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Journal of Bacteriology, September 2008, p. 6014-6025, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.00533-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cloning and Characterization of the Tetrocarcin A Gene Cluster from Micromonospora chalcea NRRL 11289 Reveals a Highly Conserved Strategy for Tetronate Biosynthesis in Spirotetronate Antibiotics{triangledown} ,{dagger}

Jie Fang, Yiping Zhang, Lijuan Huang, Xinying Jia, Qi Zhang, Xu Zhang, Gongli Tang, and Wen Liu*

State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Road, Shanghai, 200032, China

Received 18 April 2008/ Accepted 19 June 2008

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 {Delta}chlD3 and {Delta}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.

* Corresponding author. Mailing address: Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Rd., Shanghai, 200032, China. Phone: 86-21-54925111. Fax: 86-21-64166128. E-mail: wliu{at}mail.sioc.ac.cn

{triangledown} Published ahead of print on 27 June 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, September 2008, p. 6014-6025, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.00533-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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