The Mechanism Of The Acctachment Of O-(N-cyclohexanecarbonyl)-D-alaninyl Side Chain In Ansatrienins

Ansamycins is a group of type I polyketide, which started with AHBA(3-amino-5-hydroxybenzoic acid) unit, and usually shows significant bioactivities. For example, rifamycin is one of the famous clinical antituberculosis drugs. Geldanamycin and maytansine have potent antitumor activity. As an early member of ansamycins, ansatrienins show pronounced activity against filament fungi and yeasts. However, its biosynthetic reports is rare. Until 2011, Liu et al. cloned the ansatrienins biosynthetic gene cluster(myc) fortuitously and bioinformatically proposed that modular NRPS MycC, esterase MycF4 and N-acetyltransferase MycF1 is involved in the attachment of its unique O-(N-cyclohexanecarbonyl)-D-alaninyl side chain, but lack of experimental evidences. In this thesis, we identified and characterized the enzymes responsible for the attachment of the unique side chain by in vivo and in vitro experiments, and revised the attachment scheme proposed by Liu.Streptomyces sp. XZQH13 is an AHBA synthase gene positive strain, which isolated from soil sample collected at Qinghai, China. The putative ansamycin gene cluster in strain XZQH13 was predicted to encode ansatrienins(ast) derivative according to the phylogenetic analysis of its AHBA synthase gene. By constitutive expression of the positive regulator gene astGl, we succeessfully activated this cryptic gene cluster and identified two known compounds:hygdroxymycotrienins A (1) and thiazinotrienomycin G (2). After comparative analysis of ast and myc gene cluster, we proposed that two genes(astC and astFl) rather than three genes may be necessary and sufficient for the tethering of cyclohexylcarbonyl alanine moiety. In order to verify this hypothesis, we knocked out the astC and astFl gene in vivo. The disruption of astC and astFl apparently abolished the production of compound 1 and 2, and accumulated several intermediates showing similar UV-visible absorption profile to ansatrienins. The main intermediate accumulated in astC-disruption mutant was isolated and structurally elucidated to be thiazinotrienomycinol (3), which is absent of the C-11 ester group. Due to the low amount, the attempts to isolate the putative intermediates in astF1-disruption mutant failed. These in vivo results indicated that astC and astFl is indeed responsible for the attachment of O-(N-cyclohexanecarbonyl)-D-alaninyl side chain.In order to further confirm the function of astC and astFl, we expressed AstC and AstF1 recombinant protein in E. coli BL21(DE3) and purified by affinity chromotography. Then, we conducted the in vitro characterization of AstC and AstF1 using thiazinotrienomycinol (3) as substrate. Single modular NRPS AstC, comprised of A-PCP-TE domains, catalyzed the D-alanylation of 11-0 of thiazinotrienomycinol. the optimum pH and temperature is pH8.0 and 30 ℃ individually. Under the condition of 200 μM D-Alanine, by altering the concentration of thiazinotrienomycinol, we determined the Km value of AstC to thiazinotrienomycinol to be 40.21±20.60 μM, Kcat to be 0.70±0.20 min-1. Under the condition of 33.25 μM thiazinotrienomycinol, by altering the concentration of D-alanine, we determined the Km value of AstC to D-alanine to be 5.57±1.56 μM, Kcat to be 0.27±0.03 min-1. In addition, we also investigated the substrate scope of AstC, and find that besides D-Ala, D-Val, β-Ala, D-Ser, D-Thr, Gly can also be accepted and attached onto thiazinotrienomycinol. AstF1 is a N-acyltansferase, using the product of AstC as acyl acceptor and cyclohexylcarbonyl-SNAC as the acyl donor, we successfully proved that the AstF1 can catalyze the acylation of amino group of 11-O-D-alanine. Due to the large diversity of acyl group in natural ansatrienins, we tested five more acyl donors, and the LC-MS data demonstrates that AstF1 also have broad substrate scope, which can accepted all five acyl-SNACs and attached them onto amino group of 11-O-D-alanine.By means of gene knock-out, intermediates isolation and structure elucidation, protein expression and in vitro biochemical assay, we elucidated the subsequential attachment mechanism of unique O-(N-cyclohexanecarbonyl)-D-alaninyl side chain in ansatrienins biosynthesis. To the best of our knowledge, this is the first report that single modular NRPS (or its TE domain) can catalyze D-alanylation. In addition, both of AstC and AstF1 display broad substrate scope toward the acyl donors, which might be useful in manufacturing new ansatrienins or ansamycins derivatives through in vitro or in vivo synthetic biology approaches.