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Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes.

ABSTRACT: HSAF was isolated from Lysobacter enzymogenes , a bacterium used in the biological control of fungal diseases of plants. Structurally, it is a tetramic acid-containing macrolactam fused to a tricyclic system. HSAF exhibits a novel mode of action by disrupting sphingolipids important to the polarized growth of filamentous fungi. Here we describe the HSAF biosynthetic gene cluster, which contains only a single-module polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS), although the biosynthesis of HSAF apparently requires two separate polyketide chains that are linked together by one amino acid (ornithine) via two amide bonds. Flanking the PKS/NRPS are six genes that encoding a cascade of four tightly clustered redox enzymes on one side and a sterol desaturase/fatty acid hydroxylase and a ferredoxin reductase on the other side. The genetic data demonstrate that the four redox genes, in addition to the PKS/NRPS gene and the sterol desaturase/fatty acid hydroxylase gene, are required for HSAF production. The biochemical data show that the adenylation domain of the NRPS specifically activates L-ornithine and that the four-domain NRPS is able to catalyze the formation of a tetramic acid-containing product from acyl-S-ACP and ornithinyl-S-NRPS. These results reveal a previously unrecognized biosynthetic mechanism for hybrid PK/NRP in prokaryotic organisms.

SUBMITTER: Lou L

PROVIDER: S-EPMC3078565 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes.

Lou Lili L Qian Guoliang G Xie Yunxuan Y Hang Jiliang J Chen Haotong H Zaleta-Rivera Kathia K Li Yaoyao Y Shen Yuemao Y Dussault Patrick H PH Liu Fengquan F Du Liangcheng L

Journal of the American Chemical Society 20101220 4

HSAF was isolated from Lysobacter enzymogenes , a bacterium used in the biological control of fungal diseases of plants. Structurally, it is a tetramic acid-containing macrolactam fused to a tricyclic system. HSAF exhibits a novel mode of action by disrupting sphingolipids important to the polarized growth of filamentous fungi. Here we describe the HSAF biosynthetic gene cluster, which contains only a single-module polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS), although the bios ...[more]

PMID: 21171605

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Project description:Lysobacter enzymogenes is a Gram-negative, environmentally ubiquitous bacterium that produces a secondary metabolite, called heat-stable antifungal factor (HSAF), as an antifungal factor against plant and animal fungal pathogens. 4-Hydroxybenzoic acid (4-HBA) is a newly identified diffusible factor that regulates HSAF synthesis via L. enzymogenes LysR (LysRLe), an LysR-type transcription factor (TF). Here, to identify additional TFs within the 4-HBA regulatory pathway that control HSAF production, we reanalyzed the LenB2-based transcriptomic data, in which LenB2 is the enzyme responsible for 4-HBA production. This survey led to identification of three TFs (Le4806, Le4969, and Le3904). Of them, LarR (Le4806), a member of the MarR family proteins, was identified as a new TF that participated in the 4-HBA-dependent regulation of HSAF production. Our data show the following: (i) that LarR is a downstream component of the 4-HBA regulatory pathway controlling the HSAF level, while LysRLe is the receptor of 4-HBA; (ii) that 4-HBA and LysRLe have opposite regulatory effects on larR transcription whereby larR transcript is negatively modulated by 4-HBA while LysRLe, in contrast, exerts positive transcriptional regulation by directly binding to the larR promoter without being affected by 4-HBA in vitro; (iii) that LarR, similar to LysRLe, can bind to the promoter of the HSAF biosynthetic gene operon, leading to positive regulation of HSAF production; and (iv) that LarR and LysRLe cannot interact and instead control HSAF biosynthesis independently. These results outline a previously uncharacterized mechanism by which biosynthesis of the antibiotic HSAF in L. enzymogenes is modulated by the interplay of 4-HBA, a diffusible molecule, and two different TFs.IMPORTANCE Bacteria use diverse chemical signaling molecules to regulate a wide range of physiological and cellular processes. 4-HBA is an "old" chemical molecule that is produced by diverse bacterial species, but its regulatory function and working mechanism remain largely unknown. We previously found that 4-HBA in L. enzymogenes could serve as a diffusible factor regulating HSAF synthesis via LysRLe Here, we further identified LarR, an MarR family protein, as a second TF that participates in the 4-HBA-dependent regulation of HSAF biosynthesis. Our results dissected how LarR acts as a protein linker to connect 4-HBA and HSAF synthesis, whereby LarR also has cross talk with LysRLe Thus, our findings not only provide fundamental insight regarding how a diffusible molecule (4-HBA) adopts two different types of TFs for coordinating HSAF biosynthesis but also show the use of applied microbiology to increase the yield of the antibiotic HSAF by modification of the 4-HBA regulatory pathway in L. enzymogenes.

Project description:In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia coli fadD mutant JW1794. RpfB activates long-chain fatty acids (n-C16:0 and n-C18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2 In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenes IMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.

Dataset Information

Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes.

Publications

Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes.

Similar Datasets

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