ACS Synthetic Biology最新文献

P450 enzymes are promising biocatalysts and play an important role in the field of drug synthesis due to their high catalytic activity and stereoselectivity. CYP109E1 from Bacillus megaterium was used to convert VD₃ for the production of 25(OH)VD₃. However, the industrial production was still limited due to the low catalytic performance of CYP109E1. To overcome this, we constructed an engineered strain containing a modified CYP109E1 coupled with an efficient electron transfer chain and NADPH regeneration system. First, Adx_4-108T69E-Fpr was identified as the most compatible redox partner for the enzyme based on in-silico analysis. Then, targeted mutations were introduced at the substrate channel of CYP109E1, resulting in higher production efficiency. Next, the production of 25(OH)VD₃ was increased by 13.1% after introducing a double Adx_4-108T69E expression cassette. Finally, an NADPH regeneration system was introduced by overexpressing zwf, which increased the yield of 25(OH)VD₃ 48.7%. These results demonstrate that recombinant Escherichia coli BL21 (DE3) coexpressing CYP109E1_R70A-ZWF and 2Adx_4-108T69Es-Fpr is an efficient whole-cell biocatalyst for the synthesis of 25(OH)VD₃, illustrating an attractive strategy for improving the catalytic efficiency of P450 enzymes.

Advances in genome engineering of fungal strains are rapidly progressing, driven by the increasing interest in fungal biotechnology. Given the unique genomic and cellular complexity of fungi, each strain benefits from a tailored toolkit for efficient genome engineering. Here, we present a transposon-based engineering toolkit specifically optimized for Wolfiporia cocos, a species valued for its bioactive compounds. This toolkit significantly improves transformation efficiency, enabling multiplexed gene integration and facilitating rapid, flexible prototyping by assembling multiple genes into transposomes in a cocktail format, which bypasses the need for an intricate genetic circuit assembly. Engineered strains demonstrated stable expression across generations, as confirmed by successful genomic integration. Additionally, we identified six native W. cocos promoters from transcriptomic data, with two showing robust, constitutive expression in the mycelium of engineered strains. This transposon-based toolkit offers a versatile resource for synthetic biology, supporting efficient and adaptable genetic modifications within fungal systems.

Marine natural products are promising sources for drug discovery due to their unique structures and diverse biological activities. The establishment of the Global Marine Microbiome Genome Catalogue (GOMC) has significantly expanded the repository of natural products derived from marine-associated bacteria. In this study, we identified the Class I lanthipeptide biosynthetic gene cluster col from Colwellia_A sp. based on the GOMC database. Through heterologous expression in Escherichia coli and subsequent structural analysis, we characterized three novel lanthipeptides, colwesins A-C, which possess unique cyclic structures characterized by an exceptionally large number of thioether rings. To the best of our knowledge, colwesin C is the first lanthipeptide simultaneously containing locked, nonoverlapped, and nested ring topologies. These findings highlight the robust ring-forming capacity of Class I lanthipeptide synthetases. Colwesins A-C were found to exhibit anti-inflammatory activity in lipopolysaccharide-induced mouse macrophage RAW264.7 cell lines without detectable cytotoxicity. Overall, our results broaden our understanding of the structural diversity of marine-derived lanthipeptides.

Marine natural products are promising sources for drug discovery due to their unique structures and diverse biological activities. The establishment of the Global Marine Microbiome Genome Catalogue (GOMC) has significantly expanded the repository of natural products derived from marine-associated bacteria. In this study, we identified the Class I lanthipeptide biosynthetic gene cluster col from Colwellia_A sp. based on the GOMC database. Through heterologous expression in Escherichia coli and subsequent structural analysis, we characterized three novel lanthipeptides, colwesins A–C, which possess unique cyclic structures characterized by an exceptionally large number of thioether rings. To the best of our knowledge, colwesin C is the first lanthipeptide simultaneously containing locked, nonoverlapped, and nested ring topologies. These findings highlight the robust ring-forming capacity of Class I lanthipeptide synthetases. Colwesins A–C were found to exhibit anti-inflammatory activity in lipopolysaccharide-induced mouse macrophage RAW264.7 cell lines without detectable cytotoxicity. Overall, our results broaden our understanding of the structural diversity of marine-derived lanthipeptides.

Advances in genome engineering of fungal strains are rapidly progressing, driven by the increasing interest in fungal biotechnology. Given the unique genomic and cellular complexity of fungi, each strain benefits from a tailored toolkit for efficient genome engineering. Here, we present a transposon-based engineering toolkit specifically optimized for Wolfiporia cocos, a species valued for its bioactive compounds. This toolkit significantly improves transformation efficiency, enabling multiplexed gene integration and facilitating rapid, flexible prototyping by assembling multiple genes into transposomes in a cocktail format, which bypasses the need for an intricate genetic circuit assembly. Engineered strains demonstrated stable expression across generations, as confirmed by successful genomic integration. Additionally, we identified six native W. cocos promoters from transcriptomic data, with two showing robust, constitutive expression in the mycelium of engineered strains. This transposon-based toolkit offers a versatile resource for synthetic biology, supporting efficient and adaptable genetic modifications within fungal systems.

Bacteria utilize two-component system (TCS) signal transduction pathways to sense environmental and physiological stimuli and mount appropriate responses. In opportunistic pathogens such as Staphylococcus aureus, TCSs activate virulence programs in response to host defense systems. Due to their critical role in pathogenesis, TCSs are important targets for antivirulence drug discovery campaigns. However, challenges associated with screening TCSs in pathogens and in vitro have limited the output of such efforts to a small number of characterized drug candidates. Here, we functionally express the S. aureus virulence-regulating TCS SaeRS from synthetic gene regulatory elements in the model bacterium Bacillus subtilis to reliably screen this system against a small molecule library under simple culturing conditions. Our approach reveals the compound NSC97920 as a strong inhibitor of SaeRS signaling. We combine in situ, in vivo, in silico, and in vitro characterization to demonstrate that NSC97920 suppresses the critical step of autophosphorylation in the SaeS histidine kinase, resulting in strong antivirulence activity. Our work shows that heterologous expression and screening of TCSs in model bacteria could accelerate the development of therapeutics against antibiotic-resistant pathogens.

Bacteria utilize two-component system (TCS) signal transduction pathways to sense environmental and physiological stimuli and mount appropriate responses. In opportunistic pathogens such as Staphylococcus aureus, TCSs activate virulence programs in response to host defense systems. Due to their critical role in pathogenesis, TCSs are important targets for antivirulence drug discovery campaigns. However, challenges associated with screening TCSs in pathogens and in vitro have limited the output of such efforts to a small number of characterized drug candidates. Here, we functionally express the S. aureus virulence-regulating TCS SaeRS from synthetic gene regulatory elements in the model bacterium Bacillus subtilis to reliably screen this system against a small molecule library under simple culturing conditions. Our approach reveals the compound NSC97920 as a strong inhibitor of SaeRS signaling. We combine in situ, in vivo, in silico, and in vitro characterization to demonstrate that NSC97920 suppresses the critical step of autophosphorylation in the SaeS histidine kinase, resulting in strong antivirulence activity. Our work shows that heterologous expression and screening of TCSs in model bacteria could accelerate the development of therapeutics against antibiotic-resistant pathogens.

l-Fucose, a functional monosaccharide with significant commercial potential in the pharmaceutical, nutraceutical, and cosmetic industries, faces challenges in microbial production due to antibiotic-dependent plasmid maintenance systems. This study presents a dual antibiotic-free plasmid strategy in engineered Escherichia coli BL21(DE3) to achieve high-efficiency l-fucose biosynthesis. By integration of the hok/sok toxin-antitoxin system and a cysC-based auxotrophic selection into two plasmids, genetic stability and plasmid retention were ensured without antibiotics. Metabolic pathway optimization involved enhancing GDP-l-fucose supply via promoter replacements, genomic integration of key enzymes (α1,2-fucosyltransferase and α-l-fucosidase), and blocking l-fucose degradation. The engineered strain demonstrated robust performance, producing 7.99 g/L of l-fucose in shake-flask fermentation and 61.91 g/L via fed-batch cultivation─both antibiotic-free. This titer represents the highest reported l-fucose yield to date, highlighting the effectiveness of combining toxin-antitoxin and auxotrophic systems for sustainable, high-productivity microbial manufacturing.

l-Fucose, a functional monosaccharide with significant commercial potential in the pharmaceutical, nutraceutical, and cosmetic industries, faces challenges in microbial production due to antibiotic-dependent plasmid maintenance systems. This study presents a dual antibiotic-free plasmid strategy in engineered Escherichia coli BL21(DE3) to achieve high-efficiency l-fucose biosynthesis. By integration of the hok/sok toxin-antitoxin system and a cysC-based auxotrophic selection into two plasmids, genetic stability and plasmid retention were ensured without antibiotics. Metabolic pathway optimization involved enhancing GDP-l-fucose supply via promoter replacements, genomic integration of key enzymes (α1,2-fucosyltransferase and α-l-fucosidase), and blocking l-fucose degradation. The engineered strain demonstrated robust performance, producing 7.99 g/L of l-fucose in shake-flask fermentation and 61.91 g/L via fed-batch cultivation─both antibiotic-free. This titer represents the highest reported l-fucose yield to date, highlighting the effectiveness of combining toxin–antitoxin and auxotrophic systems for sustainable, high-productivity microbial manufacturing.

Synthetic biology provides a powerful approach to functional studies of viral and microbial genomes. However, in vitro, efficient and scarless DNA manipulation on large and complex human genomes remains an inevitable challenge. Here, we de novo design and successfully assemble human functional immunoglobulin heavy variable (IGHV) gene fragments up to hundred-kilobase (kb)-sized, using an iterative in vitro assembly via Escherichia coli (E. coli) based on Gibson isothermal assembly. We describe an efficient method for "scarless" (without leaving any non-native sequences) engineering of the assembled ordered functional IGHV gene fragments, which contain complex and highly repetitive regions. Our method provides a suitable way to construct bacterial artificial chromosomes (BACs) (30-100 kb) with common materials, easy manipulations, and low cost. The construction of ordered functional IGHV gene BACs expands the synthetic biologist's chassis repertoire. It is essential for the adaptive immune response and constructing immunity humanized animal models.