Construction and optimization of cordycepin-producing Saccharomyces cerevisiae / 生物工程学报

Cordycepin is the key active component of medicinal fungus Cordyceps militaris, and it shows multiple functional activities such as anti-tumor and anti-virus. Cordycepin was conventionally produced by liquid fermentation of C. militaris, but the long production cycle and the low productivity constrained its development and application. In this study, two key genes for cordycepin biosynthesis (ScCNS1 and ScCNS2) were introduced into Saccharomyces cerevisiae S288C, producing 67.32 mg/L cordycepin at 240 h. Analysis of gene expression profiles indicated that ZWF1, PRS4, ADE4, ScCNS1 and ScCNS2 which encode enzymes involved in pentose phosphate pathway, purine metabolism and cordycepin biosynthesis pathway, were significantly up-regulated in the late phage of fermentation. Optimization of fermentation medium determined that 50 g/L initial glucose followed by feeding, supplemented with 5 mmol/L Cu²⁺ and 1.0 g/L adenine were the best condition. Fed-batch fermentation using the engineered yeast in a 5 L stirred fermenter produced 137.27 mg/L cordycepin at 144 h, with a productivity up to 0.95 mg/(L·h) reached, which was 240% higher than that of the control.

Statistical optimization of kojic acid production by a UV-induced mutant strain of Aspergillus terreus

ABSTRACT The ability of four Aspergillus strains for biosynthesis of kojic acid was evaluated among which Aspergillus terreus represented the highest level (2.21 g/L) of kojic acid production. Improvement kojic acid production ability of A. terreus by random mutagenesis using different exposure time to ultraviolet light (5-40 min) was then performed to obtain a suitable mutant of kojic acid production (designated as C5-10, 7.63 g/L). Thereafter, design of experiment protocol was employed to find medium components (glucose, yeast extract, KH2PO4 (NH4)2SO4, and pH) influences on kojic acid production by the C5-10 mutant. A 25-1 fractional factorial design augmented to central composite design showed that glucose, yeast extract, and KH2PO4 were the most considerable factors within the tested levels (p < 0.05). The optimum medium composition for the kojic acid production by the C5-10 mutant was found to be glucose, 98.4 g/L; yeast extract, 1.0 g/L; and KH2PO4, 10.3 mM which was theoretically able to produce 120.2 g/L of kojic acid based on the obtained response surface model for medium optimization. Using these medium compositions an experimental maximum Kojic acid production (109.0 ± 10 g/L) was acquired which verified the efficiency of the applied method.

Enhanced production of dimethyl phthalate-degrading strain Bacillus sp. QD14 by optimizing fermentation medium

Background Integrated statistical experimental designs were applied to optimize the medium constituents for the production of a dimethyl phthalate (DMP)-degrading strain Bacillus sp. QD14 in shake-flask cultures. A Plackett-Burman design (PBD) was applied to screen for significant factors, followed by the Steepest Ascent Method (SAM) to find the nearest region of maximum response. A Box-Behnken design (BBD) of the Response Surface Methodology (RSM) was conducted to optimize the final levels of the medium components. Results After the regression equation and response surface contour plots were analyzed, the concentrations of glucose, corn meal and NaCl were found to significantly influence the biomass of DMP-degrading bacteria. A combination of 22.88 g/L of glucose, 11.74 g/L of corn meal, and 10.34 g/L of NaCl was optimum for maximum biomass production of Bacillus sp. QD14. A 57.11% enhancement of the biomass production was gained after optimization in shake-flask cultivation. The biomass production of Bacillus sp. QD14 reached 9.13 ± 0.29 × 10(8) CFU/mL, which was an excellent match for the predicted value, and the mean value of the match degree was as high as 99.30%. Conclusion In this work, the key factors affected by the fermentation of DMP-degrading strain Bacillus sp. QD14 were optimized by PBD, SAM and BBD (RSM); the yield was increased by 57,11% in the conditions in our study. We propose that the conditions optimized in the study can be applied to the fermentation for commercialization production.

Enhancement of beta-Glucosidase Activity from a Brown Rot Fungus Fomitopsis pinicola KCTC 6208 by Medium Optimization

beta-Glucosidase, which hydrolyzes cellobiose into two glucoses, plays an important role in the process of saccharification of the lignocellulosic biomass. In this study, we optimized the activity of beta-glucosidase of brown-rot fungus Fomitopsis pinicola KCTC 6208 using the response surface methodology (RSM) with various concentrations of glucose, yeast extract and ascorbic acid, which are the most significant nutrients for activity of beta-glucosidase. The highest activity of beta-glucosidase was achieved 3.02% of glucose, 4.35% of yeast extract, and 7.41% ascorbic acid where ascorbic acid was most effective. The maximum activity of beta-glucosidase predicted by the RSM was 15.34 U/mg, which was similar to the experimental value 14.90 U/mg at the 16th day of incubation. This optimized activity of beta-glucosidase was 23.6 times higher than the preliminary activity value, 0.63 U/mg, and was also much higher than previous values reported in other fungi strains. Therefore, a simplified medium supplemented with a cheap vitamin source, such as ascorbic acid, could be a cost effective mean of increasing beta-glucosidase activity.

Optimization of zofimarin production by an endophytic fungus, Xylaria sp. Acra L38

To optimize the medium for high zofimarin production, sucrose maltose, glucose, tryptone and peptone were used in an orthogonal array design experiment, where the highest value of zofimarin produced was 25.6 µg/mL. This value was about 3 times higher than that obtained with Czapek yeast extract (CzYE) culture medium. A study with Plackett-Burman design showed that sucrose, maltose, glucose and NaNO3 were significant factors in zofimarin production. Further studies using central composite design (CCD) showed the significance of glucose and the interactions of these critical components affecting zofimarin production. Multiple regression analysis of the data yielded a poor fit as shown by the mismatch of the model with these variable factors. When a polynomial equation was applied, the maximum zofimarin production was predicted to be 201.9 µg/mL. Experimental verification yielded a much lower amount of zofimarin, at around 70 µg/mL. Reconsideration of the CCD data and repetition of some runs with high zofimarin production resulted in reproducible zofimarin yield at 79.7 µ/mL. Even though the amount was lower than the predicted value, the medium optimization study was considered to be quite successful as the yield increased to around 8 times that obtained with the original CzYE culture medium.

Influence of multiple bioprocess parameters on production of lipase from Pseudomonas sp. BWS-5

The aim of the present work was to study the influence of multiple bioprocess parameters for the maximum production of lipase from Pseudomonas sp. BWS-5. The culture reached the stationary phase of growth after 36h of incubation when the maximum lipase production was obtained at flask level. The different media components such as carbon sources, nitrogen sources, trace elements and process parameters such as the pH of the medium, temperature and time of incubation, agitation/stationary conditions, etc. were optimized at flask level and at bioreactor level. The maximum enzyme production of 298 IU/mL was obtained with the use of simple medium with pH 6.5 containing glucose (1 %, w/v), peptone (3 %, w/v) and KCl (0.05 %, w/v) after 30h of incubation at 37°C under agitation (200 rpm) conditions with 0.75 vvm of air supply.

Modeling of glutamic acid production by Lactobacillus plantarum MNZ

Background: L-glutamic acid, the principal excitatory neurotransmitter in the brain and an important intermediate in metabolism acts as a precursor of γ-amino butyric acid (GABA). In the present study, culture condition for enhanced glutamic acid production by Lactobacillus plantarum MNZ was optimized and the influence of such conditions on GABA production was evaluated. Results: Results indicated that glutamic acid increased up to 3-fold (3.35) under the following condition: pH 4.5, temperature 37ºC, 12% (w/v) glucose and 0.7% (w/v) ammonium nitrate; whilst GABA production was enhanced up to 10-fold under the following condition: pH 4.5, temperature 37ºC, 6% (w/v) glucose and 0.7% (w/v) ammonium nitrate. Conclusions: This is the first report for dual biosynthesizing activities of a lactic acid bacterium for the production of glutamic acid and GABA. The results of this study can be further used for developing functional foods rich inglutamic acid and subsequently GABA as a bioactive compound.

Optimizing a culture medium for biomass and phenolic compounds production using Ganoderma lucidum

The present work was aimed at optimizing a culture medium for biomass production and phenolic compounds by using Ganoderma lucidum. The culture was optimized in two stages; a Plackett-Burman design was used in the first one for identifying key components in the medium and a central composite design was used in the second one for optimizing their concentration. Both responses (biomass and phenolic compounds) were simultaneously optimized by the latter methodology regarding desirability, and the optimal concentrations obtained were 50.00 g/L sucrose, 13.29 g/L yeast extract and 2.99 g/L olive oil. Maximum biomass production identified in these optimal conditions was 9.5 g/L and that for phenolic compounds was 0.0452 g/L, this being 100% better than that obtained in the media usually used in the laboratory. Similar patterns regarding chemical characterization and biological activity towards Aspergillus sp., from both fruiting body and mycelium-derived secondary metabolites and extracts obtained in the proposed medium were observed. It was shown that such statistical methodologies are useful for optimizing fermentation and, in the specific case of G. lucidum, optimizing processes for its production and its metabolites in submerged culture as an alternative to traditional culture.

Isolation and characterization of an Ashbya gossypii mutant for improved riboflavin production

The use of the filamentous fungus, Ashbya gossypii, to improve riboflavin production at an industrial scale is described in this paper. A riboflavin overproducing strain was isolated by ultraviolet irradiation. Ten minutes after spore suspensions of A. gossypii were irradiated by ultraviolet light, a survival rate of 5.5% spores was observed, with 10% of the surviving spores giving rise to riboflavin-overproducing mutants. At this time point, a stable mutant of the wild strain was isolated. Riboflavin production of the mutant was two fold higher than that of the wild strain in flask culture. When the mutant was growing on the optimized medium, maximum riboflavin production could reach 6.38 g/l. It has even greater promise to increase its riboflavin production through dynamic analysis of its growth phase parameters, and riboflavin production could reach 8.12 g/l with pH was adjusted to the range of 6.0-7.0 using KH2PO4 in the later growth phase. This mutant has the potential to be used for industrial scale riboflavin production.

Optimization of nutrition factors on chitinase production from a newly isolated Chitiolyticbacter meiyuanensis SYBC-H1

The present study reports statistical medial optimization for chitinase production by a novel bacterial strain isolated from soil recently, which the name Chitinolyticbacter meiyuanensis SYBC-H1 is proposed. A sequential statistical methodology comprising of Plackett-Burman and response surface methodology (RSM) was applied to enhance the fermentative production of chitinase, in which inulin was firstly used as an effective carbon source. As a result, maximum chitinase activity of 5.17 U/mL was obtained in the optimized medium, which was 15.5-fold higher than that in the basal medium. The triplicate verification experiments were performed under the optimized nutrients levels which indicated that it well agreed with the predicted value.

Enhanced exoinulinase production from Kluyveromyces marxianus YS-1 using response surface methodology

Optimization of critical medium components for exoinulinase production by Kluyveromyces marxianus YS-1 at shake-flask was investigated using response surface methodology (RSM) based on a central composite rotatable design (CCRD). A five-level with five factors CCRD was used to evaluate the influence of related factors including concentration of inulin, meat extract, calcium chloride, sodium dodecyl sulphate and medium pH. Optimum values obtained by RSM were 2 percent inulin, 2.17 percent meat extract, 0.65 mM calcium chloride, 0.10 mM sodium dodecyl sulphate and pH 5.5. Optimized medium projected a theoretical exoinulinase production of 63.61 IU/mL and biomass yield of 0.965 (OD600/10). Multiple correlation coefficient R was 0.9976 and 0.9605 for exoinulinase production and biomass yield, respectively, which being close to one, justified an excellent correlation between the predicted and experimental values. Maximum productivity of exoinulinase (64.05 IU/mL) obtained experimentally by RSM was more than double in comparison to earlier findings using classical one-variable-at-a-time technique.

Foi investigada a optimização de componentes criticos do meio de cultivo para a produção de exoinulinase por Kluyveromyces marxianus YS-1 em frascos agitados utilizando a metodologia de superficie de resposta (RSM), com base em um delineamento composto central rotativo. As variáveis independentes, com cinco niveis, foram as concentrações de inulina, de extrato de carne, de cloreto de cálcio e de dodecil sulfato de sódio, bem como o pH do meio de cultivo. Os valores ótimos, obtidos pela RSM, foram com 2 por cento de inulina, 2.17 por cento de extrato de carne, 0.65 mM de cloreto de cálcio, 0.10 mM de dodecil sulfato de sódio e pH 5.5. As estimativas de produção de exoinulinase e de rendimento em biomassa no meio otimizado foram de 63.61 UI/ml e de 9.65 (em termos de OD600/10), respectivamente. Os coeficientes de correlação múltipla R foram de 0.9976 e 0.9605 para produção de exoinulinase e biomassa, respectivamente, e, sendo próximos de um, indicam uma excelente correlação entre os valores estimados e experimentais. A máxima productividade de exoinulinase (64.05 UI/ml) obtida experimentalmente por RSM foi mais que o dobro em comparação com nossos resultados anteriores utilizando uma técnica de otimização clássica de uma variável por vez.

Optimization of medium for lipase production by Acinetobacter haemolyticus from healthy human skin.

A lipase producing Acinetobacter haemolyticus TA106 was isolated from healthy human skin of tribal population. The maximum activity of 55 U/ml was observed after medium optimization using the “one variable at a time” and the statistical approaches. The optimal composition of the medium was determined as (% w/v or v/v): tryptone - 1, yeast extract - 0.5, sodium chloride-1, olive oil-1, Tween - 80 1, manganese sulphate - 5 mM, sucrose- 1, pH-7. It was found that maximum production occurred in late log phase i.e. after 72 h and at 200 rpm. From factorial design and statistical analysis, it was found that pH, temperature, salt, inoculum density and aeration significantly affected the lipase production. It was also noted that inoculum density of 3 % (v/v), sucrose (1% w/v) and manganese sulphate (5 mM) displayed maximum lipase activity of 55 U/ml by conventional as well as statistical method. Optimization studies also indicated the increase in specific activity from 0.2 U/mg to 6.7 U/mg.

Production of alkaline protease from Cellulosimicrobium cellulans / Produção de protease alcalina porCellulosimicrobium cellulans

Cellulosimicrobium cellulans is one of the microorganisms that produces a wide variety of yeast cell walldegradingenzymes, β-1,3-glucanase, protease and chitinase. Dried cells of Saccharomyces cerevisiae were used as carbon and nitrogen source for cell growth and protease production. The medium components KH2PO4, KOH and dried yeast cells showed a significant effect (p<0.05) on the factorial fractional design. A second design was prepared using two factors: pH and percentage of dried yeast cells. The results showedthat the culture medium for the maximum production of protease was 0.2 g/l of MgSO4.7H2O, 2.0 g/l of(NH4)2SO4 and 8% of dried yeast cells in 0.15M phosphate buffer at pH 8.0. The maximum alkaline protease production was 7.0 ± 0.27 U/ml over the center point. Crude protease showed best activity at 50ºC and pH 7.0-8.0, and was stable at 50ºC.

Cellulosimicrobium cellulans é um microrganismo que produz uma variedade de enzimas que hidrolisam a parede celular de leveduras: β-1,3-glucanase, protease e quitinase. Célulasdesidratadas de Saccharomyces cerevisiae foram usadas como fonte de carbono e nitrogênio para o crescimento celular e produção de protease. Os componentes do meio de cultura: KH2PO4, KOH e células de levedura desidratadas mostraram efeitos significativos (p<0,05) no planejamento experimental fracionário. Um segundo planejamento foi preparado usandodois fatores: pH e porcentagem de células de levedura desidratadas. Os resultados mostraram que o meio de cultura para a produção máxima de protease foi 0,2 g/L de MgSO4.7H2O;2,0 g/L de (NH4)2SO4 e 8% de células de levedura desidratadas em tampão fosfato 0,15M e pH 8,0. A produção máxima de protease alcalina foi 7,0 ± 0,27 U/mL no ponto central. A proteasebruta apresentou atividade ótima a 50ºC e pH 7,0-8,0; e foi estável a 50ºC.

Screening of Poly-?-glutamic Acid High Productive Strain and Optimization of Fermentation Medium / 微生物学通报

Bacillus subtilis B6-1 was used as an original strain for mutagenic treatment and a defined medium was used as the selective medium. A mutant named B. subtilis W003 was isolated after three serial ultraviolet (UV) irradiations and one diethyl sulfate (DES) treatment. The ?-PGA yield on a rotary shaker was enhanced from 10.9 g/L in parental strain to 20.5 g/L in the mutant. It was illustrated by single factor experiments that the optimal carbon and nitrogen sources were glucose and (NH4)2SO4 respectively. The optimal fermentation medium was achieved by orthogonal test. In the optimal medium, a ?-PGA yield of 45.3 g/L was obtained after 36 h cultivation.

Optimization of Fermentation Medium with Lactose as an Inducer for High Molecular Weight Recombinant Spider Silk Protein / 中国生物工程杂志

Based on M9 culture medium,the concentration of ingredients of culture medium was optimized for the fermentation of pNSR32/BL21(DE3),the engineering bacterial with spider silk protein,and lactose as an inducer.The condition of optimum culture medium was obtained for the expression of the high molecular weight recombinant spider silk protein by using orthogonal and individual factor experimental design.The result was showed that the optimum culture medium was consisted of 0.3% glycerol,3% yeast,0.75% tryptone,0.05%(NH_4)_2SO_4 and a little inorganic salt_.It was confirmed that the optimum culture medium will benefit the growth of bacterial and expression of recombinant spider silk protein.The production level of propose protein will attain at 20% of the total proteins in the fermentation.

Screen and Optimization of Cultured Medium Constituents for Biodegradation of ?-cypermethrin,a Synthetic Pyrethroid Insecticide,by Fusarium sp. Strain HG-P-01 via Central Composite Rotatable Design / 微生物学通报

By means of comparing biomasses of biodegradation fungi,Fusarium sp.HG-P-01 for ?-cypermethrin,a synthetic pyrethroid insecticide used widely in China,in five different media,the Czapek-Dox medium was selected as the best medium for mycelia growth.Furthermore,an experiment of central composite rotatable design(CCRD) was used to optimize the content of nutrient components.The optimal composition of C,N and P in media for HG-P-01 were 20.94 g/L,1.82 g/L and 1.66 g/L,respec-tively,in which an expectant or real rate of ?-cypermethrin-degradation got to 96.34% or 93.78% by HPLC for a concentration of 50 mg/L after 24 h treatment.The predicted value in degradation rate model was con-sistent with that from HPLC method.

Study on the Optimization of Enrichment Medium of Lactobacillus casei Zhang / 微生物学通报

The effects of different carbon source,nitrogen source,proportion of carbon and nitrogen source, microelement and buffer salts on the growth of Lactobacillus casei Zhang isolated from Koumiss were studied.The enrichment medium for Lactobacillus casei Zhang was optimized by response surface methodology and its composition was glucose 20.9 g/L,soy peptone 10.45 g/L,yeast extract 10.45 g/L,K2HPO4 3.5 g/L, sodium acetate 14.6 g/L,sodium citrate 2.35 g/L,MgSO4?7H2O 1.0 g/L,MnSO4?5H2O 54 mg/L, CuSO4?5H2O 10 mg/L,tween 80 1.0 g/L.After cultivated in enrichment medium for 18h at 37℃,the living cells of Lactobacillus casei Zhang were 4.78?109 CFU/mL,which was about 10 times higher than in MRS (4.8?10 8 CFU/mL).

Medium Optimization for Antitumor Agent Mycoepoxydiene by Marine Lignicolous Fungi Diaporthe sp / 微生物学通报

Mycoepoxydiene is a novel antitumor agent extracted from marine lignicolous fungi HLY-2, which is Diaporthe phaseolorum by molecule identification. The medium optimization for mycoepoxydiene by orthogonal design and the comparison of submerged fermentation and solid state fermentation were studied. The rusult is that the maximal yield of the compound is 543mg/L, which is 43 times compared to the customary half-seawater PD medium and 15 times to the best submerged condition. This optimum culture medium included potato 250g/L, seawater 300mL/L, glucose 30g/L, lactose 50g/L, KH_ 2 PO_ 4 0.65mmol/L and (NH_ 4 )_ 2 SO_ 4 1g/L in the solid state condition. Differentiation analysis between submerged and solid state fermentation, and antitumor activity of these ferment products were also studied. The antitumor activity of products of the optimum medium approached the pure compound.

Selection of High-yield Agro-antibiotic 2-16 Producing Strain and Optimization of Its Cultivation / 微生物学通报

In order to improve the production of agro-antibiotic 2-16,the producing strain(Streptomyces ahygroscopicus var.huangshanensis) was treated by protoplast regeneration,ultraviolet radiation,NTG mutagenesis and low energy C~(+) ion implantation.At last,a high-yield strain No.515 was obtained.The production of ~()No.515 was increased by 223.10%.By using Plackett-Burman design and Response Surface Analysis provided by SAS software,the cultivation condition of No.515 was optimized.The amount of agro-antibiotic 2-16 was increased by 38.53% when the strain No.515 was cultivated in the optimum medium instead of the initial one.

Optimization of Conditions on Submerged Fermentation of Bacillus cereus / 微生物学通报

The fermentation of Bacillus cereus DLSL-2 was investigated through single-factor test. The optimized fermentation conditions are: temperature 30℃,initial pH 7.0,250 r/min. Uniform design was used in shaking flask to optimize the fermentation medium of bacillus cereus . The most suitable medium was identified as follows:4.88% defated soybean power ,1.45% maize starch, 0.106% K 2HPO 4, 1.78% yeasts extract, 5.6% inoculum. the optimized medium allowed the B.cereus biomass concentration to be increased from 3.2?109 cfu/mL to 7.1?109 cfu/mL.