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The progressive development during the evolutionary engineering was followed as a function of increased μmax and reduced lag phase time.
This indicates that adverse background mutations have been introduced in the evolved strain during the EMS treatment or spontaneously generated during the evolutionary engineering.
During the evolutionary engineering in repetitive batch cultures the maximum specific growth rate increased from 0.18 h-1 to 0.33 h-1 and the time of lag phase was decreased from 48 h to 24 h.
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The glucose-to-xylose ratio at the start of the evolutionary engineering was kept low but increased during the experiment, eventually reaching 0.57% D-xylose and 10% D-glucose.
The progress of the evolutionary engineering process was continuously monitored by evaluation of single cell clones.
The recombinant xylose utilizing S. cerevisiae TMB3400 [ 26] was used as a parental strain in the evolutionary engineering strategies.
The goal of the present paper was to develop a superior D-xylose utilizing industrial yeast strain, lacking the negative properties introduced in the background of the previously reported strain, GS1.11-26, during the mutagenesis and/or evolutionary engineering procedures used to obtain the strain.
However, due to the accumulation of background mutations during the mutagenesis and/or evolutionary engineering procedures used to develop the strain, GS1.11-26 did not retain the same tolerance to high concentrations of ethanol and acetate, and showed reduced ethanol accumulation capacity in very high-density fermentations compared to the original Ethanol Red strain.
Phenotypic characteristics during the process of evolutionary engineering evidently demonstrated the importance of evolution in repetitive batch cultures and chemostat cultures to improve the maximum specific growth rate and inhibitor conversion rates, respectively.
This result demonstrates the importance of the selection conditions during evolutionary engineering, which is in agreement with the principle, "you get what you screened for" [ 64].
Evolved cells sampled at different time points during the process of evolutionary engineering were screened by drop tests.
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