Introduction of pentose catabolic pathways into S. cerevisiae
enabled this yeast to ferment the lignocellulosic pentoses D-xylose and L-arabinose into ethanol. However, low fermentation rates compared to D-glucose and a lack of pentose utilization in the presence of high D-glucose concentrations are still major obstacles for fermentation of mixed sugar hydrolyzates [38
]. It is generally assumed that competitive inhibition of pentose uptake by D-glucose is the main problem for simultaneous co-fermentation of D-glucose and pentoses.
To investigate the influence of further D-glucose metabolism on pentose utilization independent of the competition at the uptake level we used the alternative carbon source maltose as a non-competitive transport substrate. Indeed, we found that pentose consumption was impaired by simultaneous maltose utilization (Figure ). As maltose did not impair pentose utilization in a hexo-/glucokinase mutant strain an inhibitory effect of maltose on pentose uptake can clearly be excluded. Hexose and pentose catabolism converge at the level of phosphofructokinase. Therefore, hexose catabolism might impair pentose utilization on the level of a limiting glycolytic enzyme activity. On the other hand, hexose catabolism could negatively affect regulation of enzymes of the pentose phosphate pathway used for the catabolism of pentoses. Moreover, the results do not exclude the possibility that during the utilization of D-glucose when fluxes are higher than during the utilization of maltose there might be an even stronger impact of D-glucose catabolism on pentose utilization.
Xylose isomerases are well known to also act as bona fide
glucose isomerases converting D-glucose into D-fructose [25
]. Therefore, it might be possible that intracellular D-glucose competes with D-xylose at the level of xylose isomerase. Moreover, it is known that intracellular D-glucose can be transported out of the cells via the hexose transporters [26
]. Therefore, increased levels of D-glucose within the cells might negatively influence pentose uptake. In this sense, recently it has been shown that the binding of intracellular D-glucose inhibits the yeast D-glucose sensor Snf3 which has high homologies with the hexose transporters [43
]. However, our results with hexo-/glucokinase mutant strains in the presence of maltose could prove that even increased intracellular D-glucose levels did not interfere with pentose isomerization or uptake.
Pentose consumption analyses of the hxk/glk null strains confirmed that extracellular D-glucose inhibits pentose consumption in a concentration dependent manner. Increasing concentrations of D-glucose gradually decreased pentose consumption, both in the case of D-xylose as well as L-arabinose (Figure ). Nevertheless, this experiment and also the co-consumption experiment with the hexokinase-limited mutant strain clearly show that principally co-consumption is possible as long as D-glucose concentrations are low enough.
Accordingly, overexpression of those hexose transporters with a high capacity for D-xylose uptake, Hxt7 [12
], or L-arabinose uptake, Gal2 [22
], partially relieved the inhibitory effects of extracellular D-glucose (Figure ). Especially in the case of L-arabinose consumption it turned out that constitutive overexpression of GAL2
was quite beneficial. This probably is mainly due to the strong repression of the endogenous GAL2
gene in the presence of D-glucose [44
]. Gal2 is the only hexose transporter of S. cerevisiae
that can effectively transport L-arabinose [21
]. Therefore, a constitutive overexpession of GAL2
is a prerequisite to allow efficient L-arabinose uptake in the presence of D-glucose. In the case of D-xylose, most of the D-xylose-transporting hexose transporters like HXT7
are even inducible by low concentrations of D-glucose but are repressed by high concentrations [47
]. This might explain why D-xylose consumption was less inhibited than L-arabinose consumption by low concentrations of D-glucose as these even induced higher D-xylose transport capacities. Indeed, this would also be in accordance with earlier observations demonstrating that low glucose concentrations (down to 0.1 g/L) increase xylose utilization [49
] although we could not observe this with the lowest glucose concentrations used in our study (1 g/L). It was speculated that, beside other effects, increased xylose uptake might be responsible for this.
Our results show that co-fermentation of D-glucose and pentoses can be improved either by keeping D-glucose concentrations on a low level or by the expression of specific heterologous pentose transporters that are not inhibited by D-glucose. Indeed, using prefermentation and fed-batch systems to minimize initial D-glucose concentrations D-xylose utilization could recently be increased in a simultaneous saccharification and co-fermentation (SSCF) process [50
]. Moreover, in E. coli
co-consumption of D-glucose and D-xylose could be demonstrated just by eliminating catabolite repression by D-glucose of D-xylose specific transporters xylE or xylFGH [52
]. On the other hand, Ha et al. [54
] recently engineered a xylose fermenting S. cerevisiae
strain for simultaneous fermentation of xylose and cellobiose by expressing a specific cellobiose transporter together with an intracellular β-glucosidase.
For future perspective, a pentose-fermenting hexo-/glucokinase deletion strain might be an interesting screening system to select for mutants able to ferment pentoses in the presence of increasing concentrations of D-glucose, e.g. via evolutionary engineering [55
]. Reintroduction of hexokinase activity should then result in strains able to consume pentoses even in the presence of higher concentrations of D-glucose.