11.08.2015   Advances in Biochemical Engineering/Biotechnology

Evolutionary Engineering for Improved Xylose-Isomerase-Based D-Xylose Utilisation

6.1

Evolutionary Engineering of D-Xylose-Consuming S. cerevisiae for Improved Mixed Substrate Utilisation

The sub-optimal kinetics of mixed-substrate utilisation by the genetically engineered XylA-expressing strain RWB 217 [43] suggested a low affinity (qmax/Ks) for D-xylose. Soon after the invention of the chemostat it was al­ready established that prolonged cultivation in nutrient-limited chemostats leads to selection of spontaneous mutants with an improved affinity for the growth-limiting nutrient [52,53]. This principle, which has since been demonstrated for many micro-organisms and nutrients [40,58,72,73] was applied to improve the affinity of S. cerevisiae RWB 217 for D-xylose [44].

Indeed, during prolonged anaerobic D-xylose-limited chemostat cultivation at a dilution rate of 0.06 h-1, the residual D-xylose concentration decreased threefold, indicating that cells with improved affinity for D-xylose were se­lected for [44]. After 1000 h (85 generations) of this directed evolution in chemostat cultures, single-colony isolates were tested for batch growth on a mixture of glucose and D-xylose. Although the fermentation kinetics of some of these single-cell lines, as evaluated by carbon dioxide production profiles, were already drastically improved relative to the parental strain (Fig. 6), the D-xylose phase remained slower than anticipated based on batch cultivation on D-xylose alone. A further 85 generations of chemostat cultivation resulted in only marginal improvement of the D-xylose consumption characteristics.

To select for further improvement of D-xylose fermentation kinetics, an additional evolutionary engineering strategy was applied, which involved sequential anaerobic batch cultivation on glucose-xylose mixtures [44]. To maximise the number of generations that the cells grow on D-xylose, the D-xylose concentration in the cultures was raised to 90 gL-1, with a glucose concentration of 20gL-1. After 20 cycles, the evolved culture was capable of complete anaerobic conversion of a mixture of 20 g L-1 glucose and 20 g L-1 D-xylose in about 20 h, with an inoculum size of 5% (v/v) [44].

Characterisation of the resulting strain RWB 218 (derived from single colony isolate) showed that D-xylose consumption followed the consump­tion of glucose rapidly (Fig. 7). The D-xylose consumption rate observed in these cultures was 0.9 g D-xylose (gdryweight)-1 h-1. This evolved Xl-based strain, in contrast to strains based on xylose reductase and xylitol dehydroge­nase, produced only 0.45 mM of xylitol, indicating that redox imbalance does

Fig. 6 CO2 production profiles, per litre culture, as measured in off gas of anaerobic fer­menter batch cultures with 20 g L-1 glucose and D-xylose each. Profiles have been aligned on the glucose consumption peak to eliminate variations in initial biomass. a RWB 217, b culture after chemostat selection, c RWB 218. Initial biomass concentrations were 0.20 ± 0.05 gL-1. Data from Kuyper et al. 2005 [44]

Fig. 7 Typical graph of anaerobic growth of strain RWB 218 in fermenters on synthetic medium with 20 g L-1 glucose and D-xylose each as the carbon source, duplicate experi­ments differed by less than 5%. a Glucose (•), D-xylose (O), ethanol (■), glycerol (□) and % CO2 measured in off gas per litre culture (-). b Dry weight (•), acetate (O), xylitol (■), D-lactate (□) and succinate (A). Initial biomass concentration was 0.17 gL-1. Data from Kuyper et al. 2005 [44]

not occur during alcoholic fermentation of D-xylose. The ethanol yield on total sugar in batch cultures co-fermenting glucose and D-xylose was typic­ally 0.40 g g-1, which is identical to the ethanol yield that would be achieved in glucose-grown cultures in a similar set-up. Even when tested in more concen­trated sugar mixtures (100 g L-1 glucose and 25 g L-1 D-xylose), resembling an industrial situation, this strain consumed both sugars within 24 h, starting with 1.1 gL-1 yeast dry weight as the inoculum [44].

With evolutionary engineering as a proven tool for obtaining (yeast) strains with improved properties, a full understanding of the underlying molecular changes becomes the next challenge. In an attempt to unravel the changes between the original metabolically engineered and the subsequently evolved Piromyces XI-based strains, anaerobic chemostat cultivations on D-xylose as the sole carbon source were used as the basis for transcriptome analysis with Affymetrix DNA arrays (J. T. Pronk, unpublished data). The most striking observation amongst the genes with a changed transcript level was the repre­sentation of various members of the hexose transport family, including HXT1, HXT2 and HXT4. Interestingly, HXT1 and HXT4 have been associated with D-xylose transport in previous studies [27,62]. To investigate whether the improved fermentation characteristics were indeed due to changes in sugar transport, zero trans-influx assays were performed using both the strain that was only metabolically engineered and the subsequently evolved strain [44]. The D-xylose uptake kinetics obtained for the metabolically engineered strain (Km 132 mM, Vmax 15.8 mmol (gdryweight)-1 h-1) were in agreement with other studies [22,39]. Strikingly, the D-xylose uptake kinetics of the evolved strain had changed drastically, with a 25% reduction in the Km (to 99 mM) and a twofold increase of Vmax to 32 mmol (g dryweight)-1 h-1.

6.2