Heterologous Production of Albicidin: a Promising Approach to Overproducing and Characterizing This Potent Inhibitor of DNA Gyrase

Abstract

The phytotoxin and polyketide antibiotic albicidin produced by Xanthomonas albilineans is a highly potent DNA gyrase inhibitor. Low yields of albicidin production have slowed studies of its chemical structure. Heterologous expression of albicidin biosynthetic genes in X. axonopodis pv. vesicatoria resulted in a sixfold increase in albicidin production, offering promising strategies for engineering overproduction.Albicidin is produced by Xanthomonas albilineans which is a slow-growing bacterium and the causal agent of sugarcane leaf scald (13). Albicidin is involved in the pathogenicity of X. albilineans and inhibits the replication of chloroplastic DNA (6, 7). Albicidin also inhibits DNA replication in Escherichia coli at nanomolar concentrations (4), whereas mammalian cells are unaffected at 8 g/ml (3). A recent study showed that albicidin targets DNA gyrase with features of inhibition that differ from those of other known antibiotics (11). Albicidin is synthesized by a unique hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway that does not resemble any other pathway described to date. Three genomic regions (XALB1, XALB2, and XALB3) involved in albicidin biosynthesis were cloned and sequenced (12,14,15,17). XALB1 contains the majority of albicidin biosynthetic genes in a cluster of 20 open reading frames (ORFs) (albI to albXX). ORFs albI, albIV, and albIX encode three large PKSs and NRPSs, whereas the other ORFs are resistance, modifying, and regulatory genes. XALB2 and XALB3 each contain a single biosynthetic gene, albXXI and albXXII, whose products are, respectively, a phosphopantetheinyl transferase and the heat shock protein HtpG. The antibiotic activity of albicidin against a wide range of gram-positive and gram-negative pathogenic bacteria (Enterobacter aerogenes, E. coli, Haemophilus influenzae, Klebsiella pneumoniae, Shigella sonnei, and Staphylococcus aureus) is of interest for the development of new antibacterial drugs (4, 5). Low yields of albicidin production in slow-growing X. albilineans have slowed studies of its chemical structure and potential therapeutic applications. A heterologous system for albicidin overproduction is therefore highly desirable (i) to increase the levels of expression of the biosynthetic enzymes, (ii) to obtain large quantities of albicidin, and (iii) to characterize albicidin through nuclear magnetic resonance and mass spectrometry analyses. We report here the construction of a two-plasmid expression system harboring the complete albicidin biosynthetic gene set; its transfer into a fast-growing heterologous host, X. axonopodis pv. vesicatoria; and the subsequent production of albicidin.Two wide-host-range plasmids derived from IncW vector pUFR043 (10) and IncP vector pLAFR3 (16) were used for the transfer of the complete albicidin biosynthetic machinery. The previously described IncW plasmid pALB571 (12, 14) was used to transfer albI to albIX (the insert corresponds to nucleotides 19001 to 55839 of accession no. AJ586576). Plasmid pLAFRK7 was con...

The phosphopantetheinyl transferases: catalysis of a post-translational modification crucial for life

“…vesicatoria , and showed a 6-fold increased yield of the antibiotic. 170 Since non-producing albicidin mutants are still plant pathogenic, Gabriel and Royer’s groups have systematically studied the other factors that influence pathogenicity. 165, 166 PPTase knockout mutations indeed showed no albicidin production, but did not eliminate sugar cane leaf scald.…”

Critical Intermediates Reveal New Biosynthetic Events in the Enigmatic Colibactin Pathway

“…[2] Heterologous expressions ystemsc an dramatically increase the expression levels of biosynthetic enzymes and have been widelye mployed as an effective strategy for the over-production of naturalp roducts.T he successful structure characterization of the low-yielding albicidin is ac lassic example. [16,17] In our study,t ransformation-associated recombination (TAR) cloning was used to directly capture the entire biosynthetic gene cluster of colibactin from E. coli CFT073 genomicD NA. As pecified TARc apturev ectorp CAP01-capclb that contains both Saccharomyces cerevisiae and E. coli elements wasc onstructed to facilitateT AR reassembly of this large (70 kb) gene cluster in yeast ( Figure S1 in the Supporting Information).…”

Synthesis of Albicidin Derivatives: Assessing the Role of N‐terminal Acylation on the Antibacterial Activity

“…Due to its low abundance in X. albilineans and its unusual oligo‐aromatic character, it took more than 30 years to elucidate the structure . It shows a strong antibacterial activity against Gram‐positive and Gram‐negative bacteria at nanomolar concentrations by acting primarily as a DNA gyrase inhibitor …”

“…[13] It shows as trong antibacterial activity againstG rampositive and Gram-negative bacteria at nanomolar concentrations by acting primarily as aDNA gyrase inhibitor. [14][15][16] Albicidin is an acyl pentapeptide composed of six building blocks, of which five are aromatic ( Figure 1). The acyl unit on the N-terminus is a para-coumaric acid moietyf eaturing an additional a-methyl group (MCA, A).…”