Biosynthesis of the bacterial antibiotic 3,7-dihydroxytropolone and its potential role for iron-acquisition

Natural products serve as promising drug leads due to their manifold and potent bioactivities. Tropone natural products with their unusual seven-membered aromatic ring exhibit antibacterial, antiviral and anticancer activities and therefore mark a promising group of compounds for further investigation. In the past, their biosynthesis in bacteria has been linked to phenylacetic acid degradation, where a shunt product was identified as putative universal precursor for tropone natural products such as 3,7-dihydroxytropolone (3,7-DHT). Since then, considerable efforts have been made to elucidate the downstream processing of this precursor, and although the involvement of a few individual enzymes has been demonstrated, a complete biosynthetic pathway for such a tropone natural product had so far not been elucidated.
In this work, the biosynthetic pathway for 3,7-DHT of the Gram-positive Actinobacterium Streptomyces cyaneofuscatus Soc7 could be reconstituted in vitro for the first time. Furthermore, in a collaborative effort, some insights into the biosynthesis of 3,7-DHT in Gram-negative Pseudomonas sp. Ps652 could also be obtained, which surprisingly proceeds via a different enzymatic route. Additionally, a siderophore-interacting protein (SIP), that likely facilitates iron-acquisition using tropone natural products and/or grants self-resistance against these compounds could be identified and characterized. Structural and biochemical properties of all enzymes were investigated using a combination of methods including high-performance liquid chromatography (HPLC), mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy and protein X-ray crystallography, leading to the main findings that are summarized below:
- Enzymes encoded by genes from the trl gene cluster of Gram-positive Streptomyces cyaneofuscatus Soc 7 including the putative enoyl-CoA hydratase TrlA, thioesterase TrlF, NADH- and FAD-dependent two-component flavoprotein monooxygenase (FPMO) TrlCD and NADPH- and FAD-dependent FPMO TrlE were heterologously produced in Escherichia coli BL21 pL1SL2 and subsequently purified via affinity chromatography. A protein crystal structure of TrlE could be obtained, which allowed further structural characterization. (Part I)
- HPLC and MS-measurements as well as NMR spectroscopy allowed the identification of reaction intermediates and products from in vitro assays with these enzymes. This enabled the full reconstitution of the 3,7-DHT biosynthetic pathway. Accordingly, TrlF cleaves the CoA-ester bond of the tropone natural product precursor from PAA catabolism. TrlE performs an ipso-substitution of a carboxylic acid group with a hydroxyl group (decarboxylation coupled to hydroxylation) and further catalyzes a ring-oxidation to yield tropolone. TrlCD then performs two subsequent ring-hydroxylations of tropolone to afford 7-hydroxytropolone (7-HT) and finally 3,7-DHT. Only TrlA showed no enzymatic activity in vitro under the tested conditions and was therefore substituted by a previously characterized enoyl-CoA hydratase (PaaZ-E256Q variant) from PAA catabolism. (Part I)
- In a collaboration with the group of Prof. Dr. Truman from the John Innes Center in Norwich, UK, the biosynthesis of tropone natural products in Gram-negative Pseudomonas sp. Ps652 was investigated. The results revealed that Pseudomonas sp. Ps652 is able to produce 3,7-DHT and that enzymes encoded in the tpo gene cluster play crucial roles in the biosynthesis. My contribution to this study, assisted by a master student, included the heterologous expression of the FPMO TpoE and the thiosterase TpoD from Pseudomonas sp. Ps652 in Escherichia coli BL21 and the subsequent purification via affinity chromatography. Furthermore, I conducted HPLC- and MS-measurements of the in vitro assays, which revealed that these enzymes work together to convert the tropone precursor from PAA catabolism to a likely on-pathway intermediate for 3,7-DHT biosynthesis. (Part II)
- During the investigation of the genomic environment of the trl gene cluster in Actinobacteria, a co-located gene encoding a predicted SIP could be identified in multiple bacteria. The SIP from the Gram-positive Actinobacterium Amycolatopsis regifaucium was heterologously produced in Escherichia coli BL21 pL2SL2 and subsequently purified via affinity chromatography. Thermal shift measurements as well as photometric assays revealed that this enzyme is able to reduce (di)hydroxytropolone- Fe3+ complexes and thereby provide Fe2+ for the cell. Moreover, a protein crystal structure of this SIP could be obtained and allowed further structural investigation. The combined findings from the enzymatic assays and the protein structure suggests that the current categorization of SIPs should be revisited, as the investigated enzyme showed unusual properties regarding cofactor specificity and structural composition, which do not fit the in the current categorization system similar to other recently reported SIPs. (Part III)
In summary this thesis provides insights into the biosynthesis of tropone natural products, especially 3,7-DHT, for both Gram-negative and Gram-positive bacteria. In particular, the biosynthesis in Gram-positive bacteria could be elucidated by reconstituting the complete pathway in vitro starting from phenylacetyl-CoA. Furthermore, the identification and characterization of a SIP associated with tropone natural product biosynthesis will facilitate future studies on the ecological role of 3,7-DHT and related compounds in iron-acquisition and possibly self-resistance.