High-throughput sequencing reveals miRNA effects on the primary and secondary production properties in long-term subcultured Taxus cells.

Plant-cell culture technology is a promising alternative for production of high-value secondary metabolites but is limited by the decreased metabolite production after long-term subculture. The goal of this study was to determine the effects of miRNAs on altered gene expression profiles during long-term subculture. Two Taxus cell lines, CA (subcultured for 10 years) and NA (subcultured for 6 months), were high-throughput sequenced at the mRNA and miRNA levels. A total of 265 known (78.87% of 336) and 221 novel (79.78% of 277) miRNAs were differentially expressed. Furthermore, 67.17% of the known differentially expressed (DE) miRNAs (178) and 60.63% of the novel DE-miRNAs (134) were upregulated in NA. A total of 275 inverse-related miRNA/mRNA modules were identified by target prediction analysis. Functional annotation of the targets revealed that the high-ranking miRNA targets were those implicated in primary metabolism and abiotic or biotic signal transduction. For example, various genes for starch metabolism and oxidative phosphorylation were inversely related to the miRNA levels, thereby indicating that miRNAs have important roles in these pathways. Interestingly, only a few genes for secondary metabolism were inversely related to miRNA, thereby indicating that factors other than miRNA are present in the regulatory system. Moreover, miR8154 and miR5298b were upregulated miRNAs that targeted a mass of DE genes. The overexpression of these miRNAs in CA increased the genes of taxol, phenylpropanoid, and flavonoid biosynthesis, thereby suggesting their function as crucial factors that regulate the entire metabolic network during long-term subculture. Our current studies indicated that a positive conversion of production properties from secondary metabolism to primary metabolism occurred in long-term subcultured cells. miRNAs are important regulators in the upregulation of primary metabolism.

Improved beta-carotene and lycopene production by Blakeslea trispora with ultrasonic treatment in submerged fermentation.

Response surface methodology (RSM) based on Box-Behnken design (BBD) was employed to investigate the effect of ultrasonic treatment on beta-carotene production by Blakeslea trispora. The optimized strategy involved exposing three-day-old mycelial cultures to ultrasonic treatment at a fixed frequency of 20 kHz, power of 491 W, treatment time of 3 min, working time of 3 s, and rest time of 5.8 s, repeated four times at a 24-h interval. Mycelium growth was not significantly promoted under ultrasonic stimulation; however, the glucose metabolism increased by about 10%, the average size of the aggregates significantly decreased, and the uptake rate of imidazole into cells was increased about 2.5-fold. After a 6-d culture, the technique produced 173 mg/L of beta-carotene and 82 mg/L of lycopene, which represented an increase of nearly 40.7% and 52.7%, respectively, over the yields obtained in cultures without ultrasonic treatment.

Oxidative stress response of Blakeslea trispora induced by H2O2 during ß-carotene biosynthesis.

The cellular response of Blakeslea trispora to oxidative stress induced by H2O2 in shake flask culture was investigated in this study. A mild oxidative stress was created by adding 40 µm of H2O2 into the medium after 3 days of the fermentation. The production of ß-carotene increased nearly 38 % after a 6-day culture. Under the oxidative stress induced by H2O2, the expressions of hmgr, ipi, carG, carRA, and carB involving the ß-carotene biosynthetic pathway all increased in 3 h. The aerobic metabolism of glucose remarkably accelerated within 24 h. In addition, the specific activities of superoxide dismutase and catalase were significantly increased. These changes of B. trispora were responses for reducing cell injury, and the reasons for increasing ß-carotene production caused by H2O2.

Pharmacokinetics and risk evaluation of DNA vaccine against Schistosoma japonicum.

DNA plasmid immunization is a novel approach of preventive and therapeutic vaccine. More than 100 DNA vaccines have been on preclinical or clinical phase trials, and four kinds of DNA vaccines for livestock have been approved by USDA, CFIA, and APVMA. Schistosomiasis is a worldwide parasitic disease, and vaccine immunization is supposed to be a promising approach to control the health crisis. On the basis of former preclinical studies, we further focused on the pharmacokinetics and risk evaluation of DNA vaccine in vivo. In the present study, enhanced green fluorescent protein (EGFP) report gene was fused with Schistosoma japonicum 23 kDa transmembrane protein antigen gene (Sj23) and constructed into DNA vaccine pVIVO2-Sj23.EGFP. After intramuscularly injecting 100 µg of purified DNA vaccine plasmid to immunizate BALB/c mice, we studied the tissue distribution of DNA plasmid and expressed Sj23.EGFP antigen, the persistence time of elicited antibodies, and the risk of DNA vaccine transferred into intestinal microorganisms. The results showed that DNA vaccine plasmid could be distributed into all tissues of the body after injection; however, only few organs including the injected muscle were detected DNA vaccine at postimmunization until the 100 days by PCR technology; the detection of green fluorescence protein displayed that DNA vaccine could be expressed in almost every tissue and organs; the ELISA assay indicated the immune antibody against Sj23 could persist over 70 days; and the DNA vaccine transferring intestinal flora results was negative. The results indicated that the DNA vaccine has systemic protection and long-lasting effectivity and is safe to intestinal flora.

Expression of carotenogenic genes and astaxanthin production in Xanthophyllomyces dendrorhous as a function of oxygen tension.

This report gives an insight into the specific changes in the transcription of four key carotenogenic genes [encoding geranylgeranyl diphosphate synthase (crtE), phytoene desaturase (crtI), phytoene synthase lycopene cyclase (crtYB), and astaxanthin synthase (ast), respectively] in Xanthophyllomyces dendrorhous cultures, with regard to dissolved oxygen (DO) contents of 10%, 25%, and 40% air saturation, respectively. 25% DO proved to be the most beneficial for yeast growth, transcription of carotenogenic genes, and astaxanthin content.

Construction, purification, and evaluation of multivalent DNA vaccine against Schistosoma japonicum.

DNA vaccine encoding a multivalent antigen is a novel approach of protective immunization. Four Schistosoma japonicum candidate antigen genes, glyceraldehyde-3-phosphate dehydrogenase (SjGAPDH), 23 kDa transmembrane protein (Sj23), 14 kDa fatty-acid binding protein (SjFABP) and 26 kDa glutathione-S-transferase (Sj26), are recombined into two pieces of fusion genes SjFABP.Sj23 and Sj26.SjGAPDH, respectively. Tetravalent DNA vaccine pVIVO2-SjFABP.Sj23/Sj26.SjGAPDH is constructed by co-expressing these two fusion genes. The super-coiled DNA vaccines for large-scale clinic application were purified by sequential chromatographies including group separation chromatography and affinitive chromatographies. The purified DNA vaccines were evaluated for in vivo and in vitro transfection assay. The immunoprotective properties of the different kinds of constructed DNA vaccines were appraised by pharmacological trials. The pharmacological trials results showed that tetravalent DNA vaccine has higher protective efficiency than other tested DNA vaccines.

Citrus residues isolates improve astaxanthin production by Xanthophyllomyces dendrorhous.

The wild strain and two astaxanthin-overproducing mutant strains, W618 and GNG274, of Xanthophyllomyces dendrorhous were analyzed in order to assess their ability to grow and synthesize astaxanthin in a minimal medium containing (per liter): 2 g KH2PO4, 0.5 g MgSO4, 2 g KNO3, and 1 g yeast extract, and supplemented with citrus residues isolates as a carbon source (citrus medium). The selected strain W618 was evaluated under various contents of citrus juice. At the content of 20% (v/v), the highest astaxanthin production reached 22.63 mg L(-1), which was two-fold more than that observed in yeast malt medium. Addition of 8% (v/v) n-hexadecane to the citrus medium was found to be optimal, increasing the astaxanthin yield by 21.7%.

Analysis and identification of astaxanthin and its carotenoid precursors from Xanthophyllomyces dendrorhous by high-performance liquid chromatography.

This study presents an HPLC method for simultaneous analysis of astaxanthin and its carotenoid precursors from Xanthophyllomyces dendrorhous. The HPLC method is accomplished by employing a C18 column and the mobile phase methanol/water/acetonitrile/ dichloromethane (70:4:13:13, v/v/v/v). Astaxanthin is quantified by detection at 480 nm. The carotenoid precursors are identified by LC-APCI-MS and UV-vis absorption spectra. Peaks showed in the HPLC chromatogram are identified as carotenoids in the monocyclic biosynthetic pathway or their derivatives. In the monocyclic carotenoid pathway, 3,3'-dihydroxy-beta,psi-carotene-4,4'-dione (DCD) is produced through gamma-carotene and torulene.

Microbial production of docosahexaenoic acid by a low temperature-adaptive strain Thraustochytriidae sp. Z105: screening and optimization.

As an alternative source in addition to fish oil, microbial production of docosahexaenoic acid has been recieved more and more attentions owing to their culture advantage. A unicellular eukaryotic microbe with high DHA production and capable of low temperature-adaptive growth was isolated from seawater and identified as Thraustochytriidae sp. Z105. The siginificant effect of temperature on cell growth and DHA synthesis by the strain was revealed. It could grow and produce DHA even at 4 degrees C, but hardly grow above 35 degrees C. Low temperature (15-25 degrees C) was favorable for formation of biomass, lipids and DHA, but DHA synthesis was completely blocked above 30 degrees C. Conditions for high level DHA production by Thraustochytriidae sp. Z105 in flask culture were optimized as follows: medium containing glucose 80 g/l, yeast extract 5.0 g/l, K2HPO(4) . 3 H2O 1.0 g/l, MgSO4 . 7 H2O 0.5 g/l, seawater crystal 20 g/l, pH 6.0, liquid volume 30 ml/250 ml, temperature 20 degrees C, agitation speed of 200 r/min, and culture for 120 h. Under the optimal conditions, biomass of 16.72 g/l, total lipids of 5.35 g/l, DHA yield of 1.71 g/l (accounting for 32% of the total lipids) were achieved, respectively. In flask cluture level, the DHA productivity of Thraustochytriidae sp. Z105 was higher than most reported results, which suggested the wild type strain was a potential superior candidate for industrialization of DHA production. Moreover, the strain is an unique and valuable resource for investigation of the low temperature adaptive mechanism related to DHA synthesis.

Fermentative production of L(+)-lactic acid using hydrolyzed acorn starch, persimmon juice and wheat bran hydrolysate as nutrients.

The use of hydrolyzed acorn starch as a novel carbon source for L(+)-lactic acid production was proposed. The effects of carbon-nitrogen ratio and growth factor on the fermentations were studied by single factor experiments. A lower carbon-nitrogen ratio could enhance L(+)-lactic acid production, and the expensive yeast extract could be replaced by the cheap persimmon juice providing growth factor for L(+)-lactic acid production when wheat bran hydrolysate was used as the nitrogen source. The dosages of wheat bran hydrolysate and persimmon juice in the medium were statistically optimized by response surface methodology (RSM). The yield of L(+)-lactic acid reached 45.78g/100g dry acorn with a final concentration of 57.61+/-1.37g/l and a productivity of 1.60+/-0.12g/lh when the batch fermentation was carried out in a 5l bioreactor under the optimal conditions of wheat bran hydrolysate 24.55g/l and persimmon juice 12.30g/l. Comparative batch fermentations using different raw materials such as acorn, cassava, corn and glucose showed that both the yield and the productivity of L(+)-lactic acid production were the highest when the hydrolyzed acorn starch was used as the carbon source. Therefore, the acorn could be used as a new substitute of grain raw material in L(+)-lactic acid production.

An economical approach for d-lactic acid production utilizing unpolished rice from aging paddy as major nutrient source.

In order to reduce the raw material cost of d-lactic acid fermentation, the unpolished rice from aging paddy was used as major nutrient source in this study. The unpolished rice saccharificate, wheat bran powder and yeast extract were employed as carbon source, nitrogen source and growth factors, respectively. Response surface methodology (RSM) was applied to optimize the dosages of medium compositions. As a result, when the fermentation was carried out under the optimal conditions for wheat bran powder (29.10g/l) and yeast extract (2.50g/l), the d-lactic acid yield reached 731.50g/kg unpolished rice with a volumetric production rate of 1.50g/(lh). In comparison with fresh corn and polished rice, the d-lactic acid yield increased by 5.79% and 8.71%, and the raw material cost decreased by 65% and 52%, respectively, when the unpolished rice was used as a major nutrient source. These results might provide a reference for the industrial production of d-lactic acid.

[Kinetic model for optimal feeding strategy in astaxanthin production by Xanthophyllomyces dendrorhous].

Astaxanthin is a useful pigmentation source in fish aquaculture. It has strong antioxidative activity and therefore has potential application in delaying aging and degenerative diseases in human and animals. In recent years, there is a growing demand for astaxanthin. The red yeast Xanthophyllomyces dendrorhous (called Phaffia rhodozyma before) is one of the most promising microorganisms for the commercial production of astaxanthin. During fermentation, X. dendrorhous shows the Crabtree effect. Higher glucose concentration will cause significant reductions in biomass and astaxanthin production. Therefore, fed-batch processes are particularly useful. In this paper, effects of glucose-feeding strategies on astaxanthin production by X. dendrorhous were studied. Based on the substrate inhibition model, an optimized two-stage feeding strategy for astaxanthin production of high-cell-density fermentation was proposed. Glucose concentration was first controlled at about 25 g/L during the lag phase and the early exponential phase. In such case, biomass could reach its maximum value in relatively short time. Then the glucose concentration was controlled at about 5 g/L in the later exponential phase and stationary phase. The synthesis of astaxanthin could be effectively prolonged. The results showed that the optimized two-stage feeding strategy was the best among all the feeding strategies, and could obtain the highest biomass (23.8 g/L) and astaxanthin production (29.05 mg/L), which was a significant increase (52.8% and 109% respectively) compared with a batch process.