SDS-PAGE analysis of CPO protein sizes after deglycosylation.

<p>Culture supernatants from fructose minimal medium cultures grown for 10 days on a rotary shaker at 24°C each were purified using aqueous biphasic systems and deglycosylation was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067857#s2" target="_blank">materials and methods</a> section. N+O: glycosidases were added to remove N- and O-linked oligosaccharides, O: glycosidases were added to remove O-linked oligosaccharides, N: glycosidases were added to remove N-linked oligosaccharides, ut: untreated sample, M: molecular weight marker. The numbers on the left indicate the sizes of the molecular weight marker.</p

Colony color and growth of <i>C.</i><i>fumago</i> wild type and cpo1 mutant on solid media.

<p><i>C. fumago</i> wild type strain and the cpo1 mutant were plated on fructose minimal medium plates with a piece of mycelium grown on a glucose potato agar plate and incubated at 24°C for 7 days.</p

Comparison of CPO production characteristics of wild type and cpo1 strain.

<p><b>A:</b> Comparison of CPO activities in supernatant samples of <i>C. fumago</i> wild type and cpo1 strain during cultivation in fructose minimal medium. Strain cultivation and determination of enzyme activity via the MCD assay was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067857#s2" target="_blank">materials and methods</a> section. Data is expressed as mean of values from two shake flasks ± standard deviation. <b>B:</b> Analysis of CPO protein levels in supernatant samples of <i>C. fumago</i> wild type and cpo1 strain by SDS-PAGE. SDS-PAGE analysis was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067857#s2" target="_blank">materials and methods</a> section. M: molecular weight marker. The numbers on the left indicate the sizes of the molecular weight marker.</p

Instability of cpo1 strain.

<p><b>A:</b> Analysis of CPO protein levels in supernatant samples of <i>C. fumago</i> wild type and cpo1 strain by SDS-PAGE. SDS-PAGE analysis was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067857#s2" target="_blank">materials and methods</a> section. The numbers on the left indicate the sizes of the molecular weight marker. M: molecular weight marker. CPO activity of fructose minimal medium cultures of wild type and cpo1 was determined as 60 U mL⁻¹ and 38 U mL⁻¹, respectively. <b>B:</b> UV spectra of <i>C. fumago</i> wild type and cpo1 CPO purified by aqueous biphasic systems. Culture supernatants from fructose minimal medium cultures grown for 11 days on a rotary shaker at 24°C each were purified using aqueous biphasic systems and protein concentration was equalized by dilution of the samples with 0.1 M citric acid buffer solution (pH 4).</p

UV-Vis spectra of <i>C.</i><i>fumago</i> wild type and cpo1 CPO purified by aqueous biphasic systems.

<p>Culture supernatants from fructose minimal medium cultures grown for 11 days on a rotary shaker at 24°C each were purified using aqueous biphasic systems and protein concentration was equalized by dilution of the samples with 0.1 M citric acid buffer solution (pH 4). CPO activity of the diluted samples was determined as 246.46 U mL⁻¹ and 0.03 U mL⁻¹ for wild type and cpo1, respectively.</p

Proposed reaction mechanism of C11-terpenes catalyzed by C11-TSs based on Brock et al. [22].

<p>Methyl groups introduced by the GPP-MTase are labeled with a black dot. Potential, but not detected products have a dark gray background. Products detected in this or other studies have a light gray background. Compounds that have been detected, but are no direct terpene synthase products have a hatched background.</p

Strains used in this study.

<p>Strains used in this study.</p

Proposed biosynthesis of 6-methylfarnesol in <i>E</i>.<i>coli</i> expressing the GPP-MTase of <i>S</i>. <i>coelicolor</i>.

<p>The elongation of 2-MGPP with IPP by an endogenous FPP synthase (FPPS) to 6-methylfarnesyldiphosphate (6-MFPP) and its dephosphorolation by endogenous phosphatases (probably PgpB and YbjG) are assumed.</p

Heterologous expression of 2-methylisoborneol / 2 methylenebornane biosynthesis genes in <i>Escherichia coli</i> yields novel C11-terpenes - Fig 4

<p><b>Total ion chromatogram of SBSE-GCMS analysis of the control strain 11–0 (A) and the structure and mass spectra of 6-methylfarnseol (B)</b> The spectra of the 6-methylfarnesol peak of the shown chromatogram (above) is compared with that of the reference compound (below).</p

Plasmids used in this study.

<p>Plasmids used in this study.</p