Construction of a Candida utilis strain with ratio-optimized expression of xylose-metabolizing enzyme genes by cocktail multicopy integration method
Section snippets
Strains, media and culture conditions
Escherichia coli strain DH5α (Toyobo, Osaka, Japan) transformants were grown in Luria-Bertani medium (10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of NaCl) containing 100 mg/L of ampicillin. The C. utilis strains used in this study are listed in Table 1. Cells were cultivated at 30°C in YPD2 medium (10 g/L of yeast extract, 20 g/L of peptone, and 20 g/L of glucose) unless stated otherwise. Solid media were made with 2% agar. Hygromycin B (HygB, Wako Pure Chemical Industries, Osaka,
Construction and selection of recombinant C. utilis strains by cocktail multicopy integration
A cocktail multicopy integration method based on a δ-integrative plasmid has been reported in S. cerevisiae (36), although the δ sequence of C. utilis has not been found. It has been reported that up to 90 copies of the multicopy integrative expression vector pCU155 have been integrated in tandem at the URA3 loci of C. utilis, and no notable decrease of the heterologous protein production was observed in the transformants with the plasmid after 50 generations of nonselective growth (27). The
Discussion
There have been several reports about the construction of engineered S. cerevisiae strains with a high yield of ethanol from xylose. For example, Ha et al. expressed both P. stipitis XR and its mutated XR (R276H) in S. cerevisiae, along with P. stipitis XDH and P. stipitis XK, and evolved an engineered strain by repeated subculture on xylose-containing medium (38). The resultant strain produced 27.9 g/L ethanol within 60 h on YP-based medium containing 80 g/L xylose, yielding 0.350 g produced
Acknowledgments
We wish to thank Drs. Osamu Kobayashi, Takayoshi Kirisako and Yasuyuki Tomita for valuable discussion throughout the course of this study.
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