Medium Optimization for Exopolysaccharide Production in Liquid Culture of Endophytic Fungus Berkleasmium sp. Dzf12

Berkleasmium sp. Dzf12, an endophytic fungus from Dioscorea zingiberensis, is a high producer of spirobisnaphthalenes with various bioactivities. The exopolysaccharide (EPS) produced by this fungus also shows excellent antioxidant activity. In this study, the experimental designs based on statistics were employed to evaluate and optimize the medium for EPS production in liquid culture of Berkleasmium sp. Dzf12. For increasing EPS yield, the concentrations of glucose, peptone, KH₂PO₄, MgSO₄·7H₂O and FeSO₄·7H₂O in medium were optimized using response surface methodology (RSM). Both the fractional factorial design (FFD) and central composite design (CCD) were applied to optimize the main factors which significantly affected EPS production. The concentrations of glucose, peptone and MgSO₄·7H₂O were found to be the main effective factors for EPS production by FFD experimental analysis. Based on the further CCD optimization and RSM analysis, a quadratic polynomial regression equation was derived from the EPS yield and three variables. Statistical analysis showed the polynomial regression model was in good agreement with the experimental results with the determination coefficient (adj-R²) as 0.9434. By solving the quadratic regression equation, the optimal concentrations of glucose, peptone and MgSO₄·7H₂O for EPS production were determined as 63.80, 20.76 and 2.74 g/L, respectively. Under the optimum conditions, the predicted EPS yield reached the maximum (13.22 g/L). Verification experiment confirmed the validity with the actual EPS yield as 13.97 g/L, which was 6.29-fold in comparison with that (2.22 g/L) in the original basal medium. The results provide the support data for EPS production in large scale and also speed up the application of Berkleasmium sp. Dzf12.     

Role of Aeromonas hydrophila Flagella Glycosylation in Adhesion to Hep-2 Cells,Biofilm Formation and Immune Stimulation

Abstract: Polar flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be O-glycosylated with a heterogeneous heptasaccharide glycan. Two mutants with altered (light and strong) polar flagella glycosylation still able to produce flagella were previously obtained, as well as mutants lacking the O34-antigen lipopolysaccharide (LPS) but with unaltered polar flagella glycosylation. We compared these mutants, altogether with the wild type strain, in different studies to conclude that polar flagella glycosylation is extremely important for A. hydrophila adhesion to Hep-2 cells and biofilm formation. Furthermore, the polar flagella glycosylation is an important factor for the immune stimulation of IL-8 production via toll receptor 5 (TLR5).     

Enhancement of polysaccharide production by optimization of culture conditions in shake flask submerged cultivation of Grifola umbellata

Grifola umbellata (Fries) is a traditional Chinese medicinal mushroom. In this paper, the effects of cultural condition on the mycelial growth and exopolysaccharide (EPS) production were studied. Glucose was the best carbon source for mycelia growth and EPS production. Yeast extract was the best nitrogen source for mycelia growth, whereas skim milk as nitrogen source can remarkably improve EPS production. The optimal medium constituents for EPS production were as follows: glucose 3%, skim milk 0.2%, 0.1% KH₂PO₄, 0.1% MgSO₄, 7H₂O and 0.005% vitamin B1. The initial pH value of 5 was the most efficient to EPS production. The G. umbellata culture with skim milk as nitrogen source displayed a much higher specific EPS yield of 112.35 mg/g, accounting for a 4.36 times increase compared to that with combined nitrogen source medium.     

Co-Production of Fungal Biomass Derived Constituents and Ethanol from Citrus Wastes Free Sugars without Auxiliary Nutrients in Airlift Bioreactor

The potential of two zygomycetes fungi, Mucor indicus and Rhizopus oryzae, in assimilating citrus waste free sugars (CWFS) and producing fungal chitosan, oil, and protein as well as ethanol was investigated. Extraction of free sugars from citrus waste can reduce its environmental impact by decreasing the possibility of wild microorganisms growth and formation of bad odors, a typical problem facing the citrus industries. A total sugar concentration of 25.1 g/L was obtained by water extraction of citrus waste at room temperature, used for fungal cultivation in shake flasks and airlift bioreactor with no additional nutrients. In shake flasks cultivations, the fungi were only able to assimilate glucose, while fructose remained almost intact. In contrast, the cultivation of M. indicus and R. oryzae in the four-liter airlift bioreactor resulted in the consumption of almost all sugars and production of 250 and 280 g fungal biomass per kg of consumed sugar, respectively. These biomasses correspondingly contained 40% and 51% protein and 9.8% and 4.4% oil. Furthermore, the fungal cell walls, obtained after removing the alkali soluble fraction of the fungi, contained 0.61 and 0.69 g chitin and chitosan per g of cell wall for M. indicus and R. oryzae, respectively. Moreover, the maximum ethanol yield of 36% and 18% was obtained from M. indicus and R. oryzae, respectively. Furthermore, that M. indicus grew as clump mycelia in the airlift bioreactor, while R. oryzae formed spherical suspended pellets, is a promising feature towards industrialization of the process.     

Production of cellulases and β-glucosidase in Trichoderma reesei mutated by proton beam irradiation

To obtain mutant strains producing high levels of cellulases (FPase and CMCase) and ??-glucosidase, Trichoderma reesei KCTC 6950 was mutated by proton beam irradiation. Five mutants were selected out of 1,000 mutants of T.reesei treated with proton beam irradiation, based on their ability for enzyme production on a plate screening medium. In submerged cultures containing Mandel??s fermentation medium, the mutant strain T-2 (MT-2) demonstrated a 165% increase in the activity of FPase, a 146% increase in the activity of CMCase, and a 313% increase in the activity of ??-glucosidase, compared with the wild type strain. Additionally, the properties of high level ??-glucosidase produced by MT-2 were the same as those of the wild type strain, e.g., an optimum pH of 4.8, and an optimum temperature of 65 °C. Moreover, the protein concentrations of ??-glucosidase produced by the wild type strain and MT-2 were measured by SDS-PAGE, and then ??-glucosidase activities were detected by the MUG-zymogram assay.     

Effects of Plant Growth Hormones on Mucor indicus Growth and Chitosan and Ethanol Production

The objective of this study was to investigate the effects of indole-3-acetic acid (IAA) and kinetin (KIN) on Mucor indicus growth, cell wall composition, and ethanol production. A semi-synthetic medium, supplemented with 0–5 mg/L hormones, was used for the cultivations (at 32 °C for 48 h). By addition of 1 mg/L of each hormone, the biomass and ethanol yields were increased and decreased, respectively. At higher levels, however, an inverse trend was observed. The glucosamine fraction of the cell wall, as a representative for chitosan, followed similar but sharper changes, compared to the biomass. The highest level was 221% higher than that obtained without hormones. The sum of glucosamine and N-acetyl glucosamine (chitin and chitosan) was noticeably enhanced in the presence of the hormones. Increase of chitosan was accompanied by a decrease in the phosphate content, with the lowest phosphate (0.01 g/g cell wall) being obtained when the chitosan was at the maximum (0.45 g/g cell wall). In conclusion, IAA and KIN significantly enhanced the M. indicus growth and chitosan production, while at the same time decreasing the ethanol yield to some extent. This study shows that plant growth hormones have a high potential for the improvement of fungal chitosan production by M. indicus.     

产(R)-α-基苯乙酸的芽抱杆菌B26的鉴定与诱变育种(英文)

A bacterium strain B26 capable of producing(R)-α-hydroxyphenylacetic acid [(R)-HPA](yield,47.5%;enantiomeric excess,99.1%) from phenylglyoxylic acid(PGA) with high optical purity was isolated and identified as Bacillus sp.B26 by 16S rDNA(ribosomal DNA) sequencing.Phylogenic analysis showed that the strain was most similar to Bacillus sp.enrichment culture clone SYW5(FJ601635.1) and Bacillus cereus strain FM-4(EU794727.1).Efforts were made to further improve HPA-production and PGA-tolerance by UV irradiation and UV-LiCl cooperative mutagenesis.Among viable mutants,B.sp.UV-38 and B.sp.ULi-11 exhibited better productivities than the wild type.Comparisons of HPA production and time course among wild strain and two mutants showed that B.sp.ULi-11 was more competent than B.sp.UV-38.HPA production was increased by 39.1% with B.sp.ULi-11(yield,65.4%) compared to that with B.sp.B26(yield,47.0%) when cultured in fermentation broth(pH 7.2) at 32℃ with an agitation speed of 180 r·min-1 and PGA final concentration of 15 mmol·L-1 for 25 h.     

Mutation in the pssA Gene Involved in Exopolysaccharide Synthesis Leads to Several Physiological and Symbiotic Defects in Rhizobium leguminosarum bv. trifolii

The symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum bv. trifolii 24.2 secretes large amounts of acidic exopolysaccharide (EPS), which plays a crucial role in establishment of effective symbiosis with clover. The biosynthesis of this heteropolymer is conducted by a multi-enzymatic complex located in the bacterial inner membrane. PssA protein, responsible for the addition of glucose-1-phosphate to a polyprenyl phosphate carrier, is involved in the first step of EPS synthesis. In this work, we characterize R. leguminosarum bv. trifolii strain Rt270 containing a mini-Tn5 transposon insertion located in the 3′-end of the pssA gene. It has been established that a mutation in this gene causes a pleiotropic effect in rhizobial cells. This is confirmed by the phenotype of the mutant strain Rt270, which exhibits several physiological and symbiotic defects such as a deficiency in EPS synthesis, decreased motility and utilization of some nutrients, decreased sensitivity to several antibiotics, an altered extracellular protein profile, and failed host plant infection. The data of this study indicate that the protein product of the pssA gene is not only involved in EPS synthesis, but also required for proper functioning of Rhizobium leguminosarum bv. trifolii cells.     

Antibacterial Effect of Dental Adhesive Containing Dimethylaminododecyl Methacrylate on the Development of Streptococcus mutans Biofilm

Antibacterial bonding agents and composites containing dimethylaminododecyl methacrylate (DMADDM) have been recently developed. The objectives of this study were to investigate the antibacterial effect of novel adhesives containing different mass fractions of DMADDM on Streptococcus mutans (S. mutans) biofilm at different developmental stages. Different mass fractions of DMADDM were incorporated into adhesives and S. mutans biofilm at different developmetal stages were analyzed by MTT assays, lactic acid measurement, confocal laser scanning microscopy and scanning electron microscopy observations. Exopolysaccharides (EPS) staining was used to analyze the inhibitory effect of DMADDM on the biofilm extracellular matrix. Dentin microtensile strengths were also measured. Cured adhesives containing DMADDM could greatly reduce metabolic activity and lactic acid production during the development of S. mutans biofilms (p < 0.05). In earlier stages of biofilm development, there were no significant differences of inhibitory effects between the 2.5% DMADDM and 5% DMADDM group. However, after 72 h, the anti-biofilm effects of adhesives containing 5% DMADDM were significantly stronger than any other group. Incorporation of DMADDM into adhesive did not adversely affect dentin bond strength. In conclusion, adhesives containing DMADDM inhibited the growth, lactic acid production and EPS metabolism of S. mutans biofilm at different stages, with no adverse effect on its dentin adhesive bond strength. The bonding agents have the potential to control dental biofilms and combat tooth decay, and DMADDM is promising for use in a wide range of dental adhesive systems and restoratives.     

Improvement of fermentative production of exopolysaccharides from Aureobasidium pullulans under various conditions

The optimization of exopolysaccharide (EPS) production was investigated in the polymorphic fungal strain of Aureobasidium pullulans (KCTC 6081) with varying pH, nutrients concentration, and mixing parameters in batch fermentation condition. The maximum production of EPS (～7.5 g/L) was observed at pH 4, while optimum nutrient concentration of carbon (sucrose), nitrogen (NaNO₃), phosphorous (K₂HPO₄), and ascorbic acid was 50 g/L, 5 g/L, 1 g/L and 2 g/L, respectively. Interestingly, EPS productivity under non pH controlled fermentation conditions was 0.12 g/L/h with 400 rpm mixing, while under a controlled pH of 4, the EPS productivity was 0.21 g/L/h with 600 rpm, respectively. The fed-batch fermentation increased the EPS productivity up to 0.345 g/L/h with changing mixing conditions from 200 to 600 rpm and reached 47 g/L with 88% pullulan. Thus, pH and mixing were the key parameters for enhancing EPS production from A. pullulans. It is expected that these optimized parameters can be well used for enhanced industrial production of pullulan.     

Isolation of the Autoinducer-Quenching Strain that Inhibits LasR in Pseudomonas aeruginosa

Quorum sensing (QS) has been recognized as a general phenomenon in microorganisms and plays an important role in many pathogenic bacteria. In this report, we used the Agrobacterium tumefaciens biosensor strain NT1 to rapidly screen for autoinducer-quenching inhibitors from bacteria. After initial screening 5389 isolates obtained from land and beach soil, 53 putative positive strains were identified. A confirmatory bioassay was carried out after concentrating the putative positive culture supernatant, and 22 strains were confirmed to have anti-LasR activity. Finally, we determined the strain JM2, which could completely inhibit biofilm formation of Pseudomonas aeruginosa PAO1, belonged to the genus Pseudomonas by analysis of 16S rDNA. Partially purified inhibitor factor(s) F5 derived from culture supernatants specifically inhibited LasR-controlled elastase and protease in wild type P. aeruginosa PAO1 by 68% and 73%, respectively, without significantly affecting growth; the rhl-controlled pyocyanin and rhamnolipids were inhibited by 54% and 52% in the presence of 100 μg/mL of F5. The swarming motility and biofilm of PAO1 were also inhibited by F5. Real time RT-PCR on samples from 100 μg/mL F5-treated P. aeruginosa showed downregulation of autoinducer synthase (LasRI and rhlI) and cognate receptor (lasR and rhlR) genes by 50%, 28%, 48%, and 29%, respectively. These results provide compelling evidence that the F5 inhibitor(s) interferes with the las system and significantly inhibits biofilm formation.     

Application of a New Engineered Strain of Yarrowia lipolytica for Effective Production of Calcium Ketoglutarate Dietary Supplements

The present study aimed to develop a technology for the production of dietary supplements based on yeast biomass and α-ketoglutaric acid (KGA), produced by a new transformant of Yarrowia lipolytica with improved KGA biosynthesis ability, as well to verify the usefulness of the obtained products for food and feed purposes. Transformants of Y. lipolytica were constructed to overexpress genes encoding glycerol kinase, methylcitrate synthase and mitochondrial organic acid transporter. The strains were compared in terms of growth ability in glycerol- and oil-based media as well as their suitability for KGA biosynthesis in mixed glycerol–oil medium. The impact of different C:N:P ratios on KGA production by selected strain was also evaluated. Application of the strain that overexpressed all three genes in the culture with a C:N:P ratio of 87:5:1 allowed us to obtain 53.1 g/L of KGA with productivity of 0.35 g/Lh and yield of 0.53 g/g. Finally, the possibility of obtaining three different products with desired nutritional and health-beneficial characteristics was demonstrated: (1) calcium α-ketoglutarate (CaKGA) with purity of 89.9% obtained by precipitation of KGA with CaCO₃, (2) yeast biomass with very good nutritional properties, (3) fixed biomass-CaKGA preparation containing 87.2 μg/g of kynurenic acid, which increases the health-promoting value of the product.     

球孢白僵菌催化合成R-(+)-2-(4-羟基苯氧基)丙酸的菌种选育

[目的]为采用生物转化法合成R-(+)-2-(4-羟基苯氧基)丙酸(D-HPPA)提供优良菌株及理论基础。[方法]针对球孢白僵菌,先采用紫外(UV)诱变筛选高耐受R-(+)-2-苯氧基丙酸(D-PPA)的突变株,再使用等离子体(ARTP)诱变、N-甲基-N′-硝基-N-亚硝基胍(MNNG)诱变及ARTP和MNNG复合诱变提高突变株底物转化率。[结果]得到突变株UA1052,其底物转化率为98.5%、平均每天产物积累速率为6.93 g/(L·d)(较出发菌提高了796.6%),且遗传性稳定。[结论]ARTP诱变可大幅提高球孢白僵菌催化合成D-HPPA的能力。     

Production of docosahexaenoic acid by Crypthecodinium cohnii grown in a pH-auxostat culture with acetic acid as principal carbon source

Crypthecodinium cohnii, a marine alga used for the commercial production of docosahexaenoic acid (DHA), was cultivated in medium containing sodium acetate as principal carbon source; the pH was maintained at a constant value by addition of acetic acid, which also provided an additional carbon source in a controlled manner. The accumulation of lipid by C. cohnii in this pH-auxostat culture was significantly greater than previously reported for batch cultures using glucose as principal carbon source. Of six strains tested in pH-auxostat cultures, C. cohnii ATCC 30772 was the best, with the cells reaching 20 to 30 g dry weight per liter after 98 to 144 h and containing in excess of 40% (w/w) total lipid, with DHA representing approximately half of the total fatty acids in the triacylglycerol fraction. A productivity of 36 mg DHAL⁻¹ h⁻¹ was achieved during cultivation for 98 h using a 5% (vol/vol) inoculum, and DHA production was in excess of 3 g per liter of culture. Most of the DHA was present in neutral lipids.     

Eicosapentaenoic acid and docosahexaenoic acid production potential of microalgae and their heterotrophic growth

Twenty microalgal strains were investigated in photoautotrophic flask cultures for their potential for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) production. The highest EPA proportion (% of total fatty acids) was produced by Monodus subterraneus UTEX 151 (34.2%), followed by Chlorella minutissima UTEX 2341 (31.3%) and Phaeodactylum tricornutum UTEX 642 (21.4%). The highest DHA proportion (% of total fatty acids) was obtained in Crypthecodinium cohnii UTEX L1649 (19.9%), followed by Amphidinium carterae UTEX LB 1002 (17.0%) and Thraustochytrium aureum ATCC 28211 (16.1%). Among the 20 strains screened, the EPA yield was high in M. subterraneus UTEX 151 (96.3 mg/L), P. tricornutum UTEX 642 (43.4 mg/L), Chl. minutissima UTEX 2341 (36.7 mg/L), and Por. cruentum UTEX 161 (17.9 mg/L) owing to their relatively high biomass concentrations. The DHA yield was high in C. cohnii UTEX L1649 (19.5 mg/L) and A. carterae UTEX LB 1002 (8.6 mg/L). Heterotrophic growth of these 20 microalgae was also tested on two different carbon sources, acetate and glucose. All microalgae except Nannochloropsis oculata UTEX LB 2164 showed growth on glucose (5 g/L) under heterotrophic conditions. Twelve of them could grow heterotrophically when acetate (1 g/L) was used as their sole carbon and energy source.     

Gene Overexpression and RNA Silencing Tools for the Genetic Manipulation of the S-(+)-Abscisic Acid Producing Ascomycete Botrytis cinerea

The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+)-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers) and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression of bcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing of bcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain.     

Development of an Efficient Electroporation Method for Iturin A-Producing Bacillus subtilis ZK

In order to efficiently introduce DNA into B. subtilis ZK, which produces iturin A at a high level, we optimized seven electroporation conditions and explored an efficient electroporation method. Using the optimal conditions, the electroporation efficiency was improved to 1.03 × 10⁷ transformants/μg of DNA, an approximately 10,000-fold increase in electroporation efficiency. This efficiency is the highest electroporation efficiency for B. subtilis and enables the construction of a directed evolution library or the knockout of a gene in B. subtilis ZK for molecular genetics studies. In the optimization process, the combined effects of three types of wall-weakening agents were evaluated using a response surface methodology (RSM) design, which led to a two orders of magnitude increase in electroporation efficiency. To the best of our limited knowledge, this study provides the first demonstration of using an RSM design for optimization of the electroporation conditions for B. subtilis. To validate the electroporation efficiency, a case study was performed and a gene (rapC) was inactivated in B. subtilis ZK using a suicide plasmid pMUTIN4. Moreover, we found that the rapC mutants exhibited a marked decrease in iturin A production, suggesting that the rapC gene was closely related to the iturin A production.     

Enhancement of lipid productivity by ethyl methane sulfonate-mediated random mutagenesis and proteomic analysis in Chlamydomonas reinhardtii

Microalgae-derived biomass has been considered as the most promising candidate for next generation biofuel due to its sustainability and biodegradability. In this study, microalgal strain Chlamydmonas reinhardtii was randomly mutagenized by using a chemical mutagen, ethyl methane sulfonate (EMS) to create mutants showing enhanced lipid production. We identified three random mutants that displayed high lipid production in the screening using Nile red staining. Among those, mutant #128 was selected as candidate for further studies. Our flow cytometry and confocal microscopy analysis revealed that mutant #128 contains larger and more abundant lipid bodies than that of wild-type. Moreover, mutant #128 showed 1.4-fold increased fatty acid methyl ester (FAME) content compared to wild-type under nitrogen depleted condition. In addition, mutant #128 grew faster and accumulated more biomass, resulting in high lipid production. 2D gel electrophoresis and MALDI-TOF analysis used for gene targeting revealed that β-subunit of mitochondrial ATP Synthase and two-component response regulator PilR may be involved in enhanced characteristics of mutant #128. These results show the possibilities of EMS mediated random mutagenesis in generation of mutants to produce high amount of lipid as well as further study for molecular mechanism of mutants.     

ARTP诱变钝顶螺旋藻突变体比较组学研究

微藻作为地球上重要的生物资源,为水圈提供了大量的初级代谢产物,是合成生物学和生物制造研究和应用的重要底盘微生物。其中,钝顶螺旋藻(Spirulina platensis)具有多糖含量高、营养价值高、培养工艺成熟、应用范围广等特点,其诱变育种及比较组学研究可为微藻细胞工厂系统改造技术发展提供重要依据。本课题组前期通过常压室温等离子体(atmospheric and room temperature plasmas,ARTP)诱变方法获得了三株钝顶螺旋藻突变体。本研究在连续光照培养条件下,对三株突变体的重要生理特征进行了表征,发现突变株具有高絮凝表型,且重要化合物含量也与野生型具有一定的差异。进一步,本研究通过对其主要代谢产物的代谢组学分析和全基因组测序,对突变表型产生的机理进行了初步解析。