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Poster Presentation 2-19
Chromosome Separation by CHEF Gel Electrophoresis and Construction of a Yeast Artificial Chromosome Expression Library
Min Hyung Kang, Yi Mu and Thomas W. Jeffries
Forest Products Laboratory, One Gifford, Pinchot Drive, Madison, WI 53705
Telephone: (608) 231-9453; Fax: (608) 231-9262; E-mail: twjeffries@fs.fed.us
Saccharomyces cerevisiae is used world-wide for bioethanol production but it does not grow well at high temperatures. The efficiency of simultaneous saccharification and fermentation (SSF) of cellulose increases with temperature, so thermotolerant, fermentative yeasts are of interest. The most studied thermotolerant yeast strain is K. marxianus IMB3. It is capable of producing ethanol at 50°C. The genetic evolution linkage between the K. marxianus and S. cerevisiae and the possibility of producing intergeneric hybrids1 suggested that we could use heterologous expression of thermotolerant genes from K. marxianus IMB3 in S. cerevisiae in order to clone and identify genes for thermotolerance. Because genetic studies indicate that multiple genes are involved in thermotolerance, we decided to explore large scale expression using yeast artificial chromosomes (YAC). In order to give thermotolerance to S. cerevisiae, YAC library construction was attempted with the genome of K. marxianus IMB3.
CHEF gel electrophoresis was used to separate chromosomes and construct a YAC library with several yeast strains. Chromosomes from Saccharomyces cerevisiae L2612, Pichia stipitis CBS6054 and Kluyveromyces marxianus IMB3 were separated by performing CHEF gel electrophoresis (CHEF-DR III, BIO-RAD, Hercules, CA), respectively. Well known CHEF gel electrophoresis conditions were used for S. cerevisiae as follows; 1% agarose, 0.5 x Tris-Borate-EDTA buffer, 14°C, 60-120 sec switch time, 24 hr run time, 120° angle and 6 V/cm voltage gradient. We developed and optimized conditions for chromosome separation for P. stipitis and K. marxianus IMB3. To separate chromosomes from them, the optimum conditions were as follows: 0.8 % agarose, 1 x Tris-Acetate-EDTA buffer, 14°C, 310-310 sec switch time, 48 hr run time, 113° angle and 4.5 V/cm of voltage gradient. On the CHEF gel, six chromosomes were separated from P. stipitis and seven chromosomes from K. marxianus IMB3, respectively.
The pYAC4 vector was used to construct the library and S. cerevisiae AB1380 (ura3-52, trp1, lys2-1, ade2-1, can1-100, his5, j+) was used as the recipient cell. The genomic DNA was prepared in the agarose block and partially digested with 0.6 U of Eco RI and 120 U of Eco RI methylase. The digested DNA in the agarose block was ligated directly with pYAC4 vector that was digested with Eco RI and BamHI and then dephosphorylated. After digesting agarose with b-agarase, transformation was performed with ligation mixture. Putative transformants were screened on uracil and tryptophan-deficient AHC media. Thermotolerant transformants were screened at a non-permissive temperature.
1Sakanaka, K., Yan, W., Kishida, M., and Sakai, T. 1996. Breeding a fermentative yeast at high temperature using protoplast fusion. J Ferm. Bioengineer. 81 (2): 104-108 1996
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