Revealling The Biosynthetic Mechanism Of Antibiotic Piericidin A1

Piericidin A1belongs to a class of-pyridone antibiotics that exhibitattractive biological activities, and can be produced by various Streptomycesstrains. Due to its binding capacity to mitochondrial complex I, piericidin A1is a potent inhibitory toward mitochondrial respiration. Piericidin A1showsdiverse antibacterial and antifungal activities with a minimum inhibitoryconcentration, and it can selectively kill some insects at low concentration.Furthermore, piericidin A1was identified as a highly selective antitumoragent in animal model. Previous isotope feeding experiment showed that thecarbon backbone of piericidin A1is derived from acetate and propionate,which shows stark contrast to the biosynthesis of other known-pyridonering antibiotics that involve hybrid PKS-NRPS systems. In this study, wehave got the selected mutant strain, Streptomyces piomogeues var.Hangzhouwanensis, which can highly produced a piericidin derivative withundefined chemical structure. And it should be mentioned, this strain is agood microbial resource with China's self-owned intellectual property rights. To elucidate chemical structure of targeted molecular produced by S.piomogeues var. Hangzhouwanensis, the conditions for separating andpurifying the compound were established and optimized, and10mg samplewas finally obtained and confirmed to be piericidin A1by NMR analysis. Inaddition, the conditions for the conjugal transfer of DNA from E. coli to S.piomogeues var. Hangzhouwanensis were established to further in vivo genereplacement experiment.To identify genes involved in the biosynthesis of piericidin A1, wholegenome scanning of S. piomogeues var. Hangzhouwanensis was performed onthe Roche454sequencing platform. A17kb region was indentified involvedin piericidin A1biosynthesis by large-fragement deletion experiment. Usingchromosome walking, a130kb region, in which the indentified17kb was inthe middle, was defined for piericidin A1biosynthesis. Through in silicoanalysis,12ORFs were found to be involved in piericidin A1biosynthesis: aregulatory gene (pieR), six typical modular type I PKS genes (pieA1to pieA6),an amidotransferase gene (pieD), a hypothetical gene (pieC), twomethyltransferase genes (pieB1, pieB2), and a FAD-dependentmonooxygenase gene (pieE). Gene functional analysis provides view of thebiosynthesis pathway: the PKS, amidotransferase and the hypothetical protein responsible for the-pyridone ring formation, methyltransferases andmonooxygenase catalyzing the modification steps, and the regulator related topathway-specific regulation.To probe the crucial roles of pieD, pieC and pieTE in the biosynthesis of-pyridone ring formation, they were individually inactivated by genereplacement. However, no obvious intermediate was detected either due to thelack of specific UV absorbtion of the polyketide intermediate or the instabilityand degradation of the β,-diketo carboxylic acid in the cell.To gain insights into PieTE function, we attempted to reconstitute thefunction of the last module appended with the terminal TE (module8-TE) invitro, which were biochemically characterized with β-ketoacyl-SNAC andmalonyl-CoA. In the presence of a hydrolytic TE, such as the one proposedhere, the corresponding β,-diketo acid is expected to be released. Consistentwith the detection of the putative undecane-2,4-dione, and the producedmolecular corresponding to the fluoresceinamine-conjugated3,5-dioxododecanoic acid in the fluorescein conjugation modification, PieTEis responsible to hydrolyze the highly active β,-diketoacyl-S-ACP8andrelease the linear β,-diketo carboxylic acid. Furthermore, the hydrolysisactivity of PieTE was determined by by Ellman's reagent. This showed that PieTE also exhibits a higher preference for longer chain substrates, with thelongest C10substrate being hydrolyzed at the fastest reaction rate. Besides, inthe absence of the hydrolytic TE domain, such as in the analysis ofmodule8-TE0(S148A), the β,-diketoacyl-S-ACP8would expect to undergospontaneous enolization and cyclization to form the-pyrone. Compared tothe well-known macrocyclic polyketide pathways, modular type I PKSsutilize the C-terminal TE domains to catalyze hydrolytic release of polyketideproduct are less common.According to the in vivo targeted gene inactivation study and in vitrobiochemical characterization of Pie module8, the biosynthetic pathway forthe-pyridone ring formation was proposed: the pieA1-pieA6encode for atype I PKS to afford the polyketide backbone, then to be directly hydrolyzedby PieTE for releasing linear polyketide chain, and PieD is likely to catalyzethe amidation of the terminal carboxylic group of the linear polyketideintermediate, this corresponding amide was proposed to mediate thecyclization to form the-pyridone ring. Here, the PieTE essentially preventsthe formation of pyrones as shunt products by efficiently hydrolyzing theβ,-diketo thioester from the PieA. The mechanism proposed in this study for the-pyridone ring formationis in stark contrast to the biosynthesis of other known-pyridonering-containing antibiotics that involve hybrid PKS-NRPS systems. Inpiericidin A1-pyridone ring biosynthesis, the nitrogen source is provided byammonia rather than amino acid precursor, and the PieTE efficientlyhydrolyzed the the β,-diketo thioester to afford substrate for amidation andcyclization.To invesgated post-PKS tailoring steps in the biosynthesis of piericidinA1, pieB1, pieB2and pieE were individually inactivated by gene replacement,and possible intermediates were detected in each mutant. Based on theLC-MS data, subsequent tailoring modifications on the-pyridone ring wereproposed, including one hydroxylation and two methylations. Moreover, anintermediate from pieB2mutant was accumulated and purified for furtherNMR characterization. The abovementioned investigation paves the way forunveiling post-PKS tailoring steps in piericidin A1biosynthesis.As revealed by the whole genome scanning, S. piomogeues var.Hangzhouwanensis has abundant secondary metabolic pathways, includingPKS, NRPS, hybrid NRPS-PKS, aminoglycoside, terpene, siderophore and lantibiotic, which provides wonderful oppotunity for mining various naturalproduct produced by S. piomogeues var. Hangzhouwanensis.