Mitochondrial membrane transporters as attractive targets for the fermentative production of succinic acid from glycerol in Saccharomyces cerevisiae.

Previously, we reported an engineered Saccharomyces cerevisiae CEN.PK113-1A derivative able to produce succinic acid (SA) from glycerol with net CO2 fixation. Apart from an engineered glycerol utilization pathway that generates NADH, the strain was equipped with the NADH-dependent reductive branch of the TCA cycle (rTCA) and a heterologous SA exporter. However, the results indicated that a significant amount of carbon still entered the CO2-releasing oxidative TCA cycle. The current study aimed to tune down the flux through the oxidative TCA cycle by targeting the mitochondrial uptake of pyruvate and cytosolic intermediates of the rTCA pathway, as well as the succinate dehydrogenase complex. Thus, we tested the effects of deletions of MPC1, MPC3, OAC1, DIC1, SFC1, and SDH1 on SA production. The highest improvement was achieved by the combined deletion of MPC3 and SDH1. The respective strain produced up to 45.5 g/L of SA, reached a maximum SA yield of 0.66 gSA/gglycerol, and accumulated the lowest amounts of byproducts when cultivated in shake-flasks. Based on the obtained data, we consider a further reduction of mitochondrial import of pyruvate and rTCA intermediates highly attractive. Moreover, the approaches presented in the current study might also be valuable for improving SA production when sugars (instead of glycerol) are the source of carbon.

Towards valorization of pectin-rich agro-industrial residues: Engineering of Saccharomyces cerevisiae for co-fermentation of d-galacturonic acid and glycerol.

Pectin-rich plant biomass residues represent underutilized feedstocks for industrial biotechnology. The conversion of the oxidized monomer d-galacturonic acid (d-GalUA) to highly reduced fermentation products such as alcohols is impossible due to the lack of electrons. The reduced compound glycerol has therefore been considered an optimal co-substrate, and a cell factory able to efficiently co-ferment these two carbon sources is in demand. Here, we inserted the fungal d-GalUA pathway in a strain of the yeast S. cerevisiae previously equipped with an NAD-dependent glycerol catabolic pathway. The constructed strain was able to consume d-GalUA with the highest reported maximum specific rate of 0.23 g gCDW-1 h-1 in synthetic minimal medium when glycerol was added. By means of a 13C isotope-labelling analysis, carbon from both substrates was shown to end up in pyruvate. The study delivers the proof of concept for a co-fermentation of the two 'respiratory' carbon sources to ethanol and demonstrates a fast and complete consumption of d-GalUA in crude sugar beet pulp hydrolysate under aerobic conditions. The future challenge will be to achieve co-fermentation under industrial, quasi-anaerobic conditions.

Identification of the Aldo-Keto Reductase Responsible for d-Galacturonic Acid Conversion to l-Galactonate in Saccharomyces cerevisiae.

d-galacturonic acid (d-GalUA) is the main constituent of pectin, a complex polysaccharide abundant in several agro-industrial by-products such as sugar beet pulp or citrus peel. During several attempts to valorise d-GalUA by engineering the popular cell factory Saccharomyces cerevisiae, it became obvious that d-GalUA is, to a certain degree, converted to l-galactonate (l-GalA) by an endogenous enzymatic activity. The goal of the current work was to clarify the identity of the responsible enzyme(s). A protein homology search identified three NADPH-dependent unspecific aldo-keto reductases in baker's yeast (encoded by GCY1, YPR1 and GRE3) that show sequence similarities to known d-GalUA reductases from filamentous fungi. Characterization of the respective deletion mutants and an in vitro enzyme assay with a Gcy1 overproducing strain verified that Gcy1 is mainly responsible for the detectable reduction of d-GalUA to l-GalA.