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 risk of P leaching from topsoil based on the change‐point estimated via a split‐line model between Olsen P and leachable P extracted by 0.01 M CaCl2 has been reported. However, little information is available for the assessment of P leaching from soil profiles. In this study, samples were collected at three depth profiles (0–20 cm, topsoil; 20–40 cm, subsoil; 40–60 cm, third‐layer soil) at each of 74 sites under agriculture and forest in an agroforestry area. A cascade extraction method was proposed to determine the leachable P in the subsoil, extracted by the topsoil extraction solution; a similar extracted process was followed in the third‐layer soil, and in the topsoil, it was still extracted by 0.01 M CaCl2. A positive linear correlation was found between the subsoil leachable P extracted by the topsoil extraction solution and the accumulated P obtained from the subsoil leached by topsoil leachates, and so on. Therefore, the cascade extraction method for determining leachable P from topsoils and underlying soils could be valuable for predicting the potential of P leaching from soil profiles. Approximately 81, 73, and 73% of the agricultural sampling sites were at or above the change‐points for the soil depths of 0 to 20, 20 to 40, and 40 to 60 cm (30.4, 32.9, and 18.2 mg kg−1 respectively); these values were higher than those for the forest site, implying a high risk of P leaching from agricultural soil profiles in the study area. Core Ideas A cascade extraction method for evaluating P leaching from soil profiles was proposed. The correlation of soil leachable P and accumulated P in soil leachates was confirmed. The risk of P leaching from soil profiles in an agroforestry area was calculated.

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