Cross-reactions between engineered xylose and galactose pathways in recombinant Saccharomyces cerevisiae

Abstract

Background: Overexpression of the PGM2 gene encoding phosphoglucomutase (Pgm2p) has been shown to improve galactose utilization both under aerobic and under anaerobic conditions. Similarly, xylose utilization has been improved by overexpression of genes encoding xylulokinase (XK), enzymes from the non-oxidative pentose phosphate pathway (non-ox PPP) and deletion of the endogenous aldose reductase GRE3 gene in engineered Saccharomyces cerevisiae strains carrying either fungal or bacterial xylose pathways. In the present study, we investigated how the combination of these traits affect xylose and galactose utilization in the presence or absence of glucose in S. cerevisiae strains engineered with the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway.

Results: In the absence of PGM2 overexpression, the combined overexpression of XK, the non-ox PPP and deletion of the GRE3 gene significantly delayed aerobic growth on galactose, whereas no difference was observed between the control strain and the xylose-engineered strain when the PGM2 gene was overexpressed. Under anaerobic conditions, the overexpression of the PGM2 gene increased the ethanol yield and the xylose consumption rate in medium containing xylose as the only carbon source. The possibility of Pgm2p acting as a xylose isomerase (XI) could be excluded by measuring the XI activity in both strains. The additional copy of the PGM2 gene also resulted in a shorter fermentation time during the co-consumption of galactose and xylose. However, the effect was lost upon addition of glucose to the growth medium.

Conclusions: PGM2 overexpression was shown to benefit xylose and galactose fermentation, alone and in combination. In contrast, galactose fermentation was impaired in the engineered xylose-utilizing strain harbouring extra copies of the non-ox PPP genes and a deletion of the GRE3 gene, unless PGM2 was overexpressed. These cross-reactions are of particular relevance for the fermentation of mixed sugars from lignocellulosic feedstock.

Figure 1

Aerobic growth in batch culture on YNB medium with 50 g l^-1 galactose with inocula grown in YNB medium with 20 g l^-1 glucose. S. cerevisiae strains: Control-PPP-XYL (filled square), PGM2-PPP-XYL (open square), Control-XYL (filled triangle), PGM2-XYL (open triangle).

Figure 2

Sugar consumption and product formation during anaerobic batch fermentation in defined medium. Strains: Control-PPP-XYL (A, C, E) and PGM2-PPP-XYL (B, D, F) with 20 g l^-1 xylose (A, B), 20 g l^-1 xylose and 20 g l^-1 galactose (C, D) or 20 g l^-1 xylose, 20 g l^-1 galactose and 20 g l^-1 glucose (E, F). Inocula were grown in defined medium with 20 g l^-1 glucose. Symbols: galactose (filled square), xylose (open square), glucose (filled diamond), ethanol (filled triangle), biomass (DW) (open circle), acetate (plus symbol), glycerol (multiplication symbol), xylitol (minus symbol).

Figure 3

Metabolic reactions involving the PGM2 (adapted from [41]). Abbreviations: PPP, Pentose phosphate pathway; PRPP, phosphoribosyl pyrophosphate; RPE, ribulose 5-phosphate 3-epimerase; RKI, ribose 5-phosphate ketol-isomerase; TKL, transketolase; -P, phosphate.