Production of recombinant human antithrombin by Pichia pastoris

This paper deals with the production of recombinant human antithrombin (rAT) by the methylotrophic yeast Pichia pastoris. In preliminary methanol-limited fed-batch fermentation, the rAT concentration reached 324 mg/l at 192 h of cultivation, but the specific heparin cofactor (HC) activity of rAT in the culture supernatant was 10% of that of plasma-derived antithrombin (pAT). To improve the specific HC activity of rAT, effort was first focused on the optimization of culture pH and media composition, resulting in protection of rAT against pH-dependent instability and proteolytic degradation. However, even in the optimized methanol-limited fed-batch fermentation, the specific HC activity of rAT in the culture supernatant was still 20% that of pAT. To investigate the unknown mechanisms involved in the decreased specific HC activity of rAT, the culture supernatant of mock-transfected cells was prepared by methanol-limited fed-batch fermentation. When pAT was added to this supernatant, a rapid decrease in HC activity was observed; the residual HC activity was 26% after 24 h of incubation at 25 degrees C. The loss of pAT activity was prevented by addition of a formaldehyde scavenger, amino urea, to the supernatant. In addition, alcohol oxidase activity was observed in the supernatant, resulting in the accumulation of formaldehyde in the culture broth. These results suggest that the formaldehyde produced by methanol oxidation in the culture broth of P. pastoris might decrease the HC activity of rAT during fermentation. Replacing the methanol with glycerol as the carbon source improved the specific HC activity of rAT from 20% to above 40% of that of pAT. In the glycerol-limited fed-batch fermentation, rAT is expressed at 100 mg/l under the control of the truncated mutated AOX2 promoter.     

High butanol production by Clostridium saccharoperbutylacetonicum N1-4 in fed-batch culture with pH-Stat continuous butyric acid and glucose feeding method

A pH-stat fed-batch culture by feeding butyric acid and glucose has been studied in an acetone-butanol-ethanol (ABE) fermentation using Clostridium saccharoperbutylacetonicum N1-4. The specific butanol production rate increased from 0.10 g-butanol/g-cells/h with no feeding of butyric acid to 0.42 g-butanol/g-cells/h with 5.0 g/l butyric acid. The pH value in broth decreases with butyric acid production during acidogenesis, and then butyric acid reutilization and butanol production result in a pH increase during solventogensis. The pH-stat fed-batch culture was performed to maintain a constant pH and butyric acid concentration in the culture broth, but feeding only butyric acid could not support butyric acid utilization and butanol production. Subsequently, when a mixture of butyric acid and glucose was fed, butyric acid was utilized and butanol was produced. To investigate the effect of the feeding ratio of butyric acid to glucose (B/G ratio), several B/G ratio solutions were fed. The maximum butanol production was 16 g/l and the residual glucose concentration in broth was very low at a B/G ratio of 1.4. Moreover, yields of butanol in relation to cell mass and glucose utilization were 54% and 72% higher in pH-stat fed-batch culture with butyric acid than that of conventional batch culture, respectively.     

Alteration of cellular metabolism by consecutive fed-batch cultures of mammalian cells

The metabolism of myeloma cells was altered to reduce lactate production in consecutive fed-batch cultures. The glucose concentration was maintained at low levels (0.28-0.55 mM) by employing a dynamic nutrient feeding method based on on-line measurement of the oxygen uptake rate (OUR) to estimate the metabolic demand of the cells. This strategy has been previously reported to be applied to cultures of hybridoma cells, in which the production of lactate was significantly reduced by thus maintaining the glucose concentration at low levels. However, for this cell line, a single fed-batch culture was not sufficient to alter the cellular metabolism, even at a glucose concentration of 0.28 mM. Two consecutive fed-batch cultures were employed to ensure that the cells were cultivated under a low glucose concentration for a sufficiently prolonged period of time to allow a switch of the cellular metabolism from a glycolytic (high lactate production) to oxidative (low lactate production) state.     

Enhanced 2,3-butanediol production by addition of acetic acid in Paenibacillus polymyxa

By the addition of 150 mM acetate into a batch culture at an initial pH of 6.8, the production of 2,3-butanediol (BDL) by Paenibacillus polymyxa reached 248 mM, yielding 0.87 mol.mol(-1) glucose, where the ratio of acetate consumed to glucose consumed (A/C ratio) was calculated as 0.35 mol acetate mol(-1) glucose. Therefore, a fed-batch culture was carried out by feeding glucose and acetate at a ratio of 0.35 mol acetate mol(-1) glucose. In the fed-batch culture performed at pH 6.8, BDL production reached 637 mM, yielding 0.81 mol.mol(-1) glucose, although the A/C ratio was only 0.18 mol acetate mol(-1) glucose. By decreasing pH to 6.3 in the fed-batch culture, BDL production reached 566 mM, yielding 0.88 mol.mol(-1) glucose and the A/C ratio was 0.32 mol acetate mol(-1) glucose. The optical purity of BDL, which was expressed as enantiomeric excess, was retained at more than 98% of the (R, R)-stereoisomer at the end of culture, which was comparable to that without acetate addition.     

Fed-batch coculture of Lactobacillus kefiranofaciens with Saccharomyces cerevisiae for effective production of kefiran

In a batch coculture of kefiran-producing lactic acid bacteria Lactobacillus kefiranofaciens and lactate-assimilating yeast Saccharomyces cerevisiae, lactate accumulation in the medium was observed, which inhibited kefiran production. To enhance kefiran productivity by preventing lactate accumulation, we conducted lactose-feeding batch operation with feedforward/feedback control during the coculture, so that the lactate production rate of L. kefiranofaciens was balanced with the lactate consumption rate of S. cerevisiae. The lactate concentration was maintained at less than 6 g l(-1) throughout the fed-batch coculture using a 5 l jar fermentor, although the concentration reached 33 g l(-1) in the batch coculture. Kefiran production was increased to 6.3 g in 102 h in the fed-batch coculture, whereas 4.5 g kefiran was produced in 97 h in the batch coculture. The kefiran yield on lactose basis was increased up to 0.033 g g(-1) in the fed-batch coculture, whereas that in the batch coculture was 0.027 g g(-1).     

Development of a novel method for feeding a mixture of L-lactic acid and acetic acid in fed-batch culture of Ralstonia eutropha for poly-D-3-hydroxybutyrate production

Fermentative production of poly-D-3-hydroxybutyrate [P(3HB)] from a mixture of L-lactic acid and acetic acid by Ralstonia eutropha was investigated. For fed-batch culture with cell density, it is necessary to control the concentration of these organic acids in the culture medium below the inhibitory level for cell growth. Therefore, a novel feeding method, termed the computer-controlled pH-stat substrate feeding method, was developed using the rate of increase of the pH (pH-increasing rate) of the culture medium as an indicator for feed control. The pH-increasing rate, which was calculated every minute by a pH meter-linked computer, represented secondary information regarding substrate consumption by cells. When the pH-increasing rate decreased to 5% of the maximum increasing rate, acidic substrate solution was fed into the fermentor until the pH was reduced to 7.00. Using this feeding strategy, the cell concentration and PHA content obtained in 42 h were 75.0 g/l and 73.1% (w/w), respectively, resulting in a high P(3HB) productivity of 1.30 g/l.h.     

Optimization of L-lactic acid feeding for the production of poly-D-3-hydroxybutyric acid by Alcaligenes eutrophus in fed-batch culture

We investigated optimization of the feeding of L-lactic acid for the production of poly-D-3-hydroxybutyric acid [P(3HB)] by Alcaligenes eutrophus in a fed-batch culture system. An acidic substrate solution was fed automatically so as to maintain the pH of the culture liquid at 7.0. Feeding of a substrate solution containing 45% (w/v) L-lactic acid, 6.2% (w/v) sodium L-lactate, 5.8% (w/v) ammonia water and 1.8% (w/v) potassium phosphate [at a molar ratio of carbon to nitrogen (C/N molar ratio) of 10], allowed the L-lactate concentration in the culture liquid to be maintained at approximately 2 g/l and the cell concentration reached 27.4 g/l after 15 h of cultivation. To promote P(3HB) production, a two-stage fed-batch culture consisting of a culture for cell growth and one for P(3HB) accumulation was carried out. When the substrate solution, whose C N molar ratio was 23, was fed during the P(3HB) accumulation phase, the cell concentration and the P(3HB) content in the cells reached 103 g/l and 57.6% (w/w), respectively, in 51.5 h.     

Development of a novel method for feeding a mixture of -lactic acid and acetic acid in fed-batch culture of Ralstonia eutropha for poly- -3-hydroxybutyrate production

Fermentative production of poly- -3-hydroxybutyrate [P(3HB)] from a mixture of -lactic acid and acetic acid by Ralstonia eutropha was investigated. For fed-batch culture with cell density, it is necessary to control the concentration of these organic acids in the culture medium below the inhibitory level for cell growth. Therefore, a novel feeding method, termed the computer-controlled pH-stat substrate feeding method, was developed using the rate of increase of the pH (pH-increasing rate) of the culture medium as an indicator for feed control. The pH-increasing rate, which was calculated every minute by a pH meter-linked computer, represented secondary information regarding substrate consumption by cells. When the pH-increasing rate decreased to 5% of the maximum increasing rate, acidic substrate solution was fed into the fermentor until the pH was reduced to 7.00. Using this feeding strategy, the cell concentration and PHA content obtained in 42 h were 75.0 g/l and 73.1% (w/w), respectively, resulting in a high P(3HB) productivity of 1.30 g/l·h.     

Biphasic addition strategy of hypoxanthine and thymidine for improving monoclonal antibody production

In our previous study, we demonstrated that combinatorial addition of hypoxanthine (10 mg/L) and thymidine (2 mg/L) was able to stimulate initial cell growth and elevate volumetric concentration of antibody by 22% (Chen et al., Appl. Microbiol. Biotechnol., 93, 169-178, 2012). In this study, a systematic study was carried out to investigate the effects of hypoxanthine and thymidine (H&T) on cell growth and antibody production in a much wider range of concentration. In addition, we pursued to establish a highly productive fed-batch culture via rationally designing H&T addition regime. It was found that both cell growth and antibody production in batch cultures were H&T concentration-dependent. Specifically, a low concentration stimulated cell growth while exerting no influence on specific productivity (q(mAb)), and a high concentration inhibited cell growth, however, significantly enhancing q(mAb). Subsequent experiments with fed-batch shaking flasks demonstrated the feasibility of improving antibody production using a biphasic addition strategy for H&T: supplementing a low concentration of H&T during initial cell growth phase and a high concentration of H&T at the production phase. By applying the optimized feeding regime, a maximum viable cell density (VCD) of 6.45 × 10(6)cells/mL and volumetric antibody production of 632 mg/L were achieved in a 2 L-B.Braun bioreactor. Taken together, in this study, a biphasic H&T addition strategy for cell culture was developed, which hold great promise to improve antibody production.     

Study of Cheese Associated Lactic Acid Bacteria Under Carbohydrate-Limited Conditions Using D-Stat Cultivation

The metabolic pathways alternative to glycolytic energy (ATP) during growth of starter and nonstarter lactic acid bacteria were studied simulating the depletion of carbohydrates during cheese ripening. D-stat cultivation strategy with the gradual decrease of galactose concentration in tryptone-arginine feeding medium was used. With the decrease of galactose feeding, the biomass yield calculated on carbohydrate consumption (Y_X/HEX) and acetate/lactate production ratio of all strains increased. We assume that ATP and biomass yields improved by directing the pyruvate flow from lactate to acetate and that metabolic energy could be obtained either by producing acetate from carbohydrates or from arginine metabolism in ADI-positive strains. Four LAB strains out of eight produced ornithine from arginine indicating active arginine-deiminase (ADI) pathway. These ADI-positive strains achieved 3-10 times higher Y_X/HEX than ADI-negative strains in tryptone-arginine medium. Lactobacillus plantarum also used serine as an energy source. Starters and NSLAB strains using the amino acids arginine and serine or limited amounts of carbohydrates therefore have the potential to influence flavor production in cheese more efficiently.     

Fed-batch enhancement of jenseniin G, a bacteriocin produced by Propionibacterium thoenii (jensenii) P126

Jenseniin G is an antibotulinal bacteriocin (antimicrobial peptide) produced by the dairy culture, Propionibacterium thoenii (jensenii) P126. Activity from crude jenseniin G preparations isolated from static cultures was not detected in unconcentrated cultures before day 7. Activity was not detectable until the spent culture medium was concentrated 50-100 fold. Maximum activity (21 AU/ml) was observed in concentrated supernates at day 9. The production of bacteriocin jenseniin G was increased in fed-batch fermentations for 14 d at 32 degrees C in sodium lactate broth (NLB) containing 1.2% sodium lactate. Viable cell numbers in static and fed-batch cultures reached 1.2 and 5.4x10(9), respectively, during late exponential/early stationary phase (3 d). Concentrations of viable cells in fed-batch fermentations remained constant throughout the incubation period; those in static fermentations dropped after day 6 to a final concentration of 1.5x10(7). During fed-batch fermentations, jenseniin G was directly detected at day 5. In fed-batch fermentations, maximum activity in concentrated supernates (384 AU/ml) on day 12 provided an 18 fold increase over yields in static cultures and in fermenter without pH control, and 2.4 fold increase over yields in fermenter at controlled pH at 6.4. Fed-batch fermentation shows promise as a method to obtain high concentrations of industrially significant bacteriocins from dairy propionibacteria.     

补料分批培养生产2-酮基-D-葡萄糖酸的研究

研究了补糖对荧光假单胞菌(Pseudomonas fluorescens)AR4生产2-酮基-D-葡萄糖酸的影响,并在50kL发酵罐上进行了补料分批培养工业应用性试验。研究结果表明,采用补料分批培养方法,能有效提高发酵液中的产物浓度;开始补糖时发酵液中的残糖浓度以3%～6%为宜;补料分批培养方法及AR4菌株可用于2-酮基-D-葡萄糖酸的工业化规模生产中。     

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone

Ovine leptin was cloned in the methylotrophic yeast Pichia pastoris using a pPIC9K vector. Leptin was produced and secreted into the culture medium using the Saccharomyces cerevisiae alpha-mating factor prepro signal by five clones. Expression levels of leptin varied from clone to clone, depending on the copy number of the ob gene. Highest expression was observed with the single-copy clone S27 (250 mg/l). The modifications of culture conditions in batch and fed-batch culture increase the yield of protein. The use of higher cell concentration (63 g/l) before induction of oLept associate with a regulation of pH at 3.2, which decreases the effects of proteolysis, increases the expression level of the oLept to 402 mg/l. Moreover, compared with the non-producer clone, we observed a drastic decrease in growth rate and biomass yield in the leptin-producing clones. At the end of the fed-batch phase at pH 3.2 with clone S27, mortality rate reached 17.3%. Results showed that recombinant leptin production induced metabolic stress, and a negative impact on biomass yield and growth rate. We characterized the recombinant leptin produced by clone S27. It exhibited a molecular mass of 16 kDa, an N-terminal amino acid sequence identical to that of ovine leptin but with an additional tyrosine introduced by the cloning site. Moreover, it was found to be biologically active in vitro. The available production of a large quantity of oLept will strengthen the functional study for theoretical and practical purposes.     

Study of Cheese Associated Lactic Acid Bacteria Under Carbohydrate-Limited Conditions Using D-Stat Cultivation

The metabolic pathways alternative to glycolytic energy (ATP) during growth of starter and nonstarter lactic acid bacteria were studied simulating the depletion of carbohydrates during cheese ripening. D-stat cultivation strategy with the gradual decrease of galactose concentration in tryptone-arginine feeding medium was used. With the decrease of galactose feeding, the biomass yield calculated on carbohydrate consumption (Y_X/HEX) and acetate/lactate production ratio of all strains increased. We assume that ATP and biomass yields improved by directing the pyruvate flow from lactate to acetate and that metabolic energy could be obtained either by producing acetate from carbohydrates or from arginine metabolism in ADI-positive strains. Four LAB strains out of eight produced ornithine from arginine indicating active arginine-deiminase (ADI) pathway. These ADI-positive strains achieved 3–10 times higher Y_X/HEX than ADI-negative strains in tryptone-arginine medium. Lactobacillus plantarum also used serine as an energy source. Starters and NSLAB strains using the amino acids arginine and serine or limited amounts of carbohydrates therefore have the potential to influence flavor production in cheese more efficiently.     

Enhancement of retinal production by supplementing the surfactant Span 80 using metabolically engineered Escherichia coli

The optimal temperature and pH for retinal production using metabolically engineered Escherichia coli in a 7-l fermentor were found to be 30°C and 7.0, respectively. The agitation speed was a critical factor for retinal production. The optimal agitation speed was 400 rpm (oxygen transfer coefficient, k(L)a, = 92 1/h) in batch culture and 600 rpm (k(L)a=148 1/h) in a fed-batch culture of glycerol. Span 80 was selected as a surfactant for retinal production in metabolically engineered E. coli because Span 80 had proven the most effective for increased retinal production among the tested surfactants. Under the optimal conditions in the fed-batch culture with 5 g/l Span 80, the cell mass and the concentration, content, and productivity of retinal were 24.7 g/l, 600 mg/l, 24.3mg/g-cells, and 18 mg l(-1)h(-1) after 33 h, respectively. They were 1.2-, 2.7-, 2.3-, and 2.7-fold higher than those in the fed-batch culture without Span 80, respectively. The concentration and productivity of retinal in this study were the highest ever reported. The hydrophilic portion of Span 80 (sorbitan) did not affect cell growth and retinal production, but the hydrophobic portion (oleic acid) stimulated cell growth. However, oleic acid plus sorbitan did not stimulate retinal production. Thus, Span 80, as a linked compound of oleic acid and sorbitan produced by esterification, proved to be an effective surfactant for the enhancement of retinal production.     

Analysis of hemin effect on lactate reduction in Lactococcus lactis

Lactococcus lactis is a facultative anaerobic microorganism that produces lactate as the major product, and acetate and acetoin as by-products; some strains of this species produce an antimicrobial compound, nisin. Lactate has a strong inhibitory effect on L. lactis growth. On the other hand, hemin has a suppressive effect on lactate production during L. lactis growth under aerobic condition. To achieve the optimum effect of hemin on lactate amount reduction in L. lactis ATCC11454, cultures entailing various conditions were performed with and without hemin. In the culture with hemin, L. lactis growth and lactate reduction improved compared with those in the culture without hemin; that is, lactate production was suppressed by 1.8- and 1.3-fold under batch and fed-batch cultures, respectively. In microaerobic fed-batch culture with hemin, lactate production was sufficiently suppressed. This result suggests that microaerobic fed-batch culture could be applied to the maintenance of the low lactate amount. Under this condition, metabolic shift was observed from lactate to acetoin and acetate. However, no increase in nisin production was observed even though lactate production could significantly decrease in L. lactis ATCC11454.     

Lipase production in two-step fed-batch culture of organic solvent-tolerant Pseudomonas aeruginosa LST-03

Efficient lipase production by two-step fed-batch culture of an organic solvent-tolerant bacterium, Pseudomonas aeruginosa LST-03, was investigated. When FB synthetic medium was used in flask culture, no lipase activity was detected, whereas lipase was produced at 2.3 I.U./ml in C2 complex medium. However, lipase production was induced in FB medium when a fatty acid was added to the culture broth in the stationary phase. Among fatty acids tested, long chain saturated fatty acids, such as C18 (stearic acid) and C20 (arachidic acid), were found to function as effective inducers for the production of lipase, giving an activity level almost the same as that obtained in C2 medium in flask culture. Two-step lipase production, comprised of a growth phase in fed-batch mode and a production phase in which lipase was induced by the addition of 5% (v/v) stearic acid, was carried out in a jar-fermentor. In the growth phase, the maximum cell concentration at 16 h was only 20 in terms of the optical density at 660 nm (OD660), and a low level of lipase production (8 I.U./ml) was obtained after 167 h. This was considered to be due to the exhaustion of several medium components brought about by the use of an unsuitable medium or feeding solution. After analyzing the contents of the compounds in the culture broth by inductively coupled plasma spectrometry for metal ions and HPLC for anions, a modified FB medium was designed. When this modified FB medium was used in two-step fed-batch culture, the maximum cell concentration reached an OD660 of 55 (30.2 g-dry cells/l) at 16.5 h, and lipase was produced at 96 I.U./ml after 35 h, which is approximately 40 times higher than the production level obtained in flask culture using C2 medium.     

Metabolic characterization of the bovine blastocyst,inner cell mass,trophectoderm and blastocoel fluid

The formation of a viable blastocyst is dependent upon the establishment of a correct inner cell mass (ICM):trophectoderm cell ratio but little is known about the metabolism of the two cell populations or about the composition of blastocoel fluid. In this study, the metabolism of intact bovine blastocysts, isolated ICM and trophectoderm was examined in terms of glucose and pyruvate uptake, lactate production, and amino acid consumption or production. The concentration of these nutrients in blastocoel fluid was also determined. The metabolism of glucose, pyruvate and lactate differed significantly between the isolated ICM and trophectoderm. Isolated trophectoderm had a higher pyruvate (P<0.001) and lower glucose (P<0.05) consumption, and higher lactate production (P<0.05) than did ICM. The consumption or production of amino acids by ICM and trophectoderm also differed, with the trophectoderm displaying a higher turnover (the sum of production and consumption). The ICM and trophectoderm both depleted arginine, aspartate and leucine, whereas the production of alanine was consistent. Isolated ICM depleted a further six amino acids, which appeared during trophectoderm culture; the reverse trend was observed for the remaining amino acids. The concentration of lactate in blastocoel fluid was significantly higher than in synthetic oviductal fluid supplemented with amino acids and BSA (SOFaaBSA; P<0.05). However, glucose (P<0.05) and pyruvate (P<0.001) concentrations were both lower. Aspartate, glutamate, glycine, alanine and tryptophan were present at significantly higher concentrations in blastocoel fluid than in SOFaaBSA, whereas threonine and asparagine concentrations were significantly lower. The metabolism of composite blastocysts, obtained by summing the consumption and production profiles of the ICM and trophectoderm, and taking into account their respective number of cells, was higher than that of intact blastocysts, indicating that upon isolation of the two cell populations there may be disruption to paracrine interactions or the onset of culture-induced cellular stress or both.     

Effects of pH and dissolved oxygen on cellulose production by Acetobacter xylinum BRC5 in agitated culture

Acetobacter xylinum BRC5 was cultivated in a jar fermentor using glucose as the sole carbon source. Strain BRC5 oxidized almost all of the glucose to gluconic acid; thereafter, it biosynthesized cellulose by utilizing gluconic acid accumulated in the broth. The optimal pH for metabolizing glucose to gluconic acid was 4.0, while a pH of 5.5 was preferred for cell growth and cellulose production from the accumulated gluconic acid in the medium. Shifting the pH from 4.0 to 5.5 during the cellulose production phase in batch cultures improved cellulose production and reduced the total fermentation time, compared to batch cultures at constant pH. In constant fed-batch culture, 10 g/l of cellulose was obtained from 40 g/l of glucose, a yield which was approximately 2-fold higher than in batch culture with the same initial glucose concentration, even without control of the level of dissolved oxygen. The highest cellulose yield was obtained in fed-batch cultures in which the dissolved oxygen concentration was controlled at 10% saturation. Control of pH and dissolved oxygen to optimal levels was effective for improving the production rate and yield of cellulose, to achieve a high cellulose productivity of 0.3 g cellulose/l x h. Approximately 15 g/l of cellulose was considered to be the highest yield obtainable using conventional fermentors because the culture broth then became too viscous to allow satisfactory aeration.     

葡萄糖对光滑球拟酵母发酵生产丙酮酸的影响

在30L发酵罐中研究了初始葡萄糖质量浓度和补料方式对光滑球拟酵母WSH-IP303发酵生产丙酮酸的影响．实验确定116.4g/L左右是较为适宜的初始葡萄糖质量浓度,发酵58h时丙酮酸质量浓度和产率分别为58.0g/L和0.516g/g．采用初始葡萄糖质量浓度为53.4g/L,发酵24h分批补料至葡萄糖总质量浓度为115g/L的培养方式,发酵64h时丙酮酸质量浓度和产率分别为60.2g/L和0.559g/g;采用初始葡萄糖质量浓度为62.6g/L,发酵24h开始连续补料至葡萄糖总质量浓度为115g/L的培养方式,发酵72h时丙酮酸质量浓度和产率分别为63.3g/L和0.586g/g,与葡萄糖总质量浓度相似(115g/L)的分批发酵相比,丙酮酸产量分别提高了3.8%和9.1%．实验结果表明适宜的初始葡萄糖质量浓度能促进光滑球拟酵母发酵生产丙酮酸;尽管葡萄糖补料培养可适度提高丙酮酸的产量及产率,但生产强度却有所下降．