| ECO-0501is a new kind of polyketides found from vancomycin producer Amycolatopsis orientalis. It defines a new structural class of antibiotic octaenoic acid glucuronide, which has shown activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). ECO-0501exerts its action through very rapid disruption of the bacterial cell membrane, resulting in a prompt and global effect on macromolecule synthesis. Because of the strong bacteridicial activity of ECO-0501and its low toxicity, preclinical development started on this prospective antibiotic candidate.We investigated the functions of key genes involved in ECO-0501biosynthesis and improved production traits of Amycolatopsis orientalis dA9at the genetic level. The detailed work was introduced as follows:1. Screening the ECO-0501biosynthesis cluster from cosmid library of Amycolatopsis orientalis.We screened cosmid library of Amycolatopsis orientalis by colony hybridization. Four positive cosmid clones covering the ECO-0501biosynthetic locus were identified by PCR and sequenced, including NL5B, NL9-3-1, NL3-1C and NL3-3A.2. Studies on function of regulatory gene ECO-orf4The Amycolatopsis orientalis ECO-orf4gene from the ECO-0501biosynthesis cluster was analyzed, and its deduced protein (ECO-orf4) was found to have amino acid sequence homology with large ATP-binding regulators of the LuxR (LAL) family regulators. With the database analysis, ECO-orf4could be a new member of LAL proteins and function as positive pathway-specific regulator of the corresponding biosynthetic cluster. Deletion of the corresponding gene (ECO-orf4) resulted in complete loss of ECO-0501production. Complementation by one copy of intact ECO-orf4restored the polyene biosynthesis, which demonstrated that ECO-orf4is required for ECO-0501biosynthesis. The effect of overexpression ECO-orf4on ECO-0501production indicated that it is a positive regulatory gene, while LAL-family regulators have an upper concentration limit, over which they have a negative effect on the antibiotic production. Gene expression analysis by reverse transcription PCR of the ECO-0501gene cluster showed that the transcription of ECO-orf4correlates with that of genes involved in polyketide biosynthesis, but action site of ECO-orf4can not be mapped precisely. 3. Studies on function of type Ⅱ thioesterase gene ECO-orf27ECO-orf27associated with the cluster of ECO-0501from Amycolatopsis orientalis is deduced to encode a thioesterase. The discrete thioesterase shows homology with other authentic type Ⅱ thioesterase, including conserved motifs and residues involved in catalysis. Disruption of ECO-orf27reduced the polyene production by95%. Complementation by one copy of intact ECO-orf27restored the yield to its original level, which suggested that ECO-orf27is crucial for ECO-0501biosynthesis. ECO-TE I, integral thioesterase gene from ECO-0501polyketide synthases, can not complement ECO-orf27deficient mutant, which distinguished ECO-orf27from TE I. PikAV could work as ECO-orf27in Amycolatopsis orientalis, which indicated that the two thioesterases are equivalent in their catalytic function. Overexpression of ECO-orf27resulted in a slight increase in polyketide production, which provided an alternative approach for yield improvement.4. Construction of genetically engineered Amycolatopsis orientalis strain for the biosynthesis of N-demethyl ECO-0501The method for gene disruption in Amycolatopsis orientalis was modified. We introduced an I-SceI site when the first single-crossover homologous integration took place. Expression of the I-SceI gene in Amycolatopsis orientalis resulted in promotion of homologous recombination which is18times higher than natural occurrence of homologous recombination. With this optimal approach, deletion of ECO-orf5from the genome of A. orientalis dA9resulted in A. orientalis dA9△ECO-orf5, which showed3.6times higher production of N-demethyl ECO-0501than that of A. orientalis dA9.In short, we reported the functions of ECO-orf4and ECO-orf27in A. orientalis for the first time and constructed a genetically engineered strain for the biosynthesis of N-demethyl ECO-0501. |
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