维生素在丙酮酸过量合成中的重要作用

研究了烟酸、硫胺素、吡哆醇、生物素和核黄素对一株光滑球拟酵母(\%Torulopsis glabrata\%) WSH\|IP303以葡萄糖为碳源、以氯化铵为唯一氮源生产丙酮酸的影响。利用正交试验方法,确证了硫胺素是影响WSH\|IP303生产丙酮酸的最重要因素。在硫胺素浓度一定(0.01～0.015mg/L)的前提下,提高烟酸浓度有助于加快耗糖速度。当烟酸、硫胺素、吡哆醇、生物素和核黄素的浓度分别为8、0.015、0.4、0.04和01mg/L时,摇瓶发酵48h,丙酮酸产量和产率可分别达到52.4g/L和0525g/g。采用优化的维生素组合方式,进行2.5L罐分批发酵,在初糖浓度120g/L的条件下发酵57.5h,丙酮酸产量和产率分别达到69.4g/L和0593g/g,分别比摇瓶培养的最好结果提高了32.%和13%。     

Effects of glucose, vitamins, and DO concentrations on pyruvate fermentation using Torulopsis glabrata IFO 0005 with metabolic flux analysis

The effects of glucose, vitamins, and DO concentrations on efficient pyruvic acid fermentation were investigated using Torulopsis glabrata IFO 0005, and a novel biphasic culture method was developed on the basis of the metabolic flux analysis. T. glabrata requires the four vitamins nicotinic acid (NA), thiamine hydrochloride (B(1)), pyridoxine hydrochloride, and biotin for cell growth. The deficiency of these vitamins plays an essential role in pyruvate fermentation. In the present study, we considered the effects of the first two vitamins on the pyruvate fermentation. On the basis of several batch and fed-batch experiments, it was found that, as a result of glucose inhibition of cell growth, the initial glucose concentration should be around 30-40 g/L, and the glucose concentration during fermentation should be controlled at high level around 30 g/L. On the basis of an analysis of carbon flux distribution, a biphasic fermentation method was developed where the cultivation started with a high DO (at 40-50% of air saturation) for efficient cell growth and then was reduced to 5-10% for efficient pyruvate production. Since a fair amount of ethanol was formed when the DO concentration was decreased, the addition of NA turned out to be effective in reducing the ethanol formation. This may be due to a relaxing of the requirement for NADH oxidation by the alcohol dehydrogenase pathway. Since B(1) affects both the pyruvate dehydrogenase complex and pyruvate decarboxylase, its initial concentration must be carefully determined by considering both the cell growth and pyruvate production phases.     

Production of lactic acid by Lactobacillus rhamnosus with vitamin-supplemented soybean hydrolysate

Batch fermentation studies were performed to evaluate the potentials of a complex nitrogen source, soybean, as an alternative to yeast extract for the economical production of lactic acid by Lactobacillus rhamnosus. An enzyme-hydrolysate of soybean meal, Soytone, with an adequate supplementation of vitamins was found to be highly effective in supporting lactic acid production from glucose and lactose. The effects of seven selected vitamins: d-biotin, pyridoxine, p-aminobenzoic acid, nicotinic acid, thiamine, pantothenic acid, and riboflavin, on cell growth and lactic acid production were investigated to provide the basis for the optimization of vitamin supplementation to minimize the cost. Pantothenic acid was the most required compound while the other six vitamins were also essential for high lactic acid productivity. As a result of the optimization, 15 g/l yeast extract could be successfully replaced with 19.3 g/l Soytone supplemented with the vitamins, resulting in a production of 125 g/l lactic acid from 150 g/l glucose. The volumetric productivity and lactate yield were 2.27 g/l/h and 92%, respectively, which were higher than those with 15 g/l yeast extract. The raw material cost was estimated to be 21.4 cent/kg lactic acid, which was only approximately 41% of that with yeast extract.     

Enhanced glutamic acid production by a H+-ATPase-defective mutant of Corynebacterium glutamicum

Previously we reported that a mutant of Corynebacterium glutamicum ATCC14067 with reduced H+-ATPase activity, F172-8, showed an approximately two times higher specific rate of glucose consumption than the parent, but no glutamic acid productivity under the standard biotin-limited culture conditions, where biotin concentration was set at 5.5 microg/l in the production medium (Sekine et al., Appl. Microbiol. Biotechnol., 57, 534-540 (2001)). In this study, various culture conditions were tested to check the glutamic acid productivity of strain F172-8. The mutant was found to produce glutamic acid under exhaustive biotin limitation, where the biotin concentration of the medium was set at 2.5 microg/l with much smaller inoculum size. When strain F172-8 was cultured under the same biotin-limited conditions using a jar fermentor, 53.7 g/l of glutamic acid was produced from 100 g/l glucose, while the parent produced 34.9 g/l of glutamic acid in a medium with 5.5 microg/l biotin. The glutamic acid yield of strain F172-8 also increased under Tween 40-triggered production conditions (1.2-fold higher than the parent strain). The amounts of biotin-binding enzymes were investigated by Western blot analysis. As compared to the parent, the amount of pyruvate carboxylase was lower in the mutant; however, the amount of acetyl-CoA carboxylase did not significantly change under the glutamic acid production conditions. To the best of our knowledge, this is the first report showing that the H+-ATPase-defective mutant of C. glutamicum is useful in glutamic acid production.     

Enhancement of daptomycin production in <Emphasis Type="Italic">Streptomyces roseosporus</Emphasis> LC-51 by manipulation of cofactors concentration in the fermentation culture

The effects of eight cofactors of enzymes on daptomycin production were investigated in this work, which included nicotinic acid (VPP), riboflavin (VB₂), heme, thiamine (VB₁), biotin (VH), cyanocobalamin (VB₁₂), tetrahydrofolic acid (THF) and pyridoxal 5-phosphate (VB₆). The dry cell weight (DCW), consumption of glucose, and daptomycin production were obviously improved when proper amount of exogenous cofactors were supplemented in the medium. The effects of heme, THF, VB₁₂ and VB₆ on daptomycin production were especially notable. The daptomycin yield enhanced 363, 104, 53 and 46%, respectively, when optimized amount of these four cofactors were supplemented in the broth. Moreover, the daptomycin yield further increased to 632 mg/l, which was over 4.5-fold higher than that of the control (without cofactors), at 132 h in a 7.5-l fermenter, by supplementation all of the eight cofactors at optimized concentrations (VPP 4 mg/l, VB₂ 0.5 mg/l, heme 9 mg/l, VB₁ 0.4 mg/l, VH 0.1 mg/l, VB₁₂ 0.04 mg/l, THF 6 mg/l and VB₆ 0.4 mg/l). Further, the effects of cofactors on the corresponding key enzymes and important intracellular metabolites were studied in order to elucidate the mechanism of enhancement of daptomycin production by manipulation of cofactors concentration in the fermentation culture. It is suggested that this strategy for increasing the daptomycin production in Streptomyces roseosporus LC-51 by manipulation of cofactors concentration in the fermentation culture may provide an alternative approach to enhance the production of metabolites in other Streptomyces.     

Effect of vitamin concentration on the synthesis of lactate, ethanol, pyruvate, and ethyl acetate in cells of the yeast Dipodascus magnusii

In the yeast Dipodascus magnusii, which is auxotrophic for thiamine and biotin, during cultivation on glucose with excessive thiamine concentration, pyruvate metabolism was shown to result in the synthesis of fermentation products, namely, ethanol and, to a lesser extent, lactate. Substantial synthesis of ethyl acetate was also observed under these conditions. Introduction of nicotinic acid (NA) into the medium resulted in time separation of ethanol and lactate production. It was shown that cultivation of the yeast under biotin deficiency resulted in nearly complete suppression of aerobic production of ethanol and cessation of ethyl acetate synthesis, whereas lactate synthesis was activated as early as in the first hours of cultivation. Upon introduction of NA under these conditions, lactate concentration sharply increased. These results show that the combination of thiamine and biotin with other vitamins can stimulate utilization of the pyruvate pool in yeasts towards formation of considerable amounts of lactate, which is typical only of cells of higher eukaryotes and bacteria.     

Levels of water-soluble vitamins in methanogenic and non-methanogenic bacteria

The levels of seven water-soluble vitamins in Methanobacterium thermoautotrophicum, Methanococcus voltae, Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, and Bacteroides thetaiotaomicron were compared by using a vitamin-requiring Leuconostoc strain. Both methanogens contained levels of folic acid and pantothenic acid which were approximately two orders of magnitude lower than levels in the nonmethanogens. Methanobacterium thermoautotrophicum contained levels of thiamine, biotin, nicotinic acid, and pyridoxine which were approximately one order of magnitude lower than levels in the nonmethanogens. The thiamine level in Methanococcus voltae was approximately one order of magnitude lower than levels in the nonmethanogens. Only the levels of riboflavin (and nicotinic acid and pyridoxine in Methanococcus voltae) were approximately equal in the methanogens and nonmethanogens. Folic acid may have been present in extracts of methanogens merely as a precursor, by-product, or hydrolysis product of methanopterin.     

Effect of vitamin concentration on the synthesis of lactate, ethanol, pyruvate, and ethyl acetate in cells of the yeast Dipodascus magnusii

In the yeast Dipodascus magnusii, which is auxotrophic for thiamine and biotin, during cultivation on glucose with excessive thiamine concentration, pyruvate metabolism was shown to result in the synthesis of fermentation products, namely, ethanol and, to a lesser extent, lactate. Substantial synthesis of ethyl acetate was also observed under these conditions. Introduction of nicotinic acid (NA) into the medium resulted in time separation of ethanol and lactate production. It was shown that cultivation of the yeast under biotin deficiency resulted in nearly complete suppression of aerobic production of ethanol and cessation of ethyl acetate synthesis, whereas lactate synthesis was activated as early as in the first hours of cultivation. Upon introduction of NA under these conditions, lactate concentration sharply increased. These results show that the combination of thiamine and biotin with other vitamins can stimulate utilization of the pyruvate pool in yeasts towards formation of considerable amounts of lactate, which is typical only of cells of higher eukaryotes and bacteria.     

Enhancement of pyruvate productivity in Torulopsis glabrata: Increase of NAD+ availability

This study aimed at increasing the pyruvate productivity from a multi-vitamin auxotrophic yeast Torulopsis glabrata, by increasing the availability of NAD+. We examined two strategies for increasing availability of NAD+. To supplement nicotinic acid (NA), the precursor of NAD+; and to increase the activity of alcohol dehydrogenase integrating with addition acetaldehyde as exterior electron acceptor. The addition of 8 mg l(-1) NA to the fermentation medium resulted in a significant increase in the glucose consumption rate (48.4%) and the pyruvate concentration (29%). An ethanol-utilizing mutant WSH-13 was screened and selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata CCTCC M202019. Compared with the parent strain, the alcohol dehydrogenase activity of the mutant WSH-13 increased about 110% and the mutant could utilize ethanol as the sole carbon source for growth (1.8 g l(-1) dry cell weight). When growing with glucose, the addition of 4 mg l(-1) acetaldehyde to the mutant WSH-13 culture broth led to a significant increase in the glucose consumption rate (26.3%) and pyruvate production (22.5%), but the ratio of NADH/NAD+ decreased to 0.22. Acetaldehyde did not affect the glucose and energy metabolism at high dissolved oxygen (DO) concentration. However, at lower DO concentration (20%), maintaining the acetaldehyde concentration in the mutant culture broth at 4 mg l(-1) caused an increased NAD+ concentration but a decreased NADH concentration. As a consequence, the pyruvate production rate, the pyruvate yield on glucose and the pyruvate concentration were 68, 44 and 45% higher, respectively, than the corresponding values of the control (without acetaldehyde). The strategy for increasing the glycolytic flux and the pyruvate productivity in T. glabrata by increasing the availability of NAD+ may provide an alternative approach to enhance the metabolites productivity in yeast.     

Role of 2,4-dichlorophenoxyacetic acid and of myoinositol in the formation of the callus on excised roots ofSolanum laciniatum AIT

Exeised roots ofSolanum laciniatum Ait. grown in vitro in a liquid medium will form the typical rich white callus with a high water content. Its formation is made possible by the presence of 2,4-dichlorophenoxyacetic acid and niyo-inositol in the nutrient medium. Choline, ascorbic acid, riboflavin, calcium pantothenate and biotin are inactive. A mixture of thiamine, pyridoxine and nicotinic acid will induce only slight proliferation.     

Inhibition of muscle glycogen phosphorylase b by heterocyclic vitamins and coenzymes

Inhibition of rabbit skeletal muscle glycogen phosphorylase b by biotin, pyridoxine, lipoic acid, as well as by thiamine and cobalamine vitamins and coenzymes has been found. The values of "half-saturation" concentration and Hill coefficients are determined for biotin (27 mM, 1.3), pyridoxine (19 mM, 1.7), 5'-deoxyadenosyl-cobalamine (2.5 mM, 1.5), lipoic acid (3.4 mM, 1.1), thiamine (11 mM, 1.3), thiamine diphosphate (11 mM, 1.0). Effectiveness of the enzyme inhibition by vitamins and coenzymes containing different heterocyclic groups is analysed; riboflavin and its coenzymic forms are suggested to be the most effective inhibitors.     

Screening of pyruvate-producing yeast and effect of nutritional conditions on pyruvate production

AIMS: To find a yeast strain that can overproduce pyruvate and to investigate the effect of nutrients on pyruvate production. METHODS AND RESULTS: Trichosporon cutaneum PD70, a yeast strain that can overproduce pyruvate, was isolated from shake-flask cultures of 132 yeast strains. Pyruvate was measured by the HPLC or DNP method (see Materials and methods). Pyruvate production reached approximately 30.0 +/- 1.0 g l(-1) in basal fermentation medium. Different nutrient supplements had great effects on pyruvate production. Some of the conditions that gave the highest yield are described. CONCLUSIONS: Exogenous thiamine supplement caused a decrease in pyruvate yield. Some amino acids, such as L-arginine, L-isoleucine and L-valine, caused a minor increase in pyruvate yield. Soybean peptone was the most suitable nitrogen source for pyruvate production. A glucose concentration of 15% in fermentation medium gave the highest yield (34.6 g l(-1)) and the highest yield against consumed glucose (0.429 g g(-1)). SIGNIFICANCE AND IMPACT OF THE STUDY: Nutrients have significant impacts on pyruvate production. As a pyruvate overproducing yeast strain independent of exogenous vitamins or amino acids, T. cutaneum PD70 provides an advantage for commercial pyruvate production.     

Production of arachidonic acid byMortierella alpina ATCC 32222

Summary WhenMortierella alpina ATCC 32222 was incubated in a glucose salts medium at 25°C the biomass (17.5 g/l) contained 9.62% arachidonic acid which amounted to 54% (w/w) of total biomass lipids. When the glucose concentration in the medium was varied from 0 to 150 g/l, the percentage of arachidonic acid in biomass and in lipids was highest at a glucose concentration of 30 g/l, but highest yield of arachidonic acid per litre of culture broth was observed at a glucose concentration of 100 g/l. While production of biomass reached a plateau of 17 g/l after a 3-day incubation at 25°C, the percentage of arachidonic acid in lipids and biomass increased dramatically from 3 to 6 days with a concurrent arachidonic acid yield increase from 0.89 to 1.63 g/l. Optimum initial culture pH for arachidonic acid production was in the range 6.0–6.7. By increasing the concentration of the glucose salts medium three-fold, yields of biomass and arachidonic acid were increased to 35.8 g/l and 3.73 g/l, respectively.     

Factors influencing the production of sclerotia in the wild rice (Zizania aquatica) pathogenSclerotium hydrophilum

Sclerotium hydrophilum was shown to be auxoheterotrophic for thiamine, with the addition of this vitamin being required for the induction of sclerotia on defined media, but riboflavin and pyridoxine also have a positive effect. In the absence of thiamine, an increase in glucose concentration lead to a decrease in the yield of sclerotia; however, the addition of thiamine negate this inhibition and, instead, as the glucose concentration increased a higher proportion of sclerotial initials matured. Overall it was found that thiamine, specifically the pyrimidine component of thiamine, is crucial for initiating sclerotium production, while glucose stimulates maturation. The effect of light on sclerotium production was found to be complex and dependent on the growth medium. Light is not required for either the induction or maturation of sclerotia, but continuous irradiation of developing cultures with either white light or black light induces an endogenous rhythm whereby sclerotia are formed every 48h. When exposed to alternating light/dark regimes mycelium that formed in the light does not mature sclerotia, but dark-formed mycelium does, even if it is subsequently exposed to light.     

Levels of Water-Soluble Vitamins in Methanogenic and Non-Methanogenic Bacteria

The levels of seven water-soluble vitamins in Methanobacterium thermoautotrophicum, Methanococcus voltae, Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, and Bacteroides thetaiotaomicron were compared by using a vitamin-requiring Leuconostoc strain. Both methanogens contained levels of folic acid and pantothenic acid which were approximately two orders of magnitude lower than levels in the nonmethanogens. Methanobacterium thermoautotrophicum contained levels of thiamine, biotin, nicotinic acid, and pyridoxine which were approximately one order of magnitude lower than levels in the nonmethanogens. The thiamine level in Methanococcus voltae was approximately one order of magnitude lower than levels in the nonmethanogens. Only the levels of riboflavin (and nicotinic acid and pyridoxine in Methanococcus voltae) were approximately equal in the methanogens and nonmethanogens. Folic acid may have been present in extracts of methanogens merely as a precursor, by-product, or hydrolysis product of methanopterin.     

Production of enriched in B vitamins biomass of Yarrowia lipolytica grown in biofuel waste

Yarrowia lipolytica as an oleaginous yeast is capable of growing in various non-conventional hydrophobic substrate types, especially industrial wastes. In this study, the content of thiamine (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), biotin (vitamin B7) and folic acid (vitamin B9) in the wet biomass of Y. lipolytica strains cultivated in biofuel waste (SK medium), compared to the standard laboratory YPD medium, was assessed. Additionally, the biomass of Y. lipolytica A-101 grown in biofuel waste (SK medium) was dried and examined for B vitamins concentration according to the recommended microbial methods by AOAC Official Methods. The mean values of these vitamins per 100 g of dry weight of Y. lipolytica grown in biofuel waste (SK medium) were as follows: thiamine 1.3 mg/100 g, riboflavin 5.3 mg/100 g, pyridoxine 4.9 mg/100 g, biotin 20.0 µg/100 g, and folic acid 249 µg/100 g. We have demonstrated that the dried biomass is a good source of B vitamins which can be used as nutraceuticals to supplement human diet, especially for people at risk of B vitamin deficiencies in developed countries. Moreover, the biodegradation of biofuel waste by Y. lipolytica is desired for environmental protection.     

The Effect of Succinate on Respiration,Transamination, and Pyruvate Formation in Cells of the Yeast <Emphasis Type="Italic">Dipodascus magnusii</Emphasis>

The effect of succinate on the growth and respiration of the yeast Dipodascus magnusii VKM Y-1072, which is auxotrophic for thiamine and biotin, was studied. The addition of succinate to a culture grown on glucose was found to activate the respiration of cells on various substrates by enhancing the processes related to transamination reactions. In this case, aerobic fermentation (ethanol production) decreased, whereas pyruvate production increased. When succinate was added to the medium as the sole carbon source, it supported yeast growth in the absence of one of the two vitamins, thiamine or biotin, but not both. The yeast metabolism was completely respiratory, without any signs of aerobic fermentation. A drastic rise in pyruvate production in the yeast grown on glucose in the presence of succinate and the absence of biotin are also indicative of metabolic changes.     

The effect of succinate on respiration, transamination, and pyruvate formation in cells of the yeast Dipodascus magnusii

The effect of succinate on the growth and respiration of the yeast Dipodascus magnusii VKM Y-1072, which is auxotrophic for thiamine and biotin, was studied. The addition of succinate to a culture grown on glucose was found to activate the respiration of cells on various substrates by enhancing the processes related to transamination reactions. In this case, aerobic fermentation (ethanol production) decreased, whereas pyruvate production increased. When succinate was added to the medium as the sole carbon source, it supported yeast growth in the absence of one of the two vitamins, thiamine or biotin, but not both. The yeast metabolism was completely respiratory, without any signs of aerobic fermentation. A drastic rise in pyruvate production in the yeast grown on glucose in the presence of succinate and the absence of biotin are also indicative of metabolic changes.     

Effect of medium components and time of cultivation on chitin production by Mucor circinelloides (Mucor javanicus IFO 4570) -- a factorial study

Chitin production of mycelia from Mucor circinelloides (Mucor javanicus) was studied with a two-level factorial design in all combinations of the following factors in the culture medium: time of cultivation, concentration of D-glucose, L-asparagine and thiamine. Chitin was characterised by infrared and nuclear magnetic resonance spectroscopy. The four factors showed statistically significant (95%) positive main effects on chitin production, without interactions between them. The highest chitin yield (23.9%) was obtained with the medium containing 60 g/l of glucose, 3 g/l of asparagine and 0.008 mg/l of thiamine and is comparable to the highest in the literature.     

Effect of nitrogen source and nitrogen concentration on the production of pyruvate by Torulopsis glabrata

The effect of nitrogen sources including yeast extract, peptone, soybean hydrolyzate and some inorganic nitrogen sources, as well as the nitrogen concentration on the fermentative production of pyruvate by Torulopsis glabrata WSH-IP12 was investigated. The addition of yeast extract greatly inhibited pyruvate accumulation, while peptone was shown to be the most favorable nitrogen source. In flask culture, 15 g l(-1) peptone was needed to consume 80 g l(-1) glucose with 23.4 g l(-1)of pyruvate accumulated. Pyruvate production was markedly dependent on the ratio of carbon to nitrogen (C:N), its production was improved by increasing the concentration of glucose and peptone proportionally and reduced by exclusively increasing the glucose concentration. In a glucose fed-batch culture, cell growth and pyruvate production slowed after 28 h. However, cell growth and pyruvate production recovered after further nitrogen, in the form of peptone and ammonium sulfate, was added to the culture. A final concentration of pyruvate of 54.5 g l(-1) was achieved at 64 h (yield to glucose consumed of 0.471 g g(-l)). By using aqueous ammonia instead of potassium hydroxide for pH control, 57.3 g l(-1) pyruvate with a yield of 0.498 g g(-1) was produced by 55 h. This result further indicates that nitrogen level plays an important role in the production of pyruvate.