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Targeted gene replacement in the thermotolerant yeast Kluyveromyces marxianus KCTC 17555 has been hampered by its propensity to non-homologous end joining (NHEJ). To enhance homologous recombination (HR) by blocking NHEJ, we identified and disrupted the K. marxianus KU80 gene. The ku80 deletion mutant strain (Kmku80∆) of K. marxianus KCTC 17555 did not show apparent growth defects under several conditions with the exception of exposure to tunicamycin. The targeted disruption of the three model genes, KmLEU2, KmPDC1, and KmPDC5, was increased by 13-70 % in Kmku80∆, although the efficiency was greatly affected by the length of the homologous flanking fragments. In contrast, the double HR frequency was 0-13.7 % in the wild-type strain even with flanking fragments 1 kb long. Therefore, Kmku80∆ promises to be a useful recipient strain for targeted gene manipulation.

Galacto-oligosaccharides (GOS) were synthesized from lactose by immobilized and free beta-galactosidase from Kluyveromyces lactis (Lactozym 3000 L HP-G) using either focused microwave irradiation or conventional heating. Immobilization of the beta-galactosidase on to Duolite A-568 increased the synthesis of GOS. GOS selectivity (GOS synthesis/lactose hydrolysis ratio) increased when the water activity of the media was reduced, notably with a high initial lactose concentration but also by using co-solvents in the media. The advantage of microwave heating on GOS formation was also examined. Addition of solvent and carrying out the reaction under microwave irradiation resulted an increase in the production of GOS. The selectivity for GOS synthesis can be increased by 217-fold under microwave irradiation, using immobilized beta-glucosidase and with added co-solvents such as hexanol.