DC-355/B1

This study aims to identify potential RNA polymerase (RNAP) inhibitors using a comprehensive computational approach, addressing the challenges in drug discovery related to stability, affinity, and accurate binding predictions.

The research workflow involved virtual screening to narrow down candidate compounds, molecular docking to predict optimal binding poses, molecular dynamics (MD) simulations to evaluate interaction stability over time, and MM-PBSA analysis to calculate binding energies. These steps ensured that only compounds with strong and stable binding profiles were selected for further evaluation.

The selected compounds, ZINC001286671821, ZINC000253654686, and ZINC000252693842, demonstrated varying degrees of stability and affinity. MM-PBSA analysis revealed that ZINC000252693842 had the most favourable binding energy at -106.097 ± 24.664 kJ/mol, followed by ZINC001286671821 at -89.201 ± 22.647 kJ/mol, and ZINC000253654686 at -43.832 ± 23.748 kJ/mol. Van der Waals forces were the main contributors to stability, with values of -221.032 ± 27.721 kJ/mol, -187.136 ± 23.796 kJ/mol, and -157.232 ± 19.676 kJ/mol, respectively. These findings confirm the strong binding potential of ZINC000252693842 as an RNAP inhibitor.

This study highlights the effectiveness of combining virtual screening, molecular docking, MD simulations, and MM-PBSA analysis in identifying promising RNAP inhibitors. The results establish a strong foundation for further experimental validation, advancing the development of effective therapeutic agents targeting RNA polymerase.

Modulating resource allocation in bacteria to redirect metabolic building blocks to the formation of recombinant proteins rather than biomass formation remains a grand challenge in biotechnology. Here, we present a novel approach for improved recombinant protein production (RPP) using Escherichia coli (E. coli) by decoupling recombinant protein synthesis from cell growth. We show that cell division and host mRNA transcription can be successfully inhibited by coexpression of a bacteriophage-derived E. coli RNA polymerase (RNAP) inhibitor peptide and that genes overtranscribed by the orthogonal T7 RNAP can finally account to >55% of cell dry mass (CDM). This RNAP inhibitor peptide binds the E. coli RNAP and therefore prevents σ-factor 70 mediated formation of transcriptional qualified open promoter complexes. Thereby, the transcription of σ-factor 70 driven host genes is inhibited, and metabolic resources can be exclusively utilized for synthesis of the protein of interest (POI). Here, we mimic the late phase of bacteriophage infection by coexpressing a phage-derived xenogeneic regulator that reprograms the host cell and thereby are able to significantly improve RPP under industrial relevant fed-batch process conditions at bioreactor scale. We have evaluated production of several different recombinant proteins at different scales (from microscale to 20 L fed-batch scale) and have been able to improve total and soluble proteins yields up to 3.4-fold in comparison to the reference expression system E. coli BL21(DE3). This novel approach for growth-decoupled RPP has profound implications for biotechnology and bioengineering and helps to establish more cost-effective and generic manufacturing processes for biologics and biomaterials.