High level secretion of recombinant human serum albumin by fed-batch fermentation of the methylotrophic yeast, Pichia pastoris, based on optimal methanol feeding strategy

The dynamic programming method was applied to obtain the optimal specific growth rate, mu, in the fed-batch fermentation using the recombinant human serum albumin (rHSA)-producing yeast, Pichia pastoris. Based on the relationship between the specific production rate, varrho, and the specific growth rate, mu, a simple mathematical model describing the growth and rHSA production was constructed and used for calculations. Two constraints, final volume and maximum methanol feed rate, were adopted for calculations and the optimal mu resulted as follows. That is, mu was initially at the maximum value, mu(max), then decreased gradually. Finally, mu decreased to the mu(min) that gave a maximum varrho. The decline of mu was revealed to be caused by the constraint for maximum methanol feeding rate, F(max), and F(max) was constant until mu decreased to mu(min). We tried to realize the optimal mu in the fed-batch fermentation by manipulating the methanol feeding rate and obtained it. However, the observed varrho was differed from the expected one. The discrepancy between the expected varrho and observed varrho after the change of mu suggests the inapplicability of the relationship between mu and varrho to dynamic situations where mu changes. To confirm this, simulation and fed-batch fermentation runs were carried out at a methanol feeding rate that would cause a continuous change in mu. The rHSA production was simulated well, suggesting the applicability of the relationship between mu and varrho in such situations. Discontinuity in the change in methanol feeding rate of the optimal feed pattern at the time mu changed is considered to be the cause for the discrepancy between the expected and observed varrho. Therefore, a new methanol feeding strategy that could mimic the changes in mu and varrho of the optimal strategy without a discontinuity in the feeding rate was sought using a mathematical model of fermentation by trial and error. This modification in the methanol feeding rate resulted in a considerably improved varrho and 18% increase in total rHSA production compared with those obtained by the optimal strategy.     

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.     

Oleic acid and ergosterol supplementation mitigates oxidative stress in wine strains of Saccharomyces cerevisiae

During fermentation of high-sugar-containing medium lacking lipid nutrients, wine yeasts undergo oxidative stress and oxidative damage to cell membranes and proteins. Considering that cell membranes are important stress sensors, and that under hypoxic conditions wine yeasts modulate cell membranes composition by incorporating lipids available in the growth medium, in the present work, the effects of lipid nutrition on wine yeast oxidative stress response were evaluated on two strains of Saccharomyces cerevisiae. Biomarkers of oxidative stress, oxidative damage and antioxidant response were evaluated together with viability and acetic acid production during fermentation of a synthetic must lacking lipid nutrients as compared to added oleic acid and ergosterol. The results show that the availability of lipid nutrients causes a significant reduction in the intracellular content of reactive oxygen species and in the oxidative damage to membranes and proteins, as indicated by flow cytometry of cells stained with dihydroethidum (DHE) and propidium iodide (PI) and by Western blot of protein carbonyls. Accordingly, lipid nutrients feeding results in the increase in cell viability and superoxide activity, and the reduction in trehalose accumulation, proteinase A activity and production of acetic acid. In summary, these results are compatible with the hypothesis that the supplementation of lipid nutrients mitigates oxidative stress and oxidative damage in wine strains of S. cerevisiae during growth under unfavourable conditions.     

Fed‐batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co‐substrate sorbitol

Batch‐wise sorbitol addition as a co‐substrate at the induction phase of methanol fed‐batch fermentation by Pichia pastoris (Mut⁺) was proposed as a beneficial recombinant protein production strategy and the metabolic responses to methanol feeding rate in the presence of sorbitol was systematically investigated. Adding sorbitol batch‐wise to the medium provided the following advantages over growth on methanol alone: (a) eliminating the long lag‐phase for the cells and reaching ‘high cell density production’ at t = 24 h of the process (C_X = 70 g CDW/l); (b) achieving 1.8‐fold higher recombinant human erythropoietin (rHuEPO) (at t = 18 h); (c) reducing specific protease production 1.2‐fold; (d) eliminating the lactic acid build‐up period; (e) lowering the oxygen uptake rate two‐fold; and (f) obtaining 1.4‐fold higher overall yield coefficients. The maximum specific alcohol oxidase activity was not affected in the presence of sorbitol, and it was observed that sorbitol and methanol were utilized simultaneously. Thus, in the presence of sorbitol, 130 mg/l rHuEPO was produced at t = 24 h, compared to 80 mg/l rHuEPO (t = 24 h) on methanol alone. This work demonstrates not only the ease and efficiency of incorporating sorbitol to fermentations by Mut⁺ strains of P. pastoris for the production of any bio‐product, but also provides new insights into the metabolism of the methylotrophic yeast P. pastoris. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

NY‐ESO‐1 protein glycosylated by yeast induces enhanced immune responses

Vaccine strategies that target dendritic cells to elicit potent cellular immunity are the subject of intense research. Here we report that the genetically engineered yeast Saccharomyces cerevisiae, expressing the full‐length tumour‐associated antigen NY‐ESO‐1, is a versatile host for protein production. Exposing dendritic cells (DCs) to soluble NY‐ESO‐1 protein linked to the yeast a‐agglutinin 2 protein (Aga2p) protein resulted in protein uptake, processing and MHC class I cross‐presentation of NY‐ESO‐1‐derived peptides. The process of antigen uptake and cross‐presentation was dependent on the glycosylation pattern of NY‐ESO‐1‐Aga2p protein and the presence of accessible mannose receptors. In addition, NY‐ESO‐1‐Aga2p protein uptake by dendritic cells resulted in recognition by HLA‐DP4 NY‐ESO‐1‐specific CD4⁺ T cells, indicating MHC class II presentation. Finally, vaccination of mice with yeast‐derived NY‐ESO‐1‐Aga2p protein led to an enhanced humoral and cellular immune response, when compared to the bacterially expressed NY‐ESO‐1 protein. Together, these data demonstrate that yeast‐derived full‐length NY‐ESO‐1‐Aga2p protein is processed and presented efficiently by MHC class I and II complexes and warrants clinical trials to determine the potential value of S. cerevisiae as a host for cancer vaccine development. Copyright © 2010 John Wiley & Sons, Ltd.     

Effects of methanol feeding methods on chimeric alpha-amylase expression in continuous culture of Pichia pastoris

The effects of two types of methanol feeding methods in a continuous culture of Pichia system on the cell growth and recombinant protein expression were studied using chimeric alpha-amylase as a model protein. With the feeding of methanol by a DO-stat method, the alpha-amylase concentration in the fermentation broth increased with decreasing dilution rate and reached 173 mg/l at a dilution rate of 0.013 h(-1), at which the maximum volumetric productivity of alpha-amylase was obtained. Although almost the same productivity was attained at 0.04 h(-1) with continuous methanol feeding, the alpha-amylase concentration was one third that compared with feeding by the DO-stat method, that is, 55 mg/l. Furthermore, at this dilution rate, the medium volume needed per unit time was three times that required when DO-stat was used. Therefore, continuous culture with methanol feeding by the DO-stat method may be a promising method for the production of recombinant proteins on an industrial scale by Pichia pastoris.     

Effects of feedborne Fusarium mycotoxins on the performance, metabolism, and immunity of dairy cows

Little is known about the effects of feedborne Fusarium mycotoxins on the performance, metabolism, and immunity of dairy cattle. A total mixed ration (TMR) containing a blend of feedstuffs naturally contaminated with Fusarium mycotoxins was fed for 56 d to 18 midlactation Holstein cows (average milk production, 33 kg/d) in a completely randomized design with repeated measures that included 3 treatments: 1) a control diet, 2) a contaminated diet, and 3) a contaminated diet + 0.2% polymeric glucomannan mycotoxin adsorbent (GMA). Wheat, corn, and hay were the contaminated feedstuffs used in the study. Deoxynivalenol was the major contaminant and was found in the TMR at levels of up to 3.6 μg/g of dry matter. Body weight, body condition score, dry matter intake, net energy balance, milk production, milk composition, somatic cell count, blood serum chemistry, hematology, serum Ig concentrations, and coagulation profile were measured. Dry matter intake and body weight, as well as milk production, milk composition, and SCC, were not affected by diet. Total serum protein and globulin levels increased significantly in cows fed the contaminated TMR compared with cows fed the control diet at 42 d, whereas the albumin:globulin ratio decreased. Serum urea concentrations were significantly elevated throughout the experiment in cows fed the contaminated diet compared with those fed the control diet. Serum IgA concentrations decreased significantly in cows fed the contaminated TMR at 36 d of feeding. Feeding GMA prevented these effects. Serum sodium concentration and osmolality levels were increased throughout the experiment in all cows fed the contaminated diets. We concluded that feed naturally contaminated with Fusarium mycotoxins can affect the metabolic parameters and immunity of dairy cows and that GMA can prevent some of these effects.     

Coculture with specific bacteria enhances survival of Lactobacillus plantarum NC8, an autoinducer-regulated bacteriocin producer,in olive fermentations

Bacteriocin production in Lactobacillus plantarum NC8 is activated by coculture with specific bacteriocin production-inducing bacterial strains. The system is further regulated by a three-component regulatory system involving a specific autoinducer peptide (PLNC8IF). We have used L. plantarum NC8 as a starter culture in Spanish-style green olive fermentations and examined the influence of coculturing in its survival. We found that L. plantarum NC8 greatly enhanced its growth and survival in the olive fermentations when coinoculated with two specific bacteriocin-production inducing strains, i.e. Enterococcus faecium 6T1a-20 and Pediococcus pentosaceus FBB63, when compared to singly-inoculated fermentations. In addition, a constitutive bacteriocin-producer NC8-derivative strain was used as a control in the olive fermentations and showed also better viability than the parental NC8 strain. Our results suggest the involvement of bacteriocin production in the viability enhancement found in both cases. We postulate that the presence of specific bacteria is recognized by L. plantarum NC8 as an environmental stimulus to switch a specific adaptive response on, most probably involving bacteriocin production. The design of novel bacteriocin-producing starter cultures for food fermentations should consider their constitutive versus regulated character.     

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.     

重组毕赤酵母生产外源蛋白的过程控制与生理分析的研究进展

毕赤酵母是近年来应用广泛、极受青睐的一种外源蛋白表达系统。除了构建高效、稳定的毕赤酵母生产菌株外,研究不同发酵控制条件下,细胞的生理和代谢状态变化,为毕赤酵母细胞提供最优的生长和环境条件并综合考虑发酵成本是成功实现外源蛋白规模化、商业化生产的保障。本文从培养基组分、外界环境条件、细胞比生长速率、生长期甘油流加策略和诱导期甲醇流加策略等方面阐述了发酵过程对外源蛋白表达的影响,并进一步从细胞生理和代谢层面深入讨论了不同发酵过程控制条件的影响机制。      

The use of Lactobacillus species as starter cultures for enhancing the quality of sugar cane silage

Sugar cane (Saccharum spp.) is a forage crop widely used in animal feed because of its high dry matter (DM) production (25 to 40 t/ha) and high energy concentration. The ensiling of sugar cane often incurs problems with the growth of yeasts, which leads to high losses of DM throughout the fermentative process. The selection of specific inoculants for sugar cane silage can improve the quality of the silage. The present study aimed to select strains of lactic acid bacteria (LAB) isolated from sugar cane silage and to assess their effects when used as additives on the same type of silage. The LAB strains were inoculated into sugar cane broth to evaluate their production of metabolites. The selected strains produced higher concentrations of acetic and propionic acids and resulted in better silage characteristics, such as low yeast population, lower ethanol content, and lesser DM loss. These data confirmed that facultative heterofermentative strains are not good candidates for sugar cane silage inoculation and may even worsen the quality of the silage fermentation by increasing DM losses throughout the process. Lactobacillus hilgardii strains UFLA SIL51 and UFLA SIL52 resulted in silage with the best characteristics in relation to DM loss, low ethanol content, higher LAB population, and low butyric acid content. Strains UFLA SIL51 and SIL52 are recommended as starter cultures for sugar cane silage.     

Evaluation of lactic acid bacteria and yeast diversity in traditional white pickled and fresh soft cheeses from the mountain regions of Serbia and lowland regions of Croatia

The goal of this study was the characterisation of indigenous lactic acid bacteria (LAB) and yeasts isolated from nine white pickled (BG) and nine fresh soft (ZG) artisanal cheeses collected in Serbia and Croatia. While LAB were present in all of the cheeses collected, yeasts were found in all BG cheeses but only in three ZG cheese samples. High LAB and yeast species diversity was determined (average H′_L = 0.4 and H′_Y = 0.8, respectively). The predominant LAB species in white pickled (BG) cheeses were Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides, while in fresh soft (ZG) cheeses the most dominant LAB species were L. lactis, Enterococcus faecalis, and Leuconostoc pseudomesenteroides. Among the 20 yeast species found, Debaryomyces hansenii, Candida zeylanoides, and Torulaspora delbrueckii were found to be predominant in BG cheeses, while Yarrowia lipolytica was predominant in ZG cheeses. The characterisation of metabolic and technological potentials revealed that 53.4% of LAB isolates produced antimicrobial compounds, 44.3% of LAB strains showed proteolytic activity, while most of the yeast species possessed either lipolytic or proteolytic activity. In conclusion, the results obtained in this study showed that the composition of LAB and yeast populations in white pickled and fresh soft cheeses is region specific. The knowledge gained in this study could eventually be used to select region specific LAB and yeast strains for the production of white pickled and fresh soft artisanal cheeses with geographically specific origins under controlled conditions.     

Factors influencing ethanol production rates at high-gravity brewing

The aim of this work was to investigate the influences of different fermentation factors on ethanol production rates by Saccharomyces cerevisiae (lager strain), at high-gravity brewing using response surface methodology. An empirical linear polynomial model was developed to describe the behaviour of the dependent variable as a function of significant factors. The resultant functional relationship in terms of coded values for predicting ethanol production rates was: Y=0.421+0.155X₂+0.0575X₂X_3, where Y represents the ethanol production rate (g/lh), and X₂ and X₃ are coded levels for fermentation temperature and nutrient supplementation, respectively. Patterns of yeast growth, decrease in wort gravity and ethanol production were also evaluated at the maximum ethanol production rate (0.694 g/lh). It was concluded that higher ethanol production rates could be achieved by increasing fermentation temperature and supplementing high-gravity worts with yeast extract, ergosterol and Tween 80.     

Modulation of the glycerol and ethanol syntheses in the yeast Saccharomyces kudriavzevii differs from that exhibited by Saccharomyces cerevisiae and their hybrid

In the last years there is an increasing demand to produce wines with higher glycerol levels and lower ethanol contents. The production of these compounds by yeasts is influenced by many environmental variables, and could be controlled by the choice of optimized cultivation conditions. The present work studies, in a wine model system, the effects of temperature, pH and sugar concentration on the glycerol and ethanol syntheses by yeasts Saccharomyces cerevisiae T73, the type strain of Saccharomyces kudriavzevii IFO 1802^T, and an interspecific hybrid between both species (W27), which was accomplished by the application of response surface methodology based in a central composite circumscribed design. Results show that carbon flux could be especially directed towards glycerol synthesis instead of ethanol at low pH, high sugar concentrations and low temperatures. In general, the non-wine yeast S. kudriavzevii produced higher glycerol levels and lower ethanol content than wine strains S. cerevisiae T73 and the hybrid W27, with specific and different glycerol production profiles as a function of temperature and pH. These results were congruent with the higher glycerol-3-phosphate dehydrogenase activities estimated for this species, chiefly at low temperatures (14 °C), which could explain why S. kudriavzevii is a cryotolerant yeast compared to S. cerevisiae.     

Growth performance,digestibility, blood metabolites,ruminal fermentation,and bacterial communities in response to the inclusion of gallic acid in the starter feed of preweaning dairy calves

The objective of this study was to evaluate the effects of feeding gallic acid on the growth, nutrient digestibility, plasma metabolites, rumen fermentation, and bacterial community in the rumen fluid and feces of preweaning calves. Thirty-six female Holstein calves with similar ages (means ± SD; 3.1 ± 1.39 d) and body weights (40.8 ± 2.87 kg) were randomly assigned to receive 3 treatments. Calves were fed 1 of 3 treatments as follows: basal diet with no gallic acid (control), 0.5 g/kg gallic acid in starter diet (low), and 1 g/kg gallic acid in starter diet (high). The results showed that feeding gallic acid increased growth by improving the starter intake and average daily gain of the calves. The fecal score tended to decrease in a linear manner with the addition of gallic acid. Total-tract apparent protein digestibility tended to increase linearly with feeding gallic acid. Feeding gallic acid led to a linear increase in the plasma total protein and β-hydroxybutyrate levels. In addition, feeding gallic acid linearly increased catalase and total antioxidant capacity levels and decreased malondialdehyde and tumor necrosis factor-α concentrations. The concentrations of total volatile fatty acids, propionate, butyrate, and valerate in the rumen fluid increased linearly with the addition of gallic acid, resulting in a linear pH reduction. Feeding gallic acid linearly increased the relative abundances of Prevotella_1, Saccharofermentans, and Prevotellaceae_UCG-001 and linearly decreased the relative abundance of Prevotella_7 in the rumen fluid. The Shannon index of ruminal bacterial communities linearly increased by feeding gallic acid. Feeding gallic acid linearly increased the relative abundances of Ruminococcaceae_UCG-005, Bacteroides, and Christensenellaceae_R-7_group in the feces. In summary, feeding gallic acid improved growth, antioxidant function, and rumen fermentation and altered the bacterial community in the rumen fluid and feces of preweaning dairy calves.     

Effect of grape indigenous Saccharomyces cerevisiae strains on Montepulciano d'Abruzzo red wine quality

The growth of selected, indigenous Saccharomyces cerevisiae added as starters (SRS1, MS72 and RT73) was monitored during Montepulciano d'Abruzzo wine production. In all the fermentations the addition of the starter, caused a decrease of the non-Saccharomyces yeasts. When strains MS72 and RT73 were used as starters they were detected in the first phases of fermentations, while strain SRS1 competed successfully with native yeasts during all the process. Wines obtained by fermentation with the indigenous starters showed some different characteristics, according to the chemical and sensory analyses. This study highlighted that among selected starters with high fermentative capacity, some are able to dominate better than other natural wine yeast biota, whereas some strains can interact and survive besides native yeast populations during the fermentation. As a consequence, the dominance character can have a positive or negative effect on wine quality and has to be considered in the frame of yeast selection in order to improve or characterize traditional wines. Winemakers could choose among different degrees of yeast dominance to modulate the interaction among starter and native wine yeast population.