Figures & data
Fig. 1. Effect of initial medium conditions (temperature and pH) on the growth of BD7100.
Notes: (A) The growth curve of BD7100 at 25 °C (circle), 30 °C (triangle), 35 °C (diamond), and 37 °C (square). (B) The growth curve of BD7100 at initial pH 4.1 (black square), pH 5.3 (black triangle), pH 6.2 (black diamond), pH 7.0 (black circle), pH 8.2 (white diamond), pH 9.2 (white triangle), and pH 10.1 (white square). Each value represents the mean ± SD of three independent experiments.
Fig. 2. The growth of BD7100 and BBR degradation in the growing-cell assay.
Notes: (A) The growth curve of BD7100 (black triangles) in LB medium containing 1.0 mM BBR, and the concentrations of BBR (black circles) and D-BBR (white circles) during culturing. Each value represents the mean ± SD of three independent experiments. (B) HPLC chromatograms (280 nm): Comparison of peak retention times with those of standards revealed that BD7100 degraded BBR to D-BBR after 19 h in the growing-cell cultures.
Fig. 3 The degradation of BBR by resting cells of BD7100 and the resulting metabolites.
Notes: (A) The time course of BBR (black circle) and D-BBR (black triangle) concentrations in resting-cell suspensions of BBR-induced cells, and the time course of BBR concentration in resting-cell suspension of induction-free cells (white circle). Substrate concentrations were determined by PDA-HPLC. Each value represents the mean ± SD of three independent experiments. (B) HPLC chromatogram (280 nm) of resting-cell assay with BBR. BBR was degraded to D-BBR, which was in turn further degraded to HDBA. (C) Comparison of UV spectra of HDBA and D-BBR in HPLC chromatograms. Absorption at 340 nm was observed for D-BBR but was absent for HDBA. (D) Key HMBC correlations for HDBA.
Fig. 4. The degradation of BRU by resting cells of BD7100 and the resulting metabolites.
Notes: (A) The time course of BRU (black circle) and D-BRU (black triangle) concentrations in resting-cell suspensions of BBR-induced cells, and the time course of BRU concentration in resting-cell suspension of induction-free cells (white circle). Substrate concentrations were determined by PDA-HPLC. Each value represents the mean ± SD of three independent experiments. (B) HPLC chromatogram (280 nm) of resting-cell assay with BRU. BRU was degraded to D-BRU, which was in turn further degraded to DMBA. (C) Comparison with UV spectra of DMBA and D-BRU in HPLC chromatograms. Absorption at 340 nm was observed for D-BRU but was absent for DMBA. (D) Key HMBC correlations for DMBA.
Fig. 5. The inferred pathways for degradation of BBR and its analogs in BBR-utilizing bacteria.
Notes: BBR was degraded into D-BBR as the common BBR metabolite in two distinct strains. BD3100 generated a hydroxylation product, H-BBR; in contrast, BD7100 metabolized BBR and BRU to yield HDBA and DMBA, respectively, a process that would require cleavage of the protoberberine skeleton. BD3100 degraded PAL by hydroxylation to H-PAL; BD7100 did not demonstrate detectable degradation of PAL. BBR, berberine; D-BBR, demethyleneberberine; BRU, berberrubine; D-BRU, demethyleneberberrubine; DMBA, 2,3-hydroxy-4-dimethoxybenzeneacetic acid; H-BBR, 11-hydroxyberberine; HD-BBR, 11-hydroxydemethyleneberberine; HDBA, 2-hydroxy-3,4-dimethoxybenzeneacetic acid; H-PAL, 11-hydroxypalmatine; PAL, palmatine. Dashed line indicates proposed pathway.
Supplemental material
