Ginsenoside Rh2(S) is well-known for its therapeutic potential against diverse conditions, including some cancers, inflammation, and diabetes. The enzymatic activity of uridine diphosphate glycosyltransferase 51 (UGT51) from Saccharomyces cerevisiae plays a pivotal role in the glycosylation process between UDP-glucose (donor) and protopanaxadiol (acceptor), to form ginsenoside Rh2. However, the catalytic efficiency of the UGT51 has remained a challenging task. To this end, we employed site-directed mutagenesis on UGT51 to improve its catalytic efficiency for enhanced production of ginsenoside Rh2. The mutated structure, featuring four key mutations (E805A, S998A, R1031A, and L1032A), exhibited heightened stability, binding affinity, and active site accessibility for protopanaxadiol (PPD) compared to the wild type. Under in vitro conditions, three mutants (E805A, R1031A, and L1032A) demonstrated 10%, 58%, and 65% higher enzymatic activities compared to the wild strain. Notably, the double mutant R1031A/L1032A exhibited an 85% increase in activity. Employing a fed-batch technology with PPD as the substrate yielded a Rh2 production of 4.663 g/L. The molecular dynamics (MD) simulations were employed to investigate the movements and dynamic dynamics of UGT51 mutations and PPD complexes. The root mean square deviation (RMSD) analysis revealed substantial alterations in structural conformation, particularly in the R1031A/L1032A mutations, correlating with boosted catalytic efficiency. Furthermore, the root mean square fluctuation (RMSF) simulation study aligned with both the RMSD and the solvent-accessible surface area (SASA) analyses. The computationally guided site-directed mutagenesis approach holds promise for extending its application to the development of commercially significant enzymes.

• Klíčová slova
• Fed-batch technology, Ginsenoside Rh2, MD simulations, Site-directed mutagenesis, Uridine diphosphate glycosyltransferase 51,
• Publikační typ
• časopisecké články MeSH

Given its highly innovative character and potential socioeconomic impact, Synthetic Biology is often ranked among prominent research areas and national research priorities in developed countries. The global evolution of this field is proceeding by leaps and bounds but its development at the level of individual states varies widely. Despite their current satisfactory economic status, the majority of 13, mostly post-communist, countries that entered the European Union family in and after 2004 (EU13) have long overlooked the blossoming of Synthetic Biology. Their prioritized lines of research have been directed elsewhere or "Synthetic Biology" did not become a widely accepted term to encompass their bioengineering and biotechnology domains. The Czech Republic is not an exception. The local SynBio mycelium already exists but is mainly built bottom-up through the activities of several academic labs, iGEM teams, and spin-off companies. In this article, we tell their individual stories and summarize the prerequisites that allowed their emergence in the Czech academic and business environment. In addition, we provide the reader with a brief overview of laboratories, research hubs, and companies that perform biotechnology and bioengineering-oriented research and that may be included in a notional "shadow SynBio community" but have not yet adopted Synthetic Biology as a unifying term for their ventures. We also map the current hindrances for a broader expansion of Synthetic Biology in the Czech Republic and suggest possible steps that should lead to the maturity of this fascinating research field in our country.

• Klíčová slova
• Biotechnology and bioengineering, Community, Czech Republic, EU13 countries, Public perception, Research landscape, Synthetic biology, iGEM,
• Publikační typ
• časopisecké články MeSH