 mg L^(-1), which was 14-fold higher than that of the previous studies. Our work established a platform for the biosynthesis of phloretin from the low-cost raw material 3-(4-hydroxyphenyl) propanoic acid and also illustrated the potential for industrial scale bio-manufacturing of phloretin.展开更多
基金funding from the National Key R&D Program of China(grant no.2019YFA0905700 and 2021YFC2103500)the National Natural Science Foundation of China(no.32371499)+3 种基金the China Postdoctoral Science Foundation(grant no.2019M661032)the National Natural Science Foundation of China(NSFC,grant nos.31901026 and 32171418)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(nos.TSBICIP-KJGG-002-02 and TSBICIP-CXRC-015)the Tianjin Science Fund for Distinguished Young Scholars(no.18JCJQJC48300).
文摘Although cytochrome P450 enzymes are the most versatile biocatalysts in nature,there is insufficient comprehension of the molecular mechanism underlying their functional innovation process.Here,by combining ancestral sequence reconstruction,reverse mutation assay,and progressive forward accumulation,we identified 5 founder residues in the catalytic pocket of flavone 6-hydroxylase(F6H)and proposed a"3-point fixation"model to elucidate the functional innovation mechanisms of P450s in nature.According to this design principle of catalytic pocket,we further developed a de novo diffusion model(P450Diffusion)to generate artificial P450s.Ultimately,among the 17 non-natural P450s we generated,10 designs exhibited significant F6H activity and 6 exhibited a 1.3-to 3.5-fold increase in catalytic capacity compared to the natural CYP706X1.This work not only explores the design principle of catalytic pockets of P450s,but also provides an insight into the artificial design of P450 enzymes with desired functions.
基金the National Key R&D Program of China(2020YFA0908000)National Science Fund for Excellent Young Scholars(31922047)+1 种基金Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(No.TSBICIP-KJGG-002)the China Postdoctoral Science Foundation(No.2019M661032)。
文摘Taxus,commonly known as yew,is a well-known gymnosperm with great ornamental and medicinal value.In this study,by assembling a chromosome-level genome of the Himalayan yew(Taxus wallichiana)with 10.9 Gb in 12 chromosomes,we revealed that tandem duplication acts as the driving force of gene family evolution in the yew genome,resulting in the main genes for paclitaxel biosynthesis,i.e.those encoding the taxadiene synthase,P450s,and transferases,being clustered on the same chromosome.The tandem duplication may also provide genetic resources for the nature to sculpt the core structure of taxoids at different positions and subsequently establish the complex pathway of paclitaxel by neofunctionalization.Furthermore,we confirmed that there are two genes in the cluster encoding isoenzymes of a known enzyme in the paclitaxel biosynthetic pathway.The reference genome of the Himalayan yew will serve as a platform for decoding the complete biosynthetic pathway of paclitaxel and understanding the chemodi-versity of taxoids in gymnosperms.
基金financially supported by Talents Team Construction Fund of Northwestern Polytechnical University (NWPU)the National Natural Science Foundation of China (31701722)+1 种基金the China Postdoctoral Science Foundation (2017M620471)the National Natural Science Foundation of China (31901026)。
文摘Phloretin is an important skin-lightening and depigmenting agent from the peel of apples. Although de novo production of phloretin has been realized in microbes using the natural pathway from plants, the efficiency of phloretin production is still not enough for industrial application. Here, we established an artificial pathway in the yeast to produce phloretin via assembling two genes of p-coumaroyl-CoA ligase(4CL) and chalcone synthase(CHS). CHS is a key enzyme which conventionally condenses a CoA-tethered starter with three molecules of malonyl-CoA to form the backbone of flavonoids. However, there was 33% of byproduct generated via CHS by condensing two molecules of malonyl-CoA during the fermentation process. Hence, we introduced a more efficient CHS and improved the supply of malonyl-CoA through two pathways;the by-product ratio was decreased from 33% to 17% and the production of phloretin was improved from 48 to 83.2 mg L^(-1). Finally, a fed-batch fermentation process was optimized and the production of phloretin reached 619.5 mg L^(-1), which was 14-fold higher than that of the previous studies. Our work established a platform for the biosynthesis of phloretin from the low-cost raw material 3-(4-hydroxyphenyl) propanoic acid and also illustrated the potential for industrial scale bio-manufacturing of phloretin.
