Abstract:Influenza virus assembly requires the completion of viral protein and vRNP transport to the assembly site at the plasma membrane. Therefore, efficient regulation of intracellular transport of the viral proteins and vRNPs to the surface of the host cell is especially important for virus morphogenesis. Influenza A virus uses the machineries of host cells to transport its own components including ribonucleoproteins (vRNPs) and three transmembrane proteins hemagglutinin (HA), neuraminidase (NA) and matrix 2 protein (M2). It has been shown that newly synthesized vRNPs are associated with active form of Rab11 and accumulate at recycling endosomes adjacent to the microtubule organizing center (MTOC) following nuclear export. Subsequently, they are transported along the microtubule network toward the plasma membranes in cargo vesicles. The viral transmembrane proteins are translated on the rough endoplasmic reticulum and transported to the virus assembly site at the plasma membrane. It has been found that several host factors such as ARHGAP21 and GTPase Cdc42 are involved in regulation of intracellular trafficking of influenza A virus transmembrane proteins including NA. In this review, we will highlight the current knowledge about anterograde transport and its regulation of the influenza A virus transmembrane proteins and genome in the host cytoplasm.

Abstract:In eukaryotic cells, multivesicular bodies (MVBs) are required for trafficking of membrane proteins to lysosomes for selective destruction. The sorting of ubiquitylated membrane proteins into multivesicular bodies and the biogenesis of MVBs are mediated by the endosomal sorting complex required for transport (ESCRT). Topologically equivalent to the budding of intralumenal vesicles from the limiting membrane of the MVBs, the ESCRT complex is also involved in cytokinetic abscission, phagophore formation, and enveloped virus budding. Many retroviruses and RNA viruses encode “late-domain” motifs that are able to interact with the components of the ESCRT complex, and the interactions recruit ESCRT-III and VPS4 to the viral assembly and budding sites. Recently, few studies revealed that the ESCRT complex is also required for efficient egress of some DNA viruses, including Hepatitis B, Herpes simplex virus type-1, and Autographa californica multiple nucleopolyhedrovirus. Further examination of virus-ESCRT interactions should shed light on the detailed mechanism of virus assembly and budding.

Abstract:NAD kinase catalyzes the phosphorylation of coenzyme Ⅰ [NAD(H)] to form coenzyme Ⅱ [NADP(H)], and NADPH is an important cofactor in L-isoleucine biosynthesis. In order to improve NADPH supply, ppnK, the gene encoding NAD kinase in Corynebacterium glutamicum was cloned and separately expressed in an L-isoleucine synthetic strain, Brevibacterium lactofermentum JHI3-156, by an inducible expression vector pDXW-8 and a constitutive expression vector pDXW-9. Compared with the control strain JHI3-156/pDXW-8, NAD kinase activity of the inducible ppnK-expressing strain JHI3-156/pDXW-8-ppnK was increased by 83.5%. NADP(H)/NAD(H) ratio was also increased by 63.8%. L-isoleucine biosynthesis was improved by 82.9%. Compared with the control strain JHI3-156/pDXW-9, NAD kinase activity of the constitutive ppnK-expressing strain JHI3-156/pDXW-9-ppnK was increased by 220%. NADP(H)/ NAD(H) ratio and NADPH concentration were increased by 134% and 21.7%, respectively. L-isoleucine biosynthesis was increased by 41.7%. These results demonstrate that NAD kinase can improve the coenzyme Ⅱ supply and L-isoleucine biosynthesis, which would also be useful for biosynthesis of other amino acids.

Abstract:We used ribosome engineering technology, with which antibiotic-resistant strains are resulted from mutations on microbial ribosome, to screen a high butanol-producing Clostridium strain. A novel mutant strain S3 with high butanol production and tolerance was obtained from the original Clostridium acetobutylicum L7 with the presence of mutagen of streptomycin. Butanol of 12.48 g/L and ethanol of 1.70 g/L were achieved in S3, 11.2% and 50%, respectively higher than the parent strain. The conversion rate of glucose to butanol increased from 0.19 to 0.22, and fermentation time was 9 h shorter. This caused an increase in butanol productivity by 30.5%, reaching 0.24 g/(L?h). The mutant butanol tolerance was increased from 12 g/L to 14 g/L, the viscosity of fermentation broth was dramatically decreased to 4 mPa/s, 60% lower than the parent strain. In addition, the genetic stability of mutant strain S3 was also favorable. These results demonstrate that ribosome engineering technology may be a promising process for developing high butanol-producing strains.

Abstract:Escherichia coli NZN111 is a promising strain with ldhA and pflB genes inactivated for the production of succinic acid. However, with these mutations, NAD+ could not be regenerated from NADH, and an unbalanced NADH/NAD+ ratio eliminated cell growth and glucose utilization under anaerobic conditions. Nicotinic acid mononucleotide adenylyltransferase (NAMNAT), encoded by the nadD gene, catalyzes the reaction from nicotinic acid mononucleotide (NaMN) to nicotinic acid adenine dinucleotide (NaAD) during the synthetic pathway of NAD(H). Overexpression of the nadD gene could enhance the concentration of NAD(H) and maintain a suitable NADH/NAD+ ratio. In this study, we constructed a recombinant strain E. coli NZN111/pTrc99a-nadD, and overexpressed NAMNAT with 1.0 mmol/L of IPTG under anaerobic conditions in sealed bottles. Compared to E. coli NZN111, the concentrations of NAD+ and NADH in the recombinant strain increased by 3.21-fold and 1.67-fold, respectively. The total concentration of NAD(H) was increased by 2.63-fold, and the ratio of NADH/NAD+ decreased from 0.64 to 0.42. The recombinant strain restored the cell growth and glucose utilization under anaerobic conditions. After 72 h, the recombinant strain could consume 14.0 g/L of glucose to produce 6.23 g/L of succinic acid, and the concentration of succinic acid was 19-fold higher than in E. coli NZN111.

Abstract:Some degradation products from lignocellulose pretreatment strongly inhibit the activities of cellulolytic enzymes and ethanol fermentation strains, thus the efficient removal of the inhibitor substances (“detoxification”) is the inevitable step for the biotransformation processes. In this study, the biological detoxification of furfural by a newly isolated fungus, Amorphotheca resinae ZN1, was studied and the metabolic pathways of furfural degradation was analyzed. The metabolic pathway of furfural degradation in A. resinae ZN1 was described as follows: first, furfural was quickly converted into the low toxic furfuryl alcohol; then the furfuryl alcohol was gradually converted into furfural again but under the low concentration under aerobic condition, which was not lethal to the growth of the fungi; furfural continued to be oxidized to furoic acid by A. resinae ZN1. It is likely that furoic acid was further degraded in the TCA cycle to complete the biological degradation of furfural. The present study provided the important experimental basis for speeding up the biodetoxification of furfural by A. resinae ZN1 and the rate-limiting step in the lignocellulose biotransformation to ethanol.

Abstract:In order to broaden Chinese cabbage gene pool, we conducted interspecific somatic hybridization between Chinese cabbage (Brassica campestris, 2n=20, AA) and Cabbage (B. oleracea, 2n=18, CC). Protoplasts were isolated from 10-day-old cotyledons and hypocotyls of young seedlings, and fused by 40% polyethylene glycol (PEG). Fused cells were cultured in modified K8p liquid medium supplemented with some plant growth regulators. Fusion products were characterized by their morphological, cytological and molecular biological traits. The results showed that, a total of 35 regenerated green plants were obtained from 320 calli, the plant regeneration frequency was 10.94%, and eleven of which were survived in greenhouse. All regenerants were true hybrids as confirmed by randomly amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analysis. Ploidy levels of hybrid plants were determined by chromosome counting and flow cytometry. The sum of the chromosome number (2n = 38) from the two fusion patents were found in 36.4% of regeneratns; another 36.4% had chromosomes range to 58-60; 27.2% had more chromosomes ranges to 70-76. All regenerated plants produced normal flowers. We investigated the pollen fertility and seed set after self-pollination and backcrossing with the parental species. For hybrids with chromosomes more than 38 it was possible to obtain some seeds when they after self-pollination. Within the group of hybrids with 38 chromosomes, seed set were very variable, only 0.11 seeds per pod by self-pollination, 0.23-0.76 by open-pollination, 0.02-0.04 by backcrossing with Chinese cabbage. Progeny lines obtained by self-pollination had larger leaves and leaf shapes intermediate of the parental species. Pollen fertility was gradually recovered in the first and second progenies. The backcrossing progeny lines, as a whole, exhibited morphologies were similar to Chinese cabbage. Morphological variations were observed among the somatic hybrids and their progenies.

Abstract:Protein A and protein G are two well-defined immunoglobulin (Ig)-binding proteins (IBPs), which show affinity for specific sites on Ig of mammalian hosts. Protein A and protein G contained several highly homologous IgG-binding domains which had been demonstrated to have function to bind to IgG. Whether combinations of Ig-binding domains of various IBPs could produce useful novel binding properties remains interesting. We constructed a combinatorial phage library which displayed randomly-rearranged A, B, C, D and E domains of protein A, B2 and B3 domains of protein G. Four rounds molecular evolution of this library directed by all four human IgG subclasses respectively generated a common arrangement of D-C respectively which didn’t exist in SpA. The dynamic loss of control phages and increase of the phages displaying two or more binding domains, especially the selective enrichment of D-C and strict selection of its linking peptides demonstrated the efficient molecular evolutions and the significance of the selected D-C arrangement. The phage binding assays confirmed that D-C possessed a binding advantage with four human IgG subclasses compared to SpA. In this work, a novel combination of Ig-binding domains, D-C, was obtained and presented the novel Ig binding properties which provided a novel candidate molecule for the purification, production and detection of IgG antibodies and a new approach for the further study of structures and functions of IBPs.

Abstract:The possibility of heterologous expression of human Stearoyl-CoA Desaturase (scd1) was investigated. The scd1 encoding sequence was inserted into the pNZ8149 to generate the pNZ8149-scd1 expression plasmids. Then we introduced the pNZ8149-scd1 construct into the Lactococcus lactis NZ3900 to investigate its enzyme activity. The results show that heterologous expressed SCD1 enzyme resulted in a 92%–169% increase in the C16:1n-7 and a 53-127% increase in the C18:1n-7 (P<0.05). The SCD1 enzyme was capable of producing n-7 fatty acids in Lactococcus lactis efficiently. It also suggests that the fatty acid desaturases can be heterologous expressed in Lactococcus lactis to produce the helpful fatty acids.

Abstract:Knockout is an important method for gene function study, while vector is the core of gene knockout. In order to obtain an effective vector for rapid construction of mutant and essentiality identification of the corresponding gene, we constructed a recombinant plasmid named pIDM-T based on the temperature-sensitive and replication- defective plasmid pIDM1 by inserting an Xcm I adapter into the EcoR I and Pst I sites of pIDM1. Digesting with Xcm I, pIDM-T can be prepared as a linear T-vector for PCR products cloning and be used to knockout the corresponding gene in the genome with insertion duplication mutagenesis. After the verification of temperature sensitivity of the replication of the plasmid, we cloned two Salmonella pullorum genes eno and ybdr into the constructed pIDM-T, and two recombinant plasmids pIDM-T_eno and pIDM-T_ybdr were identified. The recombinant plasmids were then transformed into S. pullorum strain CVCC527 and the IPC (Integration rate per cell) values were calculated. As a result, we identified the eno gene as an essential gene and the ybdr as a non-essential gene in CVCC527. We verified the correctness of recombination site in ybdr recombinant 527 clones (SalΔybdr) by PCR and sequencing. The pIDM-T vector can be used for gene knockout in S. pullorum, as well as the identification of essentiality of the corresponding genes, which offers an effective and rapid tool for gene function study in Salmonella.

Abstract:Routine protein assays are usually affected with various compounds, and we need to use different protein quantification protocol to deal with different interference. In order to simplify the procedure, we developed a new method, in which the components and concentrations of the reagents were modified mainly based on classic Folin-Ciocalteu’s reagent for reducing the susceptibility to interfering substances. Standard curves of the new method were established with different levels of bovine serum albumin, and then, we assessed and evaluated the detectable wavelengths and stability. In particular, the tolerability to several interfering substances was analyzed by using cytolysis solutions containing different chemicals. Our data in this study show that the new method could be applied to detecting protein concentrations accurately, even in the presence of surfactants such as 10% sodium dodecyl sulfate (SDS), 2% NP-40, or 1% TrintonX-100, chelators of 25 mmol/L EDTA or 1 mmol/L Ethylene glycol bis (2-aminoethyl) tetraacetic acid (EGTA), reductants of 1 mmol/L Dithiothretol (DTT) orβ-Mercaptoethanol (ME), or nitrogen-containing compounds of 0.5 mol/L ammonium sulphate or 4?mol/L urea. Taken together, these results indicate that the new approach significantly improves the tolerance to the interfering substances, which could be potentially useful in measuring the contents of proteins interfered with such substances.

Abstract:

Abstract: