Recent Advances in Phenazine Natural Products: Biosynthesis and Metabolic Engineering.

Phenazine natural products are a class of nitrogen-containing heterocyclic compounds produced by microorganisms. The tricyclic ring molecules show various chemical structures and extensive pharmacological activities, such as antimicrobial, anticancer, antiparasitic, anti-inflammatory, and insecticidal activities, with low toxicity to the environment. Since phenazine-1-carboxylic acid has been developed as a registered biopesticide, the application of phenazine natural products will be promising in the field of agriculture pathogenic fungi control based on broad-spectrum antifungal activity, minimal toxicity to the environment, and improvement of crop production. Currently, there are still plenty of intriguing hidden biosynthetic pathways of phenazine natural products to be discovered, and the titer of naturally occurring phenazine natural products is insufficient for agricultural applications. In this review, we spotlight the progress regarding biosynthesis and metabolic engineering research of phenazine natural products in the past decade. The review provides useful insights concerning phenazine natural products production and more clues on new phenazine derivatives biosynthesis.

Aspertides A-E: Antimicrobial Pentadepsipeptides with a Unique p-Methoxycinnamoyl Amide Group from the Marine Isolates Aspergillus tamarii MA-21 and Aspergillus insuetus SD-512.

Marine fungus-derived natural products are an important source of antimicrobial compounds against marine aquatic pathogens. Here, we describe the isolation and characterization of five new pentadepsipeptides, aspertides A-E (1-5), containing a unique p-methoxycinnamoyl amide group, from the marine fungi Aspergillus tamarii MA-21 and Aspergillus insuetus SD-512. Among them, aspertides B-E (2-5) also possessed uncommon amino acid residues, such as 3-hydroxyproline, 2,3-dihydroxyproline, or pipecolinic acid. The structures of these compounds were elucidated on the basis of NMR and mass spectroscopic analyses. The absolute configurations of them were established by chiral HPLC analyses of the acidic hydrolysates and NMR calculations with DP4+ probability analysis. In bio-activity assays, compounds 4 and 5 exhibited antibacterial activities against aquatic-pathogenic bacteria, including Edwardsiella tarda, Vibrio alginolyticus, Vibrio anguillarum, Vibrio vulnificus, and Staphylococcus aureus, with MIC values of 8-32 µg/mL.

Biosynthetic Pathway Construction and Production Enhancement of 1-Hydroxyphenazine Derivatives in Pseudomonas chlororaphis H18.

1-Hydroxyphenazine derivatives are phenazine family chemicals with broad-spectrum antibacterial and potential biological activities. However, the lack of variety and low titer hinder their applications. In this research, three enzymes PhzS (monooxygenase), NaphzNO1 (N-monooxygenase), and LaphzM (methyltransferase) were heterologously expressed in a phenazine-1-carboxylic acid generating strain Pseudomonas chlororaphis H18. Four phenazines, 1-hydroxyphenazine, 1-methoxyphenazine, 1-hydroxyphenazine N' 10-oxide, and a novel phenazine derivative 1-methoxyphenazine N' 10-oxide, were isolated, characterized in the genetically modified strains, and exhibited excellent antimicrobial activities. Next, we verified the hydroxyl methylation activity of LaphzM and elucidated the biosynthetic pathway of 1-methoxyphenazine N' 10-oxide in vitro. Moreover, the titer of 1-hydroxyphenazine derivatives was engineered. The three compounds 1-methoxyphenazine, 1-hydroxyphenazine N' 10-oxide, and 1-methoxyphenazine N' 10-oxide all reach the highest titer reported to date. This work provides a promising platform for phenazine derivatives' combinatorial biosynthesis and engineering.

Biosynthesis and metabolic engineering of 1-hydroxyphenazine in Pseudomonas chlororaphis H18.

BACKGROUND: 1-Hydroxyphenazine (1-OH-PHZ) is a phenazine microbial metabolite with broad-spectrum antibacterial activities against a lot of plant pathogens. However, its use is hampered by the low yield all along. Metabolic engineering of microorganisms is an increasingly powerful method for the production of valuable organisms at high levels. Pseudomonas chlororaphis is recognized as a safe and effective plant rhizosphere growth-promoting bacterium, and faster growth rate using glycerol or glucose as a renewable carbon source. Therefore, Pseudomonas chlororaphis is particularly suitable as the chassis cell for the modification and engineering of phenazines. RESULTS: In this study, enzyme PhzS (monooxygenase) was heterologously expressed in a phenazine-1-carboxylic acid (PCA) generating strain Pseudomonas chlororaphis H18, and 1-hydroxyphenazine was isolated, characterized in the genetically modified strain. Next, the yield of 1-hydroxyphenazine was systematically engineered by the strategies including (1) semi-rational design remodeling of crucial protein PhzS, (2) blocking intermediate PCA consumption branch pathway, (3) enhancing the precursor pool, (4) engineering regulatory genes, etc. Finally, the titer of 1-hydroxyphenazine reached 3.6 g/L in 5 L fermenter in 54 h. CONCLUSIONS: The 1-OH-PHZ production of Pseudomonas chlororaphis H18 was greatly improved through systematically engineering strategies, which is the highest, reported to date. This work provides a promising platform for 1-hydroxyphenazine engineering and production.

Two New Phenol Derivatives from the Cold Seep-Derived Fungus Aspergillus insuetus SD-512.

Two new phenol derivatives, namely insphenol A (1) and acetylpeniciphenol (2), along with seven known analogs (3-9), were isolated from the deep-sea cold seep-derived fungus, Aspergillus insuetus SD-512. The structures of 1 and 2 were established by extensive interpretation of NMR and mass spectroscopic data. The absolute configuration of 1 was determined by the combination of coupling constant analysis and acid hydrolysis. Among the isolated compounds, insphenol A (1) represents the first example of isopentenyl phenol derivative with a unique 1-glycosylation from the species Aspergillus insuetus. The isolated new compounds were evaluated for antibacterial activities against six human or aquatic pathogens, while compound 2 exhibited inhibitory effect against Edwardsiella tarda, Vibrio alginolyticus, and V. vulnificus, with MIC values of 4, 8, and 8 µg/mL, respectively.

Ophiobolin Sesterterpenoids and Farnesylated Phthalide Derivatives from the Deep Sea Cold-Seep-Derived Fungus Aspergillus insuetus SD-512.

Three new ophiobolin sesterterpenoids, (6R)-16,17,21,21-O-tetrahydroophiobolin G (1), (6R)-16,17-dihydroophiobolin H (2), and (5S,6S)-16,17-dihydroophiobolin H (3), and three new farnesylated phthalide derivatives farnesylemefuranones D-F (9-11), along with five known ophiobolin analogues (4-8), were isolated and identified from the culture extract of Aspergillus insuetus SD-512, a deep-sea-derived fungus obtained from cold seep sediments collected at a depth of 1331 m. Among them, compounds 9-11 are rare examples of phthalide derivatives linked with farnesyl moieties via ether bonds. Their structures were established on the basis of detailed interpretation of the NMR spectroscopic and mass spectrometric data. X-ray crystallographic analysis, ECD calculations, and DP4+ probability analysis were performed to confirm the structures and establish the relative and absolute configurations of compounds 1-4. Compounds 3 and 9-11 showed broad-spectrum antibacterial activities, and differences in potencies could be assigned to structural modifications. This is the first report of secondary metabolites obtained from a deep sea cold-seep-derived fungus.

Identification of 8-Azaguanine Biosynthesis-Related Genes Provides Insight Into the Enzymatic and Non-enzymatic Biosynthetic Pathway for 1,2,3-Triazole.

8-Azaguanine (1) is a special 1,2,3-triazole containing natural product that possesses potent antibacterial and antitumor activities. In the present study, the entire 8-azaguanine biosynthetic gene cluster was located from Streptomyces CGMCC4.1633. Targeted gene disruption, heterologous expression analysis, and feeding experiments identified crucial genes for 8-azaguanine production. Moreover, we characterized the structure of two novel metabolites, analyzed NO (or reactive nitrogen species) related genes 8-azgA/B and radical SAM enzyme homologous 8-AzgG, and verified the non-enzymatic ring formation reaction of 8-azaguanine 1,2,3-triazole. All of the data and presumptions provide insight into the timing and mechanism of the enzymatic and non-enzymatic pathway that produce 8-azaguanine-type 1,2,3-triazole.