Abstract
We report the development of an efficient genetic transformation system for Lipomyces starkeyi based on a modified lithium acetate transformation protocol. L. starkeyi is a highly lipogenic yeast that grows on a wide range of substrates. The initial transformation rate for this species was extremely low, and required very high concentrations of DNA. A systematic approach for optimizing the protocol resulted in an increase in the transformation efficiency by four orders of magnitude. Important parameters included cell density, the duration of incubation and recovery periods, the heat shock temperature, and the concentration of lithium acetate and carrier DNA within the transformation mixture. We have achieved efficiencies in excess of 8,000 transformants/µg DNA, which now make it possible to screen libraries in the metabolic engineering of this yeast. Metabolic engineering based on this transformation system could improve lipogenesis and enable formation of higher value products.
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Acknowledgments
This work was funded in part by the Department of Energy Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494). CHC was supported in part through a Grant entitled “Investigation of Lipid Accumulation in Lipomyces starkeyi” awarded by the Graduate School of University of Wisconsin-Madison to TWJ. CHC gratefully acknowledges Kenneth Hammel for sponsorship at the USDA Forest Products Laboratory.
Conflict of interest
TWJ is the president and owner of Xylome, a private entity. However, the research carried out in this program was conducted independently of Xylome, and the authors declare that they have no conflict of interest.
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Communicated by S. Hohmann.
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Calvey, C.H., Willis, L.B. & Jeffries, T.W. An optimized transformation protocol for Lipomyces starkeyi . Curr Genet 60, 223–230 (2014). https://doi.org/10.1007/s00294-014-0427-0
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DOI: https://doi.org/10.1007/s00294-014-0427-0