Cytochrome P450 Mining for Bufadienolide Diversification.

Bufadienolides are a class of steroids with a distinctive α-pyrone ring at C17, mostly produced by toads and consisting of over 100 orthologues. They exhibit potent cardiotonic and antitumor activities and are active ingredients of the traditional Chinese medicine Chansu and Cinobufacini. Direct extraction from toads is costly, and chemical synthesis is difficult, limiting the accessibility of active bufadienolides with diverse modifications and trace content. In this work, based on the transcriptome and genome analyses, using a yeast-based screening platform, we obtained eight cytochrome P450 (CYP) enzymes from toads, which catalyze the hydroxylation of bufalin and resibufogenin at different sites. Moreover, a reported fungal CYP enzyme Sth10 was found functioning in the modification of bufalin and resibufogenin at multiple sites. A total of 15 bufadienolides were produced and structurally identified, of which six were first discovered. All of the compounds were effective in inhibiting the proliferation of tumor cells, especially 19-hydroxy-bufalin (2) and 1ß-hydroxy-bufalin (3), which were generated from bufalin hydroxylation catalyzed by CYP46A35. The catalytic efficiency of CYP46A35 was improved about six times and its substrate diversity was expanded to progesterone and testosterone, the common precursors for steroid drugs, achieving their efficient and site-specific hydroxylation. These findings elucidate the key modification process in the synthesis of bufadienolides by toads and provide an effective way for the synthesis of unavailable bufadienolides with site-specific modification and active potentials.

Self-Assembled Saccharide-Functionalized Amphiphilic Metallacycles as Biofilms Inhibitor via "Sweet Talking".

Bacterial biofilms are troublesome in the treatment of bacterial infectious diseases due to their inherent resistance to antibiotic therapy. Exploration of alternative antibiofilm reagents provides opportunities to achieve highly effective treatments. Herein, we propose a strategy to employ self-assembled saccharide-functionalized amphiphilic metallacycles ([2+2]-Gal, [3+3]-Gal, and [6+6]-Gal) with multiple positive charges as a different type of antibacterial reagent, marrying saccharide functionalization that interact with bacteria via "sweet talking". These self-assembled glyco-metallacycles gave various nanostructures (nanoparticles, vesicles or micron-sized vesicles) with different biofilms inhibition effect on Staphylococcus aureus (S. aureus). Especially, the peculiar self-assembly mechanism, superior antibacterial effect and biofilms inhibition distinguished the [6+6]-Gal from other metallacycles. Meanwhile, in vivo S. aureus pneumonia animal model experiments suggested that [6+6]-Gal could relieve mice pneumonia aroused by S. aureus effectively. In addition, the control study of metallacycle [3+3]-EG5 confirmed the significant role of galactoside both in the self-assembly process and the antibacterial efficacy. In view of the superior effect against bacteria, the saccharide-functionalized metallacycle could be a promising candidate as biofilms inhibitor or treatment agent for pneumonia.

Efficient circular gene knockout system for Burkholderiales strain DSM 7029 and Mycobacterium smegmatis mc2 155.

Multiple gene knockouts are often employed in studies of microbial physiology and genetics. However, the selective markers that confer antibiotic resistance are generally limited, so it is necessary to remove these resistance genes before the next round of using, which is time consuming and labor intensive. Here, we created a universal circular gene knockout system for both the gram-negative bacterial Burkholderiales strain DSM 7029 and the gram-positive bacterial Mycobacterium smegmatis mc2 155, by combining the homologous recombination with multiple serine integrase-meditated site-specific recombination systems. In this system, a resistance gene and an integrase gene were constructed within the two attachment sites corresponding to a second, different integrase to form a cassette for gene disruption, which could be easily removed by the second integrase during the subsequent round of gene knockout. The sacB gene was also employed for negative selection. As the integrase-mediated deletion of the resistance/integrase gene cassette was highly efficient and concurrent with the following knockout round, the cyclic use of three cassettes could achieve multiple gene knockout in a sequential manner. Following the modularity concept in synthetic biology, common components of the knockout plasmids were retained as BioBricks, accelerating the knockout plasmids construction process. The circular gene knockout system can also be used for multiple gene insertions and applied to other microorganisms.

Site-specificity of serine integrase demonstrated by the attB sequence preference of ɸBT1 integrase.

Serine integrases mediate site-specific recombination and are extensively applied in genetic engineering and synthetic biology. However, which regions of the attachment sites determine site-specificity and how these regions function in recombination remain elusive. Here, we explored the sequence features of attB attachment sites recognized by ɸBT1 integrase, a representative serine integrase. A 34-bp DNA motif is found that displays distinct base-specific preference for every position. Further investigation of mutations at different positions within the attB sequence shows different recombination efficiencies and binding affinities. We found four conserved regions within the attB motif that coincide with the results of recombination assays, and mutations in the attB sequence that hamper recombination almost all cause reduced binding affinity.

Bxb1 integrase serves as a highly efficient DNA recombinase in rapid metabolite pathway assembly.

Phage-encoded serine integrases are widely used in genetic engineering. They also have the potential to serve as efficient DNA assemblers, demonstrated by the method of site-specific recombination-based tandem assembly (SSRTA) that can combine biological parts into devices, pathways, and systems. Here, four serine integrases, ϕBT1, TG1, ϕRv1, and Bxb1, were investigated to ascertain their in vitro DNA assembly activities. Bxb1 integrase displayed the highest efficiency to obtain final products. Thus, we conclude that Bxb1 integrase is an excellent choice for DNA assembly in vitro Using this enzyme and its recognition sites, BioBrick standards were designed that are compatible with the SSRTA method for module addition. A rapid and efficient procedure was developed for the assembly of a multigene metabolic pathway in one step, directly from non-cutting plasmids containing the gene fragments. This technique is easy and convenient, and would be of interest to the synthetic biology community.