The use of evolutionary engineering has proven to be a very successful tool for selecting for recombinant S. cerevisiae
strains capable of anaerobically utilizing sugars such as xylose and arabinose (5
). Natural selection even enabled a non-metabolically engineered S. cerevisiae
strain to utilize xylose as a sole carbon and energy source, which demonstrates that trace activities of enzymes in the targeted metabolic route can result in growth, provided that strong selection pressure is applied (4
). Hitherto, research on evolutionary engineering of pentose utilization by S. cerevisiae
has focused primarily on the use of single sugars, and there have been only a few examples of improved utilization of multiple sugars (12
). Improvement of the utilization of multiple substrates has specific challenges, as it is rather complicated to select for multiple mutations in different metabolic routes that require different kinds of (potentially conflicting) selective pressure (17
). In line with this observation, previous evolutionary engineering for anaerobic utilization of arabinose as the sole carbon source resulted in the loss of the xylose-utilizing capacities of an engineered S. cerevisiae
Given the complexity of biomass hydrolysates, evolutionary engineering strategies that enable efficient utilization of mixtures containing three (or more) different sugars are required. The sequencing batch evolution strategy with 20 g liter−1
xylose and 20 g liter−1
arabinose (Fig. ) clearly demonstrated that a slight preference for xylose over arabinose resulted in increasingly more generations on xylose and thus a shift of the selection pressure to the preferred sugar. Although, as a result, growth on xylose improved dramatically, the deteriorated kinetics of arabinose utilization resulted in overall less favorable fermentation characteristics. If the expression of a native or heterologous pathway for a less-preferred substrate confers even a slight selective disadvantage (for example, via protein burden [18
]), repeated cultivation on a fixed substrate mixture may ultimately select for strains that exhibit the well-known phenomenon of diauxic growth (15
). To minimize such unequal selection pressure on the utilization of glucose, xylose, and arabinose, a novel selection strategy involving consecutive anaerobic batch cultivation in MY-GXA, MY-XA, and MY-A was used. This selection strategy allows more even distribution of the number of generations grown on each carbon source, even when cells have a preference for one substrate over another.
The regimen involving repetitive consecutive cultivation with the three different sugar mixtures yielded an S. cerevisiae
strain (strain IMS0010) that exhibited rapid anaerobic fermentation of mixtures of glucose, xylose, and arabinose to ethanol. The underlying genetic changes involved in the improvement in xylose and arabinose utilization by the evolved strain IMS0010 remain to be investigated. The possible changes include mutations that resulted in improved codon usage of introduced genes, as codon optimization for AraA
, and AraD
in engineered S. cerevisiae
strains has been shown to result in improved arabinose conversion rates (25
). Alternatively, changes in plasmid copy numbers may have played a role in fine-tuning the levels of expression of introduced genes, as described previously for S. cerevisiae
strains evolved for lactose utilization (8
). Interestingly, the specific combined pentose consumption rate of IMS0010 nearly equals the maximum xylose consumption rate of RWB218 with glucose-xylose mixtures (12
), which might indicate that there is a common flux-controlling step during pentose utilization by these strains.
In plant biomass hydrolysates, xylose, and especially arabinose comprise only a small fraction of the total carbon. The use of such hydrolysates for evolutionary engineering would limit the selection pressure for the substrates that are less preferred by the organism of choice and therefore might not result in improved kinetics for utilization of the minor components of the medium. A simple solution would be to add these substrates to the hydrolysates in various stages of the evolutionary engineering process to obtain enough generations on the desired substrates. In this way, rapid consumption of the less predominant substrates, which often are crucial for the overall process economics, in the production environment may be achieved. Equal selection pressures for multiple sugars can be achieved in multiple ways, and the fermentation setup described above is not unique. For example, repeated cultivation with a (fixed) mixture of 5 g liter−1 glucose, 15 g liter−1 xylose, and 45 g liter−1 arabinose would also result in a more even distribution of the selection pressures. However, although the number of generations is expected to be similar, the simultaneous presence of various sugars at high (repressing) concentrations in the strategy described in this paper add selective pressure for rapid subsequent or even partially simultaneous use of the substrates.
To our knowledge, the strategy described here for improving the utilization of mixtures of three or more substrates via consecutive batch cultivation in media with alternating sugar compositions has not been described previously. Moreover, although the strategy has to be tested in practice, its applications do not seem to be limited to the selection of improved S. cerevisiae phenotypes for ethanol production; this strategy might also be applicable to other microorganisms used in (industrial) biotechnological processes based on the conversion of lignocellulosic hydrolysates or other substrate mixtures.