Streptomyces are an important source of natural products. In the post-genomic era, the exponential increase in genomic data and the continuous advancement in identifying biosynthetic gene clusters (BGCs) within genomes have significantly accelerated the discovery of natural products. Almost concurrently, the vast accumulation of genomic information has driven the rapid development of genomic enzymology. The emergence of EFI-genomic enzymology tools allows for the visualization and analysis of specific protein families, thereby facilitating the identification of promising biosynthetic gene clusters for natural product mining. The resulting natural products, in turn, contribute to a deeper understanding of enzyme functions and catalytic mechanisms.
Non-heme diiron oxygenases, a large family of metalloproteins, are widely distributed across all domains of life. These enzymes activate molecular oxygen via their diiron centers, generating peroxide or superoxide species to catalyze a variety of oxidation reactions. The reactions mediated by these proteins are highly diverse and play critical roles in various biological processes. Among these enzymes, there exists a unique subfamily of diiron proteins in Actinomycetes, comprising over 8,900 members that share greater than 25% sequence identity and are annotated as AurF-type Noxygenases. However, to date, only a small fraction of this subfamily has been functionally characterized, leaving the catalytic activities of the vast majority of its members largely unexplored. Therefore, this project utilizes bioinformatics analysis to identify novel functional AurF-like diiron proteins and elucidate the biosynthetic pathways in which they participate.
We initially performed sequence similarity network analysis on PF11583 family proteins, then analyzed and compared a large number of gene clusters from completely unknown clusters using the GNT tools. Among over 8,900 sequences, we identified 11 strains as preliminary research candidates. Subsequently, gene knockout experiments targeting PF11583 family diiron protein genes and biosynthetic backbone genes were performed in six selected strains. To investigate metabolic differences and identify potential natural products, wild-type strains Streptomyces prasinus CGMCC 4.1439 and Streptomyces griseoflavus CGMCC 4.1454 were co-fermented with their respective knockout mutants under various medium conditions. In H-P fermentation medium, the wild-type Streptomyces prasinus CGMCC 4.1439 exhibited the disappearance of two groups of cationic signals (m/z 618 and 636) compared to the mutant strain. The isolation and purification of these differential metabolites resulted in the discovery of four novel natural products.
One of the fundamental challenges in organic synthesis is the creation of molecules with specific chiral configurations, and the synthesis of enantiomerically pure compounds remains a critical focus in pharmaceutical research. By harnessing the inherent selectivity and efficiency of enzymes and other biocatalysts, biocatalytic asymmetric reactions offer a powerful approach for sustainable and efficient chemical synthesis. The enzymatic synthesis of cis-1,2-dihydrocatechol derivatives using toluene dioxygenase (TDO) has been extensively studied over decades, recognized for its high conversion rates, broad substrate tolerance, and the exclusive production of singleenantiomer products. Since the initial report on the biocatalytic production of cis-1,2-dihydrocatechol derivatives, synthetic chemists have demonstrated significant interest in this area. To date, over 100 natural products or their derivatives employ cis-1,2-dihydrocatechol derivatives as key starting materials for their total synthesis.To obtain structurally diverse starting materials for chemical total synthesis, we constructed an Escherichia coli whole-cell biocatalytic system expressing toluene dioxygenase (TDO) and further investigated its substrate recognition scope. Cycloalkyl structural units are prevalent in drug molecules, and growing evidence indicates that such structural units can enhance druggability by modulating multiple chemical and biological properties of molecules. However, no studies have reported the catalysis of cycloalkyl-substituted benzene substrates by TDO. To address this gap, we fed three cycloalkyl-monosubstituted benzene substrates individually into the TDO-based E. coli whole-cell biocatalytic system and successfully detected the corresponding cis-1,2-dihydrocatechol products. This finding not only broadens the substrate recognition range of toluene dioxygenase but also provides a novel and sustainable route for the production of chiral building blocks and starting materials for pharmaceutical synthesis.
