Cloning And Functional Analysis Of The Genes In Azalomycin F_3a Biosynthesis

Azalomycins F, which show a broad range of anti-bacterial and anti-fungal activities, is a macrolides antibiotic produced by Streptomyces sp.211726that was separated from mangrove in Hainan. Toxicity experiments also showed that the twelve of compounds except Azalomycin Fsa producted by Streptomyces sp.211726had moderate cytotoxicity against human colon tumor cell HCT-116suggesting potential applications in human tumor disease treatmention. It is note worthy that it active against Fusarium oxysporum f. sp. Cubense, and show a good potential in chemical control of banana wilt disease. Although the biological activities of Azalomycins F series compounds are obtained, its biosynthetic mechanism has not been yet revealed. Therefore, the clarification of Azalomycins F biosynthetic mechanism will provide theoretical basis for directed modification and optimization by using synthetic biology or combinatorial biosynthesis approachs.In the wide range of Azalomycins F series compounds, the structure of the Azalomycin F3a is the simplest and its yield is also higher compared with the others. Therefore, Azalomycin F3a was as a representative in this study.Firstly, the genome sequence of the Azalomycins F producing strain Streptomyces sp.211726was obtained. Based on the result of bioinformatics analysis, the structure characteristic of Azalomycins F and the catalytic properties of PKS, the putative biosynthetic gene cluster was located in the genome. The cluster was then confirmed by in-frame deletion of a key CoA ligase gene (azl4) and the boundaries of the cluster were successfully verified by the strategies of gene knock-out and LC-ESI-HRMS analysis.Second, bioinformatic analysis revealed that the cluster include five genes (azl2, azl3, azl4, azl5and azll3) might be involved in Azalomycins F backbond biosynthesis, four putative regulator genes (azl6, azl8, azl9and azl12) and three unknown genes (azl7, azl10and azl11). By in-frame deletion, seven genes were inactivated, the result of the LC-ESI-HRMS analysis revealed as follows:(1) Gene azl4is probably responsible for the activation of4-guanidinobutyric.(2) azl5may be involved in the process of transfer of the activated4-guanidinobutyric to PKS.(3) azl6and azl8may play a role of positive regulation in the Azalomycin F3a biosynthesis.(4) azl10may be related to antibacterial activity and secretion of Azalomycin F3a.(5) Gene azl7may not participate in Azalomycin F3a biosynthesis or the mechanism is unclear. Enzymatic catalysis in vitro proved that Azl13is responsible for the hydrolysis of4-Guanidinobutyramide. Thus, we proposed the biosynthetic pathway of Azalomycin F3a for the first time. This work provides opportunities to illuminate the enzymatic reactions involved in this pathway by using genetic and chemo-enzymatic methods to creat novel derivatives of Azalomycin F3a.In addition, the enzymatic function of a novel nitrilase superfamily amidase Azl13was further investigated. Bioinformatic and biochemical analysis indicated that Azl13belongs to branch13of the nitrilase superfamily. Azl13has demonstrated activities of amidase, aryl acylamidase and acyl transferase, and it displayed an unusually wide substrate spectrum. Azl13shows its highest activity on4-Guanidinobutyramide, which is its natural substrate, moderate activity on short-chain aliphatic amides and weak activity of hydrolysing aromatic and heterocyclic amides. Azl13also catalyzed acyl transfer to hydroxylamine from acetamide or the herbicide propanil. The substrate spectrum differs from that of the reprted short-chain aliphatic amidaes, probably reflecting high salinity adaptation. The broad substrate spectrum of Azl13is potentially useful for chemical synthesis and biodegradation.