Inhibition Of Yeast Growth Research Articles

Essential Oils of Rosmarinus officinalis L., Cymbopogon citratus (DC.) Stapf., and the phyto-compounds, delta-carene and alpha-pinene mediate cell cycle arrest at G2/M transition in budding yeast Saccharomyces cerevisiae

Ethnopharmacological relevance: Rosmarinus officinalis L. (Lamiaceae) is widely used in folk and traditional medicine for inflammation-related diseases due to its anti-oxidant and wound healing properties. Cymbopogon citratus (DC.) Stapf. is a widely distributed perennial herb belonging to the Poaceae family and has been extensively consumed for its traditional and ethnomedicinal, cosmetic, and nutritional effects for centuries.Aim of the study: Budding yeast is a valuable system to study cell-cycle regulation, which is the underlying defect in cancer cells. The aim of the present study was to characterize the antiproliferative effect of essential oil of Rosmarinus officinalis L. (rosemary) and Cymbopogon citratus (DC.) Stapf. (lemon grass) and their effect on cell cycle of Saccharomyces cerevisiae.Materials and methods: Rosmarinus officinalis L. and Cymbopogon citratus (DC.) Stapf. essential oil was extracted from leaves and characterized by GC–MS, TLC and FTIR. The antiproliferative activity of essential oils and their major compounds, delta-carene and α-pinene was studied by liquid growth assay. Microscopic examination of yeast cells was done to determine the budding index. Flowcytometry analysis of DNA content was analyzed to confirm the G2/M arrest of cell cycle of budding yeast.Results: Both the essential oils mediated a dose-dependent inhibition of yeast growth in liquid assays. Analysis of budding index and DNA content by flowcytometry revealed that R. officinalis L. and C. citratus (DC.) Stapf. essential oils mediate the arrest of yeast cells at G2/M phase of cell cycle. GC/MS analysis of R. officinalis oil revealed the presence of two major compounds, delta-carene (7.75%), and α-pinene (15.1%). Both delta-carene and α-pinene showed antiproliferative activity (92% and 95% of growth inhibition, respectively) and caused the arrest of yeast cell cycle at G2/M phase.Conclusions: The essential oils of R. officinalis and C. citratus, delta-carene and α-pinene can be validated in animal model for cell cycle inhibition and cancer therapeutics.

A yeast chemogenomic screen identifies pathways that modulate adipic acid toxicity

SummaryAdipic acid production by yeast fermentation is gaining attention as a renewable source of platform chemicals for making nylon products. However, adipic acid toxicity inhibits yeast growth and fermentation. Here, we performed a chemogenomic screen in Saccharomyces cerevisiae to understand the cellular basis of adipic acid toxicity. Our screen revealed that KGD1 (a key gene in the tricarboxylic acid cycle) deletion improved tolerance to adipic acid and its toxic precursor, catechol. Conversely, disrupting ergosterol biosynthesis as well as protein trafficking and vacuolar transport resulted in adipic acid hypersensitivity. Notably, we show that adipic acid disrupts the Membrane Compartment of Can1 (MCC) on the plasma membrane and impacts endocytosis. This was evidenced by the rapid internalization of Can1 for vacuolar degradation. As ergosterol is an essential component of the MCC and protein trafficking mechanisms are required for endocytosis, we highlight the importance of these cellular processes in modulating adipic acid toxicity.

Bunkers or round bales: Losses and silage quality with or without acid treatment of low dry matter grass crops

Grass clover crops were harvested with or without application of 4 L/t of a formic- and propionic acid-based silage additive and ensiled in one bunker silo and 6 round bales per treatment in each of three harvests. The study aimed to compare losses, grass silage quality and aerobic stability obtained either with round bales or precision chopped grass ensiled in bunker silos. Round bales were either sealed immediately or after delay until bunker silos were covered. Unpredicted rain showers during the three harvests gave crop DM as low as 194, 186 and 213 g/kg, respectively.Due to the lower pressure exerted on the crop by the baler than by packing vehicles in the bunkers, and the longer particle length in bales, densities in baled silage were much lower than in bunker silage, 531 vs 833 kg/m3 (P < 0.001), and 111 vs 164 kg DM/m3 (P < 0.001). Presumably due to early cell rupture and higher release of effluent caused by the applied acid, densities were higher in treated than in untreated silage, in bunkers 170 vs. 159 kg DM/m3 (P = 0.08), and in bales, 114 vs. 109 kg DM/m3 (P = 0.02).A much lower proportion of ensiled crop DM could be offered to livestock from bunkers than from round bales, 833 vs. 927 g/kg (P < 0.001). The amount of moulded, wasted silage DM was significantly higher in bunkers than in bales, 26 vs. 0.6 g/kg, (P < 0.001), and the sum of DM lost by crop respiration, effluent runoff, anaerobic fermentation, aerobic deterioration and gaseous losses was significantly higher from bunkers than bales, 141 vs. 72 g/kg (P < 0.002). Acid treatment caused only minor decreases in DM losses. It restricted acid fermentation and improved silage intake potential both in bunkers and bales (P < 0.001), and caused higher stability in bales (P < 0.009). High ethanol concentrations were found in acid treated bunker silage but not in treated bale silage. Also, a reduction in heat induced increases in fiber bound protein obtained by acid treatment in bales, but not in bunkers, suggested that the applied dosage was too low to restrict heating in bunkers, and favored yeast growth. The larger surface area susceptible to heating, and loss of additive in effluent, make higher acid dosages, or a higher proportion of ingredients that inhibit yeast growth, necessary to low DM grass crops ensiled in bunkers.

Synthesis, Spectroscopy, Single-Crystal Structure Analysis and Antibacterial Activity of Two Novel Complexes of Silver(I) with Miconazole Drug.

In a previous article, we reported on the higher toxicity of silver(I) complexes of miconazole [Ag(MCZ)2NO3 (1)] and [Ag(MCZ)2ClO4 (2)] in HepG2 tumor cells compared to the corresponding salts of silver, miconazole and cisplatin. Here, we present the synthesis of two silver(I) complexes of miconazole containing two new counter ions in the form of Ag(MCZ)2X (MCZ = 1-[2-(2,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole]; X = BF4− (3), SbF6− (4)). The novel silver(I) complexes were characterized by elemental analysis, 1H NMR, 13C NMR and infrared (IR) spectroscopy, electrospray ionization (ESI)-MS spectrometry and X-ray-crystallography. In the present study, the antimicrobial activity of all obtained silver(I) complexes of miconazole against six strains of Gram-positive bacteria, five strains of Gram-negative bacteria and yeasts was evaluated. The results were compared with those of a silver sulfadiazine drug, the corresponding silver salts and the free ligand. Silver(I) complexes exhibited significant activity against Gram-positive bacteria, which was much better than that of silver sulfadiazine and silver salts. The highest antimicrobial activity was observed for the complex containing the nitrate counter ion. All Ag(I) complexes of miconazole resulted in much better inhibition of yeast growth than silver sulfadiazine, silver salts and miconazole. Moreover, the synthesized silver(I) complexes showed good or moderate activity against Gram-negative bacteria compared to the free ligand.

Intensificación de la producción de 2-feniletanol empleando un sistema aireado asistido por una técnica de extracción de una membrana basada en solvente

The production of 2-phenylethanol (2-PE), a high-valued aroma, has been performed naturally through the bioconversion of L-phenylalanine by the Ehrlich pathway in yeast. Nevertheless, one of the main limitations of this bioconversion is the inhibition of yeast growth by 2-PE. This work evaluated the intensification of 2-PE production in a batch culture using a conventional bioreactor coupled to an in-situ product removal (ISPR) process, by means of membrane-based solvent extraction (MBSE). The culture of the native yeast Kluyveromyces marxianus ITD0090, using a shaker flask stirred at 150 rpm, produced a maximum concentration of 2-PE of 0.7 g L-1 after 24 hours. When the yeast was cultivated in a controlled aerated-stirred bioreactor operated at 0.8 vvm and 350 rpm, the maximum production of 2-PE after 48 h reached 1.49 g L-1. Intensification of the bioprocess was achieved by coupling the controlled bioreactor with a MBSE system, which allowed the continuous recovery of the 2-PE produced, reaching a final titre of 3.02 g L-1 after 56 h.

Formulation and Antifungal Activities of Syzygium Aromaticum Essential Oil Contained in Natural Bath Soap

Syzygium aromaticum essential oil (clove oil) has a promising potential as an antifungal. However, the potential has never been applied in bath soaps. This study aimed to produce an antifungal natural soap using clove oil as the additive. Natural bath soap containing clove oil was made through a cold saponification reaction of vegetable oils, sodium hydroxide, and various contents of clove oil (1, 2, and 3% w/w) as an antifungal agent. The soap quality was tested based on Indonesian National Standards (INS), including water content, total fat, insoluble matters in ethanol, free alkali, and unsaponified fatty matter. With a Kirby-Bauer diffusion method, the researchers carried out an antifungal activity est of clove oil soap on Candida albicans and Trichophyton mentagrophytes. All samples in the quality test of clove oil natural bath soap have met the standard. The antifungal activities of all tested clove oil soap performed growth inhibition of Candida albicans yeast and Trichophyton mentagrophytes mould in all treatments. However, there is no relationship between the clove oil concentration and antifungal activities in the range of clove oil content in this study. The formulation of clove oil in natural bath soap executed excellent antifungal activities against both yeast and mould. Due to the potential of clove oil as an antifungal agent, clove oil in bath soap is efficient to be used as natural cosmetics and medicinal soap in the future.

Revisiting the mode of action of the antimalarial proguanil using the yeast model

Proguanil in combination with its synergistic partner atovaquone has been used for malaria treatment and prophylaxis for decades. However its mode of action is not fully understood. Here we used yeast to investigate its activity. Proguanil inhibits yeast growth, causes cell death and acts in synergy with atovaquone. It was previously proposed that the drug would target the system that maintains the mitochondrial membrane potential when the respiratory chain is inhibited. However our data did not seem to validate that hypothesis. We proposed that proguanil would not have a specific target but accumulate in the mitochondrial to concentrations that impair multiple mitochondrial functions leading to cell death. Selection and study of proguanil resistant mutants pointed towards an unexpected resistance mechanism: the decrease of CoQ level, which possibly alters the mitochondrial membrane properties and lowers proguanil intramitochondrial level.

Improvement of 2,3-butanediol tolerance in Saccharomyces cerevisiae by using a novel mutagenesis strategy

Although the yeast Saccharomyces cerevisiae has been used to produce various bio-based chemicals, including solvents and organic acids, most of these products inhibit yeast growth at high concentrations. In general, it is difficult to rationally improve stress tolerance in yeast by modifying specific genes, because many of the genes involved in stress response remain unidentified. Previous studies have reported that various forms of stress tolerance in yeast were improved by introducing random mutations, such as DNA point mutations and DNA structural mutations. In this study, we developed a novel mutagenesis strategy that allows for the simultaneous performance of these two types of mutagenesis to construct a yeast variant with high 2,3-butanediol (2,3-BDO) tolerance. The mutations were simultaneously introduced into S.cerevisiae YPH499, accompanied by a stepwise increase in the concentration of 2,3-BDO. The resulting mutant YPH499/pol3δ/BD_392 showed 4.9-fold higher cell concentrations than the parental strain after 96h cultivation in medium containing 175g/L 2,3-BDO. Afterwards, we carried out transcriptome analysis to characterize the 2,3-BDO-tolerant strain. Gene ontology enrichment analysis with RNA sequence data revealed an increase in expression levels of genes related to amino acid metabolic processes. Therefore, we hypothesize that the yeast acquired high 2,3-BDO tolerance by amino acid function. Our research provides a novel mutagenesis strategy that achieves efficient modification of the genome for improving tolerance to various types of stressors.

Improve the production of D-limonene by regulating the mevalonate pathway of Saccharomyces cerevisiae during alcoholic beverage fermentation.

D-Limonene, a cyclized monoterpene, possesses citrus-like olfactory property and multi-physiological functions, which can be used as a bioactive compound and flavor to improve the overall quality of alcoholic beverages. In our previous study, we established an orthogonal pathway of D-limonene synthesis by introducing neryl diphosphate synthase 1 (tNDPS1) and D-limonene synthase (tLS) in Saccharomyces cerevisiae. To further increase D-limonene formation, the metabolic flux of the mevalonate (MVA) pathway was enhanced by overexpressing the key genes tHMGR1, ERG12, IDI1, and IDI1WWW, respectively, or co-overexpressing. The results showed that strengthening the MVA pathway significantly improved D-limonene production, while the best strain yielded 62.31mg/L D-limonene by co-expressing tHMGR1, ERG12, and IDI1WWW genes in alcoholic beverages. Furthermore, we also studied the effect of enhancing the MVA pathway on the growth and fermentation of engineered yeasts during alcoholic beverage fermentation. Besides, to further resolve the problem of yeast growth inhibition, we separately investigated transporter proteins of the high-yielding D-limonene yeasts and the parental strain under the stress of different D-limonene concentration, suggesting that the transporters of Aus1p, Pdr18p, Pdr5p, Pdr3p, Pdr11p, Pdr15p, Tpo1p, and Ste6p might play a more critical role in alleviating cytotoxicity and improving the tolerance to D-limonene. Finally, we verified the functions of three transporter proteins, finding that the transporter of Aus1p failed to transport D-limonene, and the others (Pdr5p and Pdr15p) could improve the tolerance of yeast to D-limonene. This study provided a valuable platform for other monoterpenes' biosynthesis in yeast during alcoholic beverage fermentation.

Antifungal activity of styrylpyridinium compounds against Candida albicans.

We synthesized a set of 13 new and earlier described styrylpyridinium compounds (N-alkyl styrylpyridinium salts with bromide or tosylate anions) in order to evaluate antifungal activity against C.albicans cells, to assay the possible synergism with fluconazole, and to estimate cytotoxicity to mammalian cells. All compounds were synthesized according to a well-known two-step procedure involving alkylation of γ-picoline with appropriate alkyl bromide and further condensation with substituted benzaldehyde. Compounds with long N-alkyl chains (C18 H37 -C20 H41 ) had no antifungal activity against the cells of all tested C.albicans strains. Other styrylpyridinium compounds were able to inhibit yeast growth at the concentrations of 0.06-16μg/ml. At fungicidal concentrations, the compound with the CN- group was least toxic to mammalian cells, showed the most effective synergism with fluconazole, and only slightly inhibited the respiration of C.albicans. The compound with the 4'-diethylamino group exhibited the strongest fungicidal properties and effectively blocked the respiration of C.albicans cells. However, toxicity to mammalian cells was also high. Summarizing, the results of our study indicate that styrylpyridinium compounds are promising candidates in the development of new antifungal drugs.

Effect of gellan gum, xylitol and natamycin on Zygosaccharomyces bailii growth and rheological characteristics in low sugar content model systems

This study evaluated the effect of some natural additives and the structure imparted by them on microbial growth and rheological characteristics in acidic model foods with reduced glycidic content. Systems were formulated using gellan gum, as gelling agent; xylitol, as aw depressor; and natamycin, as antimicrobial. Additive-free control systems were prepared. The pH was adjusted to 3.50 or 5.50 as required. Systems were inoculated with Zygosaccharomyces bailii. The effect of additives alone and combined on Z. bailii growth was studied. In some cases, the possible use of additives as yeast nutrients was evaluated. Furthermore, systems rheological characterization was performed. Additives and the structure given by gellan gum significantly affected yeast growth. Gellan gum initially slowed Z. bailii development, but as storage progressed, it acted as yeast carbon source, promoting its growth. A similar trend was observed when xylitol effect was studied. Natamycin inhibited yeast growth in all systems assayed. Additives modified the rheological characteristics of the gels and this effect depended on gellan gum concentration and pH. Obtained results emphasize the importance of considering the different effects that additives and their combinations can exert on the growth of deteriorating microorganisms and on the physical characteristics of gels.

Effects of Biosurfactants on Enzymatic Saccharification and Fermentation of Pretreated Softwood.

The enzymatic hydrolysis of cellulose is inhibited by non-productive adsorption of cellulases to lignin, and that is particularly problematic with lignin-rich materials such as softwood. Although conventional surfactants alleviate non-productive adsorption, using biosurfactants in softwood hydrolysis has not been reported. In this study, the effects of four biosurfactants, namely horse-chestnut escin, Pseudomonas aeruginosa rhamnolipid, and saponins from red and white quinoa varieties, on the enzymatic saccharification of steam-pretreated spruce were investigated. The used biosurfactants improved hydrolysis, and the best-performing one was escin, which led to cellulose conversions above 90%, decreased by around two-thirds lignin inhibition of Avicel hydrolysis, and improved hydrolysis of pretreated spruce by 24%. Red quinoa saponins (RQS) addition resulted in cellulose conversions above 80%, which was around 16% higher than without biosurfactants, and it was more effective than adding rhamnolipid or white quinoa saponins. Cellulose conversion improved with the increase in RQS addition up to 6 g/100 g biomass, but no significant changes were observed above that dosage. Although saponins are known to inhibit yeast growth, no inhibition of Saccharomyces cerevisiae fermentation of hydrolysates produced with RQS addition was detected. This study shows the potential of biosurfactants for enhancing the enzymatic hydrolysis of steam-pretreated softwood.

Mechanism of Action of Potent Boron-Containing Antifungals

Background:Boron is unusual to organic chemists, yet boron interacts greatly with organic biochemicals and has considerable bioactivity, especially as an antifungal and insecticide. The bestknown bioactive boron compounds are boric acid, its salt borax, and the closely related boronic acids. A newcomer is tavaborole (trade name Kerydin), recently developed and approved in 2014 for topical treatment of onychomycosis, a fungal infection of nails and the nail bed. It is timely to review the literature and explore the way in which these compounds may work.Methods:The focus of this review is to examine peer-reviewed literature relating to boric acid, boronic acid and tavaborole, the most bioactive boron-containing compounds, and the evidence for their proposed mechanism of antifungal action. In parallel with the literature, we have examined the fungistatic effects of boric acid on yeast.Results:All three compounds are reported to inhibit protein synthesis but their mechanism of action may differ. Chemistry studies indicate an interaction of boric acid with ribose and ribose-containing moieties such as NAD. In this review, we discuss the activity of boric acid and use both tavaborole and the boronic acids to exemplify the similar underlying mechanisms used. As there is a push to develop new antimicrobials, we demonstrate that boric acid’s fungistatic effect is alleviated with ribose, NAD and tryptophan.Conclusion:We speculate that boric acid inhibits yeast growth by disrupting tryptophan synthesis as well as downstream NAD, a rate limiting co-enzyme, essential for cellular function.

In Vitro Comparison of the Bioactivities of Japanese and Bohemian Knotweed Ethanol Extracts.

Knotweed is a flowering plant that is native to temperate and subtropical regions in the northern hemisphere. We evaluated Japanese (Reynoutria japonica Houtt.) and Bohemian (Fallopia x bohemica) knotweed rhizome and flower ethanol extracts and compared them in terms of their biological activities. The specific polyphenols were identified and quantified using HPLC/DAD, and the antioxidant activity was determined using 2,2-diphenly-1-picrylhydrazyl (DPPH) and cellular antioxidant capacity assays. The anticancer activity was evaluated as the difference between the cytotoxicity to cancer cells compared with control cells. The antimicrobial activity was determined using bacteria and yeast. The antidiabetic activity was tested as the ability of the extracts to inhibit α-amylase. Both rhizome extracts were sources of polyphenols, particularly polydatin and (−)-epicatechin; however, the cellular assay showed the highest antioxidant capacity in the flower extract of F. bohemica. The PaTu cell line was the least sensitive toward all knotweed extracts. The flower extracts of both species were less toxic than the rhizomes. However, the activity of the tested extracts was not specific for cancer cells, indicating a rather toxic mode of action. Furthermore, all used extracts decreased the α-amylase activity, and the rhizome extracts were more effective than the flower extracts. None of the extracts inhibited bacterial growth; however, they inhibited yeast growth. The results confirmed that rhizomes of Reynoutria japonica Houtt. could become a new source of bioactive compounds, which could be used for the co-treatment of diabetes and as antifungal agents.

The TIR-domain containing effectors BtpA and BtpB from Brucella abortus impact NAD metabolism.

Brucella species are facultative intracellular Gram-negative bacteria relevant to animal and human health. Their ability to establish an intracellular niche and subvert host cell pathways to their advantage depends on the delivery of bacterial effector proteins through a type IV secretion system. Brucella Toll/Interleukin-1 Receptor (TIR)-domain-containing proteins BtpA (also known as TcpB) and BtpB are among such effectors. Although divergent in primary sequence, they interfere with Toll-like receptor (TLR) signaling to inhibit the innate immune responses. However, the molecular mechanisms implicated still remain unclear. To gain insight into the functions of BtpA and BtpB, we expressed them in the budding yeast Saccharomyces cerevisiae as a eukaryotic cell model. We found that both effectors were cytotoxic and that their respective TIR domains were necessary and sufficient for yeast growth inhibition. Growth arrest was concomitant with actin depolymerization, endocytic block and a general decrease in kinase activity in the cell, suggesting a failure in energetic metabolism. Indeed, levels of ATP and NAD+ were low in yeast cells expressing BtpA and BtpB TIR domains, consistent with the recently described enzymatic activity of some TIR domains as NAD+ hydrolases. In human epithelial cells, both BtpA and BtpB expression reduced intracellular total NAD levels. In infected cells, both BtpA and BtpB contributed to reduction of total NAD, indicating that their NAD+ hydrolase functions are active intracellularly during infection. Overall, combining the yeast model together with mammalian cells and infection studies our results show that BtpA and BtpB modulate energy metabolism in host cells through NAD+ hydrolysis, assigning a novel role for these TIR domain-containing effectors in Brucella pathogenesis.

Competitive Exclusion Is a Major Bioprotective Mechanism of Lactobacilli against Fungal Spoilage in Fermented Milk Products.

A prominent feature of lactic acid bacteria (LAB) is their ability to inhibit growth of spoilage organisms in food, but hitherto research efforts to establish the mechanisms underlying bioactivity focused on the production of antimicrobial compounds by LAB. We show, in this study, that competitive exclusion, i.e., competition for a limited resource by different organisms, is a major mechanism of fungal growth inhibition by lactobacilli in fermented dairy products. The depletion of the essential trace element manganese by two Lactobacillus species was uncovered as the main mechanism for growth inhibition of dairy spoilage yeast and molds. A manganese transporter (MntH1), representing one of the highest expressed gene products in both lactobacilli, facilitates the exhaustive manganese scavenging. Expression of the mntH1 gene was found to be strain dependent, affected by species coculturing and the growth phase. Further, deletion of the mntH1 gene in one of the strains resulted in a loss of bioactivity, proving this gene to be important for manganese depletion. The presence of an mntH gene displayed a distinct phylogenetic pattern within the Lactobacillus genus. Moreover, assaying the bioprotective ability in fermented milk of selected lactobacilli from 10 major phylogenetic groups identified a correlation between the presence of mntH and bioprotective activity. Thus, manganese scavenging emerges as a common trait within the Lactobacillus genus, but differences in expression result in some strains showing more bioprotective effect than others. In summary, competitive exclusion through ion depletion is herein reported as a novel mechanism in LAB to delay the growth of spoilage contaminants in dairy products.IMPORTANCE In societies that have food choices, conscious consumers demand natural solutions to keep their food healthy and fresh during storage, simultaneously reducing food waste. The use of "good bacteria" to protect food against spoilage organisms has a long, successful history, even though the molecular mechanisms are not fully understood. In this study, we show that the depletion of free manganese is a major bioprotective mechanism of lactobacilli in dairy products. High manganese uptake and intracellular storage provide a link to the distinct, nonenzymatic, manganese-catalyzed oxidative stress defense mechanism, previously described for certain lactobacilli. The evaluation of representative Lactobacillus species in our study identifies multiple relevant species groups for fungal growth inhibition via manganese depletion. Hence, through the natural mechanism of nutrient depletion, the use of dedicated bioprotective lactobacilli constitutes an attractive alternative to artificial preservation.

Bioprocessing of shrimp wastes to obtain chitosan and its antimicrobial potential in the context of ethanolic fermentation against bacterial contamination.

This study investigated the bioprocessing of shrimp wastes to obtain chitin and its deacetylated product chitosan by a fermentation process mediated by Lactobacillus plantarum. The concentrations of glucose, bacterial inoculum, and shrimp wastes in the Man, Rogosa and Sharpe medium were optimized for the fermentation process performed in shake flasks to achieve the maximum titratable acidity to obtain chitin. The experiments were scaled up in a 700-mL working volume bioreactor, and the resulting chitin was deacetylated by the autoclave method. The bioextracted chitosan was characterized (Fourier transform infrared spectroscopy [FTIR], deacetylation degree, and molecular weight) and evaluated for its antimicrobial effects by comparing it with a commercial chitosan sample in the context of the ethanolic fermentation process for fuel alcohol production. The effect of chitosan on such a fermentation process has not been determined yet. The bacterial contaminant Lactobacillus fermentum and the main agent of ethanolic fermentation Saccharomyces cerevisiae were cultured in semi-synthetic medium and co-cultured in sugarcane juice to verify the effect of chitosan on their growth. The bioextracted chitosan (molecular weight 4.0 × 105gmol-1 and deacetylation degree 80%) was comparable to commercial chitosan, although higher concentrations of the former were required to achieve similar antimicrobial activities. Both commercial and bioextracted chitosan samples exhibited antimicrobial activity against S. cerevisiae and L. fermentum, but the concentration that caused the inhibition of yeast growth was almost tenfold higher than for the bacterium. Moreover, bioextracted chitosan showed no yeast inhibition or lethality in the range of 0.0075-0.96% while for the bacterium, growth inhibition occurred in concentrations varying from 0.24 to 0.48% and lethality of more than 99% at 0.96%. These results indicate the potential use of chitosan and especially of bioextracted chitosan in the bioethanol industry as a safer and more natural approach to combat unwanted bacterial contamination.

Inhibition of Yeast Growth by Tryptamine and Recovery with Tryptophan

Background:Tryptamine, a biogenic monoamine that is present in trace levels in the mammalian central nervous system, has probable roles as a neurotransmitter and/or a neuromodulator and may be associated with various neuropsychiatric disorders. One of the ways tryptamine may affect the body is by the competitive inhibition of the attachment of tryptophan to tryptophanyl tRNA synthetases.Methods:This study has explored the effects of tryptamine on growth of six yeast species (Saccharomyces cerevisiae, Candida glabrata, C. krusei, C. dubliniensis, C. tropicalis and C. lusitaniae) in media with glucose or ethanol as the carbon source, as well as recovery of growth inhibition by the addition of tryptophan.Results:Tryptamine was found to have an inhibitory effect on respiratory growth of all yeast species when grown with ethanol as the carbon source. Tryptamine also inhibited fermentative growth of Saccharomyces cerevisiae, C. krusei and C. tropicalis with glucose as the carbon source. In most cases the inhibitory effects were reduced by added tryptophan.Conclusion:The results obtained in this study are consistent with tryptamine competing with tryptophan to bind mitochondrial and cytoplasmic tryptophanyl tRNA synthetases in yeast: effects on mitochondrial and cytoplasmic protein synthesis can be studied as a function of growth with glucose or ethanol as a carbon source. Of the yeast species tested, there is variation in the sensitivity to tryptamine and the rescue by tryptophan. The current study suggests appropriate yeast strains and approaches for further studies.

Application of Stable Isotope Tracing to Elucidate Metabolic Dynamics During Yarrowia lipolytica α-Ionone Fermentation.

SummaryTargeted metabolite analysis in combination with 13C-tracing is a convenient strategy to determine pathway activity in biological systems; however, metabolite analysis is limited by challenges in separating and detecting pathway intermediates with current chromatographic methods. Here, a hydrophilic interaction chromatography tandem mass spectrometry approach was developed for improved metabolite separation, isotopologue analysis, and quantification. The physiological responses of a Yarrowia lipolytica strain engineered to produce ∼400 mg/L α-ionone and temporal changes in metabolism were quantified (e.g., mevalonate secretion, then uptake) indicating bottleneck shifts in the engineered pathway over the course of fermentation. Dynamic labeling results indicated limited tricarboxylic acid cycle label incorporation and, combined with a measurable ATP shortage during the high ionone production phase, suggested that electron transport and oxidative phosphorylation may limit energy supply and strain performance. The results provide insights into terpenoid pathway metabolic dynamics of non-model yeasts and offer guidelines for sensor development and modular engineering.

Characterization of the mechanism of thioredoxin-dependent activation of γ-glutamylcyclotransferase, RipAY, from Ralstonia solanacearum

A class II ChaC protein, RipAY, from phytopathogenic bacterium, Ralstonia solanacearum exhibits γ-glutamylcyclotransferase (GGCT) activity to degrade intracellular glutathione in host cells upon its interaction with host thioredoxins (Trxs). To understand the Trx-dependent activation of RipAY, we constructed various deletion mutants of RipAY and found the determinant region for GGCT activation in the N- and C-terminal sequences of RipAY by analyzing their yeast growth inhibition activity and the interaction with Trxs. Mutational analysis of the active site cysteine residues of Arabidopsis thaliana Trx-h5 (AtTrx-h5), one of the most efficiently stimulating Trxs, revealed that each active site cysteine residue of AtTrx-h5 contributes to efficient RipAY-binding and -activation activity. We also estimated that RipAY and AtTrx-h5 form a complex at a 1:2 M ratio. Furthermore, we found that the constitutive GGCT activity of Gcg1, a yeast class I ChaC protein, is also stimulated by yeast Trx1. These results indicate that class I ChaC proteins can sense the intracellular redox state and interact with Trxs to promote more efficient degradation of glutathione and regulate intracellular redox homeostasis. We hypothesize that RipAY acquired a more efficient and specific Trx-dependent activation mechanism to activate its GGCT activity only in the host eukaryotic cells during the evolution.