Compared to sugars, a major advantage of using glycerol as a feedstock for industrial bioprocesses is the fact that this molecule is more reduced than sugars. A compound whose biotechnological production might greatly profit from the substrate's higher reducing power is 1,2-propanediol (1,2-PDO). Here we present a novel metabolic engineering approach to produce 1,2-PDO from glycerol in S. cerevisiae. Apart from implementing the heterologous methylglyoxal (MG) pathway for 1,2-PDO formation from dihydroxyacetone phosphate (DHAP) and expressing a heterologous glycerol facilitator, the employed genetic modifications included the replacement of the native FAD-dependent glycerol catabolic pathway by the 'DHA pathway' for delivery of cytosolic NADH and the reduction of triosephosphate isomerase (TPI) activity for increased precursor (DHAP) supply. The choice of the medium had a crucial impact on both the strength of the metabolic switch towards fermentation in general (as indicated by the production of ethanol and 1,2-PDO) and on the ratio at which these two fermentation products were formed. For example, virtually no 1,2-PDO but only ethanol was formed in synthetic glycerol medium with urea as the nitrogen source. When nutrient-limited complex YG medium was used, significant amounts of 1,2-PDO were formed and it became obvious that the concerted supply of NADH and DHAP are essential for boosting 1,2-PDO production. Additionally, optimizing the flux into the MG pathway improved 1,2-PDO formation at the expense of ethanol. Cultivation of the best-performing strain in YG medium and a controlled bioreactor set-up resulted in a maximum titer of > 4gL 1,2-PDO which, to the best of our knowledge, has been the highest titer of 1,2-PDO obtained in yeast so far. Surprisingly, significant 1,2-PDO production was also obtained in synthetic glycerol medium after changing the nitrogen source towards ammonium sulfate and adding a buffer.

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BackgroundDue to its inevitable formation during biodiesel production and its relatively high degree of reduction, glycerol is an attractive carbon source for microbial fermentation processes. However, glycerol is catabolized in a fully respiratory manner by the eukaryotic platform organism Saccharomyces cerevisiae. We previously engineered S. cerevisiae strains to favor fermentative metabolism of glycerol by replacing the native FAD-dependent glycerol catabolic pathway with the NAD-dependent ‘DHA pathway’. In addition, a heterologous aquaglyceroporin (Fps1 homolog) was expressed to facilitate glycerol uptake. The current study was launched to scrutinize the formation of S. cerevisiae’s natural fermentation product ethanol from glycerol caused by the conducted genetic modifications. This understanding is supposed to facilitate future engineering of this yeast for fermenting glycerol into valuable products more reduced than ethanol.ResultsA strain solely exhibiting the glycerol catabolic pathway replacement produced ethanol at concentrations close to the detection limit. The expression of the heterologous aquaglyceroporin caused significant ethanol production (8.5 g L−1 from 51.5 g L−1 glycerol consumed) in a strain catabolizing glycerol via the DHA pathway but not in the wild-type background. A reduction of oxygen availability in the shake flask cultures further increased the ethanol titer up to 15.7 g L−1 (from 45 g L−1 glycerol consumed).ConclusionThe increased yield of cytosolic NADH caused by the glycerol catabolic pathway replacement seems to be a minimal requirement for the occurrence of alcoholic fermentation in S. cerevisiae growing in synthetic glycerol medium. The remarkable metabolic switch to ethanol formation in the DHA pathway strain with the heterologous aquaglyceroporin supports the assumption of a much stronger influx of glycerol accompanied by an increased rate of cytosolic NADH production via the DHA pathway. The fact that a reduction of oxygen supply increases ethanol production in DHA pathway strains is in line with the hypothesis that a major part of glycerol in normal shake flask cultures still enters the catabolism in a respiratory manner.