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Construction of a hybrid gene cluster to reveal coupled ring formation-hydroxylation in the biosynthesis of HSAF and analogues from Lysobacter enzymogenes.

ABSTRACT: HSAF and analogues are polycyclic tetramate macrolactams (PoTeMs) isolated from Lysobacter enzymogenes. Due to their antifungal activity, distinct chemical structure and new mode of action, PoTeMs have been the subject of several studies for their biosynthetic mechanism. However, polycycle formation is still not well understood. HSAF and several analogues (alteramides) carry a C20-hydroxyl, which is absent in most known PoTeMs such as combamides and pactamides. Previous studies indicated that two genes encoding NAD(P)H-dependent flavin enzymes (OX1/OX2) are responsible for the second five-membered ring formation in HSAF and alteramides. Intriguingly, the products of OX1/OX2 always carry the C20-OH. To test the hypothesis that the formation of the second five-membered ring is coupled with the C20-hydroxylation, we constructed a hybrid PoTeM gene cluster through removing OX1/OX2 in the HSAF cluster and functional complementation by CbmB, which also catalyzes the second five-membered ring formation in combamides but lacking the C20-OH. Two heterologous hosts carrying the hybrid cluster generated the same three PoTeMs, including lysobacterene B (3, the one-ring precursor of HSAF) and combamide D (4, a two-ring product lacking the C20-OH). The third product was not related to either of the clusters and was identified to be pactamide A (5) using mass spectrometry, 1D- and 2D-NMR, and ECD spectroscopy. The results demonstrate the feasibility of producing new PoTeM compounds through combinatorial biosynthesis. More importantly, this study provides the first experimental evidence to support that the second ring formation is coupled with the C20-hydroxylation in the biosynthesis of HSAF and analogues.

SUBMITTER: Li X

PROVIDER: S-EPMC6595964 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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Construction of a hybrid gene cluster to reveal coupled ring formation-hydroxylation in the biosynthesis of HSAF and analogues from Lysobacter enzymogenes.

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