Mining the Metabolic Capacity of Clostridium sporogenes Aided by Machine Learning.

Anaerobes dominate the microbiota of the gastrointestinal (GI) tract, where a significant portion of small molecules can be degraded or modified. However, the enormous metabolic capacity of gut anaerobes remains largely elusive in contrast to aerobic bacteria, mainly due to the requirement of sophisticated laboratory settings. In this study, we employed an in silico machine learning platform, MoleculeX, to predict the metabolic capacity of a gut anaerobe, Clostridium sporogenes, against small molecules. Experiments revealed that among the top seven candidates predicted as unstable, six indeed exhibited instability in C. sporogenes culture. We further identified several metabolites resulting from the supplementation of everolimus in the bacterial culture for the first time. By utilizing bioinformatics and in vitro biochemical assays, we successfully identified an enzyme encoded in the genome of C. sporogenes responsible for everolimus transformation. Our framework thus can potentially facilitate future understanding of small molecules metabolism in the gut, further improve patient care through personalized medicine, and guide the development of new small molecule drugs and therapeutic approaches.

Polyethylene Degradation by a Rhodococcous Strain Isolated from Naturally Weathered Plastic Waste Enrichment.

Polyethylene (PE) is the most widely produced synthetic polymer and the most abundant plastic waste worldwide due to its recalcitrance to biodegradation and low recycle rate. Microbial degradation of PE has been reported, but the underlying mechanisms are poorly understood. Here, we isolated a Rhodococcus strain A34 from 609 day enriched cultures derived from naturally weathered plastic waste and identified the potential key PE degradation enzymes. After 30 days incubation with A34, 1% weight loss was achieved. Decreased PE molecular weight, appearance of C-O and C═O on PE, palmitic acid in the culture supernatant, and pits on the PE surface were observed. Proteomics analysis identified multiple key PE oxidation and depolymerization enzymes including one multicopper oxidase, one lipase, six esterase, and a few lipid transporters. Network analysis of proteomics data demonstrated the close relationships between PE degradation and metabolisms of phenylacetate, amino acids, secondary metabolites, and tricarboxylic acid cycles. The metabolic roadmap generated here provides critical insights for optimization of plastic degradation condition and assembly of artificial microbial communities for efficient plastic degradation.

Conjugative type IV secretion systems enable bacterial antagonism that operates independently of plasmid transfer.

Bacterial cooperation and antagonism mediated by secretion systems are among the ways in which bacteria interact with one another. Here we report the discovery of an antagonistic property of a type IV secretion system (T4SS) sourced from a conjugative plasmid, RP4, using engineering approaches. We scrutinized the genetic determinants and suggested that this antagonistic activity is independent of molecular cargos, while we also elucidated the resistance genes. We further showed that a range of Gram-negative bacteria and a mixed bacterial population can be eliminated by this T4SS-dependent antagonism. Finally, we showed that such an antagonistic property is not limited to T4SS sourced from RP4, rather it can also be observed in a T4SS originated from another conjugative plasmid, namely R388. Our results are the first demonstration of conjugative T4SS-dependent antagonism between Gram-negative bacteria on the genetic level and provide the foundation for future mechanistic studies.

Identification of Gut Bacterial Enzymes for Keto-Reductive Metabolism of Xenobiotics.

Human gastrointestinal microbiota are known for the keto-reductive metabolism of small-molecule pharmaceuticals; however, the responsible enzymes remain poorly understood. Through in vitro biochemical assays, we report the identification of enzymes encoded in the genome of Clostridium bolteae that can reduce the ketone groups of nabumetone, hydrocortisone, and tacrolimus. The homologues to a newly identified enzyme (i.e., DesE) are potentially widely distributed in the gut microbiome. The selected enzymes display different levels of activities against additional chemicals such as two dietary compounds (i.e., raspberry ketone and zingerone), chemotherapeutic drug doxorubicin, and its aglycone metabolite doxorubicinone. Thus, our results expand the repertoire of enzymes that can reduce the ketone groups in small molecules and could serve as the basis for future personalized medicine approaches.

An E. coli-Based Biosynthetic Platform Expands the Structural Diversity of Natural Benzoxazoles.

Benzoxazoles are frequently found in synthetic pharmaceuticals and medicinally active natural products. To facilitate benzoxazole-based drug development, an eco-friendly and rapid platform for benzoxazole production is required. In this study, we have completed the biosynthesis of benzoxazoles in E. coli by coexpressing the minimal set of enzymes required for their biosynthesis. Moreover, by coupling this E. coli-based platform with precursor-directed biosynthesis, we have shown that the benzoxazole biosynthetic system is highly promiscuous in incorporating fluorine, chlorine, nitrile, picolinic, and alkyne functionalities into the scaffold. Our E. coli-based system thus paves the way for straightforward generation of novel benzoxazole analogues through future protein engineering and combinatorial biosynthesis.