Metabolic engineering of Saccharomyces cerevisiae for redox-assisted co-fermentation of l-rhamnose.

Rapid proliferation of Ulva species, commonly referred to as green tides, significantly impacts coastal regions globally, causing environmental degradation and economic disruption. Efficient utilization of Ulva biomass as a sustainable feedstock requires microbial strains capable of metabolizing its diverse sugars, including l-rhamnose, a major ulvan-derived carbohydrate constituent that industrial yeast Saccharomyces cerevisiae cannot naturally utilize. Here, we engineered S. cerevisiae for l-rhamnose utilization by integrating a heterologous l-rhamnose transporter and a complete fungal l-rhamnose catabolic pathway. Limited growth and l-rhamnose consumption under l-rhamnose-only conditions indicated that oxidative l-rhamnose catabolism imposes a net NADH burden. Introduction of the l-rhamnose module into a xylose-fermenting background enabled simultaneous consumption of 10 g/L l-rhamnose and 40 g/L xylose within 36 h under the tested co-fermentation conditions, resulting in a higher ethanol titer and detectable l-lactic acid production during mixed-sugar fermentation. Metabolite profiling further revealed production of 1,2-propanediol, which was confirmed by GC/MS and was consistent with promiscuous reduction of the l-rhamnose intermediate l-lactaldehyde by heterologous xylose reductase and/or endogenous reductases. Elementary flux mode analysis supported the stoichiometric feasibility of ethanol, l-lactate, and 1,2-propanediol formation during xylose/l-rhamnose co-utilization. Finally, alleviation of glucose repression via hexokinase mutations enabled simultaneous fermentation of glucose and l-rhamnose. Collectively, these results provide a basis for developing engineered yeast platforms to valorize Ulva-derived mixed-sugar streams.