Activation of a Cryptic Gene Cluster in <i>Lysobacter enzymogenes</i> Reveals a Module/Domain Portable Mechanism of Nonribosomal Peptide Synthetases in the Biosynthesis of Pyrrolopyrazines

<i>Lysobacter</i> are considered “peptide specialists”. However, many of the nonribosomal peptide synthetase genes are silent. Three new compounds were identified from <i>L. enzymogenes</i> upon activating the six-module-containing <i>led</i> cluster by the strong promoter <i>P</i>_HSAF. Although <i>ledD</i> was the first gene under <i>P</i>_HSAF control, the second gene <i>ledE</i> was expressed the highest. Targeted gene inactivation showed that the two-module LedE and the one-module LedF were selectively used in pyrrolopyrazine biosynthesis, revealing a module/domain portable mechanism

Yield Improvement of the Anti-MRSA Antibiotics WAP-8294A by CRISPR/dCas9 Combined with Refactoring Self-Protection Genes in <i>Lysobacter enzymogenes</i> OH11

The cyclic lipodepsipeptides WAP-8294A are antibiotics with potent activity against methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). One member of this family, WAP-8294A2 (Lotilibcin), was in clinical trials due to its high activity and distinct chemistry. However, WAP-8294A compounds are produced in a very low yield by <i>Lysobacter</i> and only under very stringent conditions. Improving WAP-8294A yield has become very critical for research and application of these anti-MRSA compounds. Here, we report a strategy to increase WAP-8294A production. We first used the CRISPR/dCas9 system to increase the expression of five cotranscribed genes (<i>orf1–5</i>) in the WAP gene cluster, by fusing the omega subunit of RNA polymerase with dCas9 that targets the operon’s promoter region. This led to the transcription of the genes increased by 5–48 folds in strain dCas9-ω3. We then refactored four putative self-protection genes (<i>orf6</i>, <i>orf7</i>, <i>orf9</i> and <i>orf10</i>) by reorganizing them into an operon under the control of a strong <i>Lysobacter</i> promoter, P_HSAF. The refactored operon was introduced into strain dCas9-ω3, and the transcription of the self-protection genes increased by 20–60 folds in the resultant engineered strains. The yield of the three main WAP-8294A compounds, WAP-8294A1, WAP-8294A2, and WAP-8294A4, increased by 6, 4, and 9 folds, respectively, in the engineered strains. The data also showed that the yield increase of WAP-8294A compounds was mainly due to the increase of the extracellular distribution. WAP-8294A2 exhibited potent (MIC 0.2–0.8 μg/mL) and specific activity against <i>S. aureus</i> among a battery of clinically relevant Gram-positive pathogens (54 isolates)

Functional and Structural Analysis of Phenazine <i>O</i>‑Methyltransferase LaPhzM from <i>Lysobacter antibioticus</i> OH13 and One-Pot Enzymatic Synthesis of the Antibiotic Myxin

Myxin is a well-known antibiotic that had been used for decades. It belongs to the phenazine natural products that exhibit various biological activities, which are often dictated by the decorating groups on the heteroaromatic three-ring system. The three rings of myxin carry a number of decorations, including an unusual aromatic <i>N</i>5,<i>N</i>10-dioxide. We previously showed that phenazine 1,6-dicarboxylic acid (PDC) is the direct precursor of myxin, and two redox enzymes (LaPhzS and LaPhzNO1) catalyze the decarboxylative hydroxylation and aromatic <i>N</i>-oxidations of PDC to produce iodinin (1.6-dihydroxy-<i>N</i>5,<i>N</i>10-dioxide phenazine). In this work, we identified the <i>LaPhzM</i> gene from <i>Lysobacter antibioticus</i> OH13 and demonstrated that <i>LaPhzM</i> encodes a SAM-dependent <i>O</i>-methyltransferase converting iodinin to myxin. The results further showed that LaPhzM is responsible for both monomethoxy and dimethoxy formation in all phenazine compounds isolated from strain OH13. LaPhzM exhibits relaxed substrate selectivity, catalyzing <i>O</i>-methylation of phenazines with non-, mono-, or di-<i>N</i>-oxide. In addition, we demonstrated a one-pot biosynthesis of myxin by <i>in vitro</i> reconstitution of the three phenazine-ring decorating enzymes. Finally, we determined the X-ray crystal structure of LaPhzM with a bound cofactor at 1.4 Å resolution. The structure provided molecular insights into the activity and selectivity of the first characterized phenazine <i>O</i>-methyltransferase. These results will facilitate future exploitation of the thousands of phenazines as new antibiotics through metabolic engineering and chemoenzymatic syntheses

Identification and Characterization of the 28‑<i>N</i>‑Methyltransferase Involved in HSAF Analogue Biosynthesis

Polycyclic tetramate macrolactams (PoTeMs) are a family of structurally intriguing bioactive natural products. Although the presence of the N-28 methyl group is known to affect bioactivities of some PoTeMs, the mechanism for this methylation remains unclear. We report here the identification and characterization of the 28-N-methyltransferase for HSAF analogues, which is encoded by a gene located outside the HSAF (heat-stable antifungal factor) cluster in Lysobacter enzymogenes C3. Our data suggested that 28-N-methyltransferase utilizes S-adenosylmethionine (SAM) to methylate HSAF analogues, and acts after the dicyclic and tricyclic ring formation and prior to C-3 hydroxylation. Kinetic analysis showed that the optimal substrate for the enzyme is 3-dehydroxy HSAF (3-deOH HSAF). Moreover, it could also accept PoTeMs bearing a 5–6 or 5–6–5 polycyclic system as substrates. This is the first N-methyltransferase identified in the family of PoTeMs, and the identification of this enzyme provides a new tool to generate new PoTeMs as antibiotic lead compounds

Unusual Activities of the Thioesterase Domain for the Biosynthesis of the Polycyclic Tetramate Macrolactam HSAF in <i>Lysobacter enzymogenes</i> C3

HSAF is an antifungal natural product with a new mode of action. A rare bacterial iterative PKS-NRPS assembles the HSAF skeleton. The biochemical characterization of the NRPS revealed that the thioesterase (TE) domain possesses the activities of both a protease and a peptide ligase. Active site mutagenesis, circular dichroism spectra, and homology modeling of the TE structure suggested that the TE may possess uncommon features that may lead to the unusual activities. The iterative PKS-NRPS is found in all polycyclic tetramate macrolactam gene clusters, and the unusual activities of the TE may be common to this type of hybrid PKS-NRPS

Heterocyclic Aromatic <i>N</i>‑Oxidation in the Biosynthesis of Phenazine Antibiotics from Lysobacter antibioticus

Heterocyclic aromatic <i>N</i>-oxides often have potent biological activities, but the mechanism for aromatic <i>N</i>-oxidation is unclear. Six phenazine antibiotics were isolated from Lysobacter antibioticus OH13. A 10 gene cluster was identified for phenazine biosynthesis. Mutation of <i>LaPhzNO1</i> abolished all <i>N</i>-oxides, while non-oxides markedly increased. LaPhzNO1 is homologous to Baeyer–Villiger flavoproteins but was shown to catazlye phenazine <i>N</i>-oxidation. LaPhzNO1 and LaPhzS together converted phenazine 1,6-dicarboxylic acid to 1,6-dihydroxyphenazine <i>N</i>5,<i>N</i>10-dioxide. LaPhzNO1 also catalyzed <i>N</i>-oxidation of 8-hydroxyquinoline

Targeted Discovery and Combinatorial Biosynthesis of Polycyclic Tetramate Macrolactam Combamides A–E

Polycyclic tetramate macrolactams (PoTeMs) are a growing class of natural products with distinct structure and diverse biological activities. By promoter engineering and heterologous expression of the cryptic <i>cbm</i> gene cluster, four new PoTeMs, combamides A–E (<b>1</b>–<b>4</b>), were identified. Additionally, two new derivatives, combamides E (<b>5</b>) and F (<b>6</b>), were generated via combinatorial biosynthesis. Together, our findings provide a sound base for expanding the structure diversities of PoTeMs through genome mining and combinatorial biosynthesis

HPLC of the yellow pigments of <i>L. enzymogenes</i> OH11.

<p>A. ΔORF5 (Pteridine-dependent dioxygenase); B. ΔORF5 fed with 3-hydroxybenzoic acid.</p

HPLC of the yellow pigment production in three ORF16 mutants of <i>L. enzymogenes</i> OH11.

<p>A. ΔORF16; B. ORF16 Q166A; C. ORF16 S120A. The phenotype of the wild type (D), ORF16 Q166A (E), and ORF16 S120A (F) is also shown.</p

HPLC of the yellow pigments of <i>L. enzymogenes</i> OH11.

<p>A. wild type; B. ΔORF17 (KAS I); C. ΔORF6 (ACP); D. ΔORF10 (outer membrane lipoprotein carrier protein); E. ΔORF13 (KR); F. xanthomonadin extract from <i>Xanthomonas campestris</i> pv. <i>campestris</i> (as a reference).</p