Hataya, S. | Search bioRxiv

bioRxiv2026

Nitrogen metabolic map of Escherichia coli mediated by multi-functional aminotransferases

Nitrogen is a key element of organic molecules essential to all life. Unlike extensively characterized carbon metabolic maps, it remains elusive how assimilated nitrogen flows through metabolic networks. Here we determined the nitrogen metabolic map of Escherichia coli and used it to construct an enzyme-constrained metabolic model that includes multi-functionality of aminotransferase enzymes responsible for nitrogen transfer reactions. We characterized substrate specificities of sixteen E. coli aminotransferase enzymes by evaluating 2,528 reactions, uncovering 56 previously unrecognized activities. The cellular concentrations of these aminotransferase enzymes were quantified and used to estimate their catalytic rates across all enzyme-substrate pairs, leading to improved predictions of nitrogen flows in E. coli. This work advances our fundamental understanding of the nitrogen metabolic map critical for metabolism and growth.

biochemistry↗

bioRxiv2026

Reducing encapsidated impurity DNA derived from plasmid backbone by modifying the p5 terminal resolution site in rAAV vector production

Recombinant adeno-associated virus (rAAV) vectors are pivotal for gene therapy; however, the encapsidation of residual DNA, particularly plasmid backbone sequences, pose significant safety risks. Recent studies have identified the p5 promoter, which contains a Rep-binding element and a terminal resolution site (TRS), as a cryptic origin of replication that facilitates packaging of upstream sequences. In this study, we investigated the effect of p5 TRS modifications on impurity DNA levels in a single-plasmid All-in-One (AiO) AAV production system. Wild-type p5 (p5wt) promoted significant packaging of upstream plasmid backbone DNA, especially when the backbone was positioned between p5wt and the inverted terminal repeat. Introducing mutations or deletions in the p5 TRS significantly reduced encapsidation of plasmid-derived sequences, including kanamycin resistance genes, and improved the ratio of full to partial particles, as seen with the p5{Delta}loop variant. Furthermore, the p5{Delta}loop-AiO system showed higher rAAV yields than both conventional triple-transfection methods and previously reported p5-spacer variants. Thus, our findings suggest a robust vector design strategy for minimizing DNA impurities, thereby enhancing the safety and efficacy of AAV-based gene therapy.

bioengineering↗