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.

Beyond Saccharomyces pastorianus for modern lager brews: Exploring non-cerevisiae Saccharomyces hybrids with heterotic maltotriose consumption and novel aroma profile.

Non-domesticated, wild Saccharomyces yeasts have promising characteristics for beer diversification, particularly when used in the generation of de novo interspecific hybrids. A major motivation for the current work was the question whether attractive novel Saccharomyces interspecific hybrids can be created for the production of exotic lager beers without using the genomic resources of the ale yeast Saccharomyces cerevisiae. Importantly, maltotriose utilization is an essential characteristic typically associated with domesticated ale/lager brewing strains. A high-throughput screening on nearly 200 strains representing all eight species of the Saccharomyces genus was conducted. Three Saccharomyces mikatae strains were able to aerobically grow on maltotriose as the sole carbon source, a trait until recently unidentified for this species. Our screening also confirmed the recently reported maltotriose utilization of the S. jurei strain D5095T. Remarkably, de novo hybrids between a maltotriose-utilizing S. mikatae or S. jurei strain and the maltotriose-negative Saccharomyces eubayanus strain CBS 12357T displayed heterosis and outperformed both parents with regard to aerobically utilizing maltotriose as the sole source of carbon. Indeed, the maximum specific growth rates on this sugar were comparable to the well-known industrial strain, Saccharomyces pastorianus CBS 1513. In lager brewing settings (oxygen-limited), the new hybrids were able to ferment maltose, while maltotriose was not metabolized. Favorable fruity esters were produced, demonstrating that the novel hybrids have the potential to add to the diversity of lager brewing.

The Dicarboxylate Transporters from the AceTr Family and Dct-02 Oppositely Affect Succinic Acid Production in S. cerevisiae.

Membrane transporters are important targets in metabolic engineering to establish and improve the production of chemicals such as succinic acid from renewable resources by microbial cell factories. We recently provided a Saccharomyces cerevisiae strain able to strongly overproduce succinic acid from glycerol and CO2 in which the Dct-02 transporter from Aspergillus niger, assumed to be an anion channel, was used to export succinic acid from the cells. In a different study, we reported a new group of succinic acid transporters from the AceTr family, which were also described as anion channels. Here, we expressed these transporters in a succinic acid overproducing strain and compared their impact on extracellular succinic acid accumulation with that of the Dct-02 transporter. The results show that the tested transporters of the AceTr family hinder succinic acid accumulation in the extracellular medium at low pH, which is in strong contrast to Dct-02. Data suggests that the AceTr transporters prefer monovalent succinate, whereas Dct-02 prefers divalent succinate anions. In addition, the results provided deeper insights into the characteristics of Dct-02, showing its ability to act as a succinic acid importer (thus being bidirectional) and verifying its capability of exporting malate.

Carbon dioxide fixation via production of succinic acid from glycerol in engineered Saccharomyces cerevisiae.

BACKGROUND: The microbial production of succinic acid (SA) from renewable carbon sources via the reverse TCA (rTCA) pathway is a process potentially accompanied by net-fixation of carbon dioxide (CO2). Among reduced carbon sources, glycerol is particularly attractive since it allows a nearly twofold higher CO2-fixation yield compared to sugars. Recently, we described an engineered Saccharomyces cerevisiae strain which allowed SA production in synthetic glycerol medium with a maximum yield of 0.23 Cmol Cmol-1. The results of that previous study suggested that the glyoxylate cycle considerably contributed to SA accumulation in the respective strain. The current study aimed at improving the flux into the rTCA pathway accompanied by a higher CO2-fixation and SA yield. RESULTS: By changing the design of the expression cassettes for the rTCA pathway, overexpressing PYC2, and adding CaCO3 to the batch fermentations, an SA yield on glycerol of 0.63 Cmol Cmol-1 was achieved (i.e. 47.1% of the theoretical maximum). The modifications in this 2nd-generation SA producer improved the maximum biomass-specific glycerol consumption rate by a factor of nearly four compared to the isogenic baseline strain solely equipped with the dihydroxyacetone (DHA) pathway for glycerol catabolism. The data also suggest that the glyoxylate cycle did not contribute to the SA production in the new strain. Cultivation conditions which directly or indirectly increased the concentration of bicarbonate, led to an accumulation of malate in addition to the predominant product SA (ca. 0.1 Cmol Cmol-1 at the time point when SA yield was highest). Off-gas analysis in controlled bioreactors with CO2-enriched gas-phase indicated that CO2 was fixed during the SA production phase. CONCLUSIONS: The data strongly suggest that a major part of dicarboxylic acids in our 2nd-generation SA-producer was formed via the rTCA pathway enabling a net fixation of CO2. The greatly increased capacity of the rTCA pathway obviously allowed successful competition with other pathways for the common precursor pyruvate. The overexpression of PYC2 and the increased availability of bicarbonate, the co-substrate for the PYC reaction, further strengthened this capacity. The achievements are encouraging to invest in future efforts establishing a process for SA production from (crude) glycerol and CO2.

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.

Complete Utilization of the Major Carbon Sources Present in Sugar Beet Pulp Hydrolysates by the Oleaginous Red Yeasts Rhodotorula toruloides and R. mucilaginosa.

Agro-industrial residues are low-cost carbon sources (C-sources) for microbial growth and production of value-added bioproducts. Among the agro-industrial residues available, those rich in pectin are generated in high amounts worldwide from the sugar industry or the industrial processing of fruits and vegetables. Sugar beet pulp (SBP) hydrolysates contain predominantly the neutral sugars d-glucose, l-arabinose and d-galactose, and the acidic sugar d-galacturonic acid. Acetic acid is also present at significant concentrations since the d-galacturonic acid residues are acetylated. In this study, we have examined and optimized the performance of a Rhodotorula mucilaginosa strain, isolated from SBP and identified at the molecular level during this work. This study was extended to another oleaginous red yeast species, R. toruloides, envisaging the full utilization of the C-sources from SBP hydrolysate (at pH 5.0). The dual role of acetic acid as a carbon and energy source and as a growth and metabolism inhibitor was examined. Acetic acid prevented the catabolism of d-galacturonic acid and l-arabinose after the complete use of the other C-sources. However, d-glucose and acetic acid were simultaneously and efficiently metabolized, followed by d-galactose. SBP hydrolysate supplementation with amino acids was crucial to allow d-galacturonic acid and l-arabinose catabolism. SBP valorization through the production of lipids and carotenoids by Rhodotorula strains, supported by complete catabolism of the major C-sources present, looks promising for industrial implementation.

Engineering Saccharomyces cerevisiae for Succinic Acid Production From Glycerol and Carbon Dioxide.

Previously, our lab replaced the endogenous FAD-dependent pathway for glycerol catabolism in S. cerevisiae by the synthetic NAD-dependent dihydroxyacetone (DHA) pathway. The respective modifications allow the full exploitation of glycerol's higher reducing power (compared to sugars) for the production of the platform chemical succinic acid (SA) via a reductive, carbon dioxide fixing and redox-neutral pathway in a production host robust for organic acid production. Expression cassettes for three enzymes converting oxaloacetate to SA in the cytosol ("SA module") were integrated into the genome of UBR2 CBS-DHA, an optimized CEN.PK derivative. Together with the additional expression of the heterologous dicarboxylic acid transporter DCT-02 from Aspergillus niger, a maximum SA titer of 10.7 g/L and a yield of 0.22 ± 0.01 g/g glycerol was achieved in shake flask (batch) cultures. Characterization of the constructed strain under controlled conditions in a bioreactor supplying additional carbon dioxide revealed that the carbon balance was closed to 96%. Interestingly, the results of the current study indicate that the artificial "SA module" and endogenous pathways contribute to the SA production in a highly synergistic manner.

Anaplerotic reactions active during growth of Saccharomyces cerevisiae on glycerol.

Anaplerotic reactions replenish TCA cycle intermediates during growth. In Saccharomyces cerevisiae, pyruvate carboxylase and the glyoxylate cycle have been experimentally identified to be the main anaplerotic routes during growth on glucose (C6) and ethanol (C2), respectively. The current study investigates the importance of the two isoenzymes of pyruvate carboxylase (PYC1 and PYC2) and one of the key enzymes of the glyoxylate cycle (ICL1) for growth on glycerol (C3) as a sole carbon source. As the wild-type strains of the CEN.PK family are unable to grow in pure synthetic glycerol medium, a reverse engineered derivative showing a maximum specific growth rate of 0.14 h-1 was used as the reference strain. While the deletion of PYC1 reduced the maximum specific growth rate by about 38%, the deletion of PYC2 had no significant impact, neither in the reference strain nor in the pyc1Δ mutant. The deletion of ICL1 only marginally reduced growth of the reference strain but further decreased the growth rate of the pyc1 deletion strain by 20%. Interestingly, the triple deletion (pyc1Δ pyc2Δ icl1Δ) did not show any growth. Therefore, both the pyruvate carboxylase and the glyoxylate cycle are involved in anaplerosis during growth on glycerol.

Saccharomyces cerevisiae exhibiting a modified route for uptake and catabolism of glycerol forms significant amounts of ethanol from this carbon source considered as 'non-fermentable'.

BACKGROUND: Due 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. RESULTS: A 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). CONCLUSION: The 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.

Glycerol as a substrate for Saccharomyces cerevisiae based bioprocesses - Knowledge gaps regarding the central carbon catabolism of this 'non-fermentable' carbon source.

Glycerol is an interesting alternative carbon source in industrial bioprocesses due to its higher degree of reduction per carbon atom compared to sugars. During the last few years, significant progress has been made in improving the well-known industrial platform organism Saccharomyces cerevisiae with regard to its glycerol utilization capability, particularly in synthetic medium. This provided a basis for future metabolic engineering focusing on the production of valuable chemicals from glycerol. However, profound knowledge about the central carbon catabolism in synthetic glycerol medium is a prerequisite for such incentives. As a matter of fact, the current assumptions about the actual in vivo fluxes active on glycerol as the sole carbon source have mainly been based on omics data collected in complex media or were even deduced from studies with other non-fermentable carbon sources, such as ethanol or acetate. A number of uncertainties have been identified which particularly regard the role of the glyoxylate cycle, the subcellular localization of the respective enzymes, the contributions of mitochondrial transporters and the active anaplerotic reactions under these conditions. The review scrutinizes the current knowledge, highlights the necessity to collect novel experimental data using cells growing in synthetic glycerol medium and summarizes the current state of the art with regard to the production of valuable fermentation products from a carbon source that has been considered so far as 'non-fermentable' for the yeast S. cerevisiae.

Involvement of the external mitochondrial NADH dehydrogenase Nde1 in glycerol metabolism by wild-type and engineered Saccharomyces cerevisiae strains.

Glycerol is an attractive substrate for microbial fermentations due to its higher degree of reduction compared to glucose. The replacement of the native FAD-dependent glycerol catabolic pathway in Saccharomyces cerevisiae by an artificial NADH-delivering dihydroxyacetone (DHA) pathway is supposed to facilitate the capturing of electrons in fermentation products. This requires that the electrons from the cytosolic NADH are not exclusively transferred to oxygen. However, the external NADH dehydrogenases (Nde1/2) and the L-glycerol 3-phosphate shuttle (composed of Gpd1/2 and Gut2), both coupled to the respiratory chain, are known to contribute to cytosolic NAD+ regeneration during growth on non-fermentable carbon sources. In order to evaluate the role of these mechanisms during growth on glycerol, we deleted GPD1/2, GUT2 as well as NDE1/2, separately and in combinations in both the glycerol-utilizing wild-type strain CBS 6412-13A and the corresponding engineered strain CBS DHA in which glycerol is catabolized by the DHA pathway. Particularly, the nde1Δ mutants showed a significant reduction in growth rate and the nde1∆ nde2∆ double deletion mutants did not grow at all in synthetic glycerol medium. The current work also demonstrates a positive impact of deleting NDE1 on the production of the fermentation product 1,2-propanediol in an accordingly engineered S. cerevisiae strain.

Forcing fermentation: Profiling proteins, peptides and polyphenols in lab-scale cocoa bean fermentation.

This study encompassed the lab-scale fermentation of cocoa beans in 300-g heaps under controlled laboratory conditions, in order to replicate the microbial dynamics and metabolomic changes that usually occur in large-scale spontaneous fermentations. Growth profiles of yeast and acetic acid bacteria (AAB) with the native assortment of microbes as well as with the use of a starter culture were very similar to those observed in literature. Greater production of acetic acid by AAB not only led to more acidic-tasting liquor but also contributed to bitterness, due to polyphenol preservation. It also brought about a drastic drop in pH leading to greater proteolytic activity. Peptides generated through proteolysis also showed incredible similarity to those reported in literature, in particular, those speculated to be involved in cocoa-specific flavour. A closer look at the naturally occurring peptide repertoires of our fermentation trials, generated by the breakdown of cocoa storage protein, pointed to a potential peptide responsible for cocoa-specific aroma.

Identification of novel genes involved in acetic acid tolerance of Saccharomyces cerevisiae using pooled-segregant RNA sequencing.

Acetic acid tolerance of the yeast Saccharomyces cerevisiae is manifested in several quantifiable parameters, of which the duration of the latency phase is one of the most studied. It has been shown recently that the latter parameter is mostly determined by a fraction of cells within the population that resumes proliferation upon exposure to acetic acid. The aim of the current study was to identify genetic determinants of the difference in this parameter between the highly tolerant strain MUCL 11987-9 and the laboratory strain CEN.PK113-7D. To this end, a combination of genetic mapping and pooled-segregant RNA sequencing was applied as a new approach. The genetic mapping data revealed four loci with a strong linkage to strain MUCL 11987-9, each containing still a large number of genes making the identification of the causal ones by traditional methods a laborious task. The genes were therefore prioritized by pooled-segregant RNA sequencing, which resulted in the identification of six genes within the identified loci showing differential expression. The relevance of the prioritized genes for the phenotype was verified by reciprocal hemizygosity analysis. Our data revealed the genes ESP1 and MET22 as two, so far unknown, genetic determinants of the size of the fraction of cells resuming proliferation upon exposure to acetic acid.

Glycerol positive promoters for tailored metabolic engineering of the yeast Saccharomyces cerevisiae.

Glycerol offers several advantages as a substrate for biotechnological applications. An important step toward using the popular production host Saccharomyces cerevisiae for glycerol-based bioprocesses has been the fact that in recent studies commonly used S. cerevisiae strains were engineered to grow in synthetic medium containing glycerol as the sole carbon source. For metabolic engineering projects of S. cerevisiae growing on glycerol, characterized promoters are missing. In the current study, we used transcriptome analysis and a yECitrine-based fluorescence reporter assay to select and characterize 25 useful promoters. The promoters of the genes ALD4 and ADH2 showed 4.2-fold and 3-fold higher activities compared to the well-known strong TEF1 promoter. Moreover, the collection contains promoters with graded activities in synthetic glycerol medium and different degrees of glucose repression. To demonstrate the general applicability of the promoter collection, we successfully used a subset of the characterized promoters with graded activities in order to optimize growth on glycerol in an engineered derivative of CEN.PK, in which glycerol catabolism exclusively occurs via a non-native DHA pathway.

A modular metabolic engineering approach for the production of 1,2-propanediol from glycerol by Saccharomyces cerevisiae.

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 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.

The sole introduction of two single-point mutations establishes glycerol utilization in Saccharomyces cerevisiae CEN.PK derivatives.

BACKGROUND: Glycerol is an abundant by-product of biodiesel production and has several advantages as a substrate in biotechnological applications. Unfortunately, the popular production host Saccharomyces cerevisiae can barely metabolize glycerol by nature. RESULTS: In this study, two evolved derivatives of the strain CEN.PK113-1A were created that were able to grow in synthetic glycerol medium (strains PW-1 and PW-2). Their growth performances on glycerol were compared with that of the previously published evolved CEN.PK113-7D derivative JL1. As JL1 showed a higher maximum specific growth rate on glycerol (0.164 h-1 compared to 0.119 h-1 for PW-1 and 0.127 h-1 for PW-2), its genomic DNA was subjected to whole-genome resequencing. Two point mutations in the coding sequences of the genes UBR2 and GUT1 were identified to be crucial for growth in synthetic glycerol medium and subsequently verified by reverse engineering of the wild-type strain CEN.PK113-7D. The growth rate of the resulting reverse-engineered strain was 0.130 h-1. Sanger sequencing of the GUT1 and UBR2 alleles of the above-mentioned evolved strains PW-1 and PW-2 also revealed one single-point mutation in these two genes, and both mutations were demonstrated to be also crucial and sufficient for obtaining a maximum specific growth rate on glycerol of ~0.120 h-1. CONCLUSIONS: The current work confirmed the importance of UBR2 and GUT1 as targets for establishing glycerol utilization in strains of the CEN.PK family. In addition, it shows that a growth rate on glycerol of 0.130 h-1 can be established in reverse-engineered CEN.PK strains by solely replacing a single amino acid in the coding sequences of both Ubr2 and Gut1.

Improvement of yeast tolerance to acetic acid through Haa1 transcription factor engineering: towards the underlying mechanisms.

BACKGROUND: Besides being a major regulator of the response to acetic acid in Saccharomyces cerevisiae, the transcription factor Haa1 is an important determinant of the tolerance to this acid. The engineering of Haa1 either by overexpression or mutagenesis has therefore been considered to be a promising avenue towards the construction of more robust strains with improved acetic acid tolerance. RESULTS: By applying the concept of global transcription machinery engineering to the regulon-specific transcription factor Haa1, a mutant allele containing two point mutations could be selected that resulted in a significantly higher acetic acid tolerance as compared to the wild-type allele. The level of improvement obtained was comparable to the level obtained by overexpression of HAA1, which was achieved by introduction of a second copy of the native HAA1 gene. Dissection of the contribution of the two point mutations to the phenotype showed that the major improvement was caused by an amino acid exchange at position 135 (serine to phenylalanine). In order to further study the mechanisms underlying the tolerance phenotype, Haa1 translocation and transcriptional activation of Haa1 target genes was compared between Haa1 mutant, overproduction and wild-type strains. While the rapid Haa1 translocation from the cytosol to the nucleus in response to acetic acid was not affected in the Haa1S135F mutant strain, the levels of transcriptional activation of four selected Haa1-target genes by acetic acid were significantly higher in cells of the mutant strain as compared to cells of the wild-type strain. Interestingly, the time-course of transcriptional activation in response to acetic acid was comparable for the mutant and wild-type strain whereas the maximum mRNA levels obtained correlate with each strain's tolerance level. CONCLUSION: Our data confirms that engineering of the regulon-specific transcription factor Haa1 allows the improvement of acetic acid tolerance in S. cerevisiae. It was also shown that the beneficial S135F mutation identified in the current work did not lead to an increase of HAA1 transcript level, suggesting that an altered protein structure of the Haa1S135F mutant protein led to an increased recruitment of the transcription machinery to Haa1 target genes.

Glycerol metabolism and transport in yeast and fungi: established knowledge and ambiguities.

There is huge variability among yeasts with regard to their efficiency in utilizing glycerol as the sole source of carbon and energy. Certain species show growth rates with glycerol comparable to those reached with glucose as carbon source; others are virtually unable to utilize glycerol, especially in synthetic medium. Most of our current knowledge regarding glycerol uptake and catabolic pathways has been gained from studying laboratory strains of the model yeast Saccharomyces cerevisiae. The growth of these strains on glycerol is dependent on the presence of medium supplements such as amino acids and nucleobases. In contrast, there is only fragmentary knowledge about S. cerevisiae isolates able to grow in synthetic glycerol medium without such supplements as well as about growth of non-Saccharomyces yeast species on glycerol. Thus, more research is required to understand why certain strains and species show superior growth performance on glycerol compared with common S. cerevisiae laboratory strains. This mini-review summarizes what is known so far about the gene products and pathways involved in glycerol metabolism and transport in yeast and fungi as well as the regulation of these processes.

Towards the exploitation of glycerol's high reducing power in Saccharomyces cerevisiae-based bioprocesses.

One advantage of using glycerol as a carbon source for industrial bioprocesses is its higher degree of reduction compared to glucose. In order to exploit this reducing power for the production of reduced compounds thereby significantly increasing maximum theoretical yields, the electrons derived from glycerol oxidation must first be saved in the form of cytosolic NAD(P)H. However, the industrial platform organism Saccharomyces cerevisiae naturally uses an FAD-dependent pathway for glycerol catabolism transferring the electrons to the respiratory chain. Here, we developed a pathway replacement strategy forcing glycerol catabolism through a synthetic, NAD+-dependent route. The required expression cassettes were integrated via CRISPR-Cas9 targeting the endogenous GUT1 locus, thereby abolishing the native FAD-dependent pathway. Interestingly, this pathway replacement even established growth in synthetic glycerol medium of strains naturally unable to grow on glycerol and an engineered derivative of CEN.PK even showed the highest ever reported maximum specific growth rate on glycerol (0.26h-1).