Abstract:Biofuel is in high demand as an alternative energy source for petroleum and diesel. Fatty acid-based biofuel has higher energy density and better compatibility with existing infrastructures. Microbial fatty acid biosynthetic pathway is important to develop biofuel. In this article, recent progresses on the modification and reconstruction of fatty acid metabolism for the production of biofuel were reviewed, with a focus on micro-diesel, long chain fatty alcohol and alkane. Problems, solutions and directions for further development of fatty acid-based biofuel were also discussed in the respect of synthetic biology.

Abstract:Wastewater resources, CO2 emission reduction and microalgae biodiesel are considered as current frontier fields of energy and environmental researches. In this paper, we reviewed the progress in system of microalgae culture for biodiesel production by wastewater and stack gas. Multiple factors including microalgal species, nutrition, culture methods and photobioreactor, which were crucial to the cultivation of microalgae for biodiesel production, were discussed in detail. A valuable culture system of microalgae for biodiesel production or other high value products combined with the treatment of wastewater by microalgae was put forward through the optimizations of algal species and culture technology. The culture system coupled with the treatment of wastewater, the reduction of CO2 emission with the cultivation of microalgae for biodiesel production will reduce the production cost of microalgal biofuel production and the treatment cost of wastewater simultaneously. Therefore, it would be a promising technology with important environmental value, social value and economic value to combine the treatment of wastewater with the cultivation of microalgae for biodiesel production.

Abstract:We investigated the enhanced immune response of a recombinant T cell immunogen as an effective cellular immune adjuvant. The T cell immunogen named TI contained several T cell epitopes from the VP1, VP4, 3A and 3D proteins of foot-and-mouth disease virus (FMDV) and two pan-T helper (TH) cell site to broaden the immunogenicity of the protein. Meanwhile, another fusion protein named OAVP1 was expressed in bacteria, which contained two VP1 proteins of O and Asia1 type FMDV. Mice were vaccinated with commercially inactivated vaccine or OAVP1 protein with or without the TI immunogen. The results show that mice inoculated with inactivated vaccine or OAVP1 protein supplemented with TI immunogen produced significantly higher level of neutralizing antibodies (P<0.01 or P<0.05) than the mice only inoculated with inactivated vaccine or OAVP1 protein by microneutralization assay. An obvious increase in T cell number by flow cytometric analysis and significantly higher concentration of IFN-γ secreted in culture media of spleen lymphocytes were observed in groups supplemented with TI immunogen (P<0.01). TI immunogen was an effective stimulator for humoral and cellular immunity and could help improve the immunogenicity of inactivated vaccine or protein subunit vaccine.

Abstract:A system coupling ethanol fermentation with microalgae culture was developed, in which CO2 produced during ethanol fermentation was used as carbon source for the growth of Tetraselmis subcordiformis, a microalgae accumulating starch intracellularly. The biomass concentration about 2.0 g DCW/L was achieved within the photobioreactor for the batch culture of 7 days, and intracellular starch accumulation was about 45%. Furthermore, ultra sonic pretreatment and enzymatic hydrolysis were applied to the microalgae biomass, and 71.1% of the intracellular starch was converted into glucose that was fermented sequently to ethanol by Saccharomyces cerevisiae with an ethanol yield of 87.6% of the theoretical value, indicating that the microalgae biomass could be an alternative feedstock for ethanol production to save grain consumption, and in the meantime mitigate the CO2 emission.

Abstract:To understand the effects of sugar whose uptake is dependent or independent on the phosphotransferase system (PTS), two-stage culture of Escherichia coli strain NZN111 that was constructed by disruption of ldhA and pflB encoding the fermentative lactate dehydrogenase (LDH) and pyruvate: formate lyase (PFL) of E. coli W1485, was carried out for organic acids production. When NZN111 was aerobically cultured on fructose (PTS dependent) or maltose (PTS independent), it fermented glucose with succinic acid and pyruvic acid as the major products in subsequent anaerobic culture. The experiments were also performed in a 5-L fermentor. The yields of succinic acid by the fructose-and maltose-grown NZN111 were 0.84 and 0.75 mol/mol, whereas the yields of pyruvic acid were 0.65 and 0.83 mol/mol, respectively. The final ratio of succinic acid to pyruvic acid in the anaerobic stage reached 1.73:1 and 1.21:1, respectively. The different behaviors in anaerobic fermentation by the fructose-, maltose- and glucose-grown NZN111 were likely caused by the regulation of catabolite repression in the aerobic culture stage.

Abstract:To efficiently use both cellulose and hemicellulose for lipid production, rice straw was hydrolyzed by a two-step process including dilute acid pretreatment and then enzymatic hydrolysis, and the hydrolysate was used as carbon source for lipid fermentation by Trichosporon fermentans HWZ004. After a simple overliming, the concentrations of acetic acid, furfural and 5-hydroxymethyl-furfural were 0.4 g/L, 0.1 g/L and 0.05 g/L, respectively. The hydrolysate could be used for lipid fermentation with T. fermentans HWZ004 without adding other nutrients except for a small amount of nitrogen source and trace CuSO4?5H2O. The optimum inoculum size, initial pH and temperature were 5.0%, 7.0 and 25 °C, respectively. A total biomass of 26.4 g/L with a lipid content of 52.2% (corresponding to a lipid yield of 13.8 g/L) was achieved after cultivation of T. fermentans HWZ004 under the above-mentioned conditions for 7 days. The lipid coefficient (lipid yield on substrate consumed) is 17.0, which is much higher than the corresponding one (11.9) obtained on detoxified rice straw hemicullulose hydrolysate by original yeast T. fermentans CICC 1368. The fatty acid composition of the lipid was found to be similar to that of vegetable oil and its unsaturated fatty acid content was over 70%, thus the lipid is a promising material for biodiesel production.

Abstract:To explore the physiological role and biocatalytic properties of short-chain dehydrogenases from Pseudomonas fluorescens GIM1.49, we cloned the structural gene pfd and characterized its over-expressed product. The length of gene pfd was 684 bp encoding a short-chain dehydrogenase with 227 amino acid residues and calculated molecular mass of 24.2 kDa. The recombinant plasmid pET28b-pfd was constructed and functionally expressed in Escherichia coli BL21(DE3), resulting in the over-production of recombinant short-chain dehydrogenase PFD with a size of 28 kDa. The enzyme could oxidize alcohols including 4-chloro-3-hydroxbutanoate ester and reduce 4-chloro-acetoacetate ester using either NAD(H) or NADP(H) as coenzyme. The enzyme showed the highest activity against 4-chloro-3-hydroxbutanoate ester as substrate, with Km of 186.90 mmol/L and Vmax of 89.56 U/mg. When catalying the oxidative reaction, its optimal temperature was 12 °C and optimal pH was 10.5, in contrast to the values of 24 °C and pH 8.8 in the reductive reaction. The enzyme had high solvent tolerance and its activity was improved by the addition of Ca2+ (1 mmol/L) or EDTA (5 mmol/L). These results indicated that the enzyme from Pseudomonas fluorescens GIM1.49 was a novel short-chain dehydrogenase and might play a role in oxidative degradation of halogenated secondary alcohols.

Abstract:We developed a method to construct a gene mutant pool in Pichia pastoris based on in vivo homologous recombination. It was an absolute PCR-dependent method (PDM) and could avoid the disadvantages of traditional mutant pool construction process such as long-experimental period, low pool capacity and inadequate abundance. The method consisted of four steps: 1) construction of recombinant expression plasmid of target gene; 2) design of long primers that have 40?70 bp of homology to expression vector fragments at both ends and amplification of target gene by error-prone PCR, DNA Shuffling or other methods; 3) PCR amplification of expression vectors fragments; 4) mixture of gene and vectors by appropriate mole ratio, electroporation, formation of expression cassette in vivo, homologous recombination with host genome and achievement of mutant pool. Screening from this library, we obtained mutants with improved enzyme activity, protein expression level and thermostability. In conclusion, PDM was very efficient and convenient with advantages of shortened pool construction cycle from 2 weeks to 3 days, enlarged pool capacity from the original 103?104 to more than 105, with a positive rate of more than 95%.

Abstract:The discovery of quorum sensing (QS) system and its critical role in bacterial virulence have revealed a new way to attack pathogenic bacterium. The pathogenecity of QS deletion mutants decreases significantly. Targeting bacterial QS system is a promising therapeutic approach to control infections and anti-microbial resistance. To obtain natural QS inhibitors from marine organisms, marine fungi (69 strains) were isolated from marine mollusca, and their extracts were screened using improved QSIS2 (Quorum Sensing Inhibitor Selector 2) assay and Chromobacterium violaceum CV026. To improve the efficiency of QSIS2 screening, 2,3,5-triphenyltetrazolium chloride (TTC) staining method was used. Extract from strain QY013 was found to have QS inhibitory activity. Further experiment indicated that pyocyanin in Pseudomonas aeruginosa PAOI and violacein in C. violaceum CV026 were reduced by QY013 extract, without affecting bacterial growth. Morphological and 18S rDNA sequence analysis revealed that strain QY013 was most closely related to Penicillium species. The above results suggest that active constituents from QY013 may be used as novel antimicrobial agents against bacterial infection.

Abstract:Early-warning of compartmentalized anaerobic reactor (CAR) was investigated in lab-scale. The performance stability of CAR at high loading rate was worse than that at common loading rate. At high loading rate, the fluctuation of effluent chemical oxygen demand (COD) concentration and volatile fatty acids (VFA) concentration was larger than that of influent COD concentration. The average relative standard deviation of effluent COD concentration and VFA concentration was 32.95% and 40.46% respectively, while that of influent COD concentration was 8.08%. The saturation of volumetric loading rate (SVLR) and VFA (SVFA) could be used to alarm the performance of anaerobic reactors. The working performance was good when the CAR was operated at normal organic loading rate (OLR), in which SVLR and SVFA were below 0.89 and 0.40 respectively. The fluctuation of performance became larger when the CAR was operated at OLR near saturation, in which SVLR and SVFA were close to 1. The performance of CAR was deteriorated when the SVLR and SVFA were more than 1.

Abstract:PeaT1, a protein elicitor from Alternaria tenuissima can promote plant growth and trigger systemic acquired resistance in plants. In order to expand the application of PeaT1, P43 promoter sequence and nprB signal peptide-encoding sequence were cloned from Bacillus subtilis 168 chromosomal DNA. The two sequences and peaT1 gene were spliced by overlapping extension. This product was cloned into the Escherichia coli-B. subtilis shuttle vector pHY300-PLK and the resultant recombinant expression vector (pHY43N- peaT1) plasmid was transformed into B. subtilis WB800. SDS-PAGE and Western blotting analysis showed that protein elicitor PeaT1 was expressed extracellularly in B. subtilis. This recombinant bacterial strain enhanced drought tolerance and promoted seedling growth in wheat.

Abstract:A fragment of chalcone synthase gene (SmCHS) was cloned from the cDNA library constructed in Saussurea medusa. The full-length cDNA sequence of SmCHS was obtained by RT-PCR. Sequence analysis showed that the full length of SmCHS was 1 313 bp, containing an open reading frame (1 170 bp) encoding 389 amino acids. The molecular weight of the protein was estimated to be 43 kDa. The prokaryotic expression plasmids pET28a(+)-SmCHS was constructed and transformed into Escherichia coli BL21(DE3) for expression. SDS-PAGE indicated that the fusion protein was expressed partially in soluble form after induction by IPTG. The recombinant protein was collected and purified by Ni-NTA affinity column. The enzymatic activity assay of the purified recombinant protein showed that the fusion protein had chalcone synthase activity. It could catalyze the condensation of a 4-coumaroyl-CoA with three malonyl-CoAs to produce naringenin chalcone.

Abstract:Hepatitis B virus X protein (HBx) has various functions and plays a crucial role in the development of hepatocellular carcinoma (HCC). However, due to different transfection efficiency levels and experimental approaches, it is difficult to correlate the exact functions of HBx to HBV-associated HCC. In this study, we constructed two prokaryotic expression vectors, pGEX-HBx-EGFP-TLM and pGEX-EGFP-TLM, which expressed HBx-EGFP-TLM and EGFP-TLM fusion proteins respectively. Both vectors contained a coding sequence of TLM transduction motif derived from the PreS2-domain of Hepatitis B Virus surface antigens. In addition, EGFP was expressed as a reporter reflecting the transduction efficiency of TLM. The fusion protein HBx-EGFP-TLM or EGFP-TLM purified from Escherichia coli BL21(DE3) by ?KTATM Purifier system was incubated with AML12 and SMMC-7721 cells. Both Western blotting and laser confocal results indicated that the translocation motif TLM could lead HBx-EGFP and EGFP into the cytoplasm. Dual-Luciferase Reporter Assay revealed that the activity of mEZH2 promoter could be up-regulated by the recombinant HBx. In conclusion, we expressed a cell-permeable HBx, which could provide a new method to study the functions of HBx.

Abstract:In order to establish a high efficient regeneration system of sunflower, we optimized the process of callus induction, differentiation and rooting by screening the optimum genotype, explant materials, hormone and cytokine concentration and additives. The results indicated that hybrid sunflowers were easier to regenerate than selfing ones; The best explant was four days cotyledon. The optimum induction medium was Murashige and Skoog (MS) +2.0 mg/L 6-benzyladenine (6-BA)+0.5 mg/L naphthaleneacetic acid (NAA)+1.0 mg/L kinetin (KT). The maximum rate of callus induction was 100%. The optimum differentiation medium was MS +0.2 mg/L 6-BA +0.5 mg/L NAA +0.3 mg/L KT +0.3 mg/L silver nitrate (AgNO3)+0.2 g/L active carbon (AC), and the buds differentiation rate was up to 71%. The best rooting culture medium was 1/2 MS +0.6 mg/L indolebutyric acid (IBA). The highest rooting rate was 77%. The analysis of variance showed that genotype, explants growth time, different kinds and concentration of hormone, AC concentration had a significant effect on sunflower regeneration.

Abstract:For studying the effects of Mycobacterium tuberculosis secretory protein ESAT-6 on the related functions of macrophages, RAW264.7 cells were transfected with pEGFP-C1-ESAT-6 and pEGFP-C1 by liposome respectively. After screening with a high level of G418, the macrophage cell lines that stably expressed EGFP-ESAT-6 fusion protein or EGFP were established. The gene and protein expression levels were further analyzed by RT-PCR, fluorescence microscopy and Western blotting. The results indicated that the EGFP-ESAT6 fusion gene was integrated into the chromosome and the protein could be stably expressed in the selected macrophage cell line. These results gave us a tool for the future study in the mechanisms of ESAT-6 protein in modulating the macrophage cells.