Production of glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma siamensis CBS 9960 and their interfacial properties

The search for a novel producer of glycolipid biosurfactants, mannosylerythritol lipids (MELs), was undertaken on the basis of the analysis of ribosomal DNA sequences of yeast strains of the genus Pseudozyma. In the course of the investigation, Pseudozyma siamensis CBS 9960, which is closely related to Pseudozyma shanxiensis, a known MEL-C producer but with a different morphology, was found to accumulate a large amount of glycolipids. On thin layer chromatography, the extracellular glycolipids showed nearly the same spots as those of the MELs produced by P. shanxiensis. However, the result of high-performance liquid chromatography analysis revealed that the present strain has a much higher glycolipid production yield than P. shanxiensis. From the structural characterization by (1)H and (13)C NMR, the major glycolipid (more than 84% of the total) was identified as a mixture of 4-O-[(2',4'-di-O-acetyl-3'-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-erythritol and 4-O-[(4'-O-acetyl-3'-O-alka(e)noyl-2'-O-butanoyl)-beta-D-mannopyranosyl]-D-erythritol, both of which are types of MEL-C. The present MEL-C possessed a short-chain acid (C(2) or C(4)) at the C-2' position and a long-chain acid (C(16)) at the C-3' position of the mannose moiety, and thus, the hydrophobic part was considerably different from that of conventional MELs, which mainly possess two medium-chain acids (C(10)) at the C-2' and C-3' positions. Under optimal growth conditions with safflower oil in a shake culture, the total amount of MELs reached approximately 19 g/l after 9 d at 25 degrees C. We further investigated the interfacial properties of the present MEL-C, considering its unique hydrophobic structure. The observed critical micelle concentration (CMC) and the surface tension at the CMC of the MEL were 4.5 x 10(-6) M and 30.7 mN/m, respectively. In addition, on a water penetration scan, the MEL efficiently formed the liquid crystal phases such as hexagonal (H) and lamella (L(a)) at a wide range of concentrations. These results demonstrated that the newly identified MEL-C produced by P. siamensis exhibits not only high surface activity but also excellent self-assembling properties, and should facilitate the development of promising yeast biosurfactants.     

Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica

The yeast Pseudozyma antarctica produces a large amount of glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surface‐active properties but also versatile biochemical actions. To investigate the biosynthesis of MELs in the yeast, we recently reported expressed sequence tag (EST) analysis and estimated genes expressing under MEL production conditions. Among the genes, a contiguous sequence of 938 bp, PA_004, showed high sequence identity to the gene emt1, encoding an erythritol/mannose transferase of Ustilago maydis, which is essential for MEL biosynthesis. The predicted translation product of the extended PA_004 containing the two introns and a stop codon was aligned with Emt1 of U. maydis. The predicted amino acid sequence shared high identity (72%) with Emt1 of U. maydis, although the amino‐terminal was incomplete. To identify the gene as PaEMT1 encoding an erythritol/mannose transferase of P. antarctica, the gene‐disrupted strain was developed by the method for targeted gene disruption, using hygromycin B resistance as the selection marker. The obtained ΔPaEMT1 strain failed to produce MELs, while its growth was the same as that of the parental strain. The additional mannosylerythritol into culture allowed ΔPaEMT1 strain to form MELs regardless of the carbon source supplied, indicating a defect of the erythritol/mannose transferase activity. Furthermore, we found that MEL formation is associated with the morphology and low‐temperature tolerance of the yeast. Copyright © 2010 John Wiley & Sons, Ltd.     

Microbial conversion of glycerol into glycolipid biosurfactants, mannosylerythritol lipids, by a basidiomycete yeast, Pseudozyma antarctica JCM 10317(T)

Microbial conversion of glycerol into functional bio-based materials was investigated, aiming to facilitate the utilization of waste glycerol. A basidiomycete yeast, Pseudozyma antarctica JCM 10317, efficiently produced mannosylerythritol lipids (MELs) as glycolipid biosurfactants from glycerol. The amount of MEL yield reached 16.3 g l(-1) by intermittent feeding of glycerol.     

Control of specific growth rate to enhance the production of a novel disintegrin,saxatilin, in recombinant Pichia pastoris

The methylotrophic yeast Pichia pastoris is one of the best hosts for the production of foreign proteins because of the presence of a strong alcohol oxidase 1 (AOX1) promoter that can be induced by methanol. Feeding the yeast, methanol induces protein production and provides an energy source for the host cells. However, excessive levels of methanol inhibit the growth of host cells, and insufficient methanol levels lead to poor growth and protein production. We have used various methanol feeding strategies to enhance the production of saxatilin. Saxatilin is a novel snake venom-derived disintegrin that inhibits tumor angiogenesis and metastasis and has been shown to suppress ovarian cancer cell invasion. A two-step increase feeding strategy to control the specific growth rate led to the best results in terms of specific protein production rates and final saxatilin amounts within the limited fermentation time.     

Ethanol yield and volatile compound content in fermentation of agave must by Kluyveromyces marxianus UMPe-1 comparing with Saccharomyces cerevisiae baker's yeast used in tequila production

In tequila production, fermentation is an important step. Fermentation determines the ethanol productivity and organoleptic properties of the beverage. In this study, a yeast isolated from native residual agave must was identified as Kluyveromyces marxianus UMPe-1 by 26S rRNA sequencing. This yeast was compared with the baker's yeast Saccharomyces cerevisiae Pan1. Our findings demonstrate that the UMPe-1 yeast was able to support the sugar content of agave must and glucose up to 22% (w/v) and tolerated 10% (v/v) ethanol concentration in the medium with 50% cells survival. Pilot and industrial fermentation of agave must tests showed that the K. marxianus UMPe-1 yeast produced ethanol with yields of 94% and 96% with respect to fermentable sugar content (glucose and fructose, constituting 98%). The S. cerevisiae Pan1 baker's yeast, however, which is commonly used in some tequila factories, showed 76% and 70% yield. At the industrial level, UMPe-1 yeast shows a maximum velocity of fermentable sugar consumption of 2.27g·L(-1)·h(-1) and ethanol production of 1.38g·L(-1)·h(-1), providing 58.78g ethanol·L(-1) at 72h fermentation, which corresponds to 96% yield. In addition, the major and minor volatile compounds in the tequila beverage obtained from UMPe-1 yeast were increased. Importantly, 29 volatile compounds were identified, while the beverage obtained from Pan1-yeast contained fewer compounds and in lower concentrations. The results suggest that the K. marxianus UMPe-1 is a suitable yeast for agave must fermentation, showing high ethanol productivity and increased volatile compound content comparing with a S. cerevisiae baker's yeast used in tequila production.     

The effect of Lactobacillus buchneri and Lactobacillus plantarum on the fermentation, aerobic stability, and ruminal degradability of low dry matter corn and sorghum silages

The effect of Lactobacillus buchneri, alone or in combination with Lactobacillus plantarum, on the fermentation, aerobic stability, and ruminal degradability of low dry matter corn and sorghum silages was studied under laboratory conditions. The inoculants were applied at 1 x 10(6) cfu/g. Silages with no additives served as control. After treatment, the chopped forages were ensiled in 1.5-L anaerobic jars. Three jars per treatment were sampled on d 2, 4, 8, 15, and 90. After 90 d of storage, the silages were subjected to an aerobic stability test lasting 5 d, in which CO2 production, as well as chemical and microbiological parameters, was measured to determine the extent of aerobic deterioration. At the end of the ensiling period (d 90), the L. buchneri- and L. buchneri + L. plantarum-inoculated silages had significantly higher levels of acetic acid than the control and L. plantarum-inoculated silages. Therefore, yeast activity was impaired in the L. buchneri- and L. buchneri + L. plantarum-inoculated silages. As a result, L. buchneri, alone or in combination with L. plantarum, improved aerobic stability of the low dry matter corn and sorghum silages. The combination of L. buchneri and L. plantarum reduced ammonia N concentrations and fermentation losses in the silages compared with L. buchneri alone. However, L. buchneri, L. plantarum, and a combination of L. buchneri + L. plantarum did not effect in situ rumen dry matter, organic matters, or neutral detergent fiber degradability of the silages. The L. buchneri was very effective in protecting the low dry matter corn and sorghum silages exposed to air under laboratory conditions. The use of L. buchneri, alone or in combination with L. plantarum, as a silage inoculant can improve the aerobic stability of low dry matter corn and sorghum silages by inhibition of yeast activity.     

Submerged culture conditions for mycelial yield and polysaccharides production by Lyophyllum decastes

The effects of various carbon and nitrogen sources, their concentrations, initial pH and fermentation duration on the production of mycelia in terms of dry weight, exo-polysaccharide (EPS) and inner polysaccharide (IPS) by Lyophyllum decastes, a culinary-medicinal mushroom, were investigated in shake-flask cultures. Lactose, glucose and fructose were the top three best carbon sources for mycelial growth with corresponding yields of 6.73 g/l, 6.36 g/l and 6.10 g/l, respectively. Glucose was the best for production of EPS and IPS with 1.65 g/l and 317 mg/g dry mycelia, respectively. Maltose also performed well for EPS production. Yeast extract was the best nitrogen source for the production of mycelia (7.03 g/l) and IPS (325 mg/g dry mycelia), whereas EPS was improved further by increasing the yeast extract concentration (2.46 g/l at 2%). Similarly, initial pH 7 and 8 were best for polysaccharides production (EPS 1.73 g/l and IPS 320 mg/g) and mycelial growth (7.10 g/l), respectively. Maximum mycelial growth peaked at 15 days of cultivation whereas polysaccharides peaked at 10 days, and then tapered off. A concentration of glucose 3% and yeast extract 1% (mycelial yield and IPS) were found to be a suitable condition for submerged culture.     

Pretreatment of the yeast antagonist, Candida oleophila, with glycine betaine increases oxidative stress tolerance in the microenvironment of apple wounds

In response to wounding, harvested fruit tissues of apple and citrus exhibit the production of reactive oxygen species (ROS). ROS production is greater when yeast antagonists used as biocontrol agents are applied in the wounds. These phenomena result in an oxidative stress environment for the yeast antagonists. It has been demonstrated that pre-exposure of some of these yeast antagonists to sublethal abiotic stress (heat or hydrogen peroxide), or stress-ameliorating compounds such as glycine betaine (GB) can induce subsequent oxidative stress tolerance in the antagonistic yeast. The increased level of oxidative stress tolerance has been demonstrated in vitro and is characterized by higher levels of antioxidant gene expression, increased production of trehalose, and lower levels of ROS when yeast are exposed to a subsequent oxidative stress. The current study determined whether or not the effects of GB on yeast antagonists determined in vitro persist and are present in planta when yeast are applied to wounded apples. The effect of exogenous GB on the production of ROS in the yeast antagonist, Candida oleophila, was determined after the yeast was placed in apple wounds. Oxidative damage to yeast cells recovered from apple wounds was also monitored. Results indicated that GB treatment improved the adaptation of C. oleophila to apple fruit wounds. Compared to untreated control yeast cells, GB-treated cells recovered from the oxidative stress environment of apple wounds exhibited less accumulation of ROS and lower levels of oxidative damage to cellular proteins and lipids. Additionally, GB-treated yeast exhibited greater biocontrol activity against Penicillium expansum and Botrytis cinerea, and faster growth in wounds of apple fruits compared to untreated yeast. The expression of major antioxidant genes, including peroxisomal catalase, peroxiredoxin TSA1, and glutathione peroxidase was elevated in the yeast by GB treatment. This study supports the premise that activation of antioxidant response in biocontrol yeast can improve biocontrol efficacy.     

Osmotic stress response in the wine yeast Dekkera bruxellensis

Dekkera bruxellensis is mainly associated with lambic beer fermentation and wine production and may contribute in a positive or negative manner to the flavor development. This yeast is able to produce phenolic compounds, such as 4-ethylguaiacol and 4-ethylphenol which could spoil the wine, depending on their concentration. In this work we have investigated how this yeast responds when exposed to conditions causing osmotic stress, as high sorbitol or salt concentrations. We observed that osmotic stress determined the production and accumulation of intracellular glycerol, and the expression of NADH-dependent glycerol-3-phosphate dehydrogenase (GPD) activity was elevated. The involvement of the HOG MAPK pathway in response to this stress condition was also investigated. We show that in D. bruxellensis Hog1 protein is activated by phosphorylation under hyperosmotic conditions, highlighting the conserved role of HOG MAP kinase signaling pathway in the osmotic stress response.     

Effect of feeding yeast culture on performance, health, and immunocompetence of dairy calves

Objectives were to determine effects of feeding a culture of Saccharomyces cerevisiae on performance, health, and immunocompetence of calves in the first 70 d of age. Holstein calves (n = 512) at 2 ± 1 d of age were randomly assigned to yeast culture (YC, 218 females and 37 males) or control (223 females and 34 males). Yeast culture was fed at 2% of the grain dry matter. All calves received colostrum during the first 24 h, pasteurized milk thereafter until 60 d of age, and grain was fed ad libitum for the first 70 d of age. Calves were housed in individual hutches, and grain intake was measured 5 d/wk. Body weight was measured at 5, 30, and 68 d of age, and attitude and fecal consistency were scored daily. Incidence and duration of health disorders and treatments were recorded. Neutrophil phagocytic and killing activities and antibody response to immunization with ovalbumin were measured. Concentrations of glucose and 3-hydroxybutyrate were measured in plasma. Grain intake did not differ between treatments and averaged 908 g/d throughout the study. Body weight change, concentrations of glucose, and 3-hydroxybutyrate did not differ between YC and control. Minor effects on neutrophil function were observed, and YC tended to increase the number of phagocytized bacteria and killing of phagocytized bacteria but did not influence humoral immune response. Attitude scores were similar between treatments throughout the study. Almost all calves experienced mild diarrhea during the study, but feeding YC improved fecal scores, reduced days with watery feces, incidence of fever and diarrhea, and risk of health disorders. Because of the high incidence of diarrhea, mortality preweaning was also high, but YC improved survival of calves by decreasing mortality rate past 13 d of age. Income at the end of the study was improved by $48/calf with YC. Feeding yeast culture in grain improved health, minimized frequency of health treatments, and reduced risk of morbidity and mortality in dairy calves.     

High cell-density expression system: a novel method for extracellular production of difficult-to-express proteins

Yeast's extracellular expression provides a cost-efficient means of producing industrially useful recombinant proteins. However, depending on the protein to be expressed, the production results in a poor yield, which is occasionally accompanied with loss of the expression plasmid and hence hampered growth of the host in the inducing medium. Here we propose an alternative approach, high cell-density expression, to improve the yield of a certain range of so-called difficult-to-express proteins. In this expression system, recombinant yeast cells resting in stationary phase (OD(660)=3-4) are suspended in a small aliquot of inducing medium to form a high cell-density culture (e.g., OD(660)=15). When applied to the yeast strains harboring Lentinula edodes laccase (Lcc1 or Lcc4) expressing plasmids, the high cell-density system allowed the host cells to synthesize elevated amounts of the laccase which resulted in >1000- to 6000-fold higher yield than those synthesized in a classical growth-associated manner. The resting cells required aerobic agitation for the maximum production. The production system also worked for other foreign enzymes but not for beta-galactosidase from Aspergillus oryzae or Escherichia coli, likely suggesting an involvement of chaperons that act on a certain range of secretory proteins.     

Isolation of Pseudozyma churashimaensis sp. nov., a novel ustilaginomycetous yeast species as a producer of glycolipid biosurfactants, mannosylerythritol lipids

An ustilaginomycetous anamorphic yeast species isolated from the leaves of Saccharum officinarum (sugarcane) in Okinawa, Japan, was identified as a novel Pseudozyma species based on morphological and physiological aspects and molecular taxonomic analysis using the D1/D2 domains of the large subunit (26S) rRNA gene and the internal transcribed spacer 1 (ITS1)-5.8S-ITS2 regions. The name Pseudozyma churashimaensis sp. nov. was proposed for the novel species, with JCM 16988(T) as the type strain. Interestingly, P. churashimaensis was found to produce glycolipid biosurfactants, a mixture of mannosylerythritol lipids (MELs), including a novel tri-acetylated derivative (MEL-A2), from glucose. The observed critical micelle concentration (CMC) and the surface tension at CMC of MEL-A2 were 1.7 × 10?? M and 29.2 mN/m, respectively. Moreover, on a water-penetration scan, MEL-A2 efficiently formed different lyotropic liquid crystalline phases, including the lamella phase at a wide range of concentrations, indicating its excellent surface-active and self-assembling properties. The novel strain of the genus Pseudozyma should thus facilitate the application of glycolipid biosurfactants in combination with other MEL producers.     

TH-AADY和生香ADY在小曲米酒生产中的应用

将耐高温酒用活性干酵母,生香活性干酵母配合糖化酶应用于小曲大饼米酒生产中,大饼用量由21％减至15％,原料出酒率提高了4.73％,酒质也较以前有所提高,取得了明显的经济效益。     

Effect of lysozyme on "flor" velum yeasts in the biological aging of sherry wines

Biological aging is a key step in the production of Sherry wine classified as “fine”. During this stage, a film of yeast referred to as “flor velum” covers the surface of the wine and substantially alters its characteristics. Other microorganisms may coexist with flor yeasts, such as lactic acid bacteria and non-Saccharomyces yeasts, whose growth may be favored under certain conditions, causing organoleptic deviations and deterioration of the wine. To prevent the development of lactic bacteria, lysozyme usage has been introduced. Lysozyme is a hydrolytic enzyme with muramidase activity that can lyse gram-positive bacteria; its use in winemaking was approved by the OIV in 1997 (resolution OENO 10/97). Thus far, the use of lysozyme during the production of Sherry wines is not widespread despite its effectiveness in controlling lactic acid bacteria. However, there have been no studies on the effect of lysozyme on flor velum. The aim of this study was to determine the influence of lysozyme on yeast growth and the formation, development and metabolism of flor velum during the biological aging process of Sherry wine. The results indicate that lysozyme does not affect the flor yeast during the fermentative stage or biofilm stage. However, if yeast inoculation is carried out under submerged culture conditions during biological aging, low doses of lysozyme (≥12.5 g/hL) affect cell multiplication and the membrane hydrophobicity of the yeast, inhibiting their aggregation and flotation and the subsequent development of flor velum. Thus, the yeast inoculation protocol and the methodology used for the addition of lysozyme influence velum development, its metabolism and the wine characteristics.     

Characteristics of the high malic acid production mechanism in Saccharomyces cerevisiae sake yeast strain No. 28

We characterized a high malic acid production mechanism in sake yeast strain No. 28. No considerable differences in the activity of the enzymes that were involved in malic acid synthesis were observed between strain No. 28 and its parent strain, K1001. However, compared with strain K1001, which actively took up rhodamine 123 during staining, the cells of strain No. 28 were only lightly stained, even when cultured in high glucose concentrations. In addition, malic acid production by the respiratory-deficient strain of K1001 was 2.5-fold higher than that of the wild-type K1001 and wild-type No. 28. The findings of this study demonstrated that the high malic acid production by strain No. 28 is attributed to the suppression of mitochondrial activity.     

Strategy for simultaneous saccharification and fermentation using a respiratory-deficient mutant of Candida glabrata for bioethanol production

High temperature and agitation are advantageous for ethanol production from insoluble feedstock when using the simultaneous saccharification and fermentation (SSF) technique. To construct an effective SSF system, a respiratory-deficient mutant was isolated from the thermotolerant yeast Candida glabrata. Our results suggest that this respiratory-deficient mutant has higher ethanol production abilities in SSF.     

活性干酵母在淀粉质酒精发酵生产中的应用

本文利用市售活性干酵母代替培养酒母,分别进行淀粉质酒精间歇发酵、半连续发酵和连续发酵大型生产,均取得了发酵速度快、出酒率高的好效果。     

Enhanced plumbagin production in elicited Plumbago indica hairy root cultures

Elicitation of Plumbago indica hairy roots with yeast carbohydrate fraction, chitosan, manganese chloride, copper chloride and methyl jasmonate exhibited significant elevation (~1.2 to 2 fold) of plumbagin production in shake flask culture as compared with control. Chitosan and methyl jasmonate elicitation also caused simultaneous plumbagin leaching into culture media. Three days' exposure of chitosan (200 mg l(-1)) and methyl jasmonate (80 μM) together synergized total plumbagin yield to its maximum 11.96 ± 0.76 mg g(-l) DW in shake flask culture. In bioreactor cultivation, a significant raise in fresh root biomass was recorded on day 20 as compared with control shake flask culture. Three days' exposure of chitosan (200 mg l(-1)) and methyl jasmonate (80 μM) with 20 days old bioreactor-culture significantly improved total plumbagin production to 13.16 ± 1.72 mg g(-l) DW with simultaneous plumbagin leaching into bioreactor media.