Domesticating a food spoilage yeast into an organic acid‐tolerant metabolic engineering host: Lactic acid production by engineered Zygosaccharomyces bailii

Lactic acid represents an important class of commodity chemicals, which can be produced by microbial cell factories. However, due to the toxicity of lactic acid at lower pH, microbial production requires the usage of neutralizing agents to maintain neutral pH. Zygosaccharomyces bailii, a food spoilage yeast, can grow under the presence of organic acids used as food preservatives. This unique trait of the yeast might be useful for producing lactic acid. With the goal of domesticating the organic acid‐tolerant yeast as a metabolic engineering host, seven Z. bailii strains were screened in a minimal medium with 10 g/L of acetic, or 60 g/L of lactic acid at pH 3. The Z. bailii NRRL Y7239 strain was selected as the most robust strain to be engineered for lactic acid production. By applying a PAN‐ARS‐based CRISPR‐Cas9 system consisting of a transfer RNA promoter and NAT selection, we demonstrated the targeted deletion of ADE2 and site‐specific integration of Rhizopus oryzae ldhA coding for lactate dehydrogenase into the PDC1 locus. The resulting pdc1::ldhA strain produced 35 g/L of lactic acid without ethanol production. This study demonstrates the feasibility of the CRISPR‐Cas9 system in Z. bailii, which can be applied for a fundamental study of the species.     

Myriocin,a Serine Palmitoyltransferase Inhibitor,Blocks Cytokinesis in Leishmania (Viannia) braziliensis Promastigotes

We studied the effect of myriocin, an inhibitor of serine palmitoyltransferase, on cultured Leishmania (Viannia) braziliensis promastigotes. Myriocin significantly reduced synthesis of inositol phosphorylceramide, the major sphingolipid expressed in promastigotes as characterized by thin layer chromatography and electrospray ionization mass spectrometry. Log‐phase promastigotes treated with 1 μM myriocin showed a 52% reduction in growth rate and morphological alterations such as more rounded shape and shorter flagellum. Promastigotes treated with myriocin also displayed a variety of aberrant cell phenotypes. The percentage of cells with one nucleus and one kinetoplast (1N1K), following treatment with 1 or 5 μM myriocin, decreased from 89% (control value) to 27% or 3%, respectively. The percentage of cells with two nuclei (2N2K) varied from 7% (control value) to 19% and 6% for 1 or 5 μM myriocin‐treated parasites, respectively. High percentage of myriocin‐treated parasites exhibited large atypical cells presenting three or more nucleus (32% and 89% for 1 or 5 μM myriocin, respectively). Transmission electron microscopy following treatment with 1 μM myriocin showed the presence of 4N parasites possibly as a result of an incomplete cytokinesis. Addition of 3‐ketodihidrosphingosine to myriocin‐treated promastigotes rescue parasite growth and morphology. Addition of ethanolamine did not rescue the myriocin effect on parasite. Our findings indicate that sphingolipids are essential for the completion of cytokinesis, and may play a major role in cell proliferation in L. (V.) braziliensis, thus, differing from data described for Leishmania major sphingolipid‐free mutant, where addition of ethanolamine rescue wild‐type parasite characteristics.     

Inhibition analysis of inhibitors derived from lignocellulose pretreatment on the metabolic activity of Zymomonas mobilis biofilm and planktonic cells and the proteomic responses

Lignocellulose pretreatment produces various toxic inhibitors that affect microbial growth, metabolism, and fermentation. Zymomonas mobilis is an ethanologenic microbe that has been demonstrated to have potential to be used in lignocellulose biorefineries for bioethanol production. Z. mobilis biofilm has previously exhibited high potential to enhance ethanol production by presenting a higher viable cell number and higher metabolic activity than planktonic cells or free cells when exposed to lignocellulosic hydrolysate containing toxic inhibitors. However, there has not yet been a systematic study on the tolerance level of Z. mobilis biofilm compared to planktonic cells against model toxic inhibitors derived from lignocellulosic material. We took the first insight into the concentration of toxic compound (formic acid, acetic acid, furfural, and 5‐HMF) required to reduce the metabolic activity of Z. mobilis biofilm and planktonic cells by 25% (IC₂₅), 50% (IC₅₀), 75% (IC₇₅), and 100% (IC₁₀₀). Z. mobilis strains ZM4 and TISTR 551 biofilm were two‐ to three fold more resistant to model toxic inhibitors than planktonic cells. Synergetic effects were found in the presence of formic acid, acetic acid, furfural, and 5‐HMF. The IC₂₅ of Z. mobilis ZM4 biofilm and TISTR 551 biofilm were 57 mm formic acid, 155 mm acetic acid, 37.5 mm furfural and 6.4 mm 5‐HMF, and 225 mm formic acid, 291 mm acetic acid, 51 mm furfural and 41 mm 5‐HMF, respectively. There was no significant difference found between proteomic analysis of the stress response to toxic inhibitors of Z. mobilis biofilm and planktonic cells on ZM4. However, TISTR 551 biofilms exhibited two proteins (molecular chaperone DnaK and 50S ribosomal protein L2) that were up‐regulated in the presence of toxic inhibitors. TISTR 551 planktonic cells possessed two types of protein in the group of 30S ribosomal proteins and motility proteins that were up‐regulated.     

Relationships among Cell Size, Membrane Permeability, and Preservative Resistance in Yeast Species

The rate of uptake of propanoic acid and the cell dimensions were measured for 23 yeasts differing in their resistance to weak-acid-type preservatives. Relationships between reciprocal uptake rate, reciprocal permeability, cell volume, cell area, volume/area, and the MICs of benzoic acid and propanoic acid for the yeasts were tested by correlation analysis on pairs of parameters. The MIC of methylparaben, which is not a weak-acid-type preservative, was included. The most significant relationships found were between both reciprocal uptake rate and reciprocal permeability and the MICs of propanoic and benzoic acids Cell volume, area, and volume/area were each individually correlated with propanoic and benzoic acid MICs, but less strongly. In multiple regression analyses, inclusion of terms for volume, area, or volume/area did not markedly increase the significance. The MIC of methylparaben was unrelated to the uptake and permeability parameters, but did show a correlation with cell volume/area. Schizosaccharomyces pombe was anomalous in having very low permeability. Exclusion of these outlying data revealed particularly strong relationships (P < 0.001) between both reciprocal uptake rate and reciprocal permeability and the benzoic acid MIC. MICs for Zygosaccharomyces bailii isolates were substantially higher than for the other species, and therefore Z. baillii isolates had a large influence on the regressions. However, the relationships observed remained significant even after removal of the Z. bailii data. In showing a correlation between the rate at which propanoic acid enters yeast cells and the ability of the cells to tolerate this and other weak-acid-type preservatives, but not methylparaben, the results suggest that the resistance mechanism, in which preservative is continuously removed from the cell, is a common and major determinant of the preservative tolerance of yeast species.     

Oxygen Requirements of the Food Spoilage Yeast Zygosaccharomyces bailii in Synthetic and Complex Media

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast Zygosaccharomyces bailii at a constant pH, pH 3.0. In aerobic, glucose-grown cultures, Z. bailii exhibited aerobic alcoholic fermentation similar to that of Saccharomyces cerevisiae and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol, S. cerevisiae exhibited rapid exponential growth. Growth of Z. bailii under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of Z. bailii in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast Candida utilis. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium, Z. bailii exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by Z. bailii. In contrast to the growth of Z. bailii, anaerobic growth of S. cerevisiae on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.     

De novo sphingolipid synthesis is essential for viability, but not for transport of glycosylphosphatidylinositol-anchored proteins, in African trypanosomes

De novo sphingolipid synthesis is required for the exit of glycosylphosphatidylinositol (GPI)-anchored membrane proteins from the endoplasmic reticulum in yeast. Using a pharmacological approach, we test the generality of this phenomenon by analyzing the transport of GPI-anchored cargo in widely divergent eukaryotic systems represented by African trypanosomes and HeLa cells. Myriocin, which blocks the first step of sphingolipid synthesis (serine + palmitate --> 3-ketodihydrosphingosine), inhibited the growth of cultured bloodstream parasites, and growth was rescued with exogenous 3-ketodihydrosphingosine. Myriocin also blocked metabolic incorporation of [3H]serine into base-resistant sphingolipids. Biochemical analyses indicate that the radiolabeled lipids are not sphingomyelin or inositol phosphorylceramide, suggesting that bloodstream trypanosomes synthesize novel sphingolipids. Inhibition of de novo sphingolipid synthesis with myriocin had no adverse effect on either general secretory trafficking or GPI-dependent trafficking in trypanosomes, and similar results were obtained with HeLa cells. A mild effect on endocytosis was seen for bloodstream trypanosomes after prolonged incubation with myriocin. These results indicate that de novo synthesis of sphingolipids is not a general requirement for secretory trafficking in eukaryotic cells. However, in contrast to the closely related kinetoplastid Leishmania major, de novo sphingolipid synthesis is essential for the viability of bloodstream-stage African trypanosomes.     

The Genome Sequence of the Highly Acetic Acid-Tolerant Zygosaccharomyces bailii-Derived Interspecies Hybrid Strain ISA1307, Isolated From a Sparkling Wine Plant

In this work, it is described the sequencing and annotation of the genome of the yeast strain ISA1307, isolated from a sparkling wine continuous production plant. This strain, formerly considered of the Zygosaccharomyces bailii species, has been used to study Z. bailii physiology, in particular, its extreme tolerance to acetic acid stress at low pH. The analysis of the genome sequence described in this work indicates that strain ISA1307 is an interspecies hybrid between Z. bailii and a closely related species. The genome sequence of ISA1307 is distributed through 154 scaffolds and has a size of around 21.2 Mb, corresponding to 96% of the genome size estimated by flow cytometry. Annotation of ISA1307 genome includes 4385 duplicated genes (∼90% of the total number of predicted genes) and 1155 predicted single-copy genes. The functional categories including a higher number of genes are ‘Metabolism and generation of energy’, ‘Protein folding, modification and targeting’ and ‘Biogenesis of cellular components’. The knowledge of the genome sequence of the ISA1307 strain is expected to contribute to accelerate systems-level understanding of stress resistance mechanisms in Z. bailii and to inspire and guide novel biotechnological applications of this yeast species/strain in fermentation processes, given its high resilience to acidic stress. The availability of the ISA1307 genome sequence also paves the way to a better understanding of the genetic mechanisms underlying the generation and selection of more robust hybrid yeast strains in the stressful environment of wine fermentations.     

Benzoate effect on a benzoate-resistant strain ofZygosaccharomyces bailii

Summary Benzoic acid inZ. bailii decreases cell yield and increases sugar uptake rate in a similar way as inS. cerevisiae. The final catabolic destiny of pyruvate, however, is a function of the respiratory capacity of each strain. On the other hand, benzoic acid inhibits oxidative metabolism inZ. bailii. This work was supported byEC project AIR2-CT-92-CO-2.     

Hydrogen Peroxide Resistance of Acetobacter pasteurianus NBRC3283 and Its Relationship to Acetic Acid Fermentation

The bacterium Acetobacter pasteurianus can ferment acetic acid, a process that proceeds at the risk of oxidative stress. To understand the stress response, we investigated catalase and OxyR in A. pasteurianus NBRC3283. This strain expresses only a KatE homolog as catalase, which is monofunctional and growth dependent. Disruption of the oxyR gene increased KatE activity, but both the katE and oxyR mutant strains showed greater sensitivity to hydrogen peroxide as compared to the parental strain. These mutant strains showed growth similar to the parental strain in the ethanol oxidizing phase, but their growth was delayed when cultured in the presence of acetic acid and of glycerol and during the acetic acid peroxidation phase. The results suggest that A. pasteurianus cells show different oxidative stress responses between the metabolism via the membrane oxidizing pathway and that via the general aerobic pathway during acetic acid fermentation.     

The Fate of Acetic Acid during Glucose Co-Metabolism by the Spoilage Yeast Zygosaccharomyces bailii

Zygosaccharomyces bailii is one of the most widely represented spoilage yeast species, being able to metabolise acetic acid in the presence of glucose. To clarify whether simultaneous utilisation of the two substrates affects growth efficiency, we examined growth in single- and mixed-substrate cultures with glucose and acetic acid. Our findings indicate that the biomass yield in the first phase of growth is the result of the weighted sum of the respective biomass yields on single-substrate medium, supporting the conclusion that biomass yield on each substrate is not affected by the presence of the other at pH 3.0 and 5.0, at least for the substrate concentrations examined. In vivo ¹³C-NMR spectroscopy studies showed that the gluconeogenic pathway is not operational and that [2−¹³C]acetate is metabolised via the Krebs cycle leading to the production of glutamate labelled on C₂, C₃ and C₄. The incorporation of [U-¹⁴C]acetate in the cellular constituents resulted mainly in the labelling of the protein and lipid pools 51.5% and 31.5%, respectively. Overall, our data establish that glucose is metabolised primarily through the glycolytic pathway, and acetic acid is used as an additional source of acetyl-CoA both for lipid synthesis and the Krebs cycle. This study provides useful clues for the design of new strategies aimed at overcoming yeast spoilage in acidic, sugar-containing food environments. Moreover, the elucidation of the molecular basis underlying the resistance phenotype of Z. bailii to acetic acid will have a potential impact on the improvement of the performance of S. cerevisiae industrial strains often exposed to acetic acid stress conditions, such as in wine and bioethanol production.     

The effect of acetic acid and specific growth rate on acetic acid tolerance and trehalose content of Saccharomyces cerevisiae

Summary The effects of acetic acid and specific growth rate on acetic acid tolerance and trehalose content of Saccharomyces cerevisiae CBS 2806 were studied using anaerobic chemostat cultures. Cells grown in the presence of acetic acid at a defined specific growth rate showed a higher acetic acid tolerance and a slightly lower trehalose content. Cells grown at a low specific growth rate showed a lower energy demand, a higher acetic acid tolerance, and a higher trehalose content. These results indicate that trehalose plays a growth rate dependent role in the tolerance of S. cerevisiae to acetic acid.     

Serine palmitoyltransferase inhibitor myriocin induces growth inhibition of B16F10 melanoma cells through G(2) /M phase arrest

Objectives: Melanoma is the most aggressive form of skin cancer, and it resists chemotherapy. Candidate drugs for effective anti‐cancer treatment have been sought from natural resources. Here, we have investigated anti‐proliferative activity of myriocin, serine palmitoyltransferase inhibitor, in the de novo sphingolipid pathway, and its mechanism in B16F10 melanoma cells. Material and methods: We assessed cell population growth by measuring cell numbers, DNA synthesis, cell cycle progression, and expression of cell cycle regulatory proteins. Ceramide, sphingomyelin, sphingosine and sphingosine‐1‐phosphate levels were analysed by HPLC. Results: Myriocin inhibited proliferation of melanoma cells and induced cell cycle arrest in the G₂/M phase. Expressions of cdc25C, cyclin B1 and cdc2 were decreased in the cells after exposure to myriocin, while expression of p53 and p21^waf1/cip1 was increased. Levels of ceramide, sphingomyelin, sphingosine and sphingosine‐1‐phosphate in myriocin‐treated cells after 24 h were reduced by approximately 86%, 57%, 75% and 38%, respectively, compared to levels in control cells. Conclusions: Our results suggest that inhibition of sphingolipid synthesis by myriocin in melanoma cells may inhibit expression of cdc25C or activate expression of p53 and p21^waf1/cip1, followed by inhibition of cyclin B1 and cdc2, resulting in G₂/M arrest of the cell cycle and cell population growth inhibition. Thus, modulation of sphingolipid metabolism by myriocin may be a potential target of mechanism‐based therapy for this type of skin cancer.     

Cloning of the <Emphasis Type="Italic">Zygosaccharomyces bailii GAS</Emphasis> 1 homologue and effect of cell wall engineering on protein secretory phenotype

Background  

Zygosaccharomyces bailii is a diploid budding yeast still poorly characterized, but widely recognised as tolerant to several stresses, most of which related to industrial processes of production. Because of that, it would be very interesting to develop its ability as a cell factory. Gas1p is a β-1,3-glucanosyltransglycosylase which plays an important role in cell wall construction and in determining its permeability. Cell wall defective mutants of Saccharomyces cerevisiae and Pichia pastoris, deleted in the GAS 1 gene, were reported as super-secretive. The aim of this study was the cloning and deletion of the GAS 1 homologue of Z. bailii and the evaluation of its deletion on recombinant protein secretion.     

Flocculating Zymomonas mobilis is a promising host to be engineered for fuel ethanol production from lignocellulosic biomass

Whereas Saccharomyces cerevisiae uses the Embden‐Meyerhof‐Parnas pathway to metabolize glucose, Zymomonas mobilis uses the Entner‐Doudoroff (ED) pathway. Employing the ED pathway, 50% less ATP is produced, which could lead to less biomass being accumulated during fermentation and an improved yield of ethanol. Moreover, Z. mobilis cells, which have a high specific surface area, consume glucose faster than S. cerevisiae, which could improve ethanol productivity. We performed ethanol fermentations using these two species under comparable conditions to validate these speculations. Increases of 3.5 and 3.3% in ethanol yield, and 58.1 and 77.8% in ethanol productivity, were observed in ethanol fermentations using Z. mobilis ZM4 in media containing ～100 and 200 g/L glucose, respectively. Furthermore, ethanol fermentation bythe flocculating Z. mobilis ZM401 was explored. Although no significant difference was observed in ethanol yield and productivity, the flocculation of the bacterial species enabled biomass recovery by cost‐effective sedimentation, instead of centrifugation with intensive capital investment and energy consumption. In addition, tolerance to inhibitory byproducts released during biomass pretreatment, particularly acetic acid and vanillin, was improved. These experimental results indicate that Z. mobilis, particularly its flocculating strain, is superior to S. cerevisiae as a host to be engineered for fuel ethanol production from lignocellulosic biomass.     

Study of amino acids as nitrogen source in Chlamydomonas reinhardtii

All five L‐amino acids tested (L‐serine, L‐lysine, L‐leucine, L‐cysteine and L‐arginine) were used by Chlamydomonas reinhardtii as sole nitrogen source. Among these, L‐Cys was special as it has not been reported before. While these amino acids could be used in the dark only in the presence of acetic acid, in conditions of light they could support the growth of C. reinhardtii without the supplementation of acetic acid. When cultured in the TAP‐N medium, the chlorophyll content was found to be lower in the dark, but higher in the light for the cells grown with L‐Arg than with other four amino acids. Exogenously supplied L‐Ser and L‐Lys did not accumulate in the cells, demonstrating that they were used by supplying ammonium to the cells from the activity of an extracellular deaminase. Further results showed that the induction of the extracellular deaminase activity required a period of nitrogen starvation, regardless of the medium containing acetic acid or not. Results also showed that the uptake of L‐Cys was similar to L‐Leu, most likely via passive diffusion. When L‐Cys and L‐Leu were supplied together to the nitrogen‐starved cells, the absorption of L‐Cys did not affect the uptake of L‐Leu.     

Effects of Ethanol and Other Alkanols on Transport of Acetic Acid in Saccharomyces cerevisiae

In glucose-grown cells of Saccharomyces cerevisiae IGC 4072, acetic acid enters only by simple diffusion of the undissociated acid. In these cells, ethanol and other alkanols enhanced the passive influx of labelled acetic acid. The influx of the acid followed first-order kinetics with a rate constant that increased exponentially with the alcohol concentration, and an exponential enhancement constant for each alkanol was estimated. The intracellular concentration of labelled acetic acid was also enhanced by alkanols, and the effect increased exponentially with alcohol concentration. Acetic acid is transported across the plasma membrane of acetic acid-, lactic acid-, and ethanol-grown cells by acetate-proton symports. We found that in these cells ethanol and butanol inhibited the transport of labelled acetic acid in a noncompetitive way; the maximum transport velocity decreased with alcohol concentration, while the affinity of the system for acetate was not significantly affected by the alcohol. Semilog plots of V_max versus alcohol concentration yielded straight lines with negative slopes from which estimates of the inhibition constant for each alkanol could be obtained. The intracellular concentration of labelled acid was significantly reduced in the presence of ethanol or butanol, and the effect increased with the alcohol concentration. We postulate that the absence of an operational carrier for acetate in glucose-grown cells of S. cerevisiae, combined with the relatively high permeability of the plasma membrane for the undissociated acid and the inability of the organism to metabolize acetic acid, could be one of the reasons why this species exhibits low tolerance to acidic environments containing ethanol.     

Fine-tuning ethanol oxidation pathway enzymes and cofactor PQQ coordinates the conflict between fitness and acetic acid production by Acetobacter pasteurianus

The very high concentrations required for industrial production of free acetic acid create toxicity and low pH values, which usually conflict with the host cell growth, leading to a poor productivity. Achieving a balance between cell fitness and product synthesis is the key challenge to improving acetic acid production efficiency in metabolic engineering. Here, we show that the synergistic regulation of alcohol/aldehyde dehydrogenase expression and cofactor PQQ level could not only efficiently relieve conflict between increased acetic acid production and compromised cell fitness, but also greatly enhance acetic acid tolerance of Acetobacter pasteurianus to a high initial concentration (3% v/v) of acetic acid. Combinatorial expression of adhA and pqqABCDE greatly shortens the duration of starting-up process from 116 to 99 h, leading to a yield of 69 g l^-1 acetic acid in semi-continuous fermentation. As a final result, average acetic acid productivity has been raised to 0.99 g l^-1 h^-1, which was 32% higher than the parental A. pasteurianus. This study is of great significance for decreasing cost of semi-continuous fermentation for producing high-strength acetic acid industrially. We envisioned that this strategy will be useful for production of many other desired organic acids, especially those involving cofactor reactions.