nystatin-a1 and tetramycin

Polyene antibiotics, including amphotericin, nystatin, pimaricin, and tetramycin, are important antifungal agents. Increasing the production of polyenes and generation of their improved analogues based on the biosynthetic pathway engineering has aroused wide concern in application researches. Herein, tetramycin and nystatin, both of which share most of acyl-CoA precursors, are produced by Streptomyces hygrospinosus var. beijingensis CGMCC 4.1123. Thus, the intracellular malonyl-CoA is found to be insufficient for PKSs (polyketide synthases) extension of tetramycin by quantitative analysis in this wild-type strain. To circumvent this problem and increase tetramycin titer, the acyl-CoA competing biosynthetic gene cluster (BGC) of nystatin was disrupted, and the biosynthetic genes of malonyl-CoA from S. coelicolor M145 were integrated and overexpressed in nys-disruption mutant strain (SY02). Moreover, in order to specifically accumulate tetramycin B from A, two copies of tetrK and a copy of tetrF were introduced, resulting in elevating tetramycin B fermentration titer by 122% to 865 ± 8 mg/L than the wild type. In this optimized strain, a new tetramycin derivative, 12-decarboxy-12-methyl tetramycin B, was generated with a titer of 371 ± 26 mg/L through inactivation of a P450 monooxygenase gene tetrG. Compared with tetramycin B, the new compound exhibited higher antifungal activity against Saccharomyces cerevisiae and Rhodotorula glutinis, but lower hemolytic toxicity to erythrocyte. This research provided a good example of employing biosynthetic engineering strategies for fermentation titer improvement of polyene and development of the derivatives for medicinal applications.

Topics: Animals; Antifungal Agents; Biosynthetic Pathways; Erythrocytes; Fermentation; Hemolysis; Horses; Macrolides; Metabolic Engineering; Multigene Family; Nystatin; Rhodotorula; Saccharomyces cerevisiae; Streptomyces

A putative regulatory gene ttmRIV located in the tetramycin biosynthetic gene cluster was found in Streptomyces ahygroscopicus. In-frame deletion of ttmRIV led to abolishment of tetramycin and significant enhancement of nystatin A1, whose production reached 2.1-fold of the H42 parental strain. Gene complementation by an integrative plasmid carrying ttmRIV restored tetramycin biosynthesis revealed that ttmRIV was indispensable to tetramycin biosynthesis. Gene expression analysis of the H42 strain and its mutant strain ΔttmRIV via reverse transcriptase-PCR of the tetramycin gene cluster demonstrated that the expression levels of most biosynthetic genes were reduced in ΔttmRIV. Results of electrophoretic mobility shift assays showed that TtmRIV bound the putative promoters of several genes in the tetramycin pathway. Thus, TtmRIV is a pathway-specific positive regulator activating the transcription of the tetramycin gene cluster in S. ahygroscopicus. Providing an additional copy of ttmRIV under the control of the ermEp* promoter in the H42 strain boosted tetramycin A production to 3.3-fold.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; Base Sequence; Bioreactors; Biosynthetic Pathways; Consensus Sequence; Gene Expression; Gene Expression Regulation, Bacterial; Genes, Bacterial; Macrolides; Molecular Sequence Data; Multigene Family; Nystatin; Plasmids; Promoter Regions, Genetic; Streptomyces