細菌代謝経路の過渡解析と工学的応用：細胞浸透ペプチド-ペプチド核酸複合体を利用して

Conventional genetic engineering approaches for bacterial metabolic pathway manipulation, although highly applicable, still face limitations including metabolic burden, irreversibility, and dependency on host cellular machinery. Cell-penetrating peptide-peptide nucleic acid conjugates (CPP-PNAs), known for their applicability as antibacterial tools and in the elucidation of protein function, offer a promising alternative to overcome such limitations. Since the application of CPP-PNA in metabolic engineering and pathway elucidation remains largely unexplored, we developed and validated a CPP-PNA platform using Synechocystis sp. PCC 6803 as a model system to demonstrate targeted metabolic pathway evaluation.

High compatibility and dose-dependent permeation efficiency in strain PCC 6803 was first observed when the amphipathic CPP (KFF)₃K was employed, achieving clear cell growth inhibition at 10 µM and above. Specific targeting of D-lactate dehydrogenase (Ddh) using CPP-Syn6803ddh conjugates achieved near-complete protein translation knockdown within 24 h, as confirmed by Western blot analysis. Metabolomics analysis using LC-MS on predetermined metabolites revealed that CPP-PNA treatment produced metabolic effects comparable to stable genetic knockout strains, with both approaches showing a significant 2.5-fold increase in pyruvate accumulation compared to wild-type controls. Further elucidating the reason for pyruvate accumulation, we observed compensatory activation of the glyoxalase pathway at 48 h post-treatment, resulting in 3-fold increased D-lactate production presumably through methylglyoxal detoxification. Validating this observation, RT-qPCR analysis confirmed 2-3-fold upregulation of the glyII gene, encoding for the glyoxalase II (GlyII) enzyme, in both CPP-PNA treated and knockout strains, while double CPP-PNA inhibition experiments targeting both Ddh and glyoxalase pathways suppressed D-lactate accumulation.

This study establishes CPP-PNAs as efficient tools for rapid, and simple metabolic pathway investigation. The approach produces results comparable to conventional genetic knockouts while offering dose-dependent control and avoiding permanent genomic alterations. Our findings reveal unexpected metabolic complexity in Synechocystis sp. PCC 6803 D-lactate synthesis under light conditions and demonstrate the utility of CPP-PNA for uncovering compensatory pathway activation. This platform represents a valuable addition to bacterial genetic engineering, addressing some of the critical limitations faced by conventional approaches, while showing potential for further understanding the biochemistry of metabolite-producing bacteria.