Production of recombinant hirudin in galactokinase-deficientSaccharomyces cerevisiae by fed-batch fermentation with continuous glucose feeding

The artificial gene coding for anticoagulant hirudin was placed under the control of theGAL10 promoter and expressed in the galactokinase-deficient strain (Δgal1) ofSaccharomyces cereivisiae, which uses galactose only as a gratuitous inducer in order to avoid its consumption. For efficient production of recombinant hirudin, a carbon source other than galactose should be provided in the medium to support growth of the Δgal1 strain. Here we demonstrate the successful use of glucose in the fed-batch fermentation of the Δgal1 strain to achieve efficient production of recombinant hirudin, with a yield of up to 400 mg hirudin/L.     

Rapid purification of recombinant human lipocortin-I secreted fromSaccharomyces cerevisiae

Human lipocortin-I was expressed as a secretory product bySaccharomyces cerevisiae harboring an expression system consisting ofGAL10 promoter, inulinase signal sequence and lipocortin-I terminator. Fed-batch fermentation was carried out to overproduce recombinant human lipocortin-I. The culture medium was desalted and concentrated by ultrafiltration, and then subjected to hydroxyapatite column chromatography. The lipocortin-I was purified to >98% purity by single-step hydroxyapatite column chromatography. However, it was found that the purified lipocortin-I was a proteolytically-cleaved form which was cleaved immediately after the basic amino acid Lys²⁶.     

Enhancement of plasmid stability and protein productivity using multi-pulse, fed-batch culture of recombinant Saccharomyces cerevisiae

The plasmid stability of a recombinant Saccharomyces cerevisiae strain, which expresses cloned -amylase, was increased when glucose or yeast extract was fed with multi-pulse mode in fed-batch culture. Using a novel strategy combining constant rate fed-batch culture and multi-pulse feeding of yeast extract, the plasmid stability was maintained over 90%, meanwhile, 36 g cells l^–1 and 208 units of cloned -amylase activity ml^–1 were obtained.     

Rapid selection of multiple gene integrant for the production of recombinant hirudin inHansenula polymorpha

For the rapid selection of higher recombinant hirudin producing strain in a methylotrophic yeastHansenula polymorpha, a multiple gene integration and dose-dependent selection vector, based on a telomere-associated ARS and a bacterial aminoglycoside 3-phosphotransferase ( (aph) gene, was adopted. Two hirudin expression cassettes (HV1 and HV2) were constructed using theMOX promoter ofH. polymorpha and the mating factor α secretion signal ofS. cerevisiae. Multiple integrants of a transformang vector containing hirudin expression cassettes were easily selected by using an antibiotic, G418. Hirudin expression level and integrated plasmid copy number of the tested transformants increased with increasing the concentration of G418 used for selection. The expression level of HV1 was consistently higher than that of HV2 under the similar conditions, suggesting that the gene context might be quite important for the high-level gene expression inH. polymorpha. The highest hirudin producing strain selected in this study produced over 96 mg/L of biologically active hirudin in a 500-mL flask and 165 mg/L in a 5-L fermentor.     

Physiological aspects of growth and recombinant DNA stability inSaccharomyces cerevisiae

Despite the fact that plasmid stability in the yeastSaccharomyces cerevisiae is influenced by both genetical and physiological parameters most attention has been focussed on the former. Physiological factors affecting the stability of plasmids have been poorly characterized despite the need for such information in order to optimize the use ofS. cerevisiae as a host for recombinant protein production processes. The physiology of wild typeS. cerevisiae differs considerably when grown using different cultivation techniques. A limited amount of phenomenological data has been reported concerning plasmid instability effects under these different conditions and in this article these have been collected together with the intention of providing an overview to instability effects and to try and propose reasons as to how the physiological response to different growth conditions can be manifested as stability/instability effects.     

Characterisation of recombinant unglycosylated human serum transferrin purified from Saccharomyces cerevisiae

Structural identity between a recombinant transferrin mutant (N413Q, N611Q) secreted from Saccharomyces cerevisiae and the native protein was shown by CD analysis and immunodiffusion assays against anti-hSTf. The ability of the recombinant protein to bind iron was confirmed by urea–PAGE and EPR analysis of the iron-saturated protein revealed the characteristic holo-transferrin spectrum, indicating conservation of both iron-binding sites. The integrity of the unglycosylated recombinant protein indicates that such protein could be a valuable tool not only for structure–function characterisation but also crystallisation assays. In addition, the recombinant transferrin was found to be as effective as native transferrin as a growth factor in cell culture medium.     

Suppression of proteolytic degradation of recombinant hirudin from Saccharomyces cerevisiae using the O2-enriched air

A novel process using O2-enriched air supply was used to suppress the C-terminal proteolytic degradation of recombinant hirudin (r-hirudin) from Saccharomyces cerevisiae. When dissolved O2 was controlled above 20% saturation level using normal air, inactive forms of C-terminally truncated hirudin were observed in culture broth from 48 h of fermentation. The use of O2-enriched air giving above 40% saturation of dissolved O2 suppressed the proteolytic degradation and hence the formation of truncated forms of inactive r-hirudin until 60 h of fermentation.     

Decrease of proteolytic degradation of recombinant hirudin produced by Pichia pastoris by controlling the specific growth rate

The effects of the specific growth rate and methanol concentration on the degradation of hirudin produced by recombinant Pichia pastoris were investigated. When a methanol-limited state and the specific growth rate of 0.02 h^–1 were maintained during the fermentation, a minimum degradation of hirudin and a maximum specific hirudin production rate were achieved. By this strategy, the production of intact recombinant hirudin Hir65 reached 0.7 g l^–1 in fed-batch fermentation. Its proportion was 35% to all forms of hirudin.     

Profiling of external metabolites during production of hantavirus nucleocapsid protein with recombinant Saccharomyces cerevisiae

Recombinant strains of Saccharomyces cerevisiae, producing hantavirus Puumala nucleocapsid protein for diagnostics and as a candidate vaccine were analyzed for uptake and excretion of intermediary metabolites during process optimization studies of fed-batch bioreactor cultures. Concentrations of glucose, maltose, galactose, pyruvate, acetaldehyde, ethanol, acetate, succinate and formaldehyde (used as a selection agent) were measured in the culture medium in order to find a metabolite pattern, indicative for the physiological state of the producer culture. When the inducer galactose was employed as a growth substrate, the metabolite profile of recombinant yeast cells was different from those of the non-recombinant original strain which excreted considerable amounts of metabolites with this substrate. In contrast, galactose-induced heterologous gene expression was indicated by the absence of excreted intermediary metabolites, except succinate. A model strain expressing a GFP fusion of hantavirus nucleocapsid protein differed in the excretion of metabolites from strains without GFP. In addition, the influence of alkali ions, employed for pH control is also demonstrated.     

Scale-up of erythritol production by an osmophilic mutant of Candida magnoliae

Erythritol production by an osmophilic mutant of Candida magnoliae was performed in fermentations of up 50 l to develop an optimized commercial process. By simultaneous feeding glucose and yeast extract, erythritol productivity of 1.2 g l(-1) h(-1) was reached giving 200 g erythritol l(-1) with a yield of 0.43 g g(-1).     

Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae lacks the ability to ferment the pentose sugar xylose that is the second most abundant sugar in nature. Therefore two different xylose catabolic pathways have been heterologously expressed in S. cerevisiae. Whereas the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway leads to the production of the by-product xylitol, the xylose isomerase (XI) pathway results in significantly lower xylose consumption. In this study, kinetic models including the reactions ranging from xylose transport into the cell to the phosphorylation of xylulose to xylulose 5-P were constructed. They were used as prediction tools for the identification of putative targets for the improvement of xylose utilization in S. cerevisiae strains engineered for higher level of the non-oxidative pentose phosphate pathway (PPP) enzymes, higher xylulokinase and inactivated GRE3 gene encoding an endogenous NADPH-dependent aldose reductase. For both pathways, the in silico analyses identified a need for even higher xylulokinase (XK) activity. In a XR-XDH strain expressing an integrated copy of the Escherichia coli XK encoding gene xylB about a six-fold reduction of xylitol formation was confirmed under anaerobic conditions. Similarly overexpression of the xylB gene in a XI strain increased the aerobic growth rate on xylose by 21%. In contrast to the in silico predictions, the aerobic growth also increased 24% when the xylose transporter gene GXF1 from Candida intermedia was overexpressed together with xylB in the XI strain. Under anaerobic conditions, the XI strains overexpressing xylB gene and the combination of xylB and GFX1 genes consumed 27% and 37% more xylose than the control strain.     

Stability studies of recombinant Saccharomyces cerevisiae in the presence of varying selection pressure

A recombinant yeast plasmid carrying the Ieu2 gene for auxotrophic complementation and a reporter gene for beta-galactosidase under the control of Gal10 promoter was studied in Saccharomyces cerevisiae. Growth, product formation, and plasmid stability were studied in defined, semi-defined, and complex media. The biomass concentration and specific activity were higher in complex medium than in defined medium, which was selective for the growth of plasmid-containing cells, leading to a 10-fold increase in volumetric activity. However, plasmid instability was very high in complex media with 50% plasmid-free cells emerging in the culture within 75 h of cultivation. In order to control instability, the growth rates of the plasmid-containing and plasmid-free cells were determined in semi-defined media, which consisted of defined medium supplemented with different concentrations of yeast extract. Below a critical concentration of yeast extract (0.05 g/L), the plasmid-containing cells had a growth rate advantage over the plasmid-free cells. This was possibly because, at this concentration of yeast extract, the availability of leucine became the rate-determining factor in the specific growth rate of plasmid-free cells. A feeding strategy was designed which maintained a low concentration of the residual yeast extract in the medium and thus continuously provided the plasmid-containing cells with a competitive advantage over the plasmid-free cells. This resulted in high stability as well as high cell density under non-selective conditions, which led to a 10-fold increase in the volumetric activity compared to that achieved in defined selective media. A simple mathematical model was formulated to verify the experimental data. The important state variables and process parameters, i.e., biomass concentration, beta-galactosidase expression, sucrose consumption, yeast extract consumption, and specific growth rates of the two cell populations, were evaluated. These variables and parameters along with the differential equations based on material balances as well as the experimental results obtained were used in a mathematical model for the fed-batch cultivation. These correctly verified the experimental data and clearly illustrated the concept behind the success of the fed-batch strategy under yeast extract starvation.     

Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives

Bioethanol production from xylose is important for utilization of lignocellulosic biomass as raw materials. The research on yeast conversion of xylose to ethanol has been intensively studied especially for genetically engineered Saccharomyces cerevisiae during the last 20 years. S. cerevisiae, which is a very safe microorganism that plays a traditional and major role in industrial bioethanol production, has several advantages due to its high ethanol productivity, as well as its high ethanol and inhibitor tolerance. However, this yeast cannot ferment xylose, which is the dominant pentose sugar in hydrolysates of lignocellulosic biomass. A number of different strategies have been applied to engineer yeasts capable of efficiently producing ethanol from xylose, including the introduction of initial xylose metabolism and xylose transport, changing the intracellular redox balance, and overexpression of xylulokinase and pentose phosphate pathways. In this review, recent progress with regard to these studies is discussed, focusing particularly on xylose-fermenting strains of S. cerevisiae. Recent studies using several promising approaches such as host strain selection and adaptation to obtain further improved xylose-utilizing S. cerevisiae are also addressed.     

Purification and characterization of human caseinomacropeptide produced by a recombinant Saccharomyces cerevisiae

Caseinomacropeptide (CMP) is a biologically active polypeptide derived from the C-terminal of milk kappa-casein. CMP is heterogeneous since it is modified differently by glycosylation and phosphorylation after translation. Recently, recombinant human CMP (hCMP) has been produced as a secretory product in yeast. The present study aimed at the purification and characterization of recombinant hCMP. By sequential molecular cut-off ultrafiltration and anion-exchange chromatography, the recombinant hCMP in the culture broth could be purified to an HPLC purity over 94%. The authenticity of the purified hCMP was confirmed by sequence analysis of N-terminal amino acids. The recombinant hCMP was estimated to be 7.0kDa by SDS-PAGE, and showed a lower glycosylation than the natural bovine CMP.     

Improvement on laboratory fermentation equipment to study Saccharomyces cerevisiae metabolism

Beside being an ordinary fermenter, the present equipment was conceived to sample the medium, to store the samples and to record photographs of the yeasts. Ten sensors were used to measure gas exchanges. During the growth of ScM1 (a Saccharomyces cerevisiae strain) on glucose, we could observe two different linear decreases of CO₂ production rates (18.17±0.12 mmol CO₂ h^–2 (g biomass)^–1 and 8.67±0.12 mmol CO₂ h^–2 (g biomass)^–1), together with a sudden variation of slope during the respiro-fermentative phase. Nomenclature F_in InletairFlowl h ^–1 F_out OutletgasFlowl h ^–1 _in Inletairtemperature°C_out Outletgastemperature°CP _atm AtmosphericPressuremmHgP _in InletairOverPressuremmHgP _out OutletgasOverPressuremmHgDODissolvedO ₂ mg l^–1 pO₂ PartialPressureO ₂ in Outlet gas % (v/v) pCO₂ PartialPressureCO ₂ in Outlet gas % (v/v) Int(t) Whole number of hours     

Influence of killer strains of Saccharomyces cerevisiae on wine fermentation

The effect of killer strains of Saccharomyces cerevisiae on the growth of sensitive strains during must fermentation was studied by using a new method to monitor yeast populations. The capability of killer yeast strains to eliminate sensitive strains depends on the initial proportion of killer yeasts, the susceptibility of sensitive strains, and the treatment of the must. In sterile filtered must, an initial proportion of 2-6% of killer yeasts was responsible for protracted fermentation and suppression of isogenic sensitive strains. A more variable initial proportion was needed to get the same effect with non-isogenic strains. The suspended solids that remain in the must after cold-settling decreased killer toxin effect. The addition of bentonite to the must avoided protracted fermentation and the suppression of sensitive strains; however, the addition of yeast dietary nutrients with yeast cell walls did not, although it decreased fermentation lag.     

Flocculation of industrial and laboratory strains ofSaccharomyces cerevisiae

Summary A comparative study has been made of different laboratory and industrial wild-type strains ofSaccharomyces cerevisiae in relation to their flocculation behavior. All strains were inhibited by mannose and only one by maltose. In regard to the stability of these characters in the presence of proteases and high salt concentrations, a relevant degree of variation was found among the strains. This was to such an extent that it did not allow their inclusion in the Flol or NewFlo phenotypes. Genetic characterization of one wild-type strain revealed that the flocculation-governing gene was allelic toFLO1 found in genetic strains.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Direct product sequestration of a recombinant cutinase from batch fermentations of Saccharomyces cerevisiae

The recovery of cutinase of Fusarium solani pisi produced by the yeast Saccharomyces cerevisiae was studied in a fluidised bed adsorption system directly integrated with a productive fermenter (so-called direct product sequestration; DPS). The relative efficiency of this system was compared with the one of a conventional purification process by discrete sequences of fermentation, broth clarification, ultrafiltration and fixed bed anion exchange chromatography. By direct product sequestration of the extracellular heterologous cutinase it was possible, through only one unit operation: (i) to perform broth clarification, (ii) to obtain a high cutinase concentration factor, and (iii) to recover cutinase with a specific activity that equalled that obtained with the conventional purification process. It was also possible (iv) to substantially reduce the total process time, (v) to improve the overall yield, and (vi) to increase cutinase productivity. Furthermore, the procedure outlined is suitable for large scale bioprocess exploitation.     

Effect of C/N ratio and starch concentration on ethanol production from sago starch using recombinant yeast

Recombinant Saccharomyces cerevisiae YKU 131 (capable of expressing glucoamylase) was used to produce ethanol from sago starch. The optimum C/N ratio for ethanol production by the recombinant yeast was 7.9, where 4.7 and 10.1 g/l ethanol was produced from 20 and 40 g/l sago starch, respectively. At sago starch concentration higher than 40 g/l and C/N ratio higher than 10.4, glucoamylase production and rate of starch hydrolysis were reduced, which in turn, reduced ethanol production significantly. The theoretical yield of ethanol based on sago starch consumed in fermentation using 40 g/l was 72.6%. This yield was slightly lower than those obtained in fermentation using soluble starch such as potato and corn starch, which ranged from 80–90% as reported in the literature. However, S. cerevisiae YKU 131 could only utilize 62% of the total amount of starch added to a medium.     

State of aggregation of recombinant hirudin in solution under physiological conditions

The state of aggregation of recombinant desulfatohirudin (r-HV1) in solution under physiological conditions (pH 7.5, 0.15N NaCl) was investigated by sedimentation equilibrium. The weight-average molecular weight ¯M _w determined by sedimentation equilibrium was found to be 6914±76 Da compared to 6964 Da expected from the amino acid sequence. The ¯M _z /¯M _w ratio was found to be 1.03, which demonstrates that under the conditions studied hirudin exists in solution as a monomer. This result is in agreement with the relative molecular weight (M _r ) of recombinant hirudin variant 3 reported by Otto and Seckler [(1991),Eur. J. Biochem. 202, 67–73], who also used equilibrium ultracentrifugation, but not with the molecular weight estimated from gel permeation chromatography of natural hirudin (51,300 Da) [Konnoet al. (1988),Arch. Biochem. Biophys. 267, 158–166]. Knowledge of the state of aggregation is essential for understanding the mechanism of interaction of thrombin and hirudin under physiological conditions.Abbreviations ¯M _w weight-average molecular weight - ¯M _z Z-average molecular weight - M _r relative molecular weight - NTSB 2-nitro-5-thiosulfobenzoic acid - Tris Tris(hydroxymethyl)aminomethane - r-HV1 recombinant desulfatohirudin - _M molar extinction coefficient