In the present work, evolutionary engineering was used to improve xylose and arabinose co-utilization in a recombinant industrial S. cerevisiae strain carrying the genes from fungal xylose and bacterial arabinose pathways chromosomally integrated in the genome. Both the aerobic growth rate and the final biomass concentration were significantly improved in the evolved strain.
Under anaerobic conditions, the evolved strain TMB3130 displayed higher total anaerobic pentose co-consumption rate as well as the highest ethanol yield. This resulted from an improved conversion of xylose to ethanol (with less xylitol and glycerol as by-products) whereas arabinose was almost stochiometrically converted to arabitol via XR enzyme (Figure 1). We suspect that the use of XR for arabinose conversion to arabitol triggered a shortage of reduced nicotinamide adenine dinucleotide phosphate (NADPH) for the XR reaction, as observed from the increased oxidized nicotinamide adenine dinucleotide phosphate (NADP+)-dependent acetate production. Lower NADPH may also have forced XR to use NADH for the conversion of xylose to xylitol, which has been shown to lower xylitol production as a result of a more balanced co-factor utilization between XR and XDH enzymes . In addition, the reduced available NADH would result in lower glycerol production. Overall ethanol production should increase since less carbon is being diverted to glycerol and xylitol by-products.
Molecular and physiological analyses of the evolved isolate TMB3130 revealed that improved transport capacity and initial sugar metabolism contributed to the increased co-utilization of xylose and arabinose. In contrast the molecular basis for the improvements of the haploid laboratory strain have so far not been reported . Comparing isolate TMB3130 and its parental strains at the gene level did not reveal any mutation in the heterologous genes of the pentose converting enzymes. However, the parental strain contained several mutations in AraA and few in AraB, as compared to the original bacterial sequence. This may indicate a high selective pressure on the first enzymes of the arabinose utilization pathway during arabinose consumption and would confirm previous indications on the pivotal role of L-arabinose isomerase in arabinose-engineered S. cerevisiae . Also two silent mutations in B. subtilis AraA were shifted towards codon usage found at increased frequencies in highly expressed genes .
In the evolved strain TMB3130, the XR and XDH activities increased 5.5- and 3.5-fold, respectively, as compared to the parental strain TMB3061. This concomitant increase in activity associated with unchanged gene sequences suggest that the higher XR and XDH activities result from one or several duplications on the genome of the integrative vector that carry XYL1 and XYL2 genes. XR and XDH levels were also higher than for the original strain TMB3400, although xylose consumption rates were in the same range. Therefore, the loss of xylose consumption capacity in strain TMB3061 was not associated with decreased XR-XDH activity whereas the recovery of xylose consumption capacity in the evolved strain was. It suggests that S. cerevisiae is able to use different strategies to recover a phenotype under selection pressure.
Under glucose limitation or absence of glucose, high-affinity glucose transporters are induced . This correlates with the higher pentose uptake rate that was observed when strains were pre-grown on xylose or arabinose instead of glucose since hexose transporters have been shown to also transport pentoses: Hxt4p, Hxt5p, Hxt7p and Gal2p can transport xylose [20, 21] whereas Gal2p can also transport arabinose . Xylose and arabinose transport was improved in the evolved strain TMB3130, 1.2- to 1.5-fold and two- to threefold, respectively. This suggests an improvement in pentose uptake rate through evolution and confirms that continuous cultivation under substrate limitation tends to generate an evolved population with improvement at the level of nutrient transport [12, 13]. Specific xylose and arabinose transporters have been reported in yeast  and overexpression of one or several of these transporters may further improve simultaneous pentose fermentation.
Despite significant improvement, the xylose and arabinose co-utilization rate of the evolved industrial strain TMB3130 was three- and fivefold lower, respectively, than what was achieved with the evolved laboratory strain . TMB3130 was derived from TMB3400 that is at least diploid and originates from a cross between wine-making strains . It has been suggested that it is more difficult to obtain substantial strain improvement by evolution of industrial S. cerevisiae strains compared to haploid laboratory strains, because of the intrinsic genetic robustness that result from the polyploidy of industrial strains [11, 14]. This would explain the limited success of evolutionary approaches with industrial S. cerevisiae strains for bioethanol production [14, 25].
The evolutionary engineering protocol used to increase the consumption rate of xylose and arabinose in the laboratory S. cerevisiae strain  and in the currently reported industrial S. cerevisiae strain differed significantly in the selection method. The laboratory strain was selected through repeated batch cultivations in media alternating combinations of glucose, xylose and arabinose sugars, after limited success in evolving the parent under chemostat conditions . In contrast, the industrial strain was improved by increasing the dilution rate in a continuous cultivation with a defined medium containing a mixture of xylose and arabinose as sole carbon sources. The industrial strain carried all genes from the heterologous pentose pathways on the genome whereas the engineered CEN.PK strain combined integrated and plasmid-born genes. On the one hand multicopy plasmids may favour fine tuning of the gene copy number to match the required expression levels, on the other hand it may be more difficult to reach a stable phenotype for mixed sugar utilization unless careful selection pressure (such as successive cultivation on alternative sugars) is applied. It has previously been reported that a strain harbouring a multicopy plasmid was more prone to evolution for improved aerobic growth on xylose than the corresponding strain with all genes integrated on the genome . Therefore, one can speculate that plasmids may increase the strain instability, thereby increasing the mutation or recombination rate and favour evolution.