In addition to tolerance and robustness, strain stability is a prerequisite when designing yeast strains for industrial use. Strains carrying mul­ticopy plasmids are generally not applicable in industry due to their instability [123,145]. Multicopy plasmids require auxotrophic or antibiotic resistance markers to be retained in the cell, both of which are not ap­plicable in industrial media containing complex nutrients and being used in large volumes. Thus, chromosomal integration is necessary for any genes to be introduced in industrially applied yeast strains. Ideally this re­quires sufficient specific activity of the introduced heterologous enzymes, so that single-copy integration supplies enough activity for metabolic func­tion. Multiple chromosomal integration has also been utilized to gener­ate stable pentose-fermenting strains with high activity of the enzymes introduced [6-8].

Metabolic engineering strategies applied on industrial strains have been limited to the introduction of the initial xylose and arabinose utilization pathways [4,5,8,101]. Only the XR-XDH pathway has been developed in in­dustrial S. cerevisiae strains [4,5,101] (strains A4 and A6, Table 1; strain F, Tables 1 and 4; strain TMB3400, Tables 1, 3 and 4; strain 1400(pLNH32), Table 2). No chromosomally integrated XI constructs have been reported. XI expression in S. cerevisiae seems to require a multicopy expression system to provide sufficient enzyme activity for xylose growth and fermentation [43]. Due to the difficulty of applying complex metabolic engineering strategies in industrial strains, procedures for random strain improvement have been relied upon to improve xylose utilization.

5.3