Tetrocarcin A (TCA), produced by Micromonospora chalcea NRRL 11289, is a spirotetronate antibiotic with potent antitumor activity and versatile modes of action. In this study, the biosynthetic gene cluster of TCA was cloned and localized to a 108-kb contiguous DNA region. In silico sequence analysis revealed 36 putative genes that constitute this cluster (including 11 for unusual sugar biosynthesis, 13 for aglycone formation, and 4 for glycosylations) and allowed us to propose the biosynthetic pathway of TCA. The formation of D-tetronitrose, L-amicetose, and L-digitoxose may begin with D-glucose-1-phosphate, share early enzymatic steps, and branch into different pathways by competitive actions of specific enzymes. Tetronolide biosynthesis involves the incorporation of a 3-C unit with a polyketide intermediate to form the characteristic spirotetronate moiety and trans-decalin system. Further substitution of tetronolide with five deoxysugars (one being a deoxynitrosugar) was likely due to the activities of four glycosyltransferases. In vitro characterization of the first enzymatic step by utilization of 1,3-biphosphoglycerate as the substrate and in vivo cross-complementation of the bifunctional fused gene tcaD3 (with the functions of chlD3 and chlD4) to ⌬chlD3 and ⌬chlD4 in chlorothricin biosynthesis supported the highly conserved tetronate biosynthetic strategy in the spirotetronate family. Deletion of a large DNA fragment encoding polyketide synthases resulted in a non-TCA-producing strain, providing a clear background for the identification of novel analogs. These findings provide insights into spirotetronate biosynthesis and demonstrate that combinatorial-biosynthesis methods can be applied to the TCA biosynthetic machinery to generate structural diversity.
The lack of a standardized database of eddy covariance observations has been an obstacle for data‐driven estimation of terrestrial CO2 fluxes in Asia. In this study, we developed such a standardized database using 54 sites from various databases by applying consistent postprocessing for data‐driven estimation of gross primary productivity (GPP) and net ecosystem CO2 exchange (NEE). Data‐driven estimation was conducted by using a machine learning algorithm: support vector regression (SVR), with remote sensing data for 2000 to 2015 period. Site‐level evaluation of the estimated CO2 fluxes shows that although performance varies in different vegetation and climate classifications, GPP and NEE at 8 days are reproduced (e.g., r2 = 0.73 and 0.42 for 8 day GPP and NEE). Evaluation of spatially estimated GPP with Global Ozone Monitoring Experiment 2 sensor‐based Sun‐induced chlorophyll fluorescence shows that monthly GPP variations at subcontinental scale were reproduced by SVR (r2 = 1.00, 0.94, 0.91, and 0.89 for Siberia, East Asia, South Asia, and Southeast Asia, respectively). Evaluation of spatially estimated NEE with net atmosphere‐land CO2 fluxes of Greenhouse Gases Observing Satellite (GOSAT) Level 4A product shows that monthly variations of these data were consistent in Siberia and East Asia; meanwhile, inconsistency was found in South Asia and Southeast Asia. Furthermore, differences in the land CO2 fluxes from SVR‐NEE and GOSAT Level 4A were partially explained by accounting for the differences in the definition of land CO2 fluxes. These data‐driven estimates can provide a new opportunity to assess CO2 fluxes in Asia and evaluate and constrain terrestrial ecosystem models.
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