Construction and Analysis of High-Ethanol-Producing Fusants with Co-Fermentation Ability through Protoplast Fusion and Double Labeling Technology

Abstract

Double labeling of resistance markers and report genes can be used to breed engineered Saccharomyces cerevisiae strains that can assimilate xylose and glucose as a mixed carbon source for ethanol fermentation and increased ethanol production. In this study Saccharomyces cerevisiae W5 and Candida shehatae 20335 were used as parent strains to conduct protoplast fusion and the resulting fusants were screened by double labeling. High performance liquid chromatography (HPLC) was used to assess the ethanol yield following the fermentation of xylose and glucose, as both single and mixed carbon sources, by the fusants. Interestingly, one fusant (ZLYRHZ7) was demonstrated to have an excellent fermentation performance, with an ethanol yield using the mixed carbon source of 0.424 g g−1, which compares with 0.240 g g−1 (W5) and 0.353 g g−1 (20335) for the parent strains. This indicates an improvement in the ethanol yield of 43.4% and 16.7%, respectively.

Molecular Genetic Characterization of Fusants from Protoplast Fusion of S. Cerevisiae and P. Stipitis ATCC 58785

“…Protoplast fusion is a conventional technique even though it has been shown to be successful for strain enhancement and it is seen as significant in constructing yeast strains as it mitigates the challenges of genetic modification enforced by traditional mating systems and supports the movement of sizeable segments of genomic DNA. Protoplast fusion is a common approach used to improve fermentation of industrial yeast strains [1][2][3]. The ease of the technical application in addition to the materials needed for this approach makes protoplast fusion the most extensively employed technique of transformation in fungi.…”