A three—component enzyme system that catalyzes in vivo the oxidation of CH_4 to CH_3OH has been purified with high specific activity from an unusual type I methanotroph through the use of stabilizing reagents.
Methanotrophic bacteria are currently used industrially for the bioconversion of methane-rich natural gas and anaerobic digestion-derived biogas to valuable products.These bacteria may also serve to mitigate the negat...Methanotrophic bacteria are currently used industrially for the bioconversion of methane-rich natural gas and anaerobic digestion-derived biogas to valuable products.These bacteria may also serve to mitigate the negative effects of climate change by capturing atmospheric greenhouse gases.Several genetic tools have previously been developed for genetic and metabolic engineering of methanotrophs.However,the available tools for use in methanotrophs are significantly underdeveloped compared to many other industrially relevant bacteria,which hinders genetic and metabolic engineering of these biocatalysts.As such,expansion of the methanotroph genetic toolbox is needed to further our understanding of methanotrophy and develop biotechnologies that leverage these unique microbes for mitigation and conversion of methane to valuable products.Here,we determined the copy number of three broad-host-range plasmids in Methylococcus capsulatus Bath and Methylosinus trichosporium OB3b,representing phylogenetically diverse Gammaproteobacterial and Alphaproteobacterial methanotrophs,respectively.Further,we show that the commonly used synthetic Anderson series promoters are functional and exhibit similar relative activity in M.capsulatus and M.trichosporium OB3b,but the synthetic series had limited range.Thus,we mutagenized the native M.capsulatus particulate methane monooxygenase promoter and identified variants with activity that expand the activity range of synthetic,constitutive promoters functional not only in M.capsulatus,but also in Escherichia coli.Collectively,the tools developed here advance the methanotroph genetic engineering toolbox and represent additional synthetic genetic parts that may have broad applicability in Pseudomonadota bacteria.展开更多
The coupled effects of nitrogen source and methane monooxygenase(MMO) on the growth and poly-β-hydroxybutyrate(PHB) accumulation capacity of methanotrophs were explored.The ammonia-supplied methanotrophs expressi...The coupled effects of nitrogen source and methane monooxygenase(MMO) on the growth and poly-β-hydroxybutyrate(PHB) accumulation capacity of methanotrophs were explored.The ammonia-supplied methanotrophs expressing soluble MMO(s MMO) grew at the highest rate, while N2-fixing bacteria expressing particulate MMO(p MMO) grew at the lowest rate. Further study showed that more hydroxylamine and nitrite was formed by ammonia-supplied bacteria containing p MMO, which might cause their slightly lower growth rate. The highest PHB content(51.0%) was obtained under nitrogen-limiting conditions with the inoculation of nitrate-supplied bacteria containing p MMO. Ammoniasupplied bacteria also accumulated a higher content of PHB(45.2%) with the expression of p MMO, while N2-fixing bacteria containing p MMO only showed low PHB production capacity(32.1%). The maximal PHB contents of bacteria expressing s MMO were low, with no significant change under different nitrogen source conditions. The low MMO activity,low cell growth rate and low PHB production capacity of methanotrophs continuously cultivated with N2 with the expression of p MMO were greatly improved in the cyclic NO3-N2 cultivation regime, indicating that long-term deficiency of nitrogen sources was detrimental to the activity of methanotrophs expressing pMMO.展开更多
Four chiral μ phenolato dinuclear (VO) 2, Cr 2, Mn 2, and Fe 2 complexes with a phenolic ligand bearing two L phenylanalines as complexing arms have been synthesized and characterized. Asymmetric epoxidations of styr...Four chiral μ phenolato dinuclear (VO) 2, Cr 2, Mn 2, and Fe 2 complexes with a phenolic ligand bearing two L phenylanalines as complexing arms have been synthesized and characterized. Asymmetric epoxidations of styrene with these complexes as chiral models of methane monooxygenase (MMO) and iodosylbenzene as an oxygen atom donor have been studied. The results reveal that the Mn 2 complex catalyzes asymmetric epoxidation of styrene with the formation of R (+) styrene oxide in 42.8% e.e. Contrarily, the (VO) 2 analogue complex gives S (-) styrene oxide in 19.2% e.e. EPR investigations suggest that the catalyzed epoxidations with the Mn 2, Cr 2 and Fe 2 complexes proceed via high valent oxometal intermediates; while in the case of the (VO) 2 complex as catalyst, the epoxidation proceeds by a pathway that involves a complex of metal iodine ligand. The catalytic activities of the complexes decrease in a sequence of Mn 2>Cr 2>(VO) 2>Fe 2.展开更多
The activation of dioxygen and incorporation into hydrocarbons have been achieved under mild conditions by a methane monooxygenase (MMO)-like system using a dinuclear iron complex [Fe2Dhist(OAc)2] BPh4 ·3H2O as t...The activation of dioxygen and incorporation into hydrocarbons have been achieved under mild conditions by a methane monooxygenase (MMO)-like system using a dinuclear iron complex [Fe2Dhist(OAc)2] BPh4 ·3H2O as the model complex, zinc powder as the electron donor, HOAc as the proton source and methylviologen as the electron transfer agent. The results show that styrene is oxygenated predominantly to styrene oxide (1 396 mol/100 mol of the Fe2 complex), benzaldehyde (16160) and acetophenone (986), and cyclohexane to cyclohexanol (9370) and cyclo-hexanone (2670). EPR studies indicate that the hypervalent ironoxo spiecs FeⅣFeⅣ=O, derived from FeⅢFeⅢ core via reduction, O2-binding and protonation, is the active intermediate which inserts the activated oxygen atom into C=C or C-H bond giving each product. The system closely resembles MMO and its close relative hemerythrin in the aspects of reaction phenomena, EPR characteristics and product distributions. The Mn2 analog cmplex、 Fe-Zn het-erodinuclear complex and mononuclear iron complex show no catalytic activity, indicating that dinuclear iron core is in-despansable to catalytic activity.展开更多
文摘A three—component enzyme system that catalyzes in vivo the oxidation of CH_4 to CH_3OH has been purified with high specific activity from an unusual type I methanotroph through the use of stabilizing reagents.
基金This work was supported by National Science Foundation MCB award#2225776.
文摘Methanotrophic bacteria are currently used industrially for the bioconversion of methane-rich natural gas and anaerobic digestion-derived biogas to valuable products.These bacteria may also serve to mitigate the negative effects of climate change by capturing atmospheric greenhouse gases.Several genetic tools have previously been developed for genetic and metabolic engineering of methanotrophs.However,the available tools for use in methanotrophs are significantly underdeveloped compared to many other industrially relevant bacteria,which hinders genetic and metabolic engineering of these biocatalysts.As such,expansion of the methanotroph genetic toolbox is needed to further our understanding of methanotrophy and develop biotechnologies that leverage these unique microbes for mitigation and conversion of methane to valuable products.Here,we determined the copy number of three broad-host-range plasmids in Methylococcus capsulatus Bath and Methylosinus trichosporium OB3b,representing phylogenetically diverse Gammaproteobacterial and Alphaproteobacterial methanotrophs,respectively.Further,we show that the commonly used synthetic Anderson series promoters are functional and exhibit similar relative activity in M.capsulatus and M.trichosporium OB3b,but the synthetic series had limited range.Thus,we mutagenized the native M.capsulatus particulate methane monooxygenase promoter and identified variants with activity that expand the activity range of synthetic,constitutive promoters functional not only in M.capsulatus,but also in Escherichia coli.Collectively,the tools developed here advance the methanotroph genetic engineering toolbox and represent additional synthetic genetic parts that may have broad applicability in Pseudomonadota bacteria.
基金supported by the National Key Scientific and Technology Project for Water Pollution Treatment of China(No.2012ZX07202006)the National Natural Science Foundation of China(No.21477014)
文摘The coupled effects of nitrogen source and methane monooxygenase(MMO) on the growth and poly-β-hydroxybutyrate(PHB) accumulation capacity of methanotrophs were explored.The ammonia-supplied methanotrophs expressing soluble MMO(s MMO) grew at the highest rate, while N2-fixing bacteria expressing particulate MMO(p MMO) grew at the lowest rate. Further study showed that more hydroxylamine and nitrite was formed by ammonia-supplied bacteria containing p MMO, which might cause their slightly lower growth rate. The highest PHB content(51.0%) was obtained under nitrogen-limiting conditions with the inoculation of nitrate-supplied bacteria containing p MMO. Ammoniasupplied bacteria also accumulated a higher content of PHB(45.2%) with the expression of p MMO, while N2-fixing bacteria containing p MMO only showed low PHB production capacity(32.1%). The maximal PHB contents of bacteria expressing s MMO were low, with no significant change under different nitrogen source conditions. The low MMO activity,low cell growth rate and low PHB production capacity of methanotrophs continuously cultivated with N2 with the expression of p MMO were greatly improved in the cyclic NO3-N2 cultivation regime, indicating that long-term deficiency of nitrogen sources was detrimental to the activity of methanotrophs expressing pMMO.
文摘Four chiral μ phenolato dinuclear (VO) 2, Cr 2, Mn 2, and Fe 2 complexes with a phenolic ligand bearing two L phenylanalines as complexing arms have been synthesized and characterized. Asymmetric epoxidations of styrene with these complexes as chiral models of methane monooxygenase (MMO) and iodosylbenzene as an oxygen atom donor have been studied. The results reveal that the Mn 2 complex catalyzes asymmetric epoxidation of styrene with the formation of R (+) styrene oxide in 42.8% e.e. Contrarily, the (VO) 2 analogue complex gives S (-) styrene oxide in 19.2% e.e. EPR investigations suggest that the catalyzed epoxidations with the Mn 2, Cr 2 and Fe 2 complexes proceed via high valent oxometal intermediates; while in the case of the (VO) 2 complex as catalyst, the epoxidation proceeds by a pathway that involves a complex of metal iodine ligand. The catalytic activities of the complexes decrease in a sequence of Mn 2>Cr 2>(VO) 2>Fe 2.
文摘The activation of dioxygen and incorporation into hydrocarbons have been achieved under mild conditions by a methane monooxygenase (MMO)-like system using a dinuclear iron complex [Fe2Dhist(OAc)2] BPh4 ·3H2O as the model complex, zinc powder as the electron donor, HOAc as the proton source and methylviologen as the electron transfer agent. The results show that styrene is oxygenated predominantly to styrene oxide (1 396 mol/100 mol of the Fe2 complex), benzaldehyde (16160) and acetophenone (986), and cyclohexane to cyclohexanol (9370) and cyclo-hexanone (2670). EPR studies indicate that the hypervalent ironoxo spiecs FeⅣFeⅣ=O, derived from FeⅢFeⅢ core via reduction, O2-binding and protonation, is the active intermediate which inserts the activated oxygen atom into C=C or C-H bond giving each product. The system closely resembles MMO and its close relative hemerythrin in the aspects of reaction phenomena, EPR characteristics and product distributions. The Mn2 analog cmplex、 Fe-Zn het-erodinuclear complex and mononuclear iron complex show no catalytic activity, indicating that dinuclear iron core is in-despansable to catalytic activity.