Production of Avilamycin A is regulated by AviC1 and AviC2, two transcriptional activators

Abstract

Streptomycetes are typical soil-dwelling bacteria with intricate morphological and biochemical differentiation of colonies, resulting in onset of secondary metabolite production. Compounds produced during these stages of differentiation comprise nearly two-thirds of bioactive molecules synthesized by microorganisms, including antibiotics, antitumor agents and immunosuppressants. 1,2 The biosynthesis of antibiotics and other secondary metabolites is controlled by interactions of both global and pathway-specific regulators. However, in all cases, the influence of the environment is reflected by the activity of pathway-specific regulatory genes, which are located within a biosynthetic gene cluster and which control the expression of biosynthetic genes. 3 On account of high structural and functional similarity, such regulators were grouped into two families, namely Streptomyces antibiotics regulatory proteins (SARP), mostly found within the aromatic polyketides clusters, and large ATP-binding regulators of LuxR family (LAL), controlling the production of macrolides and glycopeptides. [4][5][6] Recent efforts in cloning and characterization of biosynthetic gene clusters for new groups of secondary metabolites revealed novel classes of transcriptional factors that differ from typical SARPs or LAL representatives. 7,8 Avilamycins are secondary metabolites produced by Streptomyces viridochromogenes Tu57. They are active against Gram-positive bacteria. It is known that avilamycins bind to the 23S rRNA in the region proximal to the channel, where tRNA enters the A-site and blocks the protein synthesis. 9 Avilamycin resistance is mediated by 23S rRNA methylation and active avilamycin transport. 10 The avilamycin resistance genes together with the structural genes were identified and cloned as an entire biosynthetic gene cluster containing 54 open reading frames. 11 The biosynthetic steps leading to the formation of the avilamycin molecule were established by the analysis of secondary metabolites produced by mutants. 11,12 Within the cluster also, two putative regulatory genes named aviC1 and aviC2 were identified. Both genes are located in close proximity to each other and are transcribed in the same direction (Figure 1). Genes are separated by a 271-bp non-coding region. We succeeded in identifying the presence of putative bacterial promoter sequences upstream of aviC1 initiation codon by the use of BPROM bacterial promoter prediction server (Softberry Inc., Mount Kisco, NY, USA). It consists of a putative À10 box GGTTTTCAT (Score 34) and a À35 box ATGCGA (Score 12). Another putative promoter sequence is located upstream of the aviC2 translation start site consisting of a À10 box GCCCATGAT (Score 31) and a À35 box at TTTCTA (Score 34) similar to consensus Streptomycetes promoters. 13

Identification of a gene cluster for telomestatin biosynthesis and heterologous expression using a specific promoter in a clean host

“…The C-terminal sequence of TlsB contains a helix-turn-helix motif, which is widely observed in the LuxR family of transcriptional regulators. Moreover, TlsB showed homology to AviC1, which regulates avilamycin A biosynthesis 25 , and Lipreg1, which regulates lipomycin biosynthesis 26 . Consistent with our finding, deletion of the aviC1 and lipreg1 genes resulted in a significant loss in production of the corresponding metabolites.…”

Microbial Oligosaccharides with Biomedical Applications

“…It is generally acknowledged that the rarely observed orthoester linkage is necessary for the antibiotic properties of the orthosomycins. Although the orthosomycin family includes hygromycin B, everninomicin, avilamycin, flambamycin, curamycin, etc., the former three members have been extensively studied, especially in the aspect of their bioactivity and biosynthesis [ 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 ]. Herein, restricted by the space, everninomicin (EVN) and avilamycin (AVI) will be mainly discussed in this review.…”

“…Until now, twenty EVN ( 43 – 62 ) ( Figure 8 ) have been purified from wide- or mutant-type M. carbonacea var. africana [ 59 , 60 , 61 , 62 , 63 , 71 ], while thirty-seven AVI ( 63 – 99 ) ( Figure 9 ) have been isolated from the wide- or mutant-type Streptomyces viridochromogenes Tü57 [ 59 , 64 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 ]. Structurally, EVN and AVI share the same seven-sugar core (rings B to H) and characteristic substituted partner including two orthoester linkages located between rings C and D and rings G and H and a methylenedioxy bridge attached to ring H. The major differences between EVN and AVI are attributed to the presence (or absence) of the A-ring nitrosugar and the orsellinic acid in EVN (or in AVI).…”

“…Due to the multiple-ring structure of Avi A, its biosynthetic pathway is relatively complicated. Up to now, aided with the bioinformatics analysis, some of the key enzymes in the Avi A biosynthesis pathway have been successfully elucidated by analysis of the corresponding Avi derivatives, gavibamycins (Gav, 79 – 99 ), in situ gene knockout and complementation strategy, heterologous expression systems, such as S. lividans TK24, S. coelicolor CH999, Streptomyces TK66, etc., or feeding experiments [ 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 ]. Herein, four main initial precursors, including propionyl-CoA, d -glucose-1-phosphate, GDP- d -mannose, and UDP- d -glucose, are summarized in Figure 10 .…”

“…Analysis of the high-resolution crystal structures reveals that an orientation of one glycosidic linkage of oxygenase HygX is consistent with metal-catalyzed hydrogen atom abstraction from substrate, supporting that AviO1 participates the catalysis of the orthoester linkages. In addition, two resistances, rRNA-methyltransferase AviRa and AviRb, two regulators, ABC transporters AviABC1 and AviABC2 [ 80 ], and two transcriptional activators, AviC1 and AviC2 [ 81 ] are also reported. Regarding the Avi A contributes to poor water solubility and difficulties in drug development perhaps ascribed to its multiple methyl groups [ 76 , 82 ], fully understanding the Avi A biosynthetic pathway will provide useful information and contribute to produce novel derivatives with improved polarity and pharmacokinetic properties.…”