Rapid and marker-free refactoring of xylose-fermenting yeast strains with Cas9/CRISPR.

Genomic integration of expression cassettes containing heterologous genes into yeast with traditional methods inevitably deposits undesirable genetic elements into yeast chromosomes, such as plasmid-borne multiple cloning sites, antibiotic resistance genes, Escherichia coli origins, and yeast auxotrophic markers. Specifically, drug resistance genes for selecting transformants could hamper further industrial usage of the resulting strains because of public health concerns. While we constructed an efficient and rapid xylose-fermenting Saccharomyces cerevisiae, the engineered strain (SR8) might not be readily used for a large-scale fermentation because the SR8 strain contained multiple copies of drug resistance genes. We utilized the Cas9/CRISPR-based technique to refactor an efficient xylose-fermenting yeast strain without depositing any undesirable genetic elements in resulting strains. In order to integrate genes (XYL1, XYL2, and XYL3) coding for xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis, and delete both PHO13 and ALD6, a double-strand break formation by Cas9 and its repair by homologous recombination were exploited. Specifically, plasmids containing guide RNAs targeting PHO13 and ALD6 were sequentially co-transformed with linearized DNA fragments containing XYL1, XYL2, and XYL3 into S. cerevisiae expressing Cas9. As a result, two copies of XYL1, XYL2, and XYL3 were integrated into the loci of PHO13 and ALD6 for achieving overexpression of heterologous genes and knockout of endogenous genes simultaneously. With further prototrophic complementation, we were able to construct an engineered strain exhibiting comparable xylose fermentation capabilities with SR8 within 3 weeks. We report a detailed procedure for refactoring xylose-fermenting yeast using any host strains. The refactored strains using our procedure could be readily used for large-scale fermentations since they have no antibiotic resistant markers.

Simultaneous utilization of cellobiose, xylose, and acetic acid from lignocellulosic biomass for biofuel production by an engineered yeast platform.

The inability of fermenting microorganisms to use mixed carbon components derived from lignocellulosic biomass is a major technical barrier that hinders the development of economically viable cellulosic biofuel production. In this study, we integrated the fermentation pathways of both hexose and pentose sugars and an acetic acid reduction pathway into one Saccharomyces cerevisiae strain for the first time using synthetic biology and metabolic engineering approaches. The engineered strain coutilized cellobiose, xylose, and acetic acid to produce ethanol with a substantially higher yield and productivity than the control strains, and the results showed the unique synergistic effects of pathway coexpression. The mixed substrate coutilization strategy is important for making complete and efficient use of cellulosic carbon and will contribute to the development of consolidated bioprocessing for cellulosic biofuel. The study also presents an innovative metabolic engineering approach whereby multiple substrate consumption pathways can be integrated in a synergistic way for enhanced bioconversion.