Gene-Directed Generation of Unprecedented Bioactive Compounds

Bioactive compounds with previously undescribed frameworks are highly desired for the discovery and development of new drugs and agrochemicals, but very few attempts have been reported to generate such molecules in biological contexts. Here, we present a gene-directed generation of architecturally unprecedented polyketide–indole hybrids (PIHs), which was conceptualized and materialized by employing polyketide synthases expressed in a heterologous vector, with simultaneous exposure to exogenous chemicals. To make an exemplification to this generally applicable approach, the ChrA and ChrB genes of Daldinia eschscholzii IFB-TL01 were integrated into the Aspergillus oryzae (AO) cell, and the resultant ChrA/ChrB-AO transformant was cultured in the indole-3-carbinol (I3C)-supplemented medium, leading to the production of seven skeletally undescribed PIHs named aochrabines A–G. Among them, aochrabines A–C exhibited a broad spectrum in inhibiting the growth of Gram-positive bacteria, whereas aochrabines B, C, and G showed moderate antitumor activities. Unexpectedly, the construction of such aochrabine molecules was achieved by the regioselective Michael addition of 3-methyleneindolium (3MI, generated from I3C in the AO culture) to different polyketide precursors with the yields (much) higher than those in the D. eschscholzii culture where comparable. Chemically, the benzyl-methine carbons in the precursor molecules were found to be made more vulnerable to the 3MI attack by the hydrogen-bonding between the ortho-hydroxyl and meta-carbonyl groups. Collectively, this is the first report of the ortho- and meta-substituent co-driven regioselective Michael addition of electrophilic methylene compounds to heterologous PKS production platform to in situ multiply the chemodiversity of microbial cultures, thus showing great potential in producing valuable compounds with new chemical space

Elucidating the Crucial Role of Poly <i>N</i>-Acetylglucosamine from <i>Staphylococcus aureus</i> in Cellular Adhesion and Pathogenesis

<div><p><i>Staphylococcus aureus</i> is an important pathogen that forms biofilms on the surfaces of medical implants. Biofilm formation by <i>S</i>. <i>aureus</i> is associated with the production of poly <i>N</i>-acetylglucosamine (PNAG), also referred to as polysaccharide intercellular adhesin (PIA), which mediates bacterial adhesion, leading to the accumulation of bacteria on solid surfaces. This study shows that the ability of <i>S</i>. <i>aureus</i> SA113 to adhere to nasal epithelial cells is reduced after the deletion of the <i>ica</i> operon, which contains genes encoding PIA/PNAG synthesis. However, this ability is restored after a plasmid carrying the entire <i>ica</i> operon is transformed into the mutant strain, <i>S</i>. <i>aureus</i> SA113Δ<i>ica</i>, showing that the synthesis of PIA/PNAG is important for adhesion to epithelial cells. Additionally, <i>S</i>. <i>carnosus</i> TM300, which does not produce PIA/PNAG, forms a biofilm and adheres to epithelial cells after the bacteria are transformed with a PIA/PNAG-expressing plasmid, pTX<i>icaADBC</i>. The adhesion of <i>S</i>. <i>carnosus</i> TM300 to epithelial cells is also demonstrated by adding purified exopolysaccharide (EPS), which contains PIA/PNAG, to the bacteria. In addition, using a mouse model, we find that the abscess lesions and bacterial burden in lung tissues is higher in mice infected with <i>S</i>. <i>aureus</i> SA113 than in those infected with the mutant strain, <i>S</i>. <i>aureus</i> SA113Δ<i>ica</i>. The results indicate that PIA/PNAG promotes the adhesion of <i>S</i>. <i>aureus</i> to human nasal epithelial cells and lung infections in a mouse model. This study elucidates a mechanism that is important to the pathogenesis of <i>S</i>. <i>aureus</i> infections.</p></div

Effects of glucose on PIA/PNAG production and adherence of <i>S</i>. <i>aureus</i> to RPMI 2650 cells.

<p><i>S</i>. <i>aureus</i> SA113 was cultured in glucose-containing TSB for 24 h. PIA/PNAG production (A) was determined using WGA-biotin. The average number of <i>S</i>. <i>aureus</i> SA113 adhered to each RPMI 2650 cell (B) was also determined using adherence assay. Significant differences are denoted with ***<i>p</i>-value < 0.001.</p

SEM images of adhesion of <i>S</i>. <i>aureus</i> to RPMI 2650 cells.

<p>(A) RPMI 2650 cells were incubated with <i>S</i>. <i>aureus</i> SA113 (a, d), SA113Δ<i>ica</i> (b, e) and SA113Δ<i>ica</i>(pC<i>ica</i>) (c, f). The images were captured at a magnification of 1000x (a, b, c) and 3000x (d, e, f). The number of bacteria that adhered to 400 cells was enumerated, and the average number of bacteria on each cell was calculated (B). Significant differences are denoted with ***<i>p</i>-value < 0.001.</p

Gene-Inspired Mycosynthesis of Skeletally New Indole Alkaloids

Dalesindole, an antibacterial and anti-inflammatory indole alkaloid with an undescribed carbon skeleton, was stereoselectively constructed by <i>Daldinia eschscholzii</i> through class II aldolase catalyzed Michael addition of fungal chromone with 3,3′-diindolylmethane (DIM) formed in situ from indole-3-carbinol (I3C) under catalyses of monooxygenase and 8-amino-7-oxononanoate synthase (AONS). Dalesindole isomerizes via a retro-Michael reaction to give stereoisomers with bioactivities. The work provides an access to new bioactive hybrids of fungal oligoketide with microbially decorated exogenous chemistry