Table 1.
Bacterial strains and plasmids used in this study.
Fig 1.
Biotransformations with resting E. coli BL21-Gold(DE3)_pETM11-ksa14m-alkL cells limited in either nitrogen (N) or magnesium (Mg).
Microorganisms were grown in M9 medium containing 0.5% (w/v) glucose. Resting cells were prepared 5 h after induction, and biotransformations were performed as described in Materials and Methods. (A) Specific testosterone hydroxylation activity time courses for cells suspended in either N-free KPi buffer or Mg-free M9 medium, optionally supplied with 0.1 mM IPTG and 0.5 mM 5-aminolevulinic acid (inducer, dashed lines). Data points represent average values and standard deviations of biological duplicates. (B) SDS-PAGE analysis shows KSA14m (119 kDa, black arrow) and AlkL (23 kDa, grey arrow) levels during biotransformations under the indicated conditions: N-deprived cells in absence or presence of substrate (testosterone), Mg-limited cells with substrate in absence or presence of IPTG and 5-aminolevulinic acid (inducer and heme building block).
Fig 2.
Biotransformations with growing E. coli BL21-Gold(DE3)_pETM11-ksa14m-alkL cells.
Bacteria were cultivated in M9 medium with 0.5% (w/v) glucose, induced with 0.1 mM IPTG, harvested before and after 5 h of induction, and resuspended in fresh M9 medium containing 1% (w/v) glucose, 0.1 mM IPTG, and 5-aminolevulinic acid to a biomass concentration of 1 gCDW L-1. Ten mL liquid volume were transferred to 100 mL baffled, screw-capped flasks and equilibrated for 10 min at 30°C. Biotransformations were started by addition of 1 mM testosterone. (A) Time courses of specific testosterone hydroxylation activities (solid black lines), cell concentrations (dashed black lines), and steroid concentrations (dashed grey lines; products referring to the sum of 2β- and 15β-hydroxytestosterone) with 5 h of induction before and induction simultaneously to the biotransformation start. Average values and standard deviations were calculated from two biological replicates. (B) SDS-PAGE analyses showing heterologous KSA14m (119 kDa, black arrows) and AlkL (23 kDa, grey arrows) levels at different time points after biotransformation start.
Fig 3.
Performance of E. coli BL21-Gold(DE3)_pETM11-ksa14m-alkL depends on cultivation medium composition.
Cells grown and induced in M9, M9*, or RB medium containing 0.5% (w/v) glucose as carbon and energy source. Specific growth rates (A), KSA14m (119 kDa, black arrow) and AlkL (23 kDa, grey arrow) levels (SDS-PAGE, B) and resting-cell activity courses after 5 h of induction in nitrogen-deprived KPi buffer containing 1% (w/v) glucose (C) were analyzed as described in Materials and Methods. Average values and standard deviations of biological duplicates are shown.
Fig 4.
Effects of expression system and inducer concentration on the performance of E. coli BL21-Gold(DE3) strains synthesizing KSA14m and AlkL.
Left and right columns show courses of growth and specific resting-cell testosterone hydroxylation activities, respectively, upon ksa14m and alkL expression under control of different regulatory systems (S1 Fig) and with different inducer concentrations. (A) AlkS-PalkB-based system induced with DCPK; (B) LacI-PlacUV5-based system induced with IPTG; (C) LacIq-Ptac-based system induced with IPTG; (D) LacI-PT7-based system either uninduced or induced with IPTG. Cells were grown in M9 medium containing 0.5% (w/v) glucose and induced for 5 h, followed by cell harvesting and resting-cell biotransformations performed as described in Materials and Methods in nitrogen-deprived KPi buffer containing 1% (w/v) glucose with a cell concentration of 1 gCDW L-1 unless indicated otherwise. Average values and standard deviations of two biological replicates are given.
Fig 5.
Effect of amino acid exchanges in KSA14m on testosterone hydroxylation activity and stability.
E. coli BL21-Gold(DE3) cells carrying pETM11 equipped with genes encoding different KSA14m variants and AlkL were grown in M9 medium containing 0.5% (w/v) glucose. Resting cell preparation 5 h after induction with 0.1 mM IPTG and biotransformations were performed as described in Materials and Methods in nitrogen-deprived KPi buffer containing 1% (w/v) glucose. (A) Time courses of specific testosterone hydroxylation activities (solid lines) and testosterone concentrations (dashed lines). (B) Initial activities (5 min) after different equilibration times under reaction conditions. Average values and standard deviations of biological duplicates are given.
Fig 6.
P. taiwanensis VLB120_Strep hosting the heterologous synthesis of KSA14m and different hydrophobic outer membrane pores.
(A) Growth of P. taiwanensis VLB120_Strep strains equipped with different plasmids for KSA14m and hydrophobic outer membrane pore synthesis in M9 medium containing 0.5% (w/v) glucose compared to the wild type (WT). Data points represent average values and standard deviations of biological duplicates. Heterologous gene expression was induced with 0.0025% (v/v) DCPK or 0.1 mM IPTG. (B) Recombinant KSA14m levels (119 kDa, black arrow) were examined via SDS-PAGE 5 h after induction for the wild type (1) and strains harboring either palk-ksa14m-alkL (2), plac-ksa14m (3), plac-ksa14m-alkL (4), plac-ksa14m-fhuAΔ1-160 (5), or plac-ksa14m-todX (6).
Fig 7.
Resting-cell biotransformations with E. coli BL21-Gold(DE3) cells carrying pETM11 with cyp154c5, camA, camB (and optionally alkL)—One plasmid strategy.
Time courses of specific progesterone hydroxylation activities (solid lines) and product concentrations (dashed lines) are depicted. Bacteria were cultivated in M9 medium containing 0.5% (w/v) glucose. Resting cells were prepared 5 h after induction with 0.1 mM IPTG and applied as described in Materials and Methods. Average values and standard deviations of biological duplicates are presented.
Table 2.
Specific growth rates and specific activities of E. coli BL21-Gold(DE3) strains carrying genes encoding alternative CYP450sa.
Table 3.
Summary of strategies investigated to stabilize KSA14m-catalyzed testosterone hydroxylation using whole-cell systems and their effect on initial specific activity and stability.