Rapid and efficient galactose fermentation by engineered Saccharomyces cerevisiae

• Published in: Journal of biotechnology 2016-07, Vol.229, p.13-21
• Main Authors: Quarterman, Josh, Skerker, Jeffrey M., Feng, Xueyang, Liu, Ian Y., Zhao, Huimin, Arkin, Adam P., Jin, Yong-Su
• Format: Article
• Language: English
• Subjects: Cluster Analysis; Deletion; Ethanol; Ethanol - metabolism; Ethyl alcohol; Evolution; Evolutionary engineering; Fermentation; Galactose; Galactose - metabolism; Genome sequencing; Genome, Fungal - genetics; Metabolic Engineering - methods; Metabolomics; Respiration; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Strain; Systems biology; Systems Biology - methods; Yeast
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2016.04.041

•Deletion of respiratory enzyme subunit led to metabolic ‘death valley’ on galactose.•Adaptive evolution overcame inherent obstacles to galactose use without respiration.•Evolved respiration-deficient strain showed rapid, efficient galactose fermentation.•Genetic basis of evolved strain was identified by sequencing.•Fluxomics and metabolomics facilitated systems-level characterization of strains. In the important industrial yeast Saccharomyces cerevisiae, galactose metabolism requires energy production by respiration; therefore, this yeast cannot metabolize galactose under strict anaerobic conditions. While the respiratory dependence of galactose metabolism provides benefits in terms of cell growth and population stability, it is not advantageous for producing fuels and chemicals since a substantial fraction of consumed galactose is converted to carbon dioxide. In order to force S. cerevisiae to use galactose without respiration, a subunit (COX9) of a respiratory enzyme was deleted, but the resulting deletion mutant (Δcox9) was impaired in terms of galactose assimilation. Interestingly, after serial sub-cultures on galactose, the mutant evolved rapidly and was able to use galactose via fermentation only. The evolved strain (JQ-G1) produced ethanol from galactose with a 94% increase in yield and 6.9-fold improvement in specific productivity as compared to the wild-type strain. 13C-metabolic flux analysis demonstrated a three-fold reduction in carbon flux through the TCA cycle of the evolved mutant with redirection of flux toward the fermentation pathway. Genome sequencing of the JQ-G1 strain revealed a loss of function mutation in a master negative regulator of the Leloir pathway (Gal80p). The mutation (Glu348*) in Gal80p was found to act synergistically with deletion of COX9 for efficient galactose fermentation, and thus the double deletion mutant Δcox9Δgal80 produced ethanol 2.4 times faster and with 35% higher yield than a single knockout mutant with deletion of GAL80 alone. When we introduced a functional COX9 cassette back into the JQ-G1 strain, the JQ-G1-COX9 strain showed a 33% reduction in specific galactose uptake rate and a 49% reduction in specific ethanol production rate as compared to JQ-G1. The wild-type strain was also subjected to serial sub-cultures on galactose but we failed to isolate a mutant capable of utilizing galactose without respiration. We concluded that the metabolic “death valley” (i.e. no galactose utilization by the Δcox9 muta