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Investigation of culture conditions revealed that the maximal cell growth and scFv expression were achieved at pH 6.5 7.0 with 2% casamino acids after 72 h methanol induction.
Meanwhile, a maximal cell growth rate and butanol generation rate were reached by applying a two-stage pH-shift strategy, achieving 18.7 g/L butanol with a yield of 24.6% and a productivity of 0.26 g/L h.
In the shaking flask, prepared according to the arthroconidial inoculation method, the fermentation period, maximal cell growth rate, and triterpenoid productivity of SmF were 0.6-, 1.7-, and 1.6-fold of those obtained from the culture prepared according to the conventional mycelial inoculation method.
Thus it seems reasonable that the differences in the final OD can be explained either by a reduced cell growth, caused by too low ammonium concentrations, with normal fatty acid production or by a maximal cell growth but without a significant fatty acid production in the case of ammonium concentrations higher than 0.4 g l-1.
Briefly, FBA is an effective tool for the prediction of the maximal cell growth and metabolite production when given certain constraints.
A flow rate of 0.4/d was chosen for this culture system since this represented a midway value between the observed minimal and maximal cell growth rate in batch culture.
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Half-maximal cell growth inhibitory concentrations (GI50) ranged from 0.49 to 41.22 µM.
Of these derivatives, 3- 1-hydroxynaphthalen-2-yl -N- 3-methoxyphenyl -5- 4-methoxyphenyl -pyrazoline-1-carbothioamide (NPC 15) exhibited the best half-maximal cell growth inhibitory concentrations (196.3- 1-hydroxynaphthalen-2-yl -N- 3-methoxyphenyl -5- 4-methoxyphenyl -pyrazoline-1-carbothioamide
Next, a MILP problem is sequentially solved for all metabolites to attain the maximum cell growth at various flux-sum perturbations between the calculated minimal and maximal values.
Interestingly, maximal cell biomass growth remained unchanged with the increase of initial xylose content for both ScF2 and P. stipitis indicated by the constant OD600 at approximately 40, suggesting the inhibition of cell growth under high xylose concentration.
Among the genes providing resistance to ethanol, acetic acid and vanillin or furfural, Erg2, Prs3, Rpb4 and Vma8 were found, for the first time, to contribute to maximal yeast cell growth and fermentation rate in WSH, while Rav1 contributed only to reaching maximal fermentation rate.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com