7. Total carotenoid content per dry weight of mycelium is estimated using an overall extinction coefficient (EC) of
200 (1 mg ml
À1 , 1 cm), using the formula:
Carotenoids (mg/g dry weight) ¼ ([Abs max – Abs 650nm ] Â V)/
(DW Â EC Â PL).
V ¼ final volume of solvent with extracted carotenoids in
ml. DW ¼ mycelial dry weight in g. EC ¼ absorbance coefficient
in mg
À1 ml cm
À1
. PL ¼ path length in cm (see Note 27).
Mutant SG262 produced almost no carotenoids in the presence of Dox (Fig. 4a), and its spectrum analysis shows no peak of
carotenoids (Fig. 4b) while the parental strain SG39 produced
mainly neurosporaxanthin [4]. The Tet-on system is a useful tool
to modulate carotenoid biosynthesis in F. fujikuroi.
4 Notes
1. It is recommended to prepare a fresh enzyme solution for
protoplast each time because its effectiveness drops if it is
frozen at À80
C.
Fig. 4 Modulated production of carotenoids in F. fujikuroi by addition of doxycycline. Carotenoids accumulated
in the engineered strain SG262 and carS mutant SG39 were measured from mycelia cultured on Petri dishes
with either DG-Dox agar (DG with 20 μg/ml Dox) or DG agar. Cultures were grown for 7 days at 28
C under
6 W/ m
2 white light. (a) Total carotenoids calculated as described in Subheading 3.4.2. (b) Spectra of extracted
carotenoids from the carS mutant SG39 and the strain SG262 (carS gene controlled by Tet-on system).
Spectra in green show profile of carotenoids in the presence of Dox (doxycycline) and in orange represent
control samples. Spectra were relativized to extracted dry mass
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200 (1 mg ml
À1 , 1 cm), using the formula:
Carotenoids (mg/g dry weight) ¼ ([Abs max – Abs 650nm ] Â V)/
(DW Â EC Â PL).
V ¼ final volume of solvent with extracted carotenoids in
ml. DW ¼ mycelial dry weight in g. EC ¼ absorbance coefficient
in mg
À1 ml cm
À1
. PL ¼ path length in cm (see Note 27).
Mutant SG262 produced almost no carotenoids in the presence of Dox (Fig. 4a), and its spectrum analysis shows no peak of
carotenoids (Fig. 4b) while the parental strain SG39 produced
mainly neurosporaxanthin [4]. The Tet-on system is a useful tool
to modulate carotenoid biosynthesis in F. fujikuroi.
4 Notes
1. It is recommended to prepare a fresh enzyme solution for
protoplast each time because its effectiveness drops if it is
frozen at À80
C.
Fig. 4 Modulated production of carotenoids in F. fujikuroi by addition of doxycycline. Carotenoids accumulated
in the engineered strain SG262 and carS mutant SG39 were measured from mycelia cultured on Petri dishes
with either DG-Dox agar (DG with 20 μg/ml Dox) or DG agar. Cultures were grown for 7 days at 28
C under
6 W/ m
2 white light. (a) Total carotenoids calculated as described in Subheading 3.4.2. (b) Spectra of extracted
carotenoids from the carS mutant SG39 and the strain SG262 (carS gene controlled by Tet-on system).
Spectra in green show profile of carotenoids in the presence of Dox (doxycycline) and in orange represent
control samples. Spectra were relativized to extracted dry mass
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Julia Marente et al.