Abstract:During the development of bacteria-based biotechnology, bacteriophage infection is one of the constant threats and troublesome problems in industrial fermentation. The core of puzzled bacteriophage infection is a complex arm race of coevolution between bacteriophages and their hosts where bacteriophage has evolved lots of escaped ways against bacterial resistance mechanisms. The strategies of rationally designing factories and rotation of starter strains could reduce the risk of bacteriophage infection, but often fail to avoid. Genetic engineering to increase bacterial resistance is one of the strategies to prevent bacteriophage infection and more knowledge about bacteriophage and its host is needed. Recently, there are some new findings on bacterial resistance mechanisms which provide new solutions for bacteriophage infection. For example, it is possible for a rational design of resistant strains to use CRISPR-Cas based technologies just based on the sequences of bacteriophages. Moreover, it is also possible to avoid the escape of bacteriophage by iteratively building up resistance levels to generate robust industrial starter cultures. Quorum-sensing signal molecules have recently been proved to be involved in the interactions between bacteria and bacteriophages, which provides a possible way to solve bacteriophage infection from a population level. Finally, the rapid development of bacteriophage genome editing and synthetic biology will bring some new cues for preventing bacteriophage infection in industrial fermentation.

Abstract:To develop a high-yield pyruvate strain, we first engineered a pyruvate-producing Escherichia coli KLPP from wild-type E. coli MG1655 by blocking the pathways for byproduct formation via gene knockout. Then, we built a library of mutant containing 7 197 monoclones by using the pUT Mini-Tn5 transposon vector for random mutagenesis with E. coli KLPP. We developed a high-throughput method for pyruvate detection based on dinitrophenylhydrazine reaction using 96-well microplate reader. After two-round screening we successfully obtained six mutants with increased pyruvate titer using this method, the titer of pyruvate was increased by 38%, 31%, 19%, 28%, 44% and 14%, respectively. The position of transposon insertion was determined by whole genome re-sequencing, and the gene locus possibly influencing pyruvate production was analyzed, which laid the foundation for subsequent strain improvement by metabolic engineering.

Abstract:Chiral amines are important building blocks for the synthesis of pharmaceutical products and fine chemicals. Highly stereoselective synthesis of chiral amines compounds through asymmetric amination has attracted more and more attention. ω-transaminases (ω-TAs) are a promising class of natural biocatalysts which provide an efficient and environment-friendly access to production of chiral amines with stringent enantioselectivity and excellent catalytic efficiency. Compared with (S)-ω-TA, the research focused on (R)-ω-TA was relatively less. However, increasing demand for chiral (R)-amines as pharmaceutical intermediates has rendered industrial applications of (R)-ω-TA more attractive. Improving the thermostability of (R)-ω-TA with potential biotechnological application will facilitate the preparation of chiral amines. In this study, the dynamic surface loop with higher B-factor from Aspergillus terreus (R)-ω-TA was predicted by two computer softwares (PyMOL and YASARA). Then mutant enzymes were obtained by deleting amino acid residues of a dynamic surface loop using site-directed mutagenesis. The results showed that the best two mutants R131del and P132-E133del improved thermostability by 2.6 ℃ and 0.9 ℃ in T5010 (41.1 ℃ and 39.4 ℃, respectively), and 2.2-fold and 1.5-fold in half-life (t1/2) at 40 ℃ (15.0 min and 10.0 min, respectively), compared to that of wild type. Furtherly, the thermostability mechanism of the mutant enzymes was investigated by molecular dynamics (MD) simulation and intermolecular interaction analysis. R131del in the loop region has lower root mean square fluctuation (RMSF) than the wild type at 400 K for 10 ns, and mutant enzyme P132-E133del increases four hydrogen bonds in the loop region. In this study, we obtain two stability-increased mutants of (R)-ω-TA from A. terreus by deleting its dynamic surface loop and also provide methodological guidance for the use of rational design to enhance the thermal stability of other enzymes.

Abstract:Large quantity of activated sludge is generated from wastewater treatment but without yet an appropriate deposition. High temperature can lyse the activate sludge so that nitrogen and phosphorus containing nutrients are released. Halomonas CJN was found to grow on the heat lysed activated sludge and glucose for production of bioplastic poly-3-hydroxybutyrate (PHB) in the absence of yeast extract, nitrogen and phosphorus sources as well as trace elements. This reduces the PHB production cost significantly. Furthermore, acetic acid formed from anaerobic fermentation of heat lysed activated sludge can be used to replace glucose for cell growth but not much for PHB production. After construction of an additional PHB synthesis pathway in Halomonas CJN, we can produce PHB entirely from heat lysed activated sludge, reducing production cost of PHB roughly from ￥ 30 000 Yuan/ton to ￥ 20 000 Yuan/ton, thus turning waste activated sludge to valuable raw material resource.

Abstract:To enrich the resource pool of endophytic fungi from plants which produce taxol, a taxol-producing endophytic fungus TMS-26 was isolated from the stem of Taxus Media. The result of high performance liquid chromatography (HPLC) showed that TMS-26 extract exhibited similar chromatographic peaks and retention time (4.545 min) with authentic taxol. Then mass spectrometry (MS) analysis further confirmed that TMS-26 extracts contained the same mass peaks with authentic taxol ((M+Na)+=876). These indicated that the isolated endophytic fungus TMS-26 can produce taxol. According to the morphological characteristics, the molecular analysis of 18S rDNA and internal transcribed spacer nuclear rDNA gene sequence, the fungus was identified as Aspergillus fumigatus TMS-26.

Abstract:Integrins are transmembrane glycoproteins, closely related to many physiological and pathological processes. In order to explore its role in silkworm, by PCR and Rapid-amplification of cDNA ends (RACE) technology, the full-length cDNA of Bmintegrin β1 in silkworm was acquired. The domain was predicted by domain prediction website. Phylogenetic tree was constructed to analyze its evolutionary relationship. By prokaryotic expression system, protein purification method and immunizing mouse, the antibody against Bmintegrin β1 recombinant protein was obtained. The spatial-temporal expression profile of Bmintegrin β1 was investigated by semi quantitative PCR and Western blotting. Then we identified all 3 different spliceosomes, and they shared a common open reading frame of 2 502 bp, encoding 833 amino acids. Bmintegrin β1 contained all the classic domains of the integrin family, such as Integrin-B-tail, transmembrane domain etc. Phylogenetic analysis indicated that Bmintegrin β1 was close to the homologous proteins from Heliothis assulta and Danaus plexippus. In order to understand the function of Bmintegrin β1 further, we generated the antibody. In addition, Western blotting demonstrated that the antibody recognized the Bmintegrin β1 recombinant protein. Then, semi quantitative PCR and Western blotting results showed that Bmintegrin β1 was widely expressed in most of tissues, among of them, it’s exhibited the highest expression level in hemacyte. Overall, this study provides a foundation for the study of silkworm integrin family.

Abstract:By bioinformatics analysis, a putative keratinase gene gm2886 (Accession number: KY368946) was discovered in the genome of a feather-degrading strain, Streptomyces albidoflavus Fea-10. gm2886 was ligated into integrative Escherichia coli-Streptomyces shuttle vector pSET152 under the promoter PermE and added with C-terminal His-tag. The expression vector was transformed into Streptomyces pactum ACT12 by conjugal transfer and the recombinant protein GM2886-His6 was detected in fermentation broth. GM2886-His6 was purified and characterized. Its size was nearly 36 kDa. GM2886-His6 showed proteolytic activity towards a variety of substrates and could even degrade insoluble substrates, such as azure keratin and chicken feathers. The optimal pH and temperature of GM2886-His6 for proteolysis of casein was pH 10.0 and 50 ℃, respectively. The enzyme activity was inhibited by PMSF, but not EDTA, indicating that GM2886-His6 was a serine proteinase. Our results laid the foundation for the research of the molecular biological mechanism on feather-degrading and for the further utilization of Fea-10.

Abstract:This study aimed to construct prokaryotic recombinant plasmids for expression of the extracellular domains of NMDAR1 protein, purify and characterize the immunoreactivity of the recombinant proteins. Based on the mRNA sequence of human NMDAR1 gene, we predicted the structure of the antigenic domains in the extracellular part of the protein using the “phyre2” software. Primers were designed to amplify the nucleic acid fragments encoding the NMDAR1 extracellular antigenic domains by RT-PCR. The amplified gene fragments were cloned into pCold-SUMO vector to construct the recombinant plasmids which were transformed into Escherichia coli DH5α. The positive colonies harboring the recombinant plasmids were picked and verified by PCR and DNA sequencing. Then, the recombinant plasmids were transformed into E. coli BL21(DE3) strain and induced by IPTG for protein expression. The recombinant proteins were purified by Ni-NTA affinity chromatography. The target proteins were further purified by removing the 6 His-SUMO tag using enzyme excision followed by gel filtration chromatography using AKTA purifier. The purity of the recombinant proteins were evaluated by SDS-PAGE and the immunoreactivity were characterized by Western blotting. Three DNA fragments encoding the extracellular domains of NMDAR1 protein, including NR1-M1 (encoding 19–393 aa), NR1-S1 (encoding 394–544 aa) and NR1-S2 (encoding 663–800 aa), were amplified by RT-PCR. The NR1-S1 and NR1-S2 were linked with G (arginine) and T (threonine) amino acid as a combined fragment. The NR1-M1 and NR1-S1-GT-S2 fragments were cloned into pCold-SUMO vector and two recombinant plasmids, pCold-SUMO-M1 and pCold-SUMO-S1-GT-S2, were generated and expressed in E. coli. SDS-PAGE analysis showed that the recombinant plasmids expressed soluble NR1-M1 and NR1-S1-GT-S2 proteins in bacterial. After affinity chromatography and gel filtration chromatography, we obtained high purity target proteins. Western blotting assay showed that the recombinant proteins NR1-M1 and NR1-S1-GT-S2 can bind specially with their corresponding antibodies, suggesting the recombinant proteins retained antigenic reactivity. We constructed a prokaryotic expression system for expressing the NMDAR1 protein extracellular parts that had immunoreactivity successfully, and the purified proteins can be used for studying NMDAR1 function and testing the autoantibodies.

Abstract:3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) is the key enzyme to synthesize eight-carbon sugar in plant and gram-negative bacterial cell wall. To analyze the polymerization and characterization in plant KDO8PS, the candidate gene was cloned from fresh Phyllostachys edulis seedling by RT-PCR. The open reading frame of PeKDO8PS is 876 bp deduced into 291 amino acid residues. The target protein was overexpressed in Escherichia?coli induced by IPTG and then lager amount of fusion protein was purified through two-step methods with affinity chromatography and size-exclusion chromatography (SEC). SEC analysis shows that PeKDO8PS protein existed mainly in the form of dime in solution. Glutaraldehyde cross-linking experiments confirmed that the enzyme could form dimers. Further we identified that KDO8PS at high concentration two dimers could form tetramer in aqueous solution by analytical ultracentrifuge (AUC) analysis. The pH of the catalytic reaction was between 4.0 and 9.0, the optimum pH value was 8, the thermal stability range was between 25 and 65 ℃, and the optimum temperature was about 55 ℃. The enzyme activity was inhibited by some metal ions at lower concentrations, especially in the presence of Fe3+metal ion and activated by metal protease inhibitor EDTA at low concentration.

Abstract:Glutathione (GSH), a non-protein thiol product with various biological activities, has been widely used in pharmaceutical and food industries. Recently, genetic engineering becomes an important strategy for obtaining GSH-high-producing strains. However, auxotrophic selection markers used may result in reduced cell growth or GSH production. In the present study, clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9-associated-system (CRISPR-Cas), in which gRNA expression constructs and homologous DNA fragments of target genes were co-transformed into Saccharomyces cerevisiae cells, was used for the construction of the prototrophic strain derived from the engineered auxotrophic strain W303-1b/FGP. As a result, the prototrophic strain W303-1b/FGPPT showed a significantly shorter culture cycle compared with the auxotrophic strain. Furthermore, chemically defined medium could be used to culture strain W303-1b/FGPPT that might have great interests in industrial fermentation.

Abstract:Bacterial ghosts are bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology, which can be used as a new vaccine and delivery vector. In this study, a clinical isolate of avian pathogenic Escherichia coli (APEC) strain DE17 was used to prepare bacterial ghost through three different ways. The results showed that the cleavage efficiency of DE17 bacterial ghost was 99.9% with the lysis plasmid containing the PhiX174 lysis gene E. Scanning electron microscopy showed that transmembrane tunnels were formed in the middle or both ends of the cell envelope of DE17. Furthermore, the DE17 bacterial ghost was prepared with one of cell penetrating peptides (CPPs) named MAP (KLALKLALKALKAALKLA), which will completely inactivate DE17 (OD600=0.1) by 10 μmol/L MAP. The cell envelope showed a gully-like structure and obvious transmembrane tunnels were not found through the SEM. However, the DE17 could not be lysed by importing the lysis plasmid (pBV220-MAP), which was used to express MAP. The present study will benefit for research on bacterial ghost preparation methods and provide a reference for biosafety of bacterial ghost vaccines.

Abstract:Pectate lyase is widely applied in ramie degumming and fabric bioscouring in the textile industry. Compared to conventional processes that involve high alkaline and high temperature treatment, enzyme based treatments have significant advantages in fibers protectiveness, improved efficiency of refining, reduced energy consumption and pollution. Hence, it would be highly desirable to construct high-yield alkaline pectate lyase engineered strains and reduce the pectate lyase production cost. In the previous study, pectate lyase gene pel from Bacillus subtilis168 was expressed in Pichia pastoris GS115 after codon usage optimization based on the vector pHBM905A. To improve the expression level, the vector pHBM905BDM with optimized promoter and signal peptide was used to express the optimized gene pels in GS115. The transformant had increased activity from 68 U/mL to 100 U/mL with the improvement in the transcription level by 27% measured by qPCR. The transformants were further screened on pectin plates, where higher halo forming strains were picked for shake-flask fermentation and strain GS115-pHBM905BDM-pels4 showed the highest activity of 536 U/mL. Then plasmid pPIC9K-pels was constructed and electroporated into the GS115-pHBM905BDM-pels4 cells. Subsequently, high-copy transformant was screened by using the medium containing antibiotics G418, strain GS115-pHBM905BDM- pPIC9K-pels1 was identified with increased activity of 770 U/mL and the copy number of pels was 7 confirmed by qPCR. Finally, the activity of pectate lyase produced by GS115-pHBM905BDM-pPIC9K-pels1reached to 2 271 U/mL in a 5-L fermentor. The activity of pectate lyase in our study reached the highest level of expression in P. pastoris, showing good application potential in the textile industry.

Abstract:A whole-cell catalyst using Escherichia coli BL21(DE3) as a host, expressing L- threonine dehydratase from Escherichia coli, and co-expressing leucine dehydrogenase from Bacillus cereus and glucose dehydrogenase from Bacillus subtilis for cofactor regeneration, was constructed and used for one-pot production of L-2-aminobutyric acid (L-ABA) and D- gluconic acid from L-threonine and D-glucose. We used shake-flask culture to study the whole-cell catalytic condition including temperature, pH, proper permeabilization of cells and optimal wet cells amount. Moreover, the whole-cell catalyst was cultured in 5-L fermentor by fed-batch fermentation, and 164 g/L L-threonine and 248 g/L D-glucose were converted to 141.6 g/L L-ABA and 269.4 g/L D-gluconic acid. The whole-cell catalyst is promising to fulfill industrial requirements for L-ABA and D-gluconic acid.