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

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-DCT-biosynthetic 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.

由金霉素链霉菌（Streptomyces aureofaciens）产生的次级代谢产物（secondary metabolite）6-去甲基氯四环素（6-demethylchlortetracycline，6-DCT）可用作半合成四环素类药物的前体。产生6-DCT的菌株同时会合成类黑色素色素（melanin-like pigment，MP）。本研究以金霉素链霉菌NRRL 3203为对象，探究了MP合成与6-DCT合成之间的相关性。研究发现，磷酸盐或铵离子会同时抑制MP与6-DCT的合成，这表明二者的合成过程受同一套调控因子的调控。通过基因替换（gene replacement）技术，从6-DCT生产突变株NRRL 3203中获得了10株产氯四环素的重组菌株。所有重组菌株均仅合成氯四环素而不产生MP，这提示MP的合成源于6-甲基化步骤的缺陷，同时表明聚酮九酮酰胺（polyketide nonaketideamide）是同时通往MP与6-DCT的共同中间产物。为进一步验证MP是否通过6-DCT生物合成途径合成，研究人员从6-DCT生产菌株中筛选得到了6-DCT生物合成缺陷突变株。其中部分突变株仍可合成MP，而另一些突变株——包括编码脱水四环素氧化酶（anhydrotetracycline oxygenase，该酶催化该途径倒数第二步反应）的基因发生突变的菌株——则既无法合成6-DCT也无法产生MP。通过将对应的6-DCT生物合成基因进行整合转化（integrative transformation），可同时恢复6-DCT与MP的合成能力，这说明部分6-DCT生物合成酶对于MP的合成是不可或缺的。上述研究结果表明，6-甲基化步骤的缺陷会导致碳通量（carbon flux）从6-DCT生物合成途径中的特定中间产物转向分流途径（shunt pathway），最终引发MP的合成。