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.     

Production of hirudin by recombinant Saccharomyces cerevisiae in a membrane-recycle fermentor

Summary Recombinant Saccharomyces cerevisiae was employed to continuously produce hirudin in a membrane cell recycle fermentor. The gene cooing for the anticoagulant protein was combined with the GAL10 promoter for controlled expression and the MF 1 signal sequence for secretion to the fermentation broth. A dilution rate of 0.1h^–1 yielded a maximum hirudin concentration of 59mg / l with a specific hirudin concentration of 2.4 mg /g cell mass among dilution rates studied ranging from 0.05h^–1 to 0.3h^–1. Cell bleeding gave the same fermentation results as cell recycle fermentation without cell bleeding. The productivity of the cell recycle fermentation process was 6.0mg hirudin/l · hr, corresponding to a 1.7-fold increase compared with a conventional continuous culture.     

Effects of medium composition on hirudin production in recombinant Saccharomyces cerevisiae

Summary The enriched medium based on yeast nitrogen basc(YNB)increased hirudin synthesis and secretion in rccombinant Saccharomyces cerevisiae in batch and fed-batch cultures. Fed-batch fermentation with the defined medium yielded 342mg hirudin/l but supplementation of yeast extract increased the final hirudin concentration to 461mg hirudin/l. The defined medium, however, produced the product protein with higher purity of 21% and hence will allow easy separation of secreted hirudin from other contaminated polypeptides present in the growth medium. In a continuous culuture, the defined medium yielded higher concentrations of cell mass and hirudin than the complex medium.     

Effect of soybean oil on the enhanced expression of hirudin gene in Hansenula polymorpha

Summary A heterologous gene from bloodsucking leech for anticoagulant, hirudin, has been expressed in the methylotrophic yeast Hansenula polymorpha. The addition of 1%(v/v) soybean oil to the medium as a stabilizer enhanced the expression of the hirudin gene in H. polymorpha with MOX promoter. The production of hirudin in the medium with soybean oil was 320mg/L in a 5L fermenter.     

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.     

Production of antithrombotic hirudin in <Emphasis Type="Italic">GAL1</Emphasis>-disrupted <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A gratuitous strain was developed by disrupting the GAL1 gene (galactokinase) of recombinant Saccharomyces cerevisiae harboring the antithrombotic hirudin gene in the chromosome under the control of the GAL10 promoter. A series of glucose-limited fed-batch cultures were carried out to examine the effects of glucose supply on hirudin expression in the gratuitous strain. Controlled feeding of glucose successfully supported both cell growth and hirudin expression in the gratuitous strain. The optimum fed-batch culture done by feeding glucose at a rate of 0.3 g h^–1 produced a maximum hirudin concentration of 62.1 mg l^–1, which corresponded to a 4.5-fold increase when compared with a simple batch culture done with the same strain.     

Comparative recombinant protein production of eight insect cell lines

Summary A recombinantAutographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived fromSpodoptera frugiperda, Trichoplusia ni, Mamestra brassicae andEstigmene acrea. Because cell density was found to influence protein production, SEAP production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. On a per cell basis, theT. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than theS. frugiperda-derived Sf9 or Sf21 cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity with cell lines grown in serum-free and serum-containing media.     

Application of medium optimization tools for improving recombinant human interferon gamma production from Kluyveromyces lactis

The present study is focused upon improving biomass of Kluyveromyces lactis cells expressing recombinant human interferon gamma (hIFN-γ), with the aim of augmenting hIFN-γ concentration using statistical and artificial intelligence approach. Optimization of medium components viz., lactose, yeast extract, and trace elements were performed with Box–Behnken design (BBD) and artificial neural network linked genetic algorithm (ANN-GA) for maximizing biomass of recombinant K. lactis (objective function). The studies resulted over 1.5-fold improvement in the biomass concentration in a medium composed of 80?g/L lactose, 10.353?g/L yeast extract, and 15?mL/L trace elements as compared with initial biomass value. In the same study hIFN-γ concentration reached 881?µg/L which was 2.28-fold higher as compared with initial hIFN-γ concentration obtained in unoptimized medium. Further the batch fermentation study displayed mixed growth associated kinetics with the maximum hIFN-γ production rate of 1.1?mg/L. BBD and ANN-GA, both optimization techniques predicted a higher lactose concentration was clearly beneficial for augmenting K. lactis biomass which in turn increased hIFN-γ concentration.     

Production and Purification of Recombinant Hirudin Expressed in the Methylotrophic YeastPichia pastoris

A recombinant form of hirudin (HIR), a potent thrombin inhibitor derived from the leechHirudo medicinalis,was cloned and expressed in the methylotrophic yeastPichia pastoris.The HIR gene was inserted into theP. pastorispPic9K expression vector such that the gene's expression is under alcohol oxidase (AOX1) promoter control and the HIR coding sequence is fused to theSaccharomyces cerevisiaepre-pro α-mating factor signal sequence. ATn903Kan^rdeterminant andHis4⁺gene are also present on pPic9K, affording a method for selecting chromosomal integrants of the HIR gene. Following electroporation of the DNA into theP. pastorisstrain GS115 (his-4), His⁺transformants were recovered and plated on medium containing increasing concentrations of the aminoglycoside antibiotic G418. The resulting His⁺G418-resistant transformants were grown in shake flasks and screened for those that secreted recombinant hirudin (rHIR) to the growth medium. Clones exhibiting rHIR production and secretion were retained for fermentation studies where optimization of growth conditions was found to dramatically increase rHIR expression. One clone that was retained for further characterization secreted rHIR at a level of 1.5 g/liter. Using a straightforward two-step chromatography procedure, the rHIR was purified to >97% with a recovery yield of 63%. The purified rHIR had the predicted N-terminal amino acid sequence and exhibited the same thrombin inhibition kinetics as a variety of HIR isoforms produced in other heterologous systems. Based on these data,P. pastorisoffers an efficient system for production and purification of multigram quantities of biologically active rHIR for structure/function analyses.     

Contributing factor toMycoplasma pneumoniae produced stimulation of rhinovirus-RNA synthesis

It has been demonstrated thatRhinovirus ribonucleic acid (RNA) synthesis was greater inMycoplasma pneumoniae inoculated KB (Human Carcinoma of Nasopharynx) cells than in PPLO-free tissue systems (Milligan III & Fletcher, 1969). In this study,Mycoplasma pneumoniae was grown on glass to eliminate the PPLO medium. However, if PPLO medium was added to theMycoplasma pneumoniae inoculum, stimulation of viral-RNA synthesis was greater than in the presence of PPLO alone. Rhinovirus-RNA synthesis, measured by uridine-³H uptake, in the presence ofMycoplasma pneumoniae andMycoplasma pneumoniae plus medium was enhanced 1.2-fold and 1.4-fold respectively, above virus synthesis in untreated KB cell systems. PPLO medium did not appear to significantly stimulate viral RNA synthesis. However, a 5-fold increase in the concentration of the medium or its components resulted in greater viral RNA replication. The medium components, yeast extract and PPLO broth (Difco), stimulated viral RNA synthesis 4-fold and 2-fold, respectively. Our findings also demonstrated that other components, agar, phenol red, and dextrose, inhibited viral replication, whereas horse serum appeared to have no effect. This work was supported by the Life Sciences Division of the U.S. Army Research Office under contract/grant No. DAHC-20-67C-0122/DAHC 19-69-G-0011.     

Hirudin as a novel fusion tag for efficient production of lunasin in Escherichia coli

Fusion expression provides an effective means for the biosynthesis of longer peptides in Escherichia coli. However, the commonly used fusion tags are primarily suitable for laboratory scale applications due to the high cost of commercial affinity resins. Herein, a novel approach exploiting hirudin as a multipurpose fusion tag in combination with tobacco etch virus (TEV) protease cleavage has been developed for the efficient and cost-effective production of a 43-amino acid model peptide lunasin in E. coli at preparative scale. A fusion gene which allows for lunasin to be N-terminally fused to the C-terminus of hirudin through a flexible linker comprising a TEV protease cleavage site was designed and cloned in a secretion vector pTASH. By cultivation in a 7-L bioreactor, the fusion protein was excreted into the culture medium at a high yield of ~380?mg/L, which was conveniently recovered and purified by inexpensive HP20 hydrophobic chromatography at a recovery rate of ~80%. After polishing and cleavage with TEV protease, the finally purified lunasin was obtained with ≥95% purity and yield of ~86?mg/L culture medium. Conclusively, this hirudin tagging strategy is powerful in the production of lunasin and could be applicable for the production of other peptides at preparative scale.     

The growth of WI-38 cells in a serum-free,growth factor-free,medium with elevated calcium concentrations

Summary The growth of WI-38 cells in serum-free growth medium with and without hormone supplementation in the presence of elevated Ca²⁺ concentrations was investigated. At 5 mM CaCl₂, WI-38 cells seeded at low density without serum or hormone supplementation showed up to a 12-fold increased in cell number at saturation density over that obtained at day 1. Saturation densities were comparable when either 5 mM CaCl₂ or epidermal growth factor (1 mM CaCl₂) was used in the presence of insulin, dexamethasone and transferrin. Combining suboptimal doses of epidermal growth factor and CaCl₂ resulted in an additive effect on saturation density. Thus, nornal human diploid cells are capable of substantial growth in serum-free, hormone-free growth medium. In contrast, confluent cultures refed with the same medium are not responsive to elevated Ca²⁺ concentrations. In fact, elevated Ca²⁺ concentrations inhibited the proliferative response of confluent cultures to epidermal growth factor, but enhanced their response to the combined treatment of insulin, transferrin and dexamethasone. This work was supported by the United States Public Health Society grants T-32, CA09171 and AG-00378. Editor's Statement This paper rigorously dissects the interplay among external Ca²⁺ concentration, cell density and specific growth factors on fibroblast growth in defined medium. Wallace L. McKeehan     

Scale-up of recombinant hirudin production fromSaccharomyces cerevisiae

Scale-up of hirudin production fromSaccharomyces cerevisiae from bench-scale to pilot-scale was carried out based on constant volumetric oxygen transfer coefficient (K _L a). Fed-batch mode of cultivation using step-wise feeding strategy of galactose was employed for the production of hirudin in a 30-L and a 300-L pilot-scale fermentor. The final hirudin concentrations were achieved 390 mg/L and 286.1 mg/L, and the volumetric productivities were 80.4% and 90.7% with the 30-L and 300-L fermentors, respectively, compared to the productivity of the 5-L bench-scale fermentor.     

Fuel ethanol from hardwood hemicellulose hydrolysate by genetically engineeredEscherichia coli B carrying genes fromZymomonas mobilis

Summary Escherichia coli B (ATCC 11303) carrying the PET operon on plasmid pLOI 297 converted hemicellulose hydrolysate to ethanol at an efficiency of 94% theoretical maximum, which is 15% better than the highest efficiency reported for pentose utilizing yeasts in a comparable system. Aspen prehydrolysate (APH), that had been produced by theBio-Hol Process using a Wenger extruder with SO₂ as catalyst, was used as feedstock. The fermentation medium contained predominantly xylose (35g/L) with acetic acid present at about 6g/L. With the pH controlled at 7.0, this concentration of acetic acid was not inhibitory for growth or xylose fermentation. When the APH was fortified with nutrients (tryptone and yeast extract), the recombinant (inoculated at 0.5 g dry wt/L) converted 100% of the xylose to ethanol with a volumetric productivity of 0.29 g/L/hr. Overliming the APH with Ca(OH)₂, followed by neutralization to pH 7 with sulphuric acid and removal of the insolubles, resulted in a 2-fold increase in productivity. The max. productivity was 0.76 g/L/hr. The productivity in Ca(OH)₂-treated APH, fortified with only mineral salts, was 0.26 g/L/hr.     

Operon fusions of Mu d1(Ap,lac) to thel-proline biosynthetic genes ofescherichia coli K-12

Operon fusions to the promoter of either theproA,proB, orproC genes of the proline biosynthetic pathway were obtained by the use of the Mu d1(Ap,lac) bacteriophage. These fusions were further stabilized by transformation with plasmid pGW600 containing the wildtype Mu repressor gene or by transduction with phage pSG1. The level of -galactosidase in the fusion strains was not affected by the presence of exogenously addedl-proline or high concentrations of NaCl in the growth medium. A Tn5 insertion nearproBA increased -galactosidase expression 140- to 200-fold in strains carrying theproA-lac andproB-lac fusions, but the level of this enzyme was unaltered in strains carrying theproC-lac fusion. The Tn5 insertion increased intracellular proline concentrations 8- to 10-fold, suggesting that mechanisms other than allosteric inhibition may regulate proline biosynthesis, but did not confer osmotolerance to cells growing in a medium with a high concentration of salt.     

Rhamnolipid production in batch and fed-batch fermentation using<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> BYK-2 KCTC 18012P

The optimization of culture conditions for the bacteriumPseudomonas aeruginosa BYK-2 KCTC 18012P, was performed to increase its rhamnolipid production. The optimum level for carbon, nitrogen sources, temperature and pH, for rhamnolipid production in a flask, were identified as 25 g/L fish oil, 0.01% (w/v) urea, 25 and pH 7.0, respectively. Optimum conditions for batch culture, using a 7-L jar fermentor, were 200 rpm of agitation speed and a 2.0 L/min aeration rate. Under the optimum conditions, on fish oil for 216 h, the final cell and rhamnolipid concentrations were 5.3 g/L and 17.0 g/L respectively. Fed-batch fermentation, with different feeding conditions, was carried out in order to increase, cell growth and rhamnolipid production by thePseudomonas aeruginosa, BYK-2 KCTC 18012P. When 2.5 g of fish oil and 100 mL basal salts medium, containing 0.01% (w/v) urea, were fed intermittently during the fermentation, the final cell and rhamnolipid concentrations at 264 h, were 6.1 and 22.7 g/L respectively. The fed-batch culture resulted in a 1.2-fold increase in the dry cell mass and a 1.3-fold increase in rhamnolipid production, compared to the production of the batch culture. The rhamnolipid production-substrate conversion factor (0.75 g/g) was higher than that of the batch culture (0.68 g/g).     

Protoplast mutant selection of Glarea Lozoyensis and statistical optimization of medium for pneumocandin B0 yield-up

A combination of microbial strain improvement and statistical optimization is investigated to maximize pneumocandin B₀ production from Glarea lozoyensis ATCC 74030. Atmospheric and room temperature plasma (ARTP) was used to enhance G. lozoyensis ATCC 74030 in pneumocandin B₀ yield. Mutant strain G. lozoyensis Q1 exhibited 1.39-fold increase in pneumocandin B₀ production to 1134 mg/L when compared with the parent strain (810 mg/L). Further, the optimized medium provided another 1.65-fold in final pneumocandin B₀ concentration to 1873 mg/L compared to the original medium. The results of this study indicated the combined application of a classical mutation and medium optimization can improve effectively pneumocandin B₀ production from G. lozoyensis and could be a tool to improve other secondary metabolites production by fungal strains.     

Autocrine factor-independent growth of mammalian fibroblasts established in fully synthetic medium: No V-onc requirement in establishment

Summary In a previous study Chinese hamster fibroblasts carrying a partially deleted v-src were established in a synthetic medium lacking macromolecular supplements and shown to possess a particular serum-free phenotype hereafter designatedsf. In cloning efficiency assays,sf, unlike wild-type, fibroblasts required a threshold cell density to growth from single cells, suggesting autocrine stimulation. In the present study a conditioned medium harvested fromsf cells was added to the samesf cells, and the resulting cloning density was found to markedly diminish rather than increase.Sf cells were found to be unable to grow at cloning density because of trypsin damage;sf cells seeded into trypsin inhibitor-containing medium cloned with no requirement for threshold cells and were therefore independent of autocrine secretion from neighboring cells. Their cloning efficiency reached 7.7%; this value could not be improved by subcloning thesf culture, and it diminished when selenium was not added to the assay medium. To determine whether v-src is involved in thesf phenotype, five clones of the parental Chinese hamster fibroblast line not infected with Rous sarcoma virus were explanted into serum-free cultures with no macromolecular additives as in the case of v-src-containing cells. Each clone gave rise to ansf cell line growing indefinitely in synthetic medium like the v-src-containingsf cells, showing that the v-src gene is not required either for the establishment or maintenance of thesf phenotype. Supported by grants from CNRS, INSERM, contract 862015, Association Claude-Bernard, Association pour la Recherche sur le Cancer, and Fondation pour la Recherche Médicale.     

Extracellular secretion of anticoagulant peptide hirudin in Lactococcus lactis using SP310mut2 signal peptide

Hirudin can be used as an oral anticoagulant and antithrombotic agent. The hirudin variant III gene, derived from the medicinal leech, Hirudo medicinalis, was fused to SP310mut2 signal sequence and expressed by a nisin-controlled gene expression system in Lactococcus lactis which was then grown in a 7 l fermenter. After induction with 8 ng nisin ml⁻¹, the product was secreted into the culture medium and accumulated up to ~2.7 mg l⁻¹. MALDI-TOF/MS and anticoagulant activity analyses on the purified product confirmed its authenticity. This is the first demonstration that hirudin can be extracellularly secreted and correctly processed in L. lactis.