LeTetR Positively Regulates 3-Hydroxylation of the Antifungal HSAF and Its Analogs in \u3ci\u3eLysobacter enzymogenes\u3c/i\u3e OH11

The biocontrol agent Lysobacter enzymogenes OH11 produces several structurally distinct antibiotic compounds, including the antifungal HSAF (Heat Stable Antifungal Factor) and alteramides, along with their 3-dehydroxyl precursors (3-deOH). We previously showed that the 3-hydroxylation is the final step of the biosynthesis and is also a key structural moiety for the antifungal activity. However, the procedure through which OH11 regulates the 3-hydroxylation is still not clear. In OH11, the gene orf3232 was predicted to encode a TetR regulator (LeTetR) with unknown function. Here, we deleted orf3232 and found that the LeTetR mutant produced very little HSAF and alteramides, while the 3-deOH compounds were not significantly affected. The production of HSAF and alteramides was restored in orf3232-complemented mutant. qRT-PCR showed that the deletion of orf3232 impaired the transcription of a putative fatty acid hydroxylase gene, orf2195, but did not directly affect the expression of the HSAF biosynthetic gene cluster (hsaf ). When an enzyme extract from E. coli expressing the fatty acid hydroxylase gene, hsaf -orf7, was added to the LeTetR mutant, the production of HSAF and alteramides increased by 13–14 fold. This study revealed a rare function of the TetR family regulator, which positively controls the final step of the antifungal biosynthesis and thus controls the antifungal activity of the biocontrol agent

Structural basis of DNA binding by the WhiB-like transcription factor WhiB3 in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) WhiB3 is an iron–sulfur cluster-containing transcription factor belonging to a subclass of the WhiB-Like (Wbl) family that is widely distributed in the phylum Actinobacteria. WhiB3 plays a crucial role in the survival and pathogenesis of Mtb. It binds to the conserved region 4 of the principal sigma factor (σA4) in the RNA polymerase holoenzyme to regulate gene expression like other known Wbl proteins in Mtb. However, the structural basis of how WhiB3 coordinates with σA4 to bind DNA and regulate transcription is unclear. Here we determined crystal structures of the WhiB3:σA4 complex without and with DNA at 1.5 Å and 2.45 Å, respectively, to elucidate how WhiB3 interacts with DNA to regulate gene expression. These structures reveal that the WhiB3:σA4 complex shares a molecular interface similar to other structurally characterized Wbl proteins and also possesses a subclass-specific Arg-rich DNA-binding motif. We demonstrate that this newly defined Arg-rich motif is required for WhiB3 binding to DNA in vitro and transcriptional regulation in Mycobacterium smegmatis. Together, our study provides empirical evidence of how WhiB3 regulates gene expression in Mtb by partnering with σA4 and engaging with DNA via the subclass-specific structural motif, distinct from the modes of DNA interaction by WhiB1 and WhiB7

Biosynthetic Engineering and Molecular Regulation of Bioactive Natural Products in Lysobacter enzymogenes

Microbial natural products are one of the best sources for production of novel antibiotics, anticancer agents and immunosuppressants. Due to the persistent evolution of drug-resistant pathogens, the identification of novel antibiotics is extremely important. Over the past decade, Lysobacter has emerged as a rich source of bioactive natural products. This dissertation describes the regulatory mechanism and metabolic engineering of a group of natural products from Lysobacter enzymogenes for the industrial and antimicrobial applications.In the first chapter, we give a short review of structure, regulatory mechanism, and pathway engineering of HSAF and analogs, a group of polycyclic tetramate macrolactams in Lysobacter. In chapter two, we report a biosynthetic route to unsaturated odd-carbon fatty dicarboxylic acid (FDCA) based upon genetic modification of the polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene within L. enzymogenes. The newly engineered strain produces the C7 FDCA, hepta-2,4-dienedioic acid, an odd-carbon diunsaturated dicarboxylic acid. The subsequent chapters are based on regulatory mechanism of HSAF biosynthesis. In chapter three, we revealed a rare function of a TetR family regulator, which controls 3-hydroxylation of HSAF biosynthesis. We deleted the gene encoding a LeTetR regulator and found that the LeTetR mutant produced very little HSAF and alteramides, while the 3-dehydroxide compounds were not significantly affected. We assayed the activity of an enzyme extract from E. coli by expressing the fatty acid hydroxylase gene (hsaf-sd) and showed that the enzyme could restore the production of HSAF and alteramides in the LeTetR mutant for 3-hydroxylation of HSAF biosynthesis. In chapter four, we investigated regulatory mechanism for the LeTetR-regulated HSAF biosynthesis. We discovered that LeTetR binds to hsaf-sd gene encoding 3-hydroxylase. We have also determined that ligand binding to LeTetR affects its binding to the hsaf-sd gene which could ultimately 3-hydroxylation step of HSAF biosynthesis

LeTetR Positively Regulates 3-Hydroxylation of the Antifungal HSAF and Its Analogs in \u3ci\u3eLysobacter enzymogenes\u3c/i\u3e OH11

The biocontrol agent Lysobacter enzymogenes OH11 produces several structurally distinct antibiotic compounds, including the antifungal HSAF (Heat Stable Antifungal Factor) and alteramides, along with their 3-dehydroxyl precursors (3-deOH). We previously showed that the 3-hydroxylation is the final step of the biosynthesis and is also a key structural moiety for the antifungal activity. However, the procedure through which OH11 regulates the 3-hydroxylation is still not clear. In OH11, the gene orf3232 was predicted to encode a TetR regulator (LeTetR) with unknown function. Here, we deleted orf3232 and found that the LeTetR mutant produced very little HSAF and alteramides, while the 3-deOH compounds were not significantly affected. The production of HSAF and alteramides was restored in orf3232-complemented mutant. qRT-PCR showed that the deletion of orf3232 impaired the transcription of a putative fatty acid hydroxylase gene, orf2195, but did not directly affect the expression of the HSAF biosynthetic gene cluster (hsaf ). When an enzyme extract from E. coli expressing the fatty acid hydroxylase gene, hsaf -orf7, was added to the LeTetR mutant, the production of HSAF and alteramides increased by 13–14 fold. This study revealed a rare function of the TetR family regulator, which positively controls the final step of the antifungal biosynthesis and thus controls the antifungal activity of the biocontrol agent

LeTetR Positively Regulates 3-Hydroxylation of the Antifungal HSAF and Its Analogs in Lysobacter enzymogenes OH11

The biocontrol agent Lysobacter enzymogenes OH11 produces several structurally distinct antibiotic compounds, including the antifungal HSAF (Heat Stable Antifungal Factor) and alteramides, along with their 3-dehydroxyl precursors (3-deOH). We previously showed that the 3-hydroxylation is the final step of the biosynthesis and is also a key structural moiety for the antifungal activity. However, the procedure through which OH11 regulates the 3-hydroxylation is still not clear. In OH11, the gene orf3232 was predicted to encode a TetR regulator (LeTetR) with unknown function. Here, we deleted orf3232 and found that the LeTetR mutant produced very little HSAF and alteramides, while the 3-deOH compounds were not significantly affected. The production of HSAF and alteramides was restored in orf3232-complemented mutant. qRT-PCR showed that the deletion of orf3232 impaired the transcription of a putative fatty acid hydroxylase gene, orf2195, but did not directly affect the expression of the HSAF biosynthetic gene cluster (hsaf). When an enzyme extract from E. coli expressing the fatty acid hydroxylase gene, hsaf-orf7, was added to the LeTetR mutant, the production of HSAF and alteramides increased by 13–14 fold. This study revealed a rare function of the TetR family regulator, which positively controls the final step of the antifungal biosynthesis and thus controls the antifungal activity of the biocontrol agent