Evolutionarily Identified Biological Design Allowing Simultaneous Sugar Utilization in Yeast

Diauxic growth on mixtures of glucose and other sugars is a common phenotype throughout microorganisms.  Glucose is generally consumed first followed by slower growth and consumption of remaining carbon sources.  Considering that hydrolysates of renewable biomass are generally comprised of mixtures of glucose and other sugars, diauxic growth patterns are problematic for enabling efficient renewable bioconversion.   Additionally, while techniques have been developed to enable simultaneous consumption of specific mixtures of sugars, a widely-applicable technique is desirable.  In this study we report a generalizable sugar cofermentation strategy in the yeast Saccharomyces cerevisiae.  Through evolutionary engineering with the glucose analog 2-deoxyglucose, we isolate a mutant strain capable of simultaneously consuming glucose and xylose at nearly equivalent rates and identify three mutations in the yeast hexokinases sufficient to confer the phenotype.  Through analysis of deletion mutants, we rule out the role of specific yeast transporters and identify the overall cause behind the simultaneous glucose and xylose utilization: a reduced rate of glucose phosphorylation.  We then recreate the simultaneous utilization phenotype with wild-type hexokinases by controlling their expression via the yeast doxycycline induction system.  These results demonstrate that despite significant recent work in engineering xylose-specific sugar transporters, sugar mixtures can indeed be simultaneously consumed without modifications to the endogenous yeast transport machinery.  We ultimately show that this technique is also effective at enabling co-consumption of mixtures of glucose and galactose.  In the case of both glucose/xylose and glucose/galactose mixtures, tuning the rate of glucose phosphorylation allowed adjusting the relative sugar consumption rates as desired.   This is to our knowledge the first report of a broadly-applicable cofermentation strategy that works across a range of metabolic pathways and is effective without regard to competitive transport inhibition.