Identification of Kibdelomycin and Related Biosynthetic Gene Clusters and Characterization of the C-Branching of Amycolose.

Many bacterial natural products contain C-branched sugars, including components from the outer cell wall or antibiotically active metabolites. The enzymatic C-branching of keto sugars leading to longer side chains (≥C2) is catalyzed by thiamine diphosphate (ThDP)-dependent enzymes. Chiral tertiary α-hydroxy ketones are formed in this process. The ThDP-dependent enzymes that catalyze C-branching reactions belong to one of three enzymatic superfamilies: decarboxylases, transketolases, and α-ketoacid dehydrogenases 2, but branching of keto sugars has only been demonstrated for decarboxylases. In this study, we showed that an α-ketoacid dehydrogenase is responsible for C-branching of the deoxyketo sugar amycolose in the biosynthesis of kibdelomycin in Kibdelosporangium sp. MA7385. In addition, we characterized an amino transferase in the same biosynthetic gene cluster (BGC) that accepts a sterically demanding tertiary α-hydroxy ketone in a downstream reaction. Subsequently, we identified approximately 400 similar BGCs in silico, suggesting that there is a large diversity of possible ThDP-dependent enzymes catalyzing the C-branching of keto sugars and subsequent modifications.

Preparative Production of Functionalized (N- and O-Heterocyclic) Polycyclic Aromatic Hydrocarbons by Human Cytochrome P450 3A4 in a Bioreactor.

Polycyclic aromatic hydrocarbons (PAHs) and their N- and O-containing derivatives (N-/O-PAHs) are environmental pollutants and synthetically attractive building blocks in pharmaceuticals. Functionalization of PAHs can be achieved via C-H activation by cytochrome P450 enzymes (e.g., P450 CYP3A4) in an environmentally friendly manner. Despite its broad substrate scope, the contribution of CYP3A4 to metabolize common PAHs in humans was found to be small. We recently showcased the potential of CYP3A4 in whole-cell biocatalysis with recombinant yeast Komagataella phaffii (Pichia pastoris) catalysts for the preparative-scale synthesis of naturally occurring metabolites in humans. In this study, we aimed at exploring the substrate scope of CYP3A4 towards (N-/O)-PAHs and conducted a bioconversion experiment at 10 L scale to validate the synthetic potential of CYP3A4 for the preparative-scale production of functionalized PAH metabolites. Hydroxylated products were purified and characterized using HPLC and NMR analysis. In total, 237 mg of fluorenol and 48 mg of fluorenone were produced from 498 mg of fluorene, with peak productivities of 27.7 µmol/L/h for fluorenol and 5.9 µmol/L/h for fluorenone; the latter confirmed that CYP3A4 is an excellent whole-cell biocatalyst for producing authentic human metabolites.

Regiospecific 7-hydroxylation of ten-carbon monoterpenes by detoxifying CYP5035S7 monooxygenase of the white-rot fungus Polyporus arcularius.

In a previous study, we identified CYP5035S7 of the white-rot fungus Polyporus arcularius with a broad activity towards monoterpenes such as p-cymene. Therefore, in this study we aimed at further exploring the substrate scope of detoxifying CYP5035S7 towards terpenes and semi-preparatively isolating some of the products via whole-cell biotransformation, in order to obtain information about the enzyme's reactivity. We noticed a clear preference for the monoterpene skeleton and elucidated a distinct regioselectivity pattern based on key structural and electronic features of its substrates. This study illustrates how minimal characterisation effort may already suffice to provide vital information on enzymatic reactivity by the comparison of structural derivatives.

Late-Stage Functionalisation of Polycyclic (N-Hetero-) Aromatic Hydrocarbons by Detoxifying CYP5035S7 Monooxygenase of the White-Rot Fungus Polyporus arcularius.

Functionalisation of polycyclic aromatic hydrocarbons (PAHs) and their N-heteroarene analogues (NPAHs) is a tedious synthetic endeavour that requires diverse bottom-up approaches. Cytochrome P450 enzymes of white-rot fungi were shown to participate in the fungal detoxification of xenobiotics and environmental hazards via hydroxylation of PAH compounds. In this paper, the recently discovered activity of the monooxygenase CYP5035S7 towards (N)PAHs was investigated in detail, and products formed from the substrates azulene, acenaphthene, fluorene, anthracene, and phenanthrene by whole-cell biocatalysis were isolated and characterised. The observed regioselectivity of CYP5035S7 could be explained by a combination of the substrate's electron density and steric factors influencing the substrate orientation giving insight into the active-site geometry of the enzyme.

Evolution and enrichment of CYP5035 in Polyporales: functionality of an understudied P450 family.

Bioprospecting for innovative basidiomycete cytochrome P450 enzymes (P450s) is highly desirable due to the fungi's enormous enzymatic repertoire and outstanding ability to degrade lignin and detoxify various xenobiotics. While fungal metagenomics is progressing rapidly, the biocatalytic potential of the majority of these annotated P450 sequences usually remains concealed, although functional profiling identified several P450 families with versatile substrate scopes towards various natural products. Functional knowledge about the CYP5035 family, for example, is largely insufficient. In this study, the families of the putative P450 sequences of the four white-rot fungi Polyporus arcularius, Polyporus brumalis, Polyporus squamosus and Lentinus tigrinus were assigned, and the CYPomes revealed an unusual enrichment of CYP5035, CYP5136 and CYP5150. By computational analysis of the phylogeny of the former two P450 families, the evolution of their enrichment could be traced back to the Ganoderma macrofungus, indicating their evolutionary benefit. In order to address the knowledge gap on CYP5035 functionality, a representative subgroup of this P450 family of P. arcularius was expressed and screened against a test set of substrates. Thereby, the multifunctional enzyme CYP5035S7 converting several plant natural product classes was discovered. Aligning CYP5035S7 to 102,000 putative P450 sequences of 36 fungal species from Joint Genome Institute-provided genomes located hundreds of further CYP5035 family members, which subfamilies were classified if possible. Exemplified by these specific enzyme analyses, this study gives valuable hints for future bioprospecting of such xenobiotic-detoxifying P450s and for the identification of their biocatalytic potential. KEY POINTS: • The P450 families CYP5035 and CYP5136 are unusually enriched in P. arcularius. • Functional screening shows CYP5035 assisting in the fungal detoxification mechanism. • Some Polyporales encompass an unusually large repertoire of detoxification P450s.

P450 Monooxygenases Enable Rapid Late-Stage Diversification of Natural Products via C-H Bond Activation.

The biological potency of natural products has been exploited for decades. Their inherent structural complexity and natural diversity might hold the key to efficiently address the urgent need for the development of novel pharmaceuticals. At the same time, it is that very complexity, which impedes necessary chemical modifications such as structural diversification, to improve the effectiveness of the drug. For this purpose, Cytochrome P450 enzymes, which possess unique abilities to activate inert sp3-hybridised C-H bonds in a late-stage fashion, offer an attractive synthetic tool. In this review the potential of cytochrome P450 enzymes in chemoenzymatic lead diversification is illustrated discussing studies reporting late-stage functionalisations of natural products and other high-value compounds. These enzymes were proven to extend the synthetic toolbox significantly by adding to the flexibility and efficacy of synthetic strategies of natural product chemists, and scientists of other related disciplines.

Sensorimotor pathway controlling stopping behavior during chemotaxis in the Drosophila melanogaster larva.

Sensory navigation results from coordinated transitions between distinct behavioral programs. During chemotaxis in the Drosophila melanogaster larva, the detection of positive odor gradients extends runs while negative gradients promote stops and turns. This algorithm represents a foundation for the control of sensory navigation across phyla. In the present work, we identified an olfactory descending neuron, PDM-DN, which plays a pivotal role in the organization of stops and turns in response to the detection of graded changes in odor concentrations. Artificial activation of this descending neuron induces deterministic stops followed by the initiation of turning maneuvers through head casts. Using electron microscopy, we reconstructed the main pathway that connects the PDM-DN neuron to the peripheral olfactory system and to the pre-motor circuit responsible for the actuation of forward peristalsis. Our results set the stage for a detailed mechanistic analysis of the sensorimotor conversion of graded olfactory inputs into action selection to perform goal-oriented navigation.

Kinetic Resolution of 2-Substituted Indolines by N-Sulfonylation using an Atropisomeric 4-DMAP-N-oxide Organocatalyst.

The first catalytic kinetic resolution by N-sulfonylation is described. 2-Substituted indolines are resolved (s=2.6-19) using an atropisomeric 4-dimethylaminopyridine-N-oxide (4-DMAP-N-oxide) organocatalyst. Use of 2-isopropyl-4-nitrophenylsulfonyl chloride is critical to the stereodiscrimination and enables facile deprotection of the sulfonamide products with thioglycolic acid. A qualitative model that accounts for the stereodiscrimination is proposed.