Abstract:Throughout human history, pandemic bacterial diseases such as the plague and tuberculosis have posed an enormous threat to human beings. The discovery of antibiotics has provided us with powerful arsenal for the defense against bacterial infections. However, bacteria are acquiring more and more resistance genes to shield off antibiotics through mutation and horizontal gene transfer. Therefore, novel antibiotics must be produced and the arms race between bacterial pathogens and antibiotics is becoming increasingly intense. Recently, researchers have found that plasmids carrying a new metallo-β-lactamase gene, blaNDM-1, and many other antibiotics resistance genes can easily spread through bacterial populations and confer recipient stains resistance to nearly all of the current antibiotics. It is a threat to the human health and a great challenge for our medical science, which we are facing. We need to find new ways to fight and win this arms racing.

Abstract:Microbial catalase is an important industrial enzyme that catalyzes the decomposition of hydrogen peroxide to water and oxygen. This enzyme has great potential of application in food, textile and pharmaceutical industries. The production of microbial catalase has been significantly improved thanks to advances in bioprocess engineering and genetic engineering. In this paper, we review the progresses in fermentation production of microbial catalase and its application in textile industry. Among these progresses, we will highlight strain isolation, substrate and environment optimization, enzyme induction, construction of engineering strains and application process optimization. Meanwhile, we also address future research trends for microbial catalase production and its application in textile industry. Molecular modification (site-directed mutagenesis and directed revolution) will endue catalase with high pH and temperature stabilities. Improvement of catalase production, based on the understanding of induction mechanism and the process control of recombinant stain fermentation, will further accelerate the application of catalase in textile industry.

Abstract:Plant-specific type III polyketide synthase (PKS) produces a variety of plant secondary metabolites with notable structural diversity and biological activity. So far 14 plant-specific type III PKS have been identified according to their enzymatic products, and the corresponding genes have been cloned and characterized. The differences among the various PKS are mainly in their substrate specificities, the number of their condensation reactions, and the type of ring closure of their products. However, numerous studies have revealed the common features among the plant-specific type III PKS, which include sequence homology, similar gene structure, conserved amino acid residues in the reaction center, enzymatic characteristics and reaction mechanism. We briefly reviewed 14 plant-specific type III PKS to better understand genetic and metabolic engineering of plant-specific type III PKS.

Abstract:Δ8 desaturase pathway, different from common Δ6 desaturase pathway, is an alternate pathway of polyunsaturated fatty acids biosynthesis. Δ8-fatty acid desaturase is one of the key enzymes in Δ8 desaturase pathway. Two specific fragments were separately cloned from genomic DNA and cDNA of Euglena gracilis by PCR with the primers designed according to the reported sequence. Comparison of the genomic and cDNA sequences revealed that there wasn’t intron in this Δ8-fatty acid desaturase gene. This gene has an open reading frame of 1 266 bp that encodes 421 amino acids. It is 6 bp longer than the reported gene sequence, and also showed certain difference from the reported sequence in the N-terminal. The recombinant expression plasmid pYEFD by subcloning Δ8-fatty acid desaturase gene into the yeast-E. coli shuttle vector pYES2.0 was constructed and was transformed into the defective mutant INVSc1 of Saccharomyces cerevisiae by electrotransformation. The resulting strain YD8 harboring plasmid pYEFD was selected and was cultured in the induction medium with exogenous substrates ω6-eicosadienoic acid and ω3-eicosatrienoic acid for the expression of Δ8-fatty acid desaturase gene. The results indicated that high level expressed Δ8-fatty acid desaturase could convert ω6-eicosadienoic acid and ω3-eicosatrienoic acid to dihomo-γ-linolenic acid and eicosatetraenoic acid with substrate conversion ratio 31.2% and 46.3%, respectively.

Abstract:Different neutralizing agents were used as pH controller to investigate their effects on the growth and succinic acid production of Actinobacillus succinogenes NJ113. The fermentation results showed that Ca(OH)2, CaCO3 and NH4OH were not suitable for succinic acid production by A. succinogenes NJ113 because of their negative effects on cell growth. When Na-base was used, cells would flocculate and lump, and due to the sodium ion concentration reaching to a high level, OD660 dropped sharply after 12 h of fermentation. Mg-base was better because there was no significant inhibition by magnesium ion. Two combined neutralizing agents were used to maintain pH level, one with NaOH and Mg(OH)2 while the other with Na2CO3 and Mg(OH)2. The optimum ratios of the combined neutralizing agents were both 1:1(g:g) when using 100 g/L glucose. When NaOH and Mg(OH)2 were chosen with the ratio of 1:1(g:g), 69.8 g/L of the succinic acid and 74.5% of the yield was obtained.

Abstract:This study aimed to further enhance 2-keto-L-gulonic acid (2-KLG) production efficiency. A strategy for enhancing Ketogulonigenium vulgare growth and 2-KLG production by improving B. megaterium growth with sucrose was developed based on the time course of osmolality during 2-KLG industrial scale fermentation and effects of osmolality on cells growth and 2-KLG production. Results showed that the accumulation of 2-KLG and the feeding of alkaline matter led to an osmolality rise of 832 mOsmol/kg in the culture broth. High osmotic stress (1 250 mOsmol/kg) made the growth of B. megaterium and K. vulgare decreased 15.4% and 31.7%, respectively, and consequently the titer and productivity of 2-KLG reduced 67.5% and 69.3%, respectively. When supplement sucrose under high osmotic condition (1 250 mOsmol/kg), B. megaterium growth was significantly improved, with the result that 2-KLG production was increased 87%. Furthermore, by applying this sucrose addition strategy further to batch fermentation in 3 L fermentor, the productivity of 2-KLG increased 10.4%, and the duration of fermentation declined 10.8%. The results presented here provide a potential strategy for enhancing the target metabolites produced by mixed strains at environmental stress.

Abstract:Different geographic seed sources (80) of Jatropha curcas L. were collected in South China and planted in a germplasm resource garden to study their biological and agricultural properties. The average ground diameter, tree height and crown size of two-year old plants of the 80 sources was 7.6 cm, 167 cm and 114 cm, respectively, the average 1 000-seed weight was 0.676 (0.477?0.876) kg. The trees grew further to the average size of 12.6 cm diameter, 2.69 m height and 2.1 m crown at the 4th year. Among the 80 sources, six sources had higher oil yield (seed oil content of 40%?42%) and better behaving in expression of phenotype were selected for a small-scale trial of forestation to determine oil yield. Among them a provenance with outstand in expression of phenotype yielded 964.3, 2 000.6 and 2 858.7 kg/ha was achieved for two- three- and four-year old trees, respectively. Additionally, a new Jatropha mutant was found in the wild and hybridization experiments showed that its oil content increased by 6%.

Abstract:The possibility of reusing Escherichia coli cells from the broth for succinic acid production was investigated. Using succinic acid yield and productivity as criterion, we investigated the effects of cell concentration, initial glucose concentration, different neutralizers on the bioconversion. The results revealed that E. coli could convert glucose to succinic acid in a water solution of glucose and a neutralizer. According to the results, the optimal condition was as follows: the cell concentration was 50 (OD600), glucose concentration was 40 g/L and neutralizer was MgCO3. Under the optimum conditions, we carried out the consecutive batch bioconversion in 7 L fermenter. Succinic acid yield reached 91% with the productivity of 3.22 g/(L·h) for the first conversion. For the second conversion, succinic acid yield reached 86% with productivity of 2.04 g/(L·h). Furthermore, we achieved a high mass yield above 83% with the productivity of 1.82 g/(L·h) for the third bioconversion.

Abstract:Chitinases were produced by a lot of microorganisms. Chitinase gene expression in most of the chitinase producing bacteria was inducible by chitin. Low levels of chitinase were observed in the presence of glucose. To date, however, the regulation of such chitinase gene in Bacillus thuringiensis had not been well studied. In this paper, all 77 Bacillus thuringiensis strains were grown in the medium with or without chitin. We measured quantitatively the chitinase activity of the cultures. Moreover, we investigated the suppressive effect of glucose on chitinase of 4 strains. Also we studied the relationship between chitin induction and glucose suppression on chitinase. This investigation demonstrated that all tested B. thuringiensis strains could produce chitinase without chitin. After induction, the chitinolytic activity of 31 tested strains had no obvious response to the inducer, whereas 44 stains increased in different degree. Among these strains, most of them did not markedly increase the levels of chitinase, and many stains simultaneously displayed the expression mode of inducible and constitutive. The glucose inhibited the inductive effect of chitin, but it could not inhibit the basal expression of chitinase. Two strains No. 38 and No. 75 belonged to different expression types. But we just found several different bases in the regulatory region of chitinase genes chiA and chiB from them.

Abstract:To identify spatiotemporal expression patterns of vernalization genes in common wheat, we analyzed expression characteristics of several vernalization genes (VRN1, VRN2 and VRN3) in the wheat cultivars ‘Chinese spring’ and ‘Luohan 2’ by RT-PCR. The VRN1 gene was expressed at different levels in the leaves and roots at the 3-leaf stage, stems, flag leaves at the grain-filling stage, anthers, ovules, and developing seeds in ‘Chinese spring’. Expression of VRN1 increased before flowering date, then decreased after flowering time. Expression of VRN1 was not detected in dry seeds or seeds germination. Expression patterns of VRN1 in ‘Luohan 2’ were similar to those in ‘Chinese spring’, except that it was not expressed in roots or in the leaves at the 3-leaf stage in ‘Luohan 2’. Expression of VRN2 was only detected in the leaves at the 3-leaf stage and in the embryo buds during seeds germination. The spatiotemporal expression of VRN3 was similar to that of VRN1, except that VRN3 was not expressed in roots. These results improved our understanding of the molecular regulation of vernalization genes in common wheat.

Abstract:FRUITFULL (FUL) is an MADS box gene that functions early in controlling flowering time, meristem identity and cauline leaf morphology and later in carpel and fruit development in Arabidopsis thaliana. In order to clarify the regulation of FUL expression the upstream regulatory region, ?2 148 bp?+96 bp and the first intron of the FUL gene were cloned, and vectors with a series of deletion of FUL promoter, and the ones fused with the first intron were constructed. Vectors harboring the fusion of cis-acting elements with the constitutive promoters of TUBULIN and ACTIN were also constructed. β-Glucuronidase activity assays of the transgenic Arabidopsis plants showed that two cis-elements were involved in the repression of FUL expression, with one of the two being probably the binding site of the transcriptional factor AP1. And the two CArG boxes played a important role in FUL initiation particularly. Furthermore, the first intron of FUL was shown to participate in the development of carpel and stamen as an enhancer.

Abstract:In the application of therapeutic antibodies, large molecular weight of antibodies is always a problem that prevents them from penetrating into tissues or binding to antigenic determinants. To overcome this problem, we investigated the function of the heavy chain variable domain of a monoclonal anti-VEGF human IgM antibody derived from the Five-Feature Translocus Mice. We cloned the cDNA of the heavy chain variable domain, which was then inserted into pET28a vector and expressed in Escherichia coli. After purification and renaturation of the denatured recombinant protein, we obtained a 16 kDa antibody fragment, which is named as rhVVH. By immunoassaying its VEGF-binding capability in vitro, we proved that rhVVH retains this activity as the complete IgM. Importantly, rhVVH is shown to inhibit the HUVEC cell proliferation in a concentration-dependent manner. Our results indicate that the single heavy chain variable domain might inherit part of the biological function of the complete IgM antibody, which provided a valuable potential in further research on antibody miniaturisation.

Abstract:LZ-8 protein, isolated from a well known Chinese traditional medicinal fungus Ganoderma lucidum, is the first member of fungal immunomodulatory protein, members of which have been isolated from a variety of medicinal and edible mushrooms in the last two decades. The protein plays a multifunctional and important role in modulating immune system. In this report, in order to get LZ-8 protein crystals, the LZ-8 gene was expressed and purified by affinity chromatography, gel filtration chromatography and anion exchange chromatography subsequently. The protein was then crystallized using the hanging-drop vapour-diffusion method. The LZ-8 crystals were obtained and the phase information was calculated by X-ray diffraction. The resolution of LZ-8 crystals is 3.2?. This study will provide an insight into the structure of fungal immunomodulatory proteins.

Abstract:Formation and nuclear export of pre-ribosomes requires many nucleolar complexes. hNoc4L which contains a conserved Noc doman is a homolog of nucleolar complex associated 4 (S. cerevisiae), but its function is completely unclear. Here, we successfully got the recombinant lentiviral vector pll3.7-EF1-hNoc4L-Flag by replacing the U6 promoter in pll3.7 with EF1α promoter, and then inserted hNoc4L to down-stream of the EF1α prompter. We determined the transduction efficiency in different mammalian cell lines based on lentiviral packaging system. Subsequently, we analyzed the immunogenicity of the recombinant lentivirus and stable expression of hNoc4L in RAW264.7 cells. The results showed that the recombinant lentivirus characterized a high transduction efficiency, long-term expression and low immunogenicity. Therefore, we pave the way for further identification of the biological activity of hNoc4L protein during ribosome biogenesis in mammalian.

Abstract:Novel ion exchange adsorbents were synthesized by immobilizing sulfopropyl derivative onto homemade highly cross-linked agarose beads. The effects of different ligand densities (from 0.05 to 0.24 mol/L) on static and dynamic adsorption of the adsorbents were investigated using lysozyme as a model protein. Based on these results, rHLF was purified from the transgenic milk by our SP media. 1 mL high density (0.24 mol/L) adsorbent could handle 50 mL rHLF-containing milk. The mass recovery of rHLF was 86.5% and the purity was 98.5%. CD spectra demonstrated that the native structure of rHLF was not affected in the purification process. The biological functions of the purified rHLF, including iron binding, releasing and antimicrobial activities were then investigated. The results showed that rHLF had comparable iron binding and releasing activity to that of native HLF. 5 g/L concentration of rHLF significantly inhibited the growth of Escherichia coli. These studies lay a solid foundation for the wide application of our self-prepared ion exchange adsorbents in protein purification.

Abstract:Endotoxin removal is essential for the safety of biological products. To remove endotoxin efficiently, we used polymyxin B (PMB) affinity adsorbent to remove endotoxin from protein solutions by static adsorption. We studied the effects of spacer length and ligand density of the affinity adsorbent, pH, salt type and concentration, protein type and concentration, endotoxin concentration, and additive on endotoxin removal and protein recovery. Endotoxin content and protein concentration were determined by test and Lowry assay respectively. The results showed that PMB affinity adsorbent had high capacity, high adsorption speed, high removal efficiency and good reusability. In addition, ligand density, pH, salt concentration and the isoelectric point and hydrophobicity of protein all had remarkable influence on the endotoxin removal. Under the optimal conditions, the recoveries of hemoglobin, human serum albumin and lysozyme were 87.2%, 73.4% and 97.3%, respectively, and the corresponding endotoxin removal rates 99.8%, 97.9% and 99.7%, respectively. This study illustrated the effects of solution conditions on the efficiency of endotoxin removal and protein recovery, and would provide useful reference for the efficient removal of endotoxin from biological products.