Promoting Photosynthetic Production of Dammarenediol-II in Chlamydomonas reinhardtii via Gene Loading and Culture Optimization.

Ginsenosides are major bioactive compounds found in Panax ginseng that exhibit various pharmaceutical properties. Dammarenediol-II, the nucleus of dammarane-type ginsenosides, is a promising candidate for pharmacologically active triterpenes. Dammarenediol-II synthase (DDS) cyclizes 2,3-oxidosqualene to produce dammarenediol-II. Based on the native terpenoids synthetic pathway, a dammarane-type ginsenosides synthetic pathway was established in Chlamydomonas reinhardtii by introducing P. ginseng PgDDS, CYP450 enzyme (PgCYP716A47), or/and Arabidopsis thaliana NADPH-cytochrome P450 reductase gene (AtCPR), which is responsible for producing dammarane-type ginsenosides. To enhance productivity, strategies such as "gene loading" and "culture optimizing" were employed. Multiple copies of transgene expression cassettes were introduced into the genome to increase the expression of the key rate-limiting enzyme gene, PgDDS, significantly improving the titer of dammarenediol-II to approximately 0.2 mg/L. Following the culture optimization in an opt2 medium supplemented with 1.5 mM methyl jasmonate under a light:dark regimen, the titer of dammarenediol-II increased more than 13-fold to approximately 2.6 mg/L. The C. reinhardtii strains engineered in this study constitute a good platform for the further production of ginsenosides in microalgae.

Metabolic Engineering of the Isopentenol Utilization Pathway Enhanced the Production of Terpenoids in Chlamydomonas reinhardtii.

Eukaryotic green microalgae show considerable promise for the sustainable light-driven biosynthesis of high-value fine chemicals, especially terpenoids because of their fast and inexpensive phototrophic growth. Here, the novel isopentenol utilization pathway (IUP) was introduced into Chlamydomonas reinhardtii to enhance the hemiterpene (isopentenyl pyrophosphate, IPP) titers. Then, diphosphate isomerase (IDI) and limonene synthase (MsLS) were further inserted for limonene production. Transgenic algae showed 8.6-fold increase in IPP compared with the wild type, and 23-fold increase in limonene production compared with a single MsLS expressing strain. Following the culture optimization, the highest limonene production reached 117 µg/L, when the strain was cultured in a opt2 medium supplemented with 10 mM isoprenol under a light: dark regimen. This demonstrates that transgenic algae expressing the IUP represent an ideal chassis for the high-value terpenoid production. The IUP will facilitate further the metabolic and enzyme engineering to enhance the terpenoid titers by significantly reducing the number of enzyme steps required for an optimal biosynthesis.

Acuticoccus kandeliae sp. nov., isolated from rhizosphere soil of the mangrove plant Kandelia, and emended description of Acuticoccus yangtzensis.

A novel bacterial strain, J103T, was isolated from rhizosphere soil of the mangrove plant Kandelia in Mai Po Inner Deep Bay Ramsar Site, Hong Kong. The strain was aerobic, Gram-stain-negative, oval-shaped with folds in the middle, non-motile and non-spore-forming. It grew at temperatures of 20-30 °C (optimum, 25-30 °C), at pH 6.0-9.0 (optimum pH 6.0) and at NaCl concentrations of 0.5-5.0 % (w/v) (optimum 1.0-2.0 %). Strain J103T was able to reduce nitrate to nitrite, and hydrolyse urea, Tween 40 and Tween 60. The major polar lipids were aminolipid, glycolipid, phosphatidylcholine and phosphatidylglycerol. The major fatty acids were C18 : 1ω7c and C19 : 0 cyclo ω8c. The respiratory quinone was Q-10. The DNA G+C content was 68.5 mol%. Sequence analysis of the 16S rRNA gene indicated that strain J103T belongs to the genus Acuticoccus, within the family Rhodobacteraceae. The closest phylogenetic neighbour was Acuticoccus yangtzensis JL1095T, showing 96.2 % 16S rRNA gene sequence similarity. The genome size of strain J103T was 6 478 100 bp. The average nucleotide identity and digital DNA-DNA hybridization values between strain J103T and Acuticoccus yangtzensis JL1095T were 75.44 and 16.43 %, respectively. Characterization based on phylogenetic, phenotypic, chemotaxonomic and genomic evidence demonstrated that strain J103T represents a novel species of the genus Acuticoccus, for which the name Acuticoccus kandeliae sp. nov. is proposed. The type strain is J103T (=DSM 104434T=MCCC 1K03288T).

Euzebya rosea sp. nov., a rare actinobacterium isolated from the East China Sea and analysis of two genome sequences in the genus Euzebya.

Rare Actinobacteria, known as non-Streptomyces, hold great potential to produce new bioactive compounds for drug development. A strain designated DSW09T, which belongs those rare Actinobacteria, was isolated from surface seawater of the East China Sea. The cells were aerobic, Gram-positive, non-motile, non-spore-forming and rod-shaped (0.4 µm wide and 1.5-4.0 µm long). The closest relative was Euzebya tangerina F10T (96.46 % of 16S rRNA gene similarity). Cell growth occurred at 15-45 °C (optimum, 25-30 °C), at pH 6.0-9.0 (pH 6.0-7.0) and at NaCl concentrations of 0.5-5.0 % (w/v; 1.0-4.0 %). The major cellular fatty acids were summed feature 3 (comprising C16 : 1ω7c and/or C15 : 0iso 2OH), C17 : 1ω8c and C16 : 0. The predominant polar lipid was diphosphatidylglycerol. The predominant menaquinone was MK-9(H4). The cell-wall peptidoglycan was A1 γ-type, containing meso-DPA. The major cell-wall sugars were rhamnose and ribose. The genome size was 5 509 297 bp with a 71.29 mol% G+C content for strain DSW09T, while 4 781 440 bp with a 68.87 mol% G+C content for E. tangerina F10T. The average nucleotide identity and digital DNA-DNA hybridization values between strain DSW09T and E. tangerina F10T were 73.44 % and 16.43 %, respectively. Based on phylogenetic, phenotypic, chemotaxonomic evidence and genomic analyses, strain DSW09T is a novel species of genus Euzebya, for which the name Euzebya rosea sp. nov. is proposed. The type strain is DSW09T (=DMS 104446T=MCCC 1K03290T).

Emergence, circulation, and spatiotemporal phylogenetic analysis of coxsackievirus a6- and coxsackievirus a10-associated hand, foot, and mouth disease infections from 2008 to 2012 in Shenzhen, China.

Sporadic hand, foot, and mouth disease (HFMD) outbreaks and other infectious diseases in recent years have frequently been associated with certain human enterovirus (HEV) serotypes. This study explored the prevalences and genetic characteristics of non-HEV71 and non-coxsackievirus A16 (CV-A16) human enterovirus-associated HFMD infections in Shenzhen, China. A total of 2,411 clinical stool specimens were collected from hospital-based surveillance for HFMD from 2008 to 2012. The detection of HEV was performed by real-time reverse transcription-PCR (RT-PCR) and RT-seminested PCR, and spatiotemporal phylogenetic analysis was performed based on the VP1 genes. A total of 1,803 (74.8%) strains comprising 28 different serotypes were detected. In the past 5 years, the predominant serotypes were HEV71 (60.0%), followed by CV-A16 (21.2%) and two uncommon serotypes, CV-A6 (13.0%) and CV-A10 (3.3%). However, CV-A6 replaced CV-A16 as the second most common serotype between 2010 and 2012. As an emerging pathogen, CV-A6 became as common a causative agent of HFMD as HEV71 in Shenzhen in 2012. Phylogenetic analysis revealed that little variation occurred in the Chinese HEV71 and CV-A16 strains. The genetic characteristics of the Chinese CV-A6 and CV-A10 strains displayed geographic differences. The CV-A6 and CV-A10 strains circulating in Shenzhen likely originated in Europe. It was found that human enteroviruses have a high mutation rate due to evolutionary pressure and frequent recombination (3.2 × 10(-3) to 6.4 ×10(-3) substitutions per site per year for HEV71, CV-A6, CV-A16, and CV-A10). Since certain serotypes are potential threats to the public health, this study provides further insights into the significance of the epidemiological surveillance of HFMD.

[Studies on thermal denaturation of peanut allergen Ara h1 and its interaction with reducing sugars].

The thermal denaturation of peanut allergen Ara h1, its interaction with reducing sugars and the corresponding changes in allergenicity were investigated by circular dichroism (CD), fluorescence and ELISA method comprehensively. The experimental results indicate that the secondary structure of Ara h1 changes significantly along with decreasing alpha-helical structure and its allergenicity with the temperature higher than 85 degrees C, and that both xylose and fructose can stabilize Ara h1 protein structure through interacting with Ara h1 protein and decrease its allergenicity obviously. This study should be helpful to the further understanding of sensitization mechanism of food allergy and be useful for the guidance on reasonable manufacturing of peanut foods.

Cell wall breaking of Haematococcus pluvialis biomass facilitated by Baijiu jiuqu fermentation with simultaneously production of beverages.

The microalgae Haematococcus pluvialis is a commercial source of natural astaxanthin. However, mature cells develop rigid three-layer wall structures and a repulsive odor. This study applied a liquid static fermentation system to screen hydrolyzing microorganisms for cell wall hydrolysis. Baijiu jiuqu and Gutian hongqu were found to have promising potential for application. The fermentation using 2% baijiu jiuqu and 2% glucose for pre-activation achieved comparable recovery of carotenoids to homogenizer disruption methods and produced stable fragrance which may be attributed to ethyl octanoate, hexyl formate, and phenethyl butyrate, as revealed by gas chromatography-mass spectrometry analysis. The abundance of astaxanthin molecules was slightly affected by fermentation with fold change < 2, while molecules with higher fold change (>10) were mainly carbohydrates, lipids, and steroids proving the safety of the fermentation. This study provides a new scheme for the biorefining of Haematococcus. pluvialis, potentially contributing to the industrial production of natural astaxanthin.

Efficient, green, and rapid strategy for separating actinomycin D and X2 using supercritical fluid chromatography.

Actinomycin D has long been used as a first-line antitumor drug in clinical practice. Actinomycin X2, a new drug lead, is often isolated along with actinomycin D. The minor differences between the two actinomycin analogs pose a daunting challenge in separation. In this study, supercritical fluid chromatography (SFC) was successfully utilized for the purification of actinomycin X2 and actinomycin D from a marine derived Streptomyces sp. DQS-5. After one-step SFC purification, the purities of these two compounds were determined to be 97.3 % and 97.8 %, respectively. This method provides a green alternative for the separation of these pharmacologically important actinomycin antibiotics. This study also demonstrated the development of a simple and rapid method for the separation of natural products based on SFC.

Butenolide, a Marine-Derived Broad-Spectrum Antibiofilm Agent Against Both Gram-Positive and Gram-Negative Pathogenic Bacteria.

Bacterial biofilm can cause nosocomial recurrent infections and implanted device secondary infections in patients and strongly promotes development of pathogenic drug resistance in clinical treatments. Butenolide is an effective anti-macrofouling compound derived from a marine Streptomyces sp., but its antibiofilm efficacy remains largely unexplored. In the present study, the antibiofilm activities of butenolide were examined using biofilms formed by both Gram-positive and Gram-negative pathogenic model species. Four Escherichia coli strains, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA) were used as targets in antibiofilm assays that examined the effects of butenolide, including the following: (i) on bacterial growth; (ii) in inhibiting biofilm formation and eradicating mature biofilm; (iii) on biofilm structures. In addition, the synergistic effect between butenolide with tetracycline was also examined. Butenolide not only effectively inhibited the biofilm formation but also eradicated pre-formed biofilms of tested bacteria. Fractional inhibitory concentration index (FICI) indicated that butenolide was a potential tetracycline enhancer against E. coli, P. aeruginosa, and MRSA. These results indicated that butenolide may hold a great potential as an effective antibiofilm agent to control and prevent biofilm-associated infections in future clinical treatments.

A novel assessment of the traction forces upon settlement of two typical marine fouling invertebrates using PDMS micropost arrays.

Marine biofouling poses a severe threat to maritime and aquaculture industries. To prevent the attachment of marine biofouling organisms on man-made structures, countless cost and effort was spent annually. In particular, most attention has been paid on the development of efficient and environmentally friendly fouling-resistant coatings, as well as larval settlement mechanism of several major biofouling invertebrates. In this study, polydimethylsiloxane (PDMS) micropost arrays were utilized as the settlement substrata and opposite tractions were identified during early settlement of the barnacle Amphibalanus amphitrite and the bryozoan Bugula neritina The settling A. amphitrite pushed the periphery microposts with an average traction force of 376.2 nN, while settling B. neritina pulled the periphery microposts with an average traction force of 205.9 nN. These micropost displacements are consistent with the body expansion of A. amphitrite during early post-settlement metamorphosis stage and elevation of wall epithelium of B. neritina during early pre-ancestrula stage, respectively. As such, the usage of micropost array may supplement the traditional histological approach to indicate the early settlement stages or even the initiation of larval settlement of marine fouling organisms, and could finally aid in the development of automatic monitoring platform for the real-time analysis on this complex biological process.

Removal of fluoranthene and pyrene by different microalgal species.

In this work, the efficiency of four microalgal species, namely, Chlorella vulgaris, Scenedesmus platydiscus, Scenedesmus quadricauda, and Selenastrum capricornutum to remove fluoranthene (1.0 mg l(-1)), pyrene (1.0 mg l(-1)), and a mixture of fluoranthene and pyrene (each at a concentration of 0.5 mg l(-1)) was evaluated. Results showed that removal was algal species specific and was also toxicant-dependent. Se. capricornutum was the most effective species while C. vulgaris was the least efficient species in removing and transforming polycyclic aromatic hydrocarbons (PAHs). PAHs removal in 7-days of treatment was 78% and 48%, respectively by these two. All species, except S. platydiscus exhibited higher fluoranthene removal efficiency than pyrene, indicating the latter PAH was generally more stable and recalcitrant. The removal efficiency of fluoranthene and pyrene in a mixture was comparable, or higher than the respective single compound, suggesting that the presence of one PAH stimulated the removal of the other PAH.

Microbial community structures and dynamics in the O3/BAC drinking water treatment process.

Effectiveness of drinking water treatment, in particular pathogen control during the water treatment process, is always a major public health concern. In this investigation, the application of PCR-DGGE technology to the analysis of microbial community structures and dynamics in the drinking water treatment process revealed several dominant microbial populations including: α-Proteobacteria, ß-Proteobacteria, γ-Proteobacteria, Bacteroidetes, Actinobacteria Firmicutes and Cyanobacteria. α-Proteobacteria and ß-Proteobacteria were the dominant bacteria during the whole process. Bacteroidetes and Firmicutes were the dominant bacteria before and after treatment, respectively. Firmicutes showed season-dependent changes in population dynamics. Importantly, γ-Proteobacteria, which is a class of medically important bacteria, was well controlled by the O3/biological activated carbon (BAC) treatment, resulting in improved effluent water bio-safety.

[Simultaneously removal of COD, nitrogen and phosphorus from wastewater by coupling treatment system with immobilized algae-bacteria].

A coupling treatment system was developed by employing immobilized Chlamydomonas reinhardti and activated sludge to simultaneously remove COD, nitrogen and phosphorus from wastewater. The amount of wastewater treated by the system was 6 m3 per day, and hydraulic retention time was 12 h. For activated sludge section, as stirring rate of anaerobic tank was 15 r x min(-1) and DO value of aerobic tank was 5 mg x L(-1), COD decreased from about 150 mg x L(-1) to 50 mg x L(-1) and NH4+-N from 20-30 mg x L(-1) to 0.5 mg x L(-1), whereas TP only dropped from 2-3 mg x L(-1) to 1.0 mg x L(-1). For immobilized C. reinhardti section, the suitable conditions were: DO 5 mg x L(-1), illumination intensity 2000 lx, the loading ratio of immobilization pellets 20%, respectively. Under the appropriate conditions of the coupling treatment system, COD, NH4+-N and TP of the effluent were about 15 mg x L(-1), 0.5 mg x L(-1) and 0.5 mg x L(-1), respectively. During 2 months period of continuous treatment, COD, NH4+-N and TP of the effluent kept fairly constant, showing the stability of the coupling wastewater treatment system.

[Study on the removal of nitrogen and phosphorus from wastewater by Chlamydomonas reinhardtii].

Chlamydomonas reinhardtii was employed to remove nitrogen and phosphorus from wastewater. The effects of initial NH4(+) -N and TP concentrations, N/P ratios, Light/Darkness ratios, pH and immobilization on the removal of NH4(+) -N and TP were evaluated. The results showed that C. reinhardtii could almost 100% remove NH4(+) -N and TP as initial concentrations of NH4(+) -N and TP were no more than 55 mg x L(-1) and 7 mg x L(-1), respectively, whereas the removal ratio of NH4(+) -N decreased drastically to 50% with initial NH4(+) -N concentration coming up to 75 mg x L(-1). Under N/P ratios of 5:1 and 10:1, C. reinhardtii could completely remove NH4(+) -N within 3d, while 6 d was needed with N/P ratio being 25: 1. Different from NH4(+) -N removal, the removal ratio of TP could reach almost 100% within 4 d under 3 N/P ratios. With L/D ratios of 24 h: 0 h and 12 h: 12 h, 100% removal of NH4(+) -N and TP could be achieved by C. reinhardtii, but the removal rate under L/D ratio of 24 h: 0 h was relatively faster. The optimal pH range for C. reinhardtii to remove NH4(+) -N and TP was 6-7. After immobilization, the ability of C. reinhardtii to remove NH4(+) -N was significantly enhanced as the removal ratio of NH4(+) -N came up to 100% with initial NH4(+) -N concentration being 75 mg x L(-1). The ability of immobilized C. reinhardtii to remove TP kept stable, whereas the removal rate was slowed slightly.