Systematic metabolic profiling and bioactivity assays for bioconversion of Aceraceae family

<div><p>Plants are an important and inexhaustible source of bioactive molecules in food, medicine, agriculture, and industry. In this study, we performed systematic liquid chromatography–mass spectrometry (LC-MS)-based metabolic profiling coupled with antioxidant assays for indigenous plant family extracts. Partial least-squares discriminant analysis of LC-MS datasets for the extracts of 34 plant species belonging to the families Aceraceae, Asteraceae, and Rosaceae showed that these species were clustered according to their respective phylogenies. In particular, seven Aceraceae species were clearly demarcated with higher average antioxidant activities, rationalizing their application for bioconversion studies. On the basis of further evaluation of the interspecies variability of metabolic profiles and antioxidant activities among Aceraceae family plants, we found that <i>Acer tataricum</i> (TA) extracts were clearly distinguished from those of other species, with a higher relative abundance of tannin derivatives. Further, we detected a strong positive correlation between most tannin derivatives and the observed higher antioxidant activities. Following <i>Aspergillus oryzae</i>-mediated fermentative bioconversion of <i>Acer</i> plant extracts, we observed a time-correlated (0–8 days) linear increase in antioxidant phenotypes for all species, with TA having the highest activity. Temporal analysis of the MS data revealed tannin bioconversion mechanisms with a relatively higher abundance of gallic acid (m/z 169) accumulated at the end of 8 days, particularly in TA. Similarly, quercetin precursor (glycoside) metabolites were also transformed to quercetin aglycones (m/z 301) in most <i>Acer</i> plant extracts. The present study underscores the efficacy of fermentative bioconversion strategies aimed at enhancing the quality and availability of bioactive metabolites from plant extracts.</p></div

Differential metabolites identified using UHPLC-LTQ-IT-MS/MS and UPLC-Q-TOF-MS in before/after bioconversion of <i>Acer</i>.

<p>Differential metabolites identified using UHPLC-LTQ-IT-MS/MS and UPLC-Q-TOF-MS in before/after bioconversion of <i>Acer</i>.</p

High Persister Mutants in <i>Mycobacterium tuberculosis</i>

<div><p><i>Mycobacterium tuberculosis</i> forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in <i>M</i>. <i>tuberculosis</i> is not well understood. In this study, we selected for high persister (<i>hip</i>) mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the <i>hip</i> mutants obtained <i>in vitro</i> with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical <i>hip</i> isolates exhibited greater <i>ex vivo</i> survival than the low persister isolates. Our data suggest that <i>M</i>. <i>tuberculosis</i> persister formation involves multiple pathways, and <i>hip</i> mutants may contribute to the recalcitrance of the infection.</p></div

Information on the 34 native Korean plant species used in this study.

<p>Information on the 34 native Korean plant species used in this study.</p

Systematic metabolic profiling and bioactivity assays for bioconversion of Aceraceae family - Fig 4

<p>(A) Principal component analysis score plot derived from UHPLC-LTQ-IT-MS/MS datasets displaying a variance between unfermented (0 day) and fermented (8 days) samples, and (B) antioxidant activity assays, where each of the five columns indicates average 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) activity for 0-, 2-, 4-, 6-, and 8-day fermented <i>Acer</i> extracts. TR: <i>Acer triflorum</i>; PM: <i>Acer pictum</i> subsp. <i>mono</i>; BU: <i>Acer buergerianum</i>; KO: <i>Acer komarovii</i>; TA: <i>Acer tataricum</i>; PS: <i>Acer pseudosieboldianum</i>; PI: <i>Acer pictum</i>; PA: <i>Acer palmatum</i>; C: control (only broth).</p

Genetic analysis of <i>hip</i> mutants obtained <i>in vitro</i>.

<p>Representative antibiotic survival plots of 18 <i>hip</i> mutant strains, obtained from 12 independent mutageneses, are presented along with lists of genes containing non-synonymous mutations within each strain.</p

Systematic metabolic profiling and bioactivity assays for bioconversion of Aceraceae family - Fig 2

<p>(A) Partial least-square discriminant analysis (PLS-DA) score plot based on UHPLC–LTQ-IT-MS/MS datasets, (B) average antioxidant activity (2,2-diphenyl-1-picrylhydrazyl: DPPH) for seven <i>Acer</i> species plant extracts (different letters are indicative of statistically significant differences for observed bioactivities according to Duncan’s multiple-range test at <i>p</i> < 0.05), and (C) Heat map representation for the relative abundance of significantly discriminant metabolites based on the PLS-DA model (VIP > 0.7, <i>p</i> < 0.05). TR: <i>Acer triflorum</i>; PM: <i>Acer pictum</i> subsp. <i>mono</i>; BU: <i>Acer buergerianum</i>; KO: <i>Acer komarovii</i>; TA: <i>Acer tataricum</i>; PS: <i>Acer pseudosieboldianum</i>; PI: <i>Acer pictum</i>; PA: <i>Acer palmatum</i>.</p

Proposed bioconversion pathways derived for the selected metabolites in UHPLC-LTQ-IT-MS/MS datasets relevant to the time-correlated production of (A) quercetin, and (B) gallic acid during the fermentative bioconversion process.

<p>The inset graphs indicate the metabolite peak area plotted along the y-axis, whereas the incubation period (0, 4, and 8 days) during fermentation is plotted along the x-axis. The seven <i>Acer</i> species are indicated with different color codes in the inset graph.</p

Systematic metabolic profiling and bioactivity assays for bioconversion of Aceraceae family - Fig 1

<p>(A) Partial least-square discriminant analysis score plot based on UHPLC–LTQ-IT-MS/MS datasets, and (B) average antioxidant activity (2,2-diphenyl-1-picrylhydrazyl: DPPH), for the metabolite extracts derived from plant species belonging to the families Aceraceae, Rosaceae, and Asteraceae. The different letters are indicative of statistically significant differences for observed bioactivities according to Duncan’s multiple-range test at <i>p</i> < 0.05.</p

Characterization of <i>hip</i> mutants obtained <i>in vitro</i>.

<p>Persister assays, performed by antibiotic treatment with streptomycin (10 μg/ml) and rifampicin (1 μg/ml) for 14 days, reveal the number of drug tolerant persister cells based on CFU counts. Exponential (A) and stationary phase (B) treatment of mutagenized strain mc²6020 at each stage of the <i>hip</i> mutant selection process. Time-dependent persister assays in exponential (C) and stationary phase (D) with independent mutants KL2801, KL2825, KL2849, and wild type strain (mc²6020). Late exponential phase cultures were treated with various concentrations of streptomycin (E) or rifampicin (F) or with antibiotics not used in the selection process, kanamycin (50 μg/ml) or ofloxacin (10 μg/ml) (G). Cultures grown in minimal media with glycerol, butyrate, or propionate as the sole carbon source were treated in exponential phase (H). Data represent the average of three biological replicates and the error bars represent standard deviation.</p