Yi Mu,
Jose M. Laplaza and Thomas W. Jeffries*
Phone: (608)231-9453
E-mail: twjeffri@facstaff.wisc.edu
Kluyveromyces marxianus is an industrially interesting yeast because of its ability to grow at high temperature on a wide range of substrates including inulin and galactose. Disruption of the cytochrome C gene (CYC1) has been used in a variety of yeasts to interrupt the electron transfer system in mitochondria. This has been shown to produce strains with higher fermentative capacity in some cases. A 333-bp open reading frame constituting the CYC1 gene in K. marxianus CBS6556 was cloned along with 601 bp upstream and 328 bp downstream.
A transformation system was developed for K. marxianus
CBS 6556 by inserting an ARS fragment from K.
marxianus Y610 into the plasmid pTEF1/Zeo
containing the zeocin resistance gene (sh ble). Successful transformation of K. marxianus
was obtained using a modified LiAc method. CYC1 was
disrupted using the sh ble. The
knock-out mutant (Dcyc1) could
not grow on non-fermentable carbon sources such as glycerol or xylose. In addition,
the Dcyc1 strain could not grow on cellobiose. The permissive temperature of the Dcyc1 strain was lower by 2° C when
compared to the parental strain. The Dcyc1 strain did not show an
improvement over the CYC1 strain in
ethanol production when grown in rich media with 5% xylose.
After 142 hr of fermentation in xylose, the cell viability percentages of the host and the
knock-out strain were 95.6%, 75.3% respectively.