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.

Cell wall synthesis and central carbohydrate metabolism are interconnected by the SNF1/Mig1 pathway in Kluyveromyces lactis.

The trimeric AMP-activated kinase complex (AMPK) is conserved from yeast to humans and is best known for its role in balancing energy metabolism. Additional functions, including the regulation of cell wall biosynthesis, have been proposed for the SNF1 complex, the baker's yeast homolog of AMPK. We here demonstrate that this function is conserved in the Crabtree-negative milk yeast Kluyveromyces lactis. Deletion mutants in the genes encoding the subunits of the trimeric complex (Klsnf1, Klgal83, Klsnf4) displayed increased sensitivities towards cell wall stress agents and a mutant lacking the kinase subunit had a thinner cell wall in transmission electron micrographs as compared to wild type. Epistasis analyses demonstrated that the KlSNF1 complex acts in parallel to cell wall integrity (CWI) signaling and stress sensitivities of Klsnf1 deletions can be suppressed by additional deletions in glycolytic genes (KlPFK1, KlPFK2, KlPGI1) or by a Klmig1 mutant. Western blot analyses of an HA-tagged KlMig1p revealed its phosphorylation on ethanol medium similar to its S. cerevisiae ortholog, but a substantial amount of protein remained phosphorylated even with high glucose concentrations. Application of cell wall stress shifted this equilibrium towards the non-phosphorylated fraction of KlMig1p. We conclude that KlMig1p may exert a negative regulatory function on cell wall biosynthesis.

Investigation of the role of four mitotic septins and chitin synthase 2 for cytokinesis in Kluyveromyces lactis.

Septins are key components of the cell division machinery from yeast to humans. The model yeast Saccharomyces cerevisiae has five mitotic septins, Cdc3, Cdc10, Cdc11, Cdc12, and Shs1. Here we characterized the five orthologs from the genetically less-redundant milk yeast Kluyveromyces lactis. We found that except for KlSHS1 all septin genes are essential. Klshs1 deletions displayed temperature-sensitive growth and morphological defects. Heterologous complementation analyses revealed that all five K. lactis genes encode functional orthologs of their S. cerevisiae counterparts. Fluorophore-tagged versions of the K. lactis septins localized to a ring at the incipient bud site and split into two separate rings at the bud neck later in cytokinesis. One of the key proteins recruited to the bud neck by septins in S. cerevisiae is the chitin synthase Chs2, which synthesizes the primary septum. KlCHS2 was found to be essential and deletions showed cytokinetic defects upon spore germination. KlChs2-GFP also localized to the bud neck and to punctate structures in K. lactis. We conclude that cytokinesis in K. lactis is similar to S. cerevisiae and chimeric septin complexes are fully functional in both yeasts. In contrast to some S. cerevisiae strains, KlChs2 and KlCdc10 were found to be essential.

Regulation of cytokinesis in the milk yeast Kluyveromyces lactis.

Cytokinesis in yeast and mammalian cells is a highly coordinated process mediated by the constriction of an actomyosin ring. In yeasts, it is accompanied by the formation of a chitinous primary septum. Although much is known about the regulation of cytokinesis in budding yeast, overlapping functions of redundant genes complicates genetic analyses. Here, we investigated the effects of various deletion mutants on cytokinesis in the milk yeast Kluyveromyces lactis. To determine the spatiotemporal parameters of cytokinesis components, live-cell imaging of fluorophor-tagged KlMyo1 and a new Lifeact probe for KlAct1 was employed. In contrast to Saccharomyces cerevisiae, where deletion of ScMYO1 is lethal, Klmyo1 deletion was temperature-sensitive. Transmission and scanning electron microscopy demonstrated that the Klmyo1 deletion cells had a defect in the formation of the primary septum and in cell separation; this result was confirmed by FACS analyses. Deletion of KlCYK3 was lethal, whereas in S. cerevisiae a cyk3 deletion is synthetically lethal with hof1 deletion. Growth of Klhof1 mutants was osmoremedial at 25°C, as it is in S. cerevisiae. CYK3 and HOF1 genes cross-complemented in both species, suggesting that they are functional homologs. Inn1, a common interactor for these two regulators, was essential in both yeasts and the encoding genes did not cross-complement. The C2 domain of the Inn1 homologs conferred species specificity. Thus, our work establishes K. lactis as a model yeast to study cytokinesis with less genetic redundancy than S. cerevisiae. The viability of Klmyo1 deletions provides an advantage over budding yeast to study actomyosin-independent cytokinesis. Moreover, the lethality of Klcyk3 null mutants suggests that there are fewer functional redundancies with KlHof1 in K. lactis.

Mutations in SNF1 complex genes affect yeast cell wall strength.

The trimeric SNF1 complex from Saccharomyces cerevisiae, a homolog of mammalian AMP-activated kinase, has been primarily implicated in signaling for the utilization of alternative carbon sources to glucose. We here find that snf1 deletion mutants are hypersensitive to different cell wall stresses, such as the presence of Calcofluor white, Congo red, Zymolyase or the glucan synthase inhibitor Caspofungin in the growth medium. They also have a thinner cell wall. Caspofungin treatment triggers the phosphorylation of the catalytic Snf1 kinase subunit at Thr210 and removal of this phosphorylation site by mutagenesis (Snf1-T210A) abolishes the function of Snf1 in cell wall integrity. Deletion of the PFK1 gene encoding the α-subunit of the heterooctameric yeast phosphofructokinase suppresses the cell wall phenotypes of a snf1 deletion, which suggests a compensatory effect of central carbohydrate metabolism. Epistasis analyses with mutants in cell wall integrity (CWI) signaling confirm that the SNF1 complex and the CWI pathway independently affect yeast cell integrity.