Production of l-Methionine-enriched cells of a mutant derived from a methylotrophic yeast,Candida boidinii

l-Methionine-enriched cells production of an ethionine-resistant mutant of Candida boidinii no. 2201 was greatly improved by the control of pH and by feeding of methanol and other medium components during cultivation in a jar fermentor. Under the optimal conditions, 38.5 g (as dry weight)_of cells abd 282 mg of pool methionine (intracellular pool of free l-methionine) per l of culture broth were obtained after 11 d of cultivation.The culture conditions for production of l-methionine-enriched cells in continuous culture were investigated. With limited methanol in continuous cultivation, pool methionine productivity reached a maximum value of 1.14 mg·l⁻¹·h⁻¹ at a dilution rate of 0.05·h⁻¹. During methanol-limited growth in continuous cultivation, the pool methionine content of the mutant was about 20–35% higher than that in batch cultivation.     

Production of Cytochrome c by an Obligate Methylotroph,Methylomonas sp. YK 1

An obligate methanol-utilizing bacterium, Methylomonas sp. YK 1, was isolated and used as a cytochrome c producer. The strain was mutagenized so as to be resistant to metabolic inhibitors related to the function of cytochrome c. The strain, YK 56, which was derived as a KCN-resistant mutant contained 3 times the cellular level of cytochrome c compared to the parent strain. Optimization of the culture conditions for the mutant to enhance the cytochrome c productivity was performed. Peptone, succinate, l-malate or FeSO₄ · 7H₂O increased the productivity when added to the culture medium. Under the optimal culture conditions, strain YK 56 produced about 60 mg cytochrome c per liter when methanol and peptone were fed to the medium during the cultivation.     

Overproduction of glutathione by <Emphasis Type="SmallCaps">l</Emphasis>-cysteine addition and a temperature-shift strategy

The present study investigated the effects of three constituent amino acids on glutathione production in flask culture of Candida utilis. Although l-glutamic acid and glycine had little impact on cell growth and glutathione biosynthesis, l-cysteine positively influenced glutathione production, despite inhibiting cell growth when it was added prior to stationary phase. Adding 8 mmol/L of l-cysteine to the culture broth at 16 h boosted glutathione production by 91%, increasing the intracellular glutathione content by 106% compared to untreated controls. A temperature-shift strategy, in which we shifted batch and fed-batch cultures of C. utilis from 30 to 26°C, also significantly enhanced glutathione production. Applying both strategies (i.e. adding 20 mmol/L l-cysteine and shifting the temperature from 30 to 26°C) at 33 h enhanced the glutathione concentration and the intracellular glutathione content to 1,312 mg/L and 3.75%, respectively, during fed-batch cultivation (glucose feeding at a constant rate of 18.3 g/h). The average specific glutathione production rate under this condition was 129% higher than that of the control without strategy.     

Optimal conditions for the production of exopolysaccharide by marine microorganismHahella chejuensis

A marine microorganism, strain 96CJ10356 produced exopolysaccharide, designated as EPS-R. To optimize culture conditions for the production of EPS-R, carbon and nitrogen sources, mineral salts, temperature, and pH were examined. From this study, STN medium for the production of EPS-R was suggested as follows; sucrose 20 g, tryptone 10 g, NaCl 10 g, MgSO₄ 5 g, CaCl₂ 1 g, KH₂PO₄ 76 mg, K₂HPO₄ 83 mg, FeCl₂ 5 mg, MnCl₂ 1 mg, NaMoO₄ 1 mg, and ZnCl₂ 1 mg per liter at pH 7.0. About 9.23 g/L of EPS-R was obtained from STN medium after cultivation for 120 h at 25°C in a 5-liter jar fermentor with an aeration rate of 0.17 vvm. Apparent viscosity and flocculation activity of the culture broth were increased with the production of EPS-R and the maximal values were 415 cP and 1400 unit/mL against 0.5% activated carbon, respectively.     

A Cytochrome c from a Lupanine-Transforming Pseudomonas putida Strain Is Expressed in Escherichia coli during Aerobic Cultivation and Efficiently Exported and Assembled in the Periplasm

We have cloned, sequenced, and heterologously expressed a periplasmic cytochrome c from a lupanine-utilizing Pseudomonas putida strain. Aerobic batch cultivation of Escherichia coli TB1 harboring the cytochrome c gene placed downstream of the lac promoter in pUC9 vector resulted in significant production of the holo-cytochrome c in the periplasm (~4 mg of hemoprotein/liter of culture). The recombinant cytochrome c was purified to homogeneity and was found to be functional in accepting electrons from lupanine hydroxylase while catalyzing hydroxylation of lupanine. Comparison of the N-terminal amino acid sequence of the isolated cytochrome c with that deduced from the DNA sequence indicated that the signal sequence was processed at the bond position predicted by the SigPep program. The molecular size of the cytochrome c determined by electrospray mass spectrometry (9,595) was in precise agreement with that predicted from the nucleotide sequence.     

Absolute Configuration of (+)-1-Acetoxy-p-menth-3-one Isolated from Essential Oil of Mentha gentilis (L.)

The most effective electro-energizing fermentation (E-E F) conditions for l-glutamate (l-Glu) production by Brevibacterium flavum No. 2247 were determined. The adding of 0.01 mm neutral red at the beginning of cultivation was found most effective. A 1.5 V direct current was applied to the culture broth at 6~8 hr after inoculation in the cathode compartment, l-Glu was produced at 51.0 mg per ml, and this is about a 15 % increase in yield compared to the yield of the not electro-energizing (E-E) control (44.3 mg/ml).     

Fed-batch culture with a modified DO-stat method

To support a high growth rate of microorganism in fed-batch culture with high cell density, a modified DO-stat method was developed. In this method, an exponential substrate feed was coupled with the usual DO-stat method, i.e., a fixed amount of substrate per DO signal was exponentially fed to the culture based on the estimation of the substrate consumption rate and thereafter the feed was stopped in order to prevent the oversupply of substrate until an abrupt increase in the concentration of dissolved oxygen (DO) in the broth appeared. After that, the feed was started again and this cyclic operation was repeated throughout the cultivation. This method was applied to the fed-batch cultivation of ethanol utilizing yeast, Candida brassicae. At high cell densities (> 10 g/l), this modified method was more effective than the usual one in keeping a higher growth rate.     

Formation of autodiploid strains in Aspergillus niger and their application to citric acid production from starch

Autodiploid strains were induced by colchicine treatment of Aspergillus niger WU-2223L, a citric acid-producing strain. In shaking culture, a representative autodiploid strain, L-d1, yielded higher citric acid than the parental strain, WU-2223L. When glucose was used as a carbon source, L-d1 and WU-2223L produced 67.2 g/l and 62.0 g/l of citric acid, respectively, from 120 g/l of glucose in 9 d-cultivation. Furthermore, the autodiploid strain L-d1 produced 49.6 g/l of citric acid, 1.4 times as much as that produced by WU-2223L from 120 g/l of soluble starch. During the whole period of cultivation with starch, the extracellular glucoamylase activity of L-d1 was on the same level as that of WU-2223L, but the extracellular acid-protease activity of L-d1 was much higher. The addition of pepstatin, an inhibitor of acid protease, to the culture broth at 2 d greatly increased the extracellular glucoamylase activity, and citric acid production by L-d1 reached a level of 59.0 g/l. During several subcultivations on both minimal and complete agar media, the autodiploid strains were genetically stable since they formed diploid conidia in their uniform colonies without producing sectors, and maintained citric acid productivity. However, when cultivated on minimal and complete agar media containing benomyl as a haploidizing reagent, the autodiploid strains readily formed sectors of haploid segregants. The properties of the haploid strains obtained by the benomyl treatment of the autodiploid strains were similar in morphology and citric acid productivity to those of the parental strain, WU-2223L. These results indicated that the enhanced production of citric acid from soluble starch by the autodiploid strains was due to autodiploid formation but not to gene mutation caused by the colchicine treatment.     

Continuous production of an antibiotic polypeptide (nisin) by Lactococcus lactis using a bioreactor coupled to a microfiltration module

The continuous production of nisin, an antibiotic polypeptide, by Lactococcus lactis in a bioreactor system coupled to a microfiltration module is described. Nisin productivity with respect to both cultivation time (ND) and the quantity of glucose consumed (ND/S_f) in continuous production was enhanced by maintaining a low concentration of lactic acid in the broth. A maximum ND of 7.80 × 10⁴ U·l⁻¹·h⁻¹ and ND/S_f of 5.20 × 10³ U·g⁻¹·h⁻¹ were obtained when the glucose concentration in the feed medium was 15 g/l. These values represent about 4.1- and 4.5-fold increases, respectively, over those obtained in batch culture.     

Extracellular production of menaquinone-4 by a mutant of Flavobacterium sp. 238-7 with a detergent-supplemented culture

Culture conditions for the extracellular production of menaquinone (MK) by a mutant strain, K₃-15, of Flavobacterium sp. 238-7 were investigated. A detergent-supplemented culture medium consisted of 60 g of glycerol, 23 g of peptone, 3 g of yeast extract, 7 g of K₂HPO₄, 5 g of NaCl, 0.8 g of MgSO₄ · 7H₂O, and 0.5 g of Rikanon UA 5012 in 1 l of tap water, pH 7.0, was constructed. Amounts of MK-4, MK-6, and total MK in 1 l of the medium were 101 mg, 39 mg, and 140 mg, respectively, after 7 d of cultivation at 28°C. Further studies with some additives showed that the addition of cedar wood oil increased the productivity of MK, especially MK-4. In the presence of 0.1% Rikanon UA 5012 and 0.1% cedar wood oil, mutant strain K₃-15 produced 155 mg/l intracellularly and 105 mg/l extracellularly) of MK-4 and 27 mg/l (16 mg/l intracellularly and 11 mg/l extracellularly) of MK-6, and the total amount of MK reached 182 mg/l.     

Fuzzy neural network for the control of high cell density cultivation of recombinant Escherichia coli

A five-layer fuzzy neural network (FNN) was developed for the control of fed-batch cultivation of recombinant Escherichia coli JM103 harboring plasmid pUR 2921. The FNN was believed to represent the membership functions of the fuzzy subsets and to implement fuzzy inference using previous experimental data. This FNN was then used for compensating the exponential feeding rate determined by the feedforward control element. The control system is therefore a feedforward-feedback type. The change in pH of the culture broth and the specific growth rate were used as the inputs to FNN to calculate the glucose feeding rate. A cell density of 84 g DWC/l in the fed-batch cultivation of the recombinant E. coli was obtained with this control strategy. Two different FNNs were then employed before and after induction to enhance plasmid-encoded β-galactosidase production. Before induction the specific growth rate was set as 0.31 h⁻¹, while it was changed to 0.1 h⁻¹ after induction. Compared to when only one FNN was used, the residual glucose concentration could be tightly controlled at an appropriate level by employing two FNNs, resulting in an increase in relative activity of β-galactosidase which was about four times greater. The present investigation demonstrates that a feedforward-feedback control strategy with FNN is a promising control strategy for the control of high cell density cultivation and high expression of a target gene in fed-batch cultivation of a recombinant strain.     

Vitamin B12 production by isopropanol-assimilating microorganisms

We isolated about 500 isopropanol(IPA)-assimilating bacteria from many soil samples, among which 23 strains produced vitamin B₁₂. Taxonomical studies of the best producer, designated strain Hi16.3, showed that it belonged to the genus Arthrobacter. Vitamin B₁₂ production by the strain was higher than that by 12 other authentic Arthrobacter spp. using glucose as a sole carbon source. In fed-batch culture, the maximum production yield with strain Hi16.3 (named A. hyalinus) was 2 mg/l in the culture broth, when 80 ml of IPA/l broth was consumed.     

Effect of mixed organic substrate on α-tocopherol production by <Emphasis Type="Italic">Euglena gracilis</Emphasis> in photoheterotrophic culture

Effects of organic carbon sources on cell growth and alpha-tocopherol productivity in wild and chloroplast-deficient W14ZUL strains of Euglena gracilis under photoheterotrophic culture were investigated. In both strains, the increase in cell growth was particularly high when glucose was added as the sole organic carbon source. On the other hand, alpha-tocopherol production per dry cell weight was enhanced by adding ethanol. Ethanol addition also increased the chlorophyll concentration in wild strain and mitochondria activity in W14ZUL strain. For effective alpha-tocopherol production, the effects of mixture of glucose and ethanol were investigated. The results showed that, when a mixture of glucose (6 g/l) and ethanol (4 g/l) was used, alpha-tocopherol productivity per culture broth was 3.89 x 10(-2) mg l(-1) h(-1), which was higher than the value obtained without addition of organic carbon source (0.92 x 10(-2) mg l(-1) h(-1)). In addition, under fed-batch cultivation using an internally illuminated photobioreactor, the alpha-tocopherol production per culture broth was 23.43 mg/l, giving a productivity of 16.27 x 10(-2) mg l(-1) h(-1).     

Production of poly-β-hydroxybutyric acid from carbon dioxide by Alcaligenes eutrophus ATCC 17697T

The characteristics of PHB production from carbon dioxide by autotrophic culture of Alcaligenes eutrophus ATCC 17697^T using a recycled gas closed circuit culture system under the condition of oxygen limitation were investigated. Cell concentration increased to more than 60 g/l after 60 h of cultivation, while the PHB concentration reached 36 g/l. PHB accumulation in the oxygen-limited culture was superior than that in an ammonium-deficient culture. The PHB produced was identified as a homopolymer of d-3-hydroxybutyrate by ¹H and ¹³C NMR analysis. The stoichiometry for PHB production from CO₂ under the oxygen limitation condition was indicated to be as follows: 33H₂ + 12O₂ + 4CO₂ → C₄H₆O₂ + 30H₂O. This stoichiometry shows that the hydrogen consumption per one mole of CO₂ for PHB production is larger than that for cell formation.     

Control of the syntheses of bacteriochlorophyll,c- and b-type cytochromes,and coproporphyrin III in Rhodopseudomonas sphaeroides mutant H5

Rhodopseudomonas sphaerodes mutant H5 lacking 5-aminolevulinic acid synthase was grown phototrophically in chemostat cultures limited by malate. Tetrapyrrole formation was limited by 5-aminolevulinic acid. With variation of dilution rates the cultures exhibited two regions of almost constant cell protein, dry weight and bacteriochlorophyll levels suggesting the formation of two physiological modifications of the strain. These modifications were further characterized by differences in the rates of 5-aminolevulinic acid consumption, the production of reserve material, the stoichiometries of 5-aminolevulinic acid consumption and bacteriochlorophyll or cytochrome production, specific bacteriochlorophyll and cytochrome contents as well as the ratio of bacteriochlorophyll protein complexes. In contrast, cellular levels of coproporphyrin II stayed almost constant over the entire range of dilution rates employed. Bacteriochlorophyll and b-type cytochrome cellular levels exhibited hyperbolic dependencies on the specific rate of 5-aminolevulinic acid consumption, and c-type cytochrome levels a signmoidal dependency. Bacteriochlorophyll cellular levels showed a biphasic dependency with half maximal saturations at 2.6 and 15.4 nmol of 5-aminolevulinic acid consumed per mg of protein and h, and maximal levels of 15.2 and 21 nmol bacteriochlorophyll per mg of protein. Cellular levels of c- and b-type cytochromes were half maximally saturated at 19.5 and 14.5 nmol 5-aminolevulinic acid consumed per mg protein and h while maximal levels were reached at 0.5 and 0.17 nmol of c- and b-type cytochromes, respectively, per mg of protein.The data suggest that within the cell bacteriochlorophyll as well as c- and b-type cytochrome units are assembled according to a defined pattern of kinetics characteristic of each group of compounds. Under otherwise constant external conditions the expression of the pattern is controlled by the rate of 5-aminolevulinic acid supply.     

Control of glucose feeding using exit gas data and its application to the production of PHB from tapioca hydrolysate byAlcaligenes eutrophus

Summary A method to estimate the glucose concentration in the culture broth using CO₂ evolution rate (CER) data from a mass spectrometer was developed.Alcaligenes eutrophus was cultivated to produce poly(3-hydroxybutyric acid) (PHB) from tapioca hydrolysate using this method. Thek value (g glucose/mol CO₂), defined as the glucose consumption per CO₂ evolution, decreased with culture time and was automatically changed using CER data. The glucose concentration in the culture broth could be controlled at 10 to 20 g/L. A final cell concentration of 106 g/L, PHB concentration of 61 g/L. and PHB content of 58 % of dry cell weight were obtained after 59 h of cultivation.     

Definition of culture conditions for Arxula adeninivorans,a rational basis for studying heterologous gene expression in this dimorphic yeast

The yeast Arxula adeninivorans is considered to be a promising producer of recombinant proteins. However, growth characteristics are poorly investigated and no industrial process has been established yet. Though of vital interest for strain screening and production processes, rationally defined culture conditions remain to be developed. A cultivation system was evolved based on targeted sampling and mathematical analysis of rationally designed small-scale cultivations in shake flasks. The oxygen and carbon dioxide transfer rates were analyzed as conclusive online parameters. Oxygen limitation extended cultivation and led to ethanol formation in cultures supplied with glucose. Cultures were inhibited at pH-values below 2.8. The phosphorus demand was determined as 1.55 g phosphorus per 100 g cell dry weight. Synthetic SYN6 medium with 20 g glucose l^?1 was optimized for cultivation in shake flasks by buffering at pH 6.4 with 140 mmol MES l^?1. Optimized SYN6 medium and operating conditions provided non-limited cultivations without by-product formation. A maximal specific growth rate of 0.32 h^?1 and short fermentations of 15 h were achieved. A pH optimum curve was derived from the oxygen transfer rates of differently buffered cultures, showing maximal growth between pH 2.8 and 6.5. Furthermore, it was shown that the applied medium and cultivation conditions were also suitable for non-limiting growth and product formation of a genetically modified A. adeninivorans strain expressing a heterologous phytase.     

Selection of mutants of Aspergillus niger showing enhanced productivity of citric acid from starch in shaking culture

Mutants with enhanced citric acid production from soluble starch were induced from Aspergillus niger WU-2223L. After UV-irradiation of a conidial suspension of strain WU-2223L, mutants were selected on modified starch-methyl red agar plates on the basis of higher amylolytic activity and acid productivity. The 8 mutants selected showed enhanced citric acid production from soluble starch in shaking culture. Among them, a representative mutant strain, 2M-43, produced 48.0gg/l of citric acid from 120 g/l of soluble starch in 9 d of cultivation in shaking culture, whereas strain WU-2223L produced 35.1 g/l. Glucoamylase activities in the culture filtrates of strains 2M-43 and WU-2223L reached maximum levels of 3.62 U/ml and 2.11 U/ml, respectively, both at 3 d of cultivation, and thereafter decreased.     

Xylitol production by a methanol yeast,Candida boidinii (Kloeckera sp.) No. 2201

A methanol yeast, Candida boidinii no. 2201, could produce xylitol and also d-xylulose during cultivation on d-xylose medium. These fermentative products were identified by high performance liquid chromatography. A large amount of xylitol was obtained from a d-xylose medium containing ammonium acetate and yeast extract at the initial pH of 7.0. Maximum productivities of xylitol and enzymes concerned with the production were observed after 4–5 d of cultivation. A d-xylose (100 g/l) medium supplemented with 2% (v/v) methanol gave higher amounts of xylitol (48.5 g/l) and d-xylulose (3.3 g/l). Enzyme activities for d-xylose reduction, d-xylulose reduction, d-xylose isomerization, and d-xylulose phosphorylation, which could be involved in the xylitol production, were measured in cell-free extracts during cultivation and a possible pathway of xylitol production was discussed.     

Enzymatic production of l-tryptophan from dl-5-indolylmethylhydantoin by Flavobacterium sp.

An enzymatic production of l-tryptophan from dl-5-indolylmethylhydantoin by the action of hydantoinase and carbamoylase has been investigated. A strain identified as (Flavobacterium) sp. I-3 isolated from soil was found to form l-tryptophan from dl-5-indolylmethylhydantoin. Cultural conditions for the formation of the l-tryptophan-forming activity were investigated, and the highest activity of 0.81 μmol min⁻¹of l-tryptophan formed per 1 ml of culture broth (hydantoinase, 3.6 μmol min⁻¹of N-carbamoyl-l-tryptophan formed per 1 ml of culture broth; carbamoylase, 0.92 μmol min⁻¹of l-tryptophan formed per 1 ml of culture broth) was obtained. These activities were found to be inducible and intracellular. Optimization of the parameters of the conversion reaction resulted in accumulation of 50 mg of l-tryptophan per 1 ml of cultural broth per day. The conversion yield from dl-5-indolylmethylhydantoin was about 100%. Accumulated l-tryptophan was readily isolated in pure form by ordinary procedures.