Abstract:Microbial transglutaminase, which could catalyze the cross-linking of many proteins or non-protein materials, has been widely used in food, pharmaceutical and textile industry. To enhance the yield of the enzyme and establish corresponding platform for molecular modification, the researchers of Japanese Ajinomoto began to construct the recombinant strain producing transglutaminase in the 1990s. So far, the enzyme has been successfully expressed in different expression systems. Some of the recombinant strains are more productive than wild strains. Recently, progress has been made in the molecular modification of microbial transglutaminase, and the activity, thermo-stability and specificity of the enzyme are improved. This review briefly summarized and analyzed the strategies involved in these studies, and noted its trends.

Abstract:With the development of low-carbon economy and renewable resource, fermentation of the pentose sugar xylose to produce ethanol becomes a very hot topic. The recombinant Saccharomyces cerevisiae can be constructed by expressing heterologous xylose isomerase (XI). Because Thermus thermophilus XI (TthXI) does not need cofactor, it has been developed for establishing the utilization pathway of xylose in S. cerevisiae. In this article, we reviewed the progress on xylose isomerase. We first introduced the primary properties, sequence and structure characters of xylose isomerase, and discussed its thermostability. The molecular modification of xylose isomerase, including of substrate specificity and thermostability were discussed in detail. Meanwhile, combined with our own research, we also discussed how to improve the xylose isomerase activity at room temperature. Finally, we suggested perspectives of xylose isomerase.

Abstract:Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is an important ubiquitous cytosol enzyme that fixes HCO3- together with phosphoenolpyruvate (PEP) and yields oxaloacetate that can be converted to intermediates of the citric acid cycle. In plant cells, PEPC participates in CO2 assimilation and other important metabolic pathways, and it has broad functions in different plant tissues. PEPC is also involved in the regulation of storage product synthesis and metabolism in seeds, such as affecting the metabolic fluxes from sugars/starch towards the synthesis of fatty acids or amino acids and proteins. In this review, we introduced the progress in classification, structure and regulation of PEPC in plant tissues. We discussed the potential applications of plant PEPCs in genetic engineering. The researches in functions and regulation mechanism of plant PEPCs will provide beneficial approaches to applications of plant PEPCs in high-yield crops breeding, energy crop and microbe genetic engineering.

Abstract:The objective of the study was to clone avian β-defensin (AvBD) 3 gene from goose tissues, express the recombinant AvBD3 protein in Escherichia coli, and determine its antimicrobial activity. The mRNA of goose AvBD3 was cloned from spleen and bursa of Fabricius of the gooses by RT-PCR. The sequence analysis showed that the genefragment of AvBD3 contained 182 bp, and encoded 60 amino acids. Homology analysis showed that goose AvBD3 shared the highest percentage of amino acid homology (100%) with chicken AvBD3. The cDNA of goose AvBD3 was sub-cloned into BamH I and Sal I sites of pGEX-6p-1 vector to construct recombinant plasmid pGEX-goose AvBD3. The recombinant plasmid was transformed into E. coli BL21 and the bacteria was induced with IPTG. It was demonstrated by SDS-PAGE that a 31 kDa protein which was equal to goose AvBD3 protein in molecular weight was highly expressed. The purified recombinant goose AvBD3 exhibited extensive antimicrobial activity against twelve bacteria strains, including Gram-positive and Gram-negative investigated. At high salt ions conditions, antimicrobial activity of recombinant goose AvBD3 protein against both Staphylococcus aureus and Pasteurella multocida decreased significantly. In addition, hemolysis activity of the recombinant protein was extremely low, and the recombinant protein remained antimicrobial activity under different pH values.

Abstract:Dermonecrotic toxin (DNT) is identified as one of the most important virulence factor of Bordetella bronchiseptica. The complete coding sequence (4 356 bp) of the dnt gene was cloned into the prokaryotic expression vector pET-28a, and expressed in the Eschierichia coli BL21 (DE3) under IPTG (Isopropyl-β-D-thiogalactopyranoside) induction. The recombinant His6-DNT protein showed immunological reactivity in the Western-blot analysis. The recombinant protein was purified from crude lysates of BL21 harboring pET-DNT with the purity of 93.2%. His6-DNT showed the dermonecrotic effects in the infant mouse assay. However, rabbit anti-serum against recombinant DNT protein could neutralize the dermonecrotic effects of native DNT to the infant mice in vivo. These findings suggest that the recombinant DNT protein retained the characteristics and immunogenicity of native DNT. Furthermore, this approach could be used to induce active immunity and serum immunoglobulin for production of a passive therapeutic reagent. In this study, we have shown that the recombinant His6-DNT protein retained the characteristics of native DNT of B. bronchiseptica, which built a good foundation for the further research on the structure and function of DNT.

Abstract:The N-terminal of porcine parvovirus (PPV) viral protein 2 (VP2) links a glycine-rich domain which is a cleavage site of PPV VP3.In order to confirm that the glycine-rich domain was essential for the self-assembling of virus-like particles (VLPs).The VP2 gene with glycine-rich domain deleted and the complete VP2 gene were inserted to eukaryotic expression vector pCI-neo and were named pCI-△VP2 and pCI-VP2. Then, pCI-△VP2, pCI-VP2 and pCI-neo were transferred into Vero Cells by liposome and the VLPs was detected by SDS-PAGE, Western blotting, indirect immunofluorescence and immunoelectron microscopy. Furthermore, 56 female Kunming mice were divided into 5 groups and injected intramuscularly with pCI-△VP2, pCI-VP2 and pCI-neo as DNA vaccine, PPV inactivated vaccine and normal saline separately. The peripheral blood of the mice was collected to analyze the subgroups of the peripheral blood mononuclear cell by flow cytometry, to detect the antibody and lymphocyte proliferation by indirect-ELISA and MTT assay separately. The results show that the VLPs were observed both in the pCI-△VP2 and pCI-VP2 transferred Vero Cells. The two VLPs could agglutinate guinea pig erythrocytes. The results also show that both the pCI-△VP2 and pCI-VP2 vaccine induced special cellular and humoral immunity effectively. Those results revealed that the glycine-rich domain is not essential for the VPL’s self-assembling. This study provides a new theoretical evidence for the relationship between the gene structure and protein function of VP2.

Abstract:The production of 2, 3-butanediol and succinic acid by a moderate halophile under anaerobic condition was investigated. This halophile, termed Salinivibrio YS, was isolated from the solid samples collected from Aydingkol Lake. Based on the single factor experiment, the parameters and their values for the production were obtained. Then, the optimum values of these parameters by the orthogonal experiments were obtained: temperature, 33 °C; initial pH of fermentation, 8.0; the pH during fermentation, 7.0; the concentration of acetic acid was 3 g/L and NaCl was 10 g/L. Finally, a 3-L fermentation based on these conditions was carried out. After 108 h of fermentation under anaerobic condition, 35.05 g/L of 2, 3-butanediol and 22.46 g/L of succinic acid were obtained. About 50% of total glucose conversion was achieved. The study on 2, 3-butanediol and succinic acid by a halophile under anaerobic condition will expand the applications of halophiles and open a new area of production of 2, 3-butanediol and succinic acid.

Abstract:Besides medical application, 4-hydroxybutyrate (4-HB) is a precursor of P3HB4HB, a bioplastic showing excellent physical properties and degradability. Escherichia coli S17-1 (pZL-dhaT-aldD) can transform 1, 4-butanediol (1,4-BD) into 4HB with participation of cofactor NAD. To enhance productivity, nicotinic acid phosphoribosyltransferase (PncB) and nicotinamide adenine dinucleotide synthetase (NadE) were overexpressed to increase intracellular nicotinamide adenine dinucleotide concentration and promote reaction process. The shake flask fermentation result showed that the conversion rate increased by 13.03% with help of PncB-NadE, leading to 4.87 g/L 4HB from 10 g/L 1,4-BD, and productivity was increased by 40.91% to1.86 g/g. These results demonstrated that expression of PncB and NadE is beneficial for conversion of 1,4-BD to 4HB.

Abstract:Yarrowia lipolytica lipase Lip2 (YlLip2) is an important industrial enzyme with many potential applications. To alleviate the dissolved oxygen (DO) limitation and improve YlLip2 production during high-cell density fermentation, the YlLip2 gene lip2 and Vitreoscilla hemoglobin (VHb) gene vgb were co-expressed in Pichia pastoris under the control of AOX1 and PsADH2 promoter, respectively. The PsADH2 promoter from Pichia stipitis could be activated under oxygen limitation. The SDS-PAGE and CO-difference spectrum analysis indicated that VHb and YlLip2 had successfully co-expressed in recombinant strains. Compared with the control cells (VHb?, GS115/9Klip2), the expression levels of YlLip2 in VHb-expressing cells (VHb+, GS115/9Klip2-pZPVT) under oxygen limitation were improved 25% in shake-flask culture and 83% in a 10 L fermentor. Moreover, the VHb+ cells displayed higher biomass than VHb? cells at lower DO levels in a 10 L fermentor. In this study, we also achieved a VHb-expressing clone harboring multicopy lip2 gene (GS115/9Klip2-pZPVTlip2 49#), which showed the maximum lipolytic activity of 33 900 U/mL in a 10 L fermentor under lower DO conditions. Therefore, it can be seen that expression of VHb with PsADH2 promoter in P. pastoris combined with increasing copies of lip2 gene is an effective strategy to improve YlLip2 production.

Abstract:In Escherichia coli, uptake of L-tryptophan is done by three distinct permeases, encoded by mtr, tnaB, and aroP. Based on the mtr single-gene knockout, we constructed the mtr.tnaB and mtr.aroP double-gene knockout mutants and the mtr.tnaB.aroP triple-gene knockout mutant. The fermentation results showed that the mtr.tnaB and mtr.aroP knockout mutants produced 1.38 g/L and 1.27 g/L L-tryptophan, respectively, which was 17% and 9% higher than that of the mtr knockout mutant. However, the mtr.tnaB.aroP knockout mutant was significantly affected on cell growth and only produced 0.63 g/L L-tryptophan. During the fed-batch fermentation in a 3-L fermentor, the mtr.tnaB knockout mutant produced 12.2 g/L L-tryptophan, which was 27% higher than that of the mtr knockout mutant. This study demonstrates the effect of multi-gene knockouts of L-tryptophan transport system of Escherichia coli on the biosynthesis of L-tryptophan.

Abstract:To illustrate the complex fermentation process of submerged culture of Antrodia camphorata ATCC 200183, we observed the morphology change of this filamentous fungus. Then we used two optimization models namely response surface methodology (RSM) and artificial neural network (ANN) to model the fermentation process of Antrodia camphorata. By genetic algorithm (GA), we optimized the inoculum size and medium components for Antrodia camphorata production. The results show that fitness and prediction accuracy of ANN model was higher when compared to those of RSM model. Using GA, we optimized the input space of ANN model, and obtained maximum biomass of 6.2 g/L at the GA-optimized concentrations of spore (1.76×105 /mL) and medium components (glucose, 29.1 g/L; peptone, 9.3 g/L; and soybean flour, 2.8 g/L). The biomass obtained using the ANN-GA designed medium was (6.1±0.2) g/L which was in good agreement with the predicted value. The same optimization process may be used to improve the production of mycelia and bioactive metabolites from potent medicinal fungi by changing the fermentation parameters.

Abstract:Rhizopus oryzae lipase (ROL) is not only a biocatalyst used in a broad range of biotechnological fields, but also a model to investigate the function of intramolecular chaperone in the post-translational processing of lipase. In this study, we cloned and expressed the mature lipase gene (m-ROL) containing the pre-sequence (pro-ROL) of R. oryzae HU3005 in Pichia pastoris GS115 and characterized their enzymatic activities. m-ROL exhibited higher hydrolysis activity towards middle-chain substrates (C10 and C12) at pH 9.0, whereas pro-ROL preferred short-chain substrates (C4) and displayed maximal activity at pH 8.0. Moreover, pro-ROL possessed better thermal stability than m-ROL. This enzymatic discrepancy between m-ROL and p-ROL may be due to the pre-sequence that affects the folding and conformation of the mature lipase domain. To improve the expression level of m-ROL in P. pastoris, overlap extension PCR was conducted to substitute eight less-frequently used codons of m-ROL with frequently used codons. After methanol-induced expression for 72 h, the activity and protein content of the codon optimized m-ROL reached 132.7 U/mL and 50.4 mg/L, while the activity of the parental m-ROL and pro-ROL are 28.7 U/mL and 14.4 mg/L, 29.6 U/mL and 14.1 mg/L, respectively.

Abstract:We observed the influence of methanol concentration on purification recovery of recombinant human consensus interferon-α (cIFN) produced by Pichia pastoris. Fermentations controlled at 0.75% (W/V) methanol showed a 24% increase in cIFN expression compared to using 0.25% methanol. However, the purification recovery rate of cIFN at 0.25% methanol was 3.75-fold higher than that at 0.75% methanol. To seek the reason, we analyzed the stability of cIFN by SDS-PAGE and Native-PAGE as well as Western blotting. The electrophoresis results revealed that cIFN formed a lot of aggregates in media when the induction was controlled at 0.75% methanol, and two different aggregate forms were found: disulfide bond covalent aggregates and non-covalent aggregates. However, these aggregates almost disappeared when the methanol concentration was controlled at 0.25%, at the same time, cIFN bioactivity of supernatant increased almost 4.48-fold. Finally, 0.73g monomer cIFN was obtained after purification from 1 liter supernatant at 0.25% methanol induction, showed a 2.84-fold increase compare to the induction at 0.75% methanol.

Abstract:Directed evolution was used to improve the performance of β-1,3-1,4-glucanase (designated as PtLic16A) from Paecilomyces thermophila J18 under acidic condition. A mutant library was constructed by error-prone PCR and DNA shuffling, and positive clones were screened by Congo red staining. More than 1 500 mutants were selected. One mutant (PtLic16AM1) exhibited an optimal activity at pH 5.5, while the optimal pH of the wild-type enzyme was 7.0. The mutant PtLic16AM1 kept the high specific activity and thermotolerence of the wild-type enzyme. Sequence analysis revealed that the mutant enzyme has four sense substitutions which caused four amino acid substitutions - namely T58S, Y110N, G195E and D221G.. Homology modeling showed that among the four amino acid substitutions, Y110N was near the active site of the enzyme, while the other three was distant. T58S and G195E may play key roles in the change of optimal pH. This study provided a new perspective of obtaining applicable β-1,3-1,4-glucanase for industrial use.