Metabolic engineering of bakers yeast for more efficient respiratory and fermentative glycerol utilization

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Glycerol is an abundant carbon source and an attractive feedstock for microbial fermentations due to its relatively high degree of reduction (DOG) compared to neutral sugars. This higher DOG is accompanied with higher theoretical yields of reduced small molecules used as fuels, bulk and fine chemicals. Moreover, it allows a much higher theoretical carbon dioxide fixation yield in mixed substrate conversions for the microbial production of oxidized products such as organic acids. The yeast S. cerevisiae is a popular fungal cell factory in industrial biotechnology due to its robustness in industrial settings. Particularly the ability of fungal organisms to grow at relatively low pH is highly attractive for an economically feasible organic acid production since it facilitates downstream processing. Within a prior DFG-funded project, we made significant progress in improving growth and fermentation of S. cerevisiae with glycerol as the sole source of carbon. Particularly, the equipment of S. cerevisiae with a heterologous transport protein (an aquaglyceroporin that facilitates glycerol transport) and the replacement of the endogenous FAD-dependent L-G3P pathway by an NADH-generating DHA pathway enabled the popular strain CEN.PK to grow in synthetic glycerol medium with a µmax of about 0.26 h-1. Apart from improving growth on glycerol, the engineered glycerol catabolic pathway delivering cytosolic NADH theoretically allows redox-factor neutral succinic acid (SA) production via the reverse TCA (rTCA) pathway which is combined with net fixation of CO2. We equipped the glycerol-utilizing strain with the rTCA pathway, an increased pyruvate decarboxylase activity, and a heterologous organic acid transporter. The best strain produced SA (and a minor portion of malate) from glycerol with an organic acid yield corresponding to 61.5 % of the maximum theoretical yield. Offgas analysis in controlled bioreactors with CO2 enriched gas-phase indicated that, over the course of the fermentation, our best strain led to net CO2 consumption (CO2-negativ process). Although our results are highly encouraging to test anaerobic SA fermentation, a remaining challenge is the fact that the current SA fermentation pathway consumes net ATP. Moreover, exporting SA and keeping it outside the cells is very energy-expensive. Thus, a certain portion of carbon has to enter the oxidative TCA cycle (oxTCA cycle) to supply ATP for maintenance and growth. The current proposal has two major goals. First, the general ATP balance has to be improved in order to decrease the dependency of the cells on the oxTCA cycle and respiration. A particular focus will be put on ATP-consuming transport processes and the anaplerotic reactions. A second goal is to fine-tune the flux into the oxTCA cycle in order to meet the energy and anabolic requirements of the cells without losing more carbon than necessary in the form of carbon dioxide and biomass.