Complete In Vitro Reconstitution of the Apramycin Biosynthetic Pathway Demonstrates the Unusual Incorporation of a ß-d-Sugar Nucleotide in the Final Glycosylation Step.

Apramycin is a widely used aminoglycoside antibiotic with applications in veterinary medicine. It is composed of a 4-amino-4-deoxy-d-glucose moiety and the pseudodisaccharide aprosamine, which is an adduct of 2-deoxystreptamine and an unusual eight-carbon bicyclic dialdose. Despite its extensive study and relevance to medical practice, the biosynthetic pathway of this complex aminoglycoside nevertheless remains incomplete. Herein, the remaining unknown steps of apramycin biosynthesis are reconstituted in vitro, thereby leading to a comprehensive picture of its biological assembly. In particular, phosphomutase AprJ and nucleotide transferase AprK are found to catalyze the conversion of glucose 6-phosphate to NDP-ß-d-glucose as a critical biosynthetic intermediate. Moreover, the dehydrogenase AprD5 and transaminase AprL are identified as modifying this intermediate via introduction of an amino group at the 4″ position without requiring prior 6″-deoxygenation as is typically encountered in aminosugar biosynthesis. Finally, the glycoside hydrolase family 65 protein AprO is shown to utilize NDP-ß-d-glucose or NDP-4"-amino-4"-deoxy-ß-d-glucose to form the 8',1″-O-glycosidic linkage of saccharocin or apramycin, respectively. As the activated sugar nucleotides in all known natural glycosylation reactions involve either NDP-α-d-hexoses or NDP-ß-l-hexoses, the reported chemistry expands the scope of known biological glycosylation reactions to NDP-ß-d-hexoses, with important implications for the understanding and repurposing of aminoglycoside biosynthesis.

Biosynthetic Origin of the Octose Core and Its Mechanism of Assembly during Apramycin Biosynthesis.

Apramycin is an aminoglycoside antibiotic isolated from Streptoalloteichus tenebrarius and S. hindustanus that has found clinical use in veterinary medicine. The apramycin structure is notable for its atypical eight-carbon bicyclic dialdose (octose) moiety. While the apramycin biosynthetic gene cluster (apr) has been identified and several of the encoded genes functionally characterized, how the octose core itself is assembled has remained elusive. Nevertheless, recent gene deletion studies have hinted at an N-acetyl aminosugar being a key precursor to the octose, and this hypothesis is consistent with the additional feeding experiments described in the present report. Moreover, bioinformatic analysis indicates that AprG may be structurally similar to GlcNAc-2-epimerase and hence recognize GlcNAc or a structurally similar substrate suggesting a potential role in octose formation. AprG with an extended N-terminal sequence was therefore expressed, purified, and assayed in vitro demonstrating that it does indeed catalyze a transaldolation reaction between GlcNAc or GalNAc and 6'-oxo-lividamine to afford 7'-N-acetyldemethylaprosamine with the same 6'-R and 7'-S stereochemistry as those observed in the apramycin product. Biosynthesis of the octose core in apramycin thus proceeds in the [6 + 2] manner with GlcNAc or GalNAc as the two-carbon donor, which has not been previously reported for biological octose formation, as well as novel inverting stereochemistry of the transferred fragment. Consequently, AprG appears to be a new transaldolase that lacks any apparent sequence similarity to the currently known aldolases and catalyzes a transaldolation for which there is no established biological precedent.

Changing Fates of the Substrate Radicals Generated in the Active Sites of the B12-Dependent Radical SAM Enzymes OxsB and AlsB.

OxsB is a B12-dependent radical SAM enzyme that catalyzes the oxidative ring contraction of 2'-deoxyadenosine 5'-phosphate to the dehydrogenated, oxetane containing precursor of oxetanocin A phosphate. AlsB is a homologue of OxsB that participates in a similar reaction during the biosynthesis of albucidin. Herein, OxsB and AlsB are shown to also catalyze radical mediated, stereoselective C2'-methylation of 2'-deoxyadenosine monophosphate. This reaction proceeds with inversion of configuration such that the resulting product also possesses a C2' hydrogen atom available for abstraction. However, in contrast to methylation, subsequent rounds of catalysis result in C-C dehydrogenation of the newly added methyl group to yield a 2'-methylidene followed by radical addition of a 5'-deoxyadenosyl moiety to produce a heterodimer. These observations expand the scope of reactions catalyzed by B12-dependent radical SAM enzymes and emphasize the susceptibility of radical intermediates to bifurcation along different reaction pathways even within the highly organized active site of an enzyme.

Metabolic engineering of E. coli for ß-alanine production using a multi-biosensor enabled approach.

ß-alanine is an important biomolecule used in nutraceuticals, pharmaceuticals, and chemical synthesis. The relatively eco-friendly bioproduction of ß-alanine has recently attracted more interest than petroleum-based chemical synthesis. In this work, we developed two types of in vivo high-throughput screening platforms, wherein one was utilized to identify a novel target ribonuclease E (encoded by rne) as well as a redox-cofactor balancing module that can enhance de novo ß-alanine biosynthesis from glucose, and the other was employed for screening fermentation conditions. When combining these approaches with rational upstream and downstream module engineering, an engineered E. coli producer was developed that exhibited 3.4- and 6.6-fold improvement in ß-alanine yield (0.85 mol ß-alanine/mole glucose) and specific ß-alanine production (0.74 g/L/OD600), respectively, compared to the parental strain in a minimal medium. Across all of the strains constructed, the best yielding strain exhibited 1.08 mol ß-alanine/mole glucose (equivalent to 81.2% of theoretic yield). The final engineered strain produced 6.98 g/L ß-alanine in a batch-mode bioreactor and 34.8 g/L through a whole-cell catalysis. This approach demonstrates the utility of biosensor-enabled high-throughput screening for the production of ß-alanine.

Two Radical SAM Enzymes Are Necessary and Sufficient for the In Vitro Production of the Oxetane Nucleoside Antiviral Agent Albucidin.

Oxetanocin A and albucidin are two oxetane natural products. While the biosynthesis of oxetanocin A has been described, less is known about albucidin. In this work, the albucidin biosynthetic gene cluster is identified in Streptomyces. Heterologous expression in a nonproducing strain demonstrates that the genes alsA and alsB are necessary and sufficient for albucidin biosynthesis confirming a previous study (Myronovskyi et al. Microorganisms 2020, 8, 237). A two-step construction of albucidin 4'-phosphate from 2'-deoxyadenosine monophosphate (2'-dAMP) is shown to be catalyzed in vitro by the cobalamin dependent radical S-adenosyl-l-methionine (SAM) enzyme AlsB, which catalyzes a ring contraction, and the radical SAM enzyme AlsA, which catalyzes elimination of a one-carbon fragment. Isotope labelling studies show that AlsB catalysis begins with stereospecific H-atom transfer of the C2'-pro-R hydrogen from 2'-dAMP to 5'-deoxyadenosine, and that the eliminated one-carbon fragment originates from C3' of 2'-dAMP.

Identification of the Enzymes Mediating the Maturation of the Seryl-tRNA Synthetase Inhibitor SB-217452 during the Biosynthesis of Albomycins.

Albomycin δ2 is a sulfur-containing sideromycin natural product that shows potent antibacterial activity against clinically important pathogens. The l-serine-thioheptose dipeptide partial structure, known as SB-217452, has been found to be the active seryl-tRNA synthetase inhibitor component of albomycin δ2 . Herein, it is demonstrated that AbmF catalyzes condensation between the 6'-amino-4'-thionucleoside with the d-ribo configuration and seryl-adenylate supplied by the serine adenylation activity of AbmK. Formation of the dipeptide is followed by C3'-epimerization to produce SB-217452 with the d-xylo configuration, which is catalyzed by the radical S-adenosyl-l-methionine enzyme AbmJ. Gene deletion suggests that AbmC is involved in peptide assembly linking SB-217452 with the siderophore moiety. This study establishes how the albomycin biosynthetic machinery generates its antimicrobial component SB-217452.

Fluorogenic Aptamer Optimizations on a Massively Parallel Sequencing Platform.

F luorogenic ap tamers (FAPs) have become an increasingly important tool in cellular sensing and pathogen diagnostics. However, fine-tuning FAPs for enhanced performance remains challenging even with the structural details provided by X-ray crystallography. Here we present a novel approach to optimize a DNA-based FAP (D-FAP), Lettuce, on repurposed Illumina next-generation sequencing (NGS) chips. When substituting its cognate chromophore, DFHBI-1T, with TO1-biotin, Lettuce not only shows a red-shifted emission peak by 53 nm (from 505 to 558 nm), but also a 4-fold bulk fluorescence enhancement. After screening 8,821 Lettuce variants complexed with TO1-biotin, the C14T mutation is found to exhibit an improved apparent dissociated constant ( vs. 0.82 µM), an increased quantum yield (QY: 0.62 vs. 0.59) and an elongated fluorescence lifetime (τ: 6.00 vs. 5.77 ns), giving 45% more ensemble fluorescence than the canonical Lettuce/TO1-biotin complex. Molecular dynamic simulations further indicate that the π-π stacking interaction is key to determining the coordination structure of TO1-biotin in Lettuce. Our screening-and-simulation pipeline can effectively optimize FAPs without any prior structural knowledge of the canonical FAP/chromophore complexes, providing not only improved molecular probes for fluorescence sensing but also insights into aptamer-chromophore interactions.