Survival of Kluyveromyces marxianus with stigmasterol as subjected to freezing stress.

Kluyveromyces marxianus is an aerobic yeast and is interested to be applied in many industries. This research was aimed to study the effect of the sterol alternative to ergosterol on the freezing stress of K. marxianus UBU1-11, a thermotolerant yeast. The 0-9 mgL-1 stigmasterol were added to the YM broth and applied for culturing. The growth of all conditions were not interfered by the addition of stigmasterol. The intra-cellular sterol content was detected in the medium with 5 mgL-1 stigmasterol and higher, where the maximum content was 0.32 mg g-1 cell dry weight. After frozen and thawed, the cultures contained stigmasterol had significantly higher viability than those without. It was found that the amount of stigmasterol contained in cells did not affect the number of survival. The stigmasterol provided a significant protection to the yeast cell when subjected to slow freezing. It also increased the survival rate of the culture subjected to subzero temperature storage.

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae.

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.