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Biotechnology and Applied Biochemistry (2008) 49, (113–120) (Printed in Great Britain)

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Genome shuffling in the ethanologenic yeast Candida krusei to improve acetic acid tolerance

Pingying Wei*†, Zilong Li*, Peng He*, Yuping Lin* and Ning Jiang*¹

*Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China, and †Graduate School, Chinese Academy of Sciences, Beijing, People's Republic of China

Key words: acetic acid tolerance, Candida krusei, ethanol fermentation, genome shuffling, membrane integrity, non-haploid organism.

Abbreviations used: CAB, 0.1 mol·l^-1 citric acid buffer of pH 5.8; CABK, CAB with 0.8 mol·l^-1 KCl; EFM, ethanol fermentation medium; YNBX, yeast nitrogen base/xylose; YNB, YNBX without xylose; YPD, yeast extract, peptone and dextrose medium; YPDK, YPD with 0.8 mol·l^-1 KCl.

¹To whom correspondence should be addressed (email jiangn@im.ac.cn).

Genome shuffling was used to improve the acetic acid tolerance of an ethanologenic yeast, Candida krusei GL560. A mutant, S4-3, was isolated and selected after four rounds of genome shuffling. It was found that the mutant S4-3 had a higher viability in the YNBX (yeast nitrogen base/xylose) medium with acetic acid and grew better in the YPD (yeast extract, peptone and dextrose) medium [1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose] with acetic acid than the parent strain GL560. The mutant S4-3 also improved its multiple stress tolerance to ethanol, H₂O₂, heat and freeze–thaw. Furthermore, S4-3 showed higher ethanol production than GL560 in EFM (ethanol fermentation medium) with or without acetic acid. The DNA content of S4-3 was similar to its parent strains in the genome shuffling. This suggested that gene exchange, as caused by homologous recombination, may have occurred during the process. Higher membrane integrity and intracellular catalase activity were two possible reasons for the higher acid-tolerance phenotype of S4-3. These results indicated that genome shuffling is a powerful means of rapidly improving the complex traits of non-haploid organisms, while still maintaining robust growth.

Received 2 April 2007/29 May 2007; accepted 16 July 2007

Published as Immediate Publication 16 July 2007, doi:10.1042/BA20070072

© 2008 Portland Press Ltd