高产DHA破囊壶菌Aurantiochytrium sp. PKU#SW7诱变株的筛选

【目的】对野生菌株Aurantiochytrium sp.PKU#SW7诱变育种,筛选高产DHA突变株。【方法】采用UV诱变和化学药物胁迫筛选方式,以菌株的生物量、油脂产量、DHA产量作为筛选指标,获得高产DHA突变株。【结果】经鉴定获得一株DHA高产突变株PKU#PM003,该菌株传代4次后仍保持较好的遗传稳定性。摇瓶发酵后,PKU#PM003生物量产量高达6.62 g/L,比原始菌株5.95 g/L提高了11.26%,脂肪酸含量高达4.01 g/L,比原始菌株3.18 g/L提高了26.1%,DHA在脂肪酸中所占比例由29.97%增加到33.43%,产量提高了41.01%,油脂突变效果显著。【结论】突变株PKU#PM003可作为性状优良的工业化发酵生产菌种,并在DHA产量提升上仍具有巨大的空间。     

Screening of lipid high producing mutant from rhodotorula glutinis by low ion implantation and study on optimization of fermentation medium

In order to obtain lipid producing strain with high-yield, the wild type stain Rhodotorula glutinis was treated by low ion implantation, and optimization of fermentation medium for higher lipid yield was carried out using mutant strain. It was found that the strain had a higher positive mutation rate when the output power was 10 keV and the dose of N⁺ implantation was 80 × 2.6 × 10¹³ ions/cm². Then a high-yield mutant strain D30 was obtained through cid-heating coupling ultrasonic method and lipid yield was 3.10 g/L. Additionally, the surface response method was used to optimize fermentation medium. The three significant factors (glucose, peptone, KH₂PO₄) were optimized using response surface methodology (RSM), and the optimized parameters of fermentation medium were as follows: glucose 73.40 g/L, peptone 1.06 g/L and KH₂PO₄ 3.56 g/L. Finally the fermentation characteristic of high-yield mutation strain D30 was studied, when fermentation time was 10 days, which lipid yield increased to 7.81 g/L. Fatty acid composition of the lipid was determined by GC, and the most represented fatty acids of mutant D30 were C16:0 (11.4 %), C16:1 (5.66 %), C18:1 (49.3 %), and C18:2 (27.0 %).     

Mutagenesis of echinocandin B overproducing Aspergillus nidulans capable of using starch as main carbon source

Abstract

Echinocandin B, a kind of antimycotic with cyclic lipo-hexapeptides, was produced by fermentation with Aspergillus nidulans using fructose as main carbon source. The objective of this study was to screen a high-yield mutant capable of using cheap starch as main carbon source by atmospheric and room temperature plasma (ARTP) treatment in order to decrease the production cost of echinocandin B. A stable mutant A. nidulans ZJB19033, which can use starch as optimal carbon source instead of expensive fructose, was selected from two thousands isolates after several cycles of ARTP mutagenesis. To further increase the production of echinocandin B, the optimization of fermentation medium was performed by response surface methodology (RSM), employing Plackett-Burman design (PBD) followed by Box-Behnken design (BBD). The optimized fermentation medium provided the optimal yield of echinocandin B, 2425.9?±?43.8?mg/L, 1.3-fold compared to unoptimized medium. The results indicated that the mutant could achieve high echinocandin B production using cheap starch as main carbon source, and the cost of carbon sources in fermentation medium reduced dramatically by about 45%.     

Strain development and medium optimization for fumaric acid production

Rhizopus oryzae RUR709 mutant was isolated based on halo size from selection medium via mutagenesis with UV and γ-rays, and the production of fumaric acid in the submerged fermentation was assessed. The maximum concentration of fumaric acid was obtained using 0.5% corn steep liquor (CSL) as the nitrogen source. Organic nitrogen sources were shown to be more effective in fumaric acid production than inorganic nitrogen sources. Using optimum medium obtained by response surface methodology (RSM), the maximum concentration of fumaric acid achieved in flask culture was 26.2 g/L, which is fairly close to the 27.4 g/L predicted by the model. The highest concentration of fumaric acid in the stirred-tank reactor generated by the R. oryzae RUR709 mutant was 32.1 g/L and yield (0.45 g/g) and productivity (0.32 g/L/h) were highest at 4 days.     

高产吩嗪-1-羧酸（PCA）假单胞菌株M18G发酵优化模型的构建

吩嗪-1-羧酸（phenazine-1-carboxylic acid, PCA）是促进根际生长假单胞菌分泌的重要抗菌物质。采用Plackett-Burman（PB）设计和响应面法（response surface method, RSM）对假单胞菌株M18（Pseudomonas sp. M18）的gacA基因突变株M18G的次级代谢产物PCA发酵的营养条件进行建模。运用Plackett-Burman（PB）设计试验,从12个营养成分中筛选出4个关键的组分,进而采用RSM 法对这4个因素进行中心组合设计试验,建立回归方程并进行统计学分析,绘制各营养因子之间的关系图。实验结果表明：建立的模型能合理地模拟并优化发酵中各参数及其浓度,确定发酵培养基的成分和浓度为：黄豆粉33.4g/L,葡萄糖12.7g/L,大豆蛋白胨10.9g/L和乙醇13.8 g/L, M18G菌株经60 h发酵培养,最高PCA产率能达到1.89 g/L,比优化前提高了6倍左右。各营养因子的等值线图表明黄豆粉和乙醇在PCA高产发酵中起到更为关键的作用,因此提供了提高PCA发酵产量的有效方法,并为其未来商业化应用奠定了基础。     

玉米原料高产γ-聚谷氨酸优良菌株的选育及发酵条件优化

以实验室筛选到的一株枯草芽孢杆菌（Bacillus subtilis）B-1为出发菌株,采用紫外诱变技术对出发菌株进行反复诱变,得到一株能够利用玉米原料生产γ-聚谷氨酸的优良高产菌株B-115,摇瓶发酵γ-聚谷氨酸的产量由原菌株的12.5g/L提高到19.5g/L。再以该菌株为研究对象利用响应面法进行碳源、氮源、谷氨酸钠、金属离子等发酵条件的优化实验,经48h摇瓶发酵,γ-聚谷氨酸产量达到40.98g/ L。     

基于人工神经网络-遗传算法的樟芝发酵培养基优化

采用优化模型对药用丝状真菌樟芝的复杂发酵过程进行建模,并获得最优发酵培养基组成.对樟芝发酵过程中的形态变化过程进行了观察,并分别采用人工神经网络(ANN)和响应面法(RSM)对樟芝发酵过程进行建模,同时采用遗传算法(GA)优化了发酵培养基组成.结果表明,ANN模型比RSM模型具有更好的实验数据拟合能力和预测能力,GA计算得到樟芝生物量理论最大值为6.2 g/L,并获得发酵最佳接种量及培养基组成:孢子浓度1.76× 105个/mL,葡萄糖29.1 g/L,蛋白胨9.4 g/L,黄豆粉2.8 g/L.在最佳培养条件下,樟芝生物量为(6.1±0.2)g/L.基于ANN-GA的优化方法可用于优化其他丝状真菌的复杂发酵过程,从而获得生物量或活性代谢产物.     

Combining classical, genetic, and process strategies for improved precursor-directed production of 6-deoxyerythronolide B analogues

A process for the production of erythromycin aglycone analogues has been developed by combining classical strain mutagenesis techniques with modern recombinant DNA methods and traditional process improvement strategies. A Streptomyces coelicolor strain expressing the heterologous 6-deoxyerythronolide B (6-dEB) synthase (DEBS) for the production of erythromycin aglycones was subjected to random mutagenesis and selection. Several strains exhibiting 2-fold higher productivities and reaching >3 g/L total macrolide aglycones were developed. These mutagenized strains were cured of the plasmid carrying the DEBS genes and a KS1 degrees mutant DEBS operon was introduced for the production of novel analogues when supplemented with a synthetic diketide precursor. The strains expressing the mutant DEBS were screened for improved 15-methyl-6-dEB production, and the best clone, strain B9, was found to be 50% more productive as compared to the parent host strain used for 15-methyl-6-dEB production. Strain B9 was evaluated in 5-L fermenters to confirm productivity in a scalable process. Although peak titers of 0.85 g/L 15-methyl-6-dEB by strain B9 confirmed improved productivity, it was hypothesized that the low solubility of 15-methyl-6-dEB limited productivity. The solubility of 15-methyl-6-dEB in water was determined to be 0.25-0.40 g/L, although higher titers are possible in fermentation medium. The incorporation of the hydrophobic resin XAD-16HP resulted in both the in situ adsorption of the product and the slow release of the diketide precursor. The resin-containing fermentation achieved 1.3 g/L 15-methyl-6-dEB, 50% higher than the resin-free process. By combining classical mutagenesis, recombinant DNA techniques, and process development, 15-methyl-6-dEB productivity was increased by over 100% in a scalable fermentation process.     

阿维菌素生产菌的常压室温等离子体诱变育种及培养基优化

【目的】通过诱变筛选技术选育阿维菌素高产突变株,对其发酵培养基进行响应面优化,提高阿维菌素产量。【方法】采用常压室温等离子体(ARTP)诱变技术,结合链霉抗性和卡那霉素抗性筛选法及96深孔板高通量筛选法,筛选阿维菌素高产株。在单因素实验的基础上,应用响应面分析法对其发酵培养基进行优化,最后确定最佳培养基配方。【结果】获得一株遗传性状稳定的阿维菌素高产株K-1A6,其阿维菌素产量达到4.22 g/L,比出发菌株9-39提高了23.4%,在最佳培养基中阿维菌素产量达到5.36 g/L,较优化前提高了27.01%。【结论】通过对阿维链霉菌9-39菌株进行ARTP诱变筛选及发酵培养基优化研究能显著提高阿维菌素的产量。     

Production, characterization and antioxidant activity of exopolysaccharides from submerged culture of Morchella crassipes

The aim of this work was to investigate the fermentation optimization, molecular characterization, and antioxidant activity in vitro of exopolysaccharides (EPS) from Morchella crassipes in submerged culture. Firstly, an optimal medium for EPS production was obtained by single-factor experiment and central composite design as follows: maltose 44.79?g/L and tryptone 4.21?g/L. Then, one fraction of EPS was obtained from the culture filtrates by size exclusion chromatography and the molecular characteristics were examined by a multi-angle laser light scattering and refractive index detector system. The weight-average molar mass and the polydispersity ratio of the EPS fraction were revealed to be 1.961?×?10(4)?g/mol and 1.838, respectively. FT-IR spectroscopy was used for obtaining vibrational spectra of the purified EPS fraction. Finally, the antioxidant activity of EPS was investigated and the relationship with molecular properties was discussed as well.     

Development of a New <Emphasis Type="Italic">Amycolatopsis orientalis</Emphasis> Strain for High-Yield Eremomycin Production

A multistep random UV mutagenesis of an eremomycin-producing strain Amycolatopsis orientalis VKPM Ac-1125 with rather low productivity (0.5 g/L) and the further selection of the most high-yield mutant strains have resulted in a highly productive E 13-26 strain. After optimization of the fermentation medium by multifactor analysis, the most efficient composition for the fermentation medium was established, %: glycerol, 8; soybean meals, 2.0; MgCl₂, 0.02; KH₂PO₄, 0.02; KNO₃, 0.6; CaCl₂, 0.3; skim milk powder, 0.46; and glucose, 0.8. The maximum productivity of this strain on the optimized medium reached 3.42 ± 0.17 g/L. Therefore, the obtained strain is considered to be very promising for further use in the selection and development of technology for the large-scale eremomycin production.     

Production of 2,3-Butanediol from Sucrose by a <Emphasis Type="Italic">Klebsiella</Emphasis> Species

Chemical 2,3-butanediol is an important platform compound possessing diverse industrial applications. So far, it is mainly produced by using petrochemical feedstock which is associated with high cost and adverse environmental impacts. Hence, finding alternative routes (e.g., via fermentation using renewable carbon sources) to produce 2,3-butanediol are urgently needed. In this study, we report a wild-type Klebsiella sp. strain XRM21, which is capable of producing 2,3-butanediol from a wide variety of carbon sources including glucose, sucrose, xylose, and glycerol. Among them, fermentation of sucrose leads to the highest production of 2,3-butanediol. To maximize the production of 2,3-butanediol, fermentation conditions were first optimized for strain XMR21 by using response surface methodology (RSM) in batch reactors. Subsequently, a fed-batch fermentation strategy was designed based on the optimized parameters, where 91.2 g/L of 2,3-butanediol could be produced from substrate sucrose dosing in 100 g/L for three times. Moreover, random mutagenesis of stain XMR21 resulted in a highly productive mutant strain, capable of producing 119.4 and 22.5 g/L of 2,3-butanediol and ethanol under optimized fed-batch fermentation process within 65 h with a total productivity of 2.18 g/L/h, which is comparable to the reported highest 2,3-butanediol concentration produced by previous strains. This study provides a potential strategy to produce industrially important 2,3-butanediol from low-cost sucrose.     

Response surface methods for optimizingSaccharopolyspora spinosa,a novel macrolide producer

Summary Strain A83543, recently identified asSaccharopolyspora spinosa, was cultured in a variety of media to optimize macrolide titer. Response surface methodology (RSM) was used to improve the fermentation medium and to characterize the microorganism's response to systematic variations in medium composition. Three sequential RSM studies on wild-type A83543 and two high macrolide-producing mutants showed that each strain produced maximum titers in nearly identical fermentation media. No obvious differences in nutrient requirements were evident in the three strains indicating little interaction between mutational change and medium composition through at least two cycles of mutagenesis. The overall increase in macrolide titer starting from the wild-type organism in the original fermentation medium to the second-generation mutant in the optimized medium was over 25-fold.     

Citric acid production by selected mutants of Aspergillus niger from cane molasses

The present investigation deals with citric acid production by some selected mutant strains of Aspergillus niger from cane molasses in 250 ml Erlenmeyer flasks. For this purpose, a conidial suspension of A. niger GCB-75, which produced 31.1 g/l citric acid from 15% (w/v) molasses sugar, was subjected to UV-induced mutagenesis. Among the 3 variants, GCM-45 was found to be a better producer of citric acid (50.0 +/- 2a) and it was further improved by chemical mutagenesis using N-methyl, N-nitro-N-nitroso-guanidine (MNNG). Out of 3,2-deoxy-D-glucose resistant variants, GCMC-7 was selected as the best mutant, which produced 96.1 +/- 1.5 g/l citric acid 168 h after fermentation of potassium ferrocyanide and H2SO4 pre-treated blackstrap molasses in Vogel's medium. On the basis of kinetic parameters such as volumetric substrate uptake rate (Qs), and specific substrate uptake rate (qs), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and produced more citric acid. The mutant GCMC-7 has greater commercial potential than the parental strain with regard to citrate synthase activity. The addition of 2.0 x 10(-5) M MgSO4 x 5H2O into the fermentation medium reduced the Fe2+ ion concentration by counter-acting its deleterious effect on mycelial growth. The magnesium ions also induced a loose-pelleted form of growth (0.6 mm, diameter), reduced the biomass concentration (12.5 g/l) and increased the volumetric productivity of citric acid monohydrate (113.6 +/- 5 g/l).     

Optimization of liquid-state fermentation conditions for the glyphosate degradation enzyme production of strain Aspergillus oryzae by ultraviolet mutagenesis

This study aimed to obtain strains with high glyphosate-degrading ability and improve the ability of glyphosate degradation enzyme by the optimization of fermentation conditions. Spore from Aspergillus oryzae A-F02 was subjected to ultraviolet mutagenesis. Single-factor experiment and response surface methodology were used to optimize glyphosate degradation enzyme production from mutant strain by liquid-state fermentation. Four mutant strains were obtained and named as FUJX 001, FUJX 002, FUJX 003, and FUJX 004, in which FUJX 001 gave the highest total enzyme activity. Starch concentration at 0.56%, GP concentration at 1,370?mg/l, initial pH at 6.8, and temperature at 30°C were the optimum conditions for the improved glyphosate degradation endoenzyme production of A. oryzae FUJX 001. Under these conditions, the experimental endoenzyme activity was 784.15?U/100?ml fermentation liquor. The result (784.15?U/100?ml fermentation liquor) was approximately 14-fold higher than that of the original strain. The result highlights the potential of glyphosate degradation enzyme to degrade glyphosate.     

黄嘌呤氧化酶产生菌的离子注入诱变及其二步发酵的研究

采用剂量 1.0× 10 1 2 ～ 1.0× 10 1 6ions cm2 的N+注入产黄嘌呤氧化酶的球形节杆菌ATCC80 10 ,研究其诱变效果。结果表明 :细菌的存活曲线呈现特殊的“马鞍形” ,菌落形态和颜色发生了变化 ,且变化与产酶量有关 ,经过筛选获得了遗传性能稳定的高酶活突变株 ,使酶活较出发菌提高了 43.0 %。改进发酵工艺 ,采用二步发酵其酶得率是一步发酵的 4.75倍。     

N+ ion beam implantation of tannase-producing Aspergillus niger and optimization of its process parameters under submerged fermentation

Low-energy ion implantation as a novel mutagen has been increasingly applied in the microbial mutagenesis for its higher mutation frequency and wider mutation spectra. In this work, N⁺ ion beam implantation was used to enhance Aspergillus niger TA9701 in tannase yield. The optimization of process parameters under submerged fermentation was carried out to further improve the tannase yield of the mutant, Aspergillus niger J-T18. The results indicate that an excellent mutant J-T18 with a yield of 38.5 IU/mL, that is five times that of the original strain, was achieved by nine successive implantations under the conditions of 10 keV and 30–40 (×2.6?×?10¹³) ions/cm². This optimization further increased the yield of the mutant by 42 %, i.e. 53.6 U/mL which occurred in the mutant cultivated in the optimal fermentation culture medium composed of: rice flour 5 %; ammonium sulfate 1 %; tannic acid 2 %; calcium carbonate 0.5 %; manganese sulfate 0.1 %; and dipotassium phosphate 0.3 %; incubated at 30°C and 180 rpm for 72 h.     

Ethanol production from xylose by Fusarium oxysporum and the optimization of culture conditions

Bioethanol is the most commonly used renewable biofuel as an alternative to fossil fuels. Many microbial strains can convert lignocellulosics into bioethanol. However, very few natural strains with a high capability of fermenting pentose sugars and simultaneously utilizing various sugars have been reported. In this study, fermentation of sugar by Fusarium oxysporum G was performed for the production of ethanol to improve the performance of the fermentation process. The influences of pH, substrate concentration, temperature, and rotation speed on ethanol fermentation are investigated. The three significant factors (pH, substrate concentration, and temperature) are further optimized by quadratic orthogonal rotation regression combination design and response surface methodology (RSM). The optimum conditions are pH 4, 40?g/L of xylose, 32?°C, and 110?rpm obtained through single factor experiment design. Finally, it is found that the maximum ethanol production (10.0?g/L) can be achieved after 7 d of fermentation under conditions of pH 3.87, 45.2?g/L of xylose, and 30.4?°C. Glucose is utilized preferentially for the glucose–xylose mixture during the initial fermentation stage, but glucose and xylose are synchronously consumed without preference in the second period. These findings are significant for the potential industrial application of this strain for bioethanol production.     

Optimization of fermentation parameters in phage production using response surface methodology

Previously, we used computer-controlled fermentation technology to improve the yield of filamentous phage produced in Escherichia coli by 10-fold (Grieco et?al., Bioprocess Biosyst Eng 32:773-779, 2009). In the current study, three major fermentation parameters (temperature, dissolved oxygen [DO], and pH) were investigated using design of experiments (DOE) methodology. Response surface methodology (RSM) was employed to create a process model and determine the optimal conditions for maximal phage production. The experimental data fitted best to a quadratic model (p?相似文献   

Improved thermostable α-amylase activity of Bacillus amyloliquefaciens by low-energy ion implantation

Thermostable α-amylase is of great importance in the starch fermentation industry; it is extensively used in the manufacture of beverages, baby foods, medicines, and pharmaceuticals. Bacillus amyloliquefaciens produces thermostable α-amylase; however, production of thermostable α-amylase is limited. Ion-beam implantation is an effective method for mutation breeding in microbes. We conducted ion-beam implantation experiments using two different ions, Ar(+) and N(+), to determine the survival rate of and dose effect on a high α-amylase activity strain of B. amyloliquefaciens that had been isolated from soil samples. N(+) implantation resulted in a higher survival rate than Ar(+) implantation. The optimum implantation dose was 2.08 × 10(15) ions/cm(2). Under this implantation condition, we obtained a thermally and genetically stable mutant α-amylase strain (RL-1) with high enzyme activity for degrading α-amylase. Compared to the parental strain (RL), the RL-1 strain had a 57.1% increase in α-amylase activity. We conclude that ion implantation in B. amyloliquefaciens can produce strains with increased production of thermostable α-amylase.