Cell-free expression of NO synthase and P450 enzyme for the biosynthesis of an unnatural amino acid L-4-nitrotryptophan

Cell-free system has emerged as a powerful platform with a wide range of in vitro applications and recently has contributed to express metabolic pathways for biosynthesis.Here we report in vitro construction of a native biosynthetic pathway for L-4-nitrotryptophan(L-4-nitro-Trp)synthesis using an Escherichia coli-based cell-free protein synthesis(CFPS)system.Naturally,a nitric oxide(NO)synthase(TxtD)and a cytochrome P450 enzyme(TxtE)are responsible for synthesizing L-4-nitro-Trp,which serves as one substrate for the biosynthesis of a nonribosomal peptide herbicide thaxtomin A.Recombinant coexpression of TxtD and TxtE in a heterologous host like E.coli for L-4-nitro-Trp production has not been achieved so far due to the poor or insoluble expression of TxtD.Using CFPS,TxtD and TxtE were successfully expressed in vitro,enabling the formation of L-4-nitro-Trp.After optimization,the cell-free system was able to synthesize approximately 360μM L-4-nitro-Trp within 16 h.Overall,this work expands the application scope of CFPS for study and synthesis of nitro-containing compounds,which are important building blocks widely used in pharmaceuticals,agrochemicals,and industrial chemicals.

Contribution of Winter SSTA in the Tropical Eastern Pacific to Changes of Tropical Cyclone Precipitation over Southeast China

Tropical cyclone precipitation(TCP)accounts for 10%-40%of the boreal summer precipitation that occurs over Southeast China(SEC),causing flood disasters and serious damage.On the decadal scale,TCP increases significantly in SEC while TC frequency decreases in the western North Pacific(WNP)during 1980-2019.Therefore,variations in TCP and the corresponding physical mechanism are investigated in this study.First,an empirical statistical method is introduced to quantify the TCP amount based on accumulated cyclone energy(ACE)and TC frequency with the TCP anomaly decomposed into three items(rainfall frequency,rainfall intensity,and nonlinear item).ACE,as the integration of TC intensity and frequency,is a more effective index than TC frequency for depicting the characteristics of TCP because the contribution of rainfall frequency represented by ACE is higher than that of TC frequency.Then,the physical mechanism affecting the WNP TC activities and TCP in SEC are inspected.Positive sea surface temperature anomaly(SSTA)over the tropical eastern Pacific(TEP)in winter can trigger variations of air-sea interaction over the tropical Pacific,including low-level divergent winds,mid-tropospheric descent flows,high-level convergent winds coupled with negative anomalies of vorticity and humidity over the tropical western Pacific(TWP)in the next summer.These dynamic conditions provide unfavorable environments for TC activities in the WNP and constrain TCP in SEC.Furthermore,more significantly negative SSTA events in the TEP facilitate enhanced ACE along with positive relative vorticity,relative humidity,and upwelling vertical winds anomalies over the coast of SEC after 1998,which is a reasonable explanation for the increasing TCP in SEC.