08.08.2015   Advances in Biochemical Engineering/Biotechnology

Evolutionary Engineering of S. cerevisiae only Containing Fungal Xylose Isomerase

After the proof of principle of XI expression in S. cerevisiae, not only metabolic engineering, but also evolutionary engineering was applied to im­prove the rate of D-xylose utilisation of a strain solely over-expressing XI [44]. Since improvement of the aerobic consumption rate was initially the target of this selection experiment, serial transfer in a shake flask was chosen as the cultivation condition of this evolution run. Indeed, after 30 serial transfers, the specific growth rate of this culture improved drastically (24-fold) from 0.005 h-1 to 0.12 h-1 (Fig. 8). However, a strain isolated from this selection ex­periment was not yet capable of anaerobic growth. Therefore, an additional ten selection rounds were performed in oxygen-limited batch cultures, fi­nally followed by ten cycles in an anaerobic sequencing batch reactor. From this culture a single colony was isolated (named RWB 202-AFX, for anaero­bic fermentation of D-xylose based on strain RWB 202) and used for further characterisation of the end product of this evolutionary engineering.

It was shown that only the expression of a XI, followed by evolutionary en­gineering for anaerobic growth, can also result in a S. cerevisiae strain that can grow on 2% D-xylose as the sole carbon source, with a growth rate of 0.03 h-1 in anaerobic batch fermentations [45]. However, although this strain displayed a good ethanol yield on D-xylose (0.42 gg-1) and very low pro­duction of xylitol (2.8 mM), the obtained growth rate, and therefore ethanol

Fig. 8 Doubling time during serial transfer of S. cerevisiae RWB 202 in shake-flask cul­tures on synthetic medium with xylose. Each data point represents the doubling time of a single serial-transfer flask estimated from the OD660 measured at inoculation and at the time of the next transfer. Occasional transfer of cultures after they had reached stationary phase probably accounts for the unexpectedly high estimated doubling times in some of the cultures. Data from Kuyper et al. 2004 [45]

production rate, were insufficient to allow economically viable industrial ap­plication. During these batch cultivations, small amounts of D-xylulose (up to 8 mM) were still excreted into the broth, indicating that evolutionary engin­eering alone did not fully overcome the metabolic limitations downstream of this metabolite. This result indicates that although evolutionary engineering is a very powerful tool, it has limitations and, in this case, the combination of knowledge-based metabolic engineering (Sect. 5) combined with evolution­ary engineering (Sect. 6.1) resulted in more desirable attributes and higher ethanol production rates.

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