Mechanism of the Incidental Production of a Melanin-Like Pigment during 6-Demethylchlortetracycline Production in Streptomyces aureofaciens

Abstract

The secondary metabolite 6-demethylchlortetracycline (6-DCT), which is produced by Streptomyces aureofaciens, is used as a precursor of semisynthetic tetracyclines. Strains that produce 6-DCT also produce a melanin-like pigment (MP). The correlation between MP production and 6-DCT production was investigated by using S. aureofaciens NRRL 3203. Production of both MP and 6-DCT was repressed by phosphate or ammonium ions, suggesting that syntheses of these compounds are controlled by the same regulators. Ten chlortetracycline-producing recombinants were derived from 6-DCT-producing mutant NRRL 3203 by gene replacement. All of the recombinants produced chlortetracycline but not MP, indicating that MP production is the results of a defect in the 6-methylation step and suggesting that the polyketide nonaketideamide is a common intermediate leading to MP as well as 6-DCT. To further examine the possibility that MP might be synthesized via the 6-DCT-biosynthetic pathway, mutants defective in 6-DCT biosynthesis were derived from a 6-DCT producer. Some of these mutants were able to produce MP, while others, including mutants with mutations in the gene encoding anhydrotetracycline oxygenase, an enzyme catalyzing the penultimate step in the pathway, produced neither 6-DCT nor MP. Production of 6-DCT and production of MP were restored simultaneously by integrative transformation with the corresponding 6-DCT-biosynthetic genes, indicating that some of 6-DCTbiosynthetic enzymes are indispensable for MP production. These findings suggest that a defect in the 6-methylation step results in redirection of carbon flux from a certain intermediate in the 6-DCT-biosynthetic pathway to a shunt pathway and results in MP production.

Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase

“…1) (9,37,50). The gene clusters responsible for oxytetracycline (compound 1; oxy and otc genes) and chlorotetracycline have been located on the genomes of Streptomyces rimosus (2,9) and Streptomyces aureofaciens (43), respectively. For the oxy (otc) cluster, gene sequences for the minimal PKS (28), a putative D-ring cyclase (46), and a downstream anhydrotetracycline (ATC)-oxygenase (45) have been deposited in GenBank.…”

Identification and Cloning of the Gene Involved in the Final Step of Chlortetracycline Biosynthesis inStreptomyces aureofaciens

“…S. aureofaciens HP77 (6-DMT producer; [chl]) derived from H-7591 3) was used in this study. Escherichia coli JM110 9) was used in the preparation of a gene bank of S. aureofaciens HP77 and in the isolation of the dam y To whom correspondence should be addressed.…”

“…Fax: +81-42-726-8330; E-mail: t.nakano@kyowa.co.jp Abbreviations: CTC, chlortetracycline; 6-DCT, 6-demethyl-7-chlortetracycline; DHDMT, 5a,11a-dehydro-6-demethyltetracycline; DMATC, 6-demethylanhydrotetracycline; 6-DMT, 6-demethyltetracycline; TB, tetramid blue; TCDH, tetracycline dehydrogenase and dcm plasmid DNAs that were used for the transformation of S. aureofaciens strains. Integrative vector pSE119 3) and E. coli-Streptomyces shuttle vector pSE101 10) were used for integrative transformation and plasmid transformation of S. aureofaciens respectively. Plasmid pBR322 11) was used to recover a recombinant DNA fragment that was integrated into the S. aureofaciens genome.…”

“…SK2 medium 12) was used for the growth of S. aureofaciens strains. For tetracycline production, SK2 and PK2 medium 3) were used as the seed and the production medium respectively. The culture conditions and procedures have been described previously.…”

“…During these biosynthetic processes, coordinate reactions by biosynthetic enzymes were required for the complete formation of CTC, because a disorder of the coordinated reactions, usually caused by a mutation, resulted in the generation of at least nineteen CTCrelated shunt compounds. [1][2][3] Among these CTC-related compounds, only four compounds, tetracycline, CTC, 6-demethylchlortetracycline (6-DCT), and 6-demethyltetracycline (6-DMT), have antibacterial activity. These four compounds were characterized by the presence of a single bond at the 5a,11a-position, in contrast to other shunt products, which have a double bond at this position.…”