Construction of a Candida utilis strain with ratio-optimized expression of xylose-metabolizing enzyme genes by cocktail multicopy integration method

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We previously reported the construction of a recombinant Candida utilis strain expressing mXYL1, XYL2 and XYL3, which encode mutated Candida shehatae xylose reductase K275R/N277D, C. shehatae xylitol dehydrogenase and Pichia stipitis xylulokinase to produce ethanol from xylose. However, its productivity was low. In this study, to breed a strain with higher productivity of ethanol from xylose, we used a cocktail multicopy integration method to attain optimized gene dosage of the three enzymes. Gene expression cassettes of the xylose-metabolizing enzymes were simultaneously integrated into C. utilis chromosomes in one step. Measurement of integrated gene copy number and xylose fermentability in all of the resulting integrant strains revealed that the copy number ratio of XYL2/mXYL1 in strains with higher ethanol yield was higher than that in strains with lower ethanol yield, whereas the copy number ratio of mXYL1/XYL3 was lower in strains with higher ethanol yield. The resultant strain CIS35, which was found to be the best producer of ethanol from xylose produced 29.2 g/L of ethanol, yielding 0.402 g ethanol/g xylose. This result provides that C. utilis may be a good candidate as a host for ethanol production from xylose.

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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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