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1.
Lifei Chen Chunling Ma Ruiming Wang Jianlou Yang Haijie Zheng 《Biotechnology letters》2016,38(10):1769-1774
Objectives
To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.Results
Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.Conclusion
Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.2.
Sen Miao Hao Wu Yue Zhao Qinggele Caiyin Yanni Li Jianjun Qiao 《Biotechnology letters》2018,40(6):941-948
3.
Jinxiang Zhu Qiaoyun Zhu Ruiqing Gong Qin Xu Menghao Cai Tianyi Jiang Xiangshan Zhou Mian Zhou Yuanxing Zhang 《Biotechnology letters》2018,40(9-10):1365-1376
Objective
Around one-fourth of the Komagataella phaffii genes encode hypothetical proteins with unknown functions. However, lack of powerful tools for genetic screening in K. phaffii significantly limits the functional analysis of these unknown genes. Transposon mutagenesis has been utilized as an insertional mutagenesis tool in many other organisms and would be extremely valuable if it could be applied in K. phaffii.Results
In this study, we investigated in K. phaffii the transposition activity and efficiency of piggyBac (PB) transposon, a DNA transposon from the cabbage looper moth Trichoplusia ni through the integrated-plasmid system. We also designed a binary-plasmid system which could generate stable mutants. Finally we evaluated the quality of this mutagenesis system by a simple screening for functional genes involved in K. phaffii carbon catabolite repression.Conclusions
Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.4.
Andrelisse Arruda Viviane Castelo Branco Reis Vinícius Daniel Ferreira Batista Bruno Sahim Daher Luiza Cesca Piva Janice Lisboa De Marco Lidia Maria Pepe de Moraes Fernando Araripe Gonçalves Torres 《Biotechnology letters》2016,38(3):509-517
Objectives
To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.Results
P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.Conclusions
A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.5.
Objectives
To characterize the genes responsible for ethanol utilization in Pichia pastoris.Results
ADH3 (XM_002491337) and ADH (FN392323) genes were disrupted in P. pastoris. The ADH3 mutant strain, MK115 (Δadh3), lost its ability to grow on minimal ethanol media but produced ethanol in minimal glucose medium. ADH3p was responsible for 92 % of total Adh enzyme activity in glucose media. The double knockout strain MK117 (Δadh3Δadh) also produced ethanol. The Adh activities of X33 and MK116 (Δadh) strains were not different. Thus, the ADH gene does not play a role in ethanol metabolism.Conclusion
The PpADH3 is the only gene responsible for consumption of ethanol in P. pastoris.6.
F. Cheikhrouhou R. Guidara A. Masmoudi H. Trabelsi S. Neji H. Sellami F. Makni A. Ayadi 《Mycopathologia》2017,182(5-6):583-589
Aim
Malassezia folliculitis is caused by the invasion of hair follicles by large numbers of Malassezia cells. Several Malassezia researches still use cultures, morphology and biochemical techniques. The aim of this study was to identify Malassezia species isolated from patients diagnosed with folliculitis, at the Parasitology and Mycology Laboratory of Sfax University Hospital, and to explore the genetic diversity of Malassezia by using PCR-RFLP and PCR-sequencing targeting the rDNA region of the Malassezia genome.Patients and Methods
Specimens were taken from 27 patients with Malassezia folliculitis. For the molecular identification, PCR amplification of the 26S rDNAD1/D2 region was carried out using the Malup and Maldown primers and three restriction enzymes (BanI, MspI and HeaII) for RFLP analysis. The nucleotide sequences of each isolate were compared to those in the NCBI GenBank by using BLASTIN algorithm.Results
Three species of Malassezia yeasts were identified among the 31 Malassezia strains isolated: M. globosa (83.9%), M. sympodialis (12. 9%) and M. furfur (3.2%). The sequence analysis of M. globosa showed six genotypes.Conclusion
There is a high genotypic variability of M. globosa colonizing patients with folliculitis.7.
Yuko Matsuda Otomi Cho Takashi Sugita Daiki Ogishima Satoru Takeda 《Mycopathologia》2018,183(4):691-700
8.
Background
DNA double-strand breaks (DSBs) are highly cytotoxic and mutagenic. MRE11 plays an essential role in repairing DNA by cleaving broken ends through its 3′ to 5′ exonuclease and single-stranded DNA endonuclease activities.Methods
The present study aimed to in silico characterization and molecular modeling of MRE11 from Phoenix dactylifera L cv deglet nour (DnMRE11) by various bioinformatic approaches. To identify DnMRE11 cDNA, assembled contigs from our cDNA libraries were analysed using the Blast2GO2.8 program.Results
The DnMRE11 protein length was 726 amino acids. The results of HUMMER show that DnMRE11 is formed by three domains: the N-terminal core domain containing the nuclease and capping domains, the C-terminal half containing the DNA binding and coiled coil region. The structure of DnMRE11 is predicted using the Swiss-Model server, which contains the nuclease and capping domains. The obtained model was verified with the structure validation programs such as ProSA and QMEAN servers for reliability. Ligand binding studies using COACH indicated the interaction of DnMRE11 protein with two Mn2+ ions and dAMP. The ConSurf server predicted that residues of the active site and Nbs binding site have high conservation scores between plant species.Conclusions
A model structure of DnMRE11 was constructed and validated with various bioinformatics programs which suggested the predicted model to be satisfactory. Further validation studies were conducted by COACH analysis for active site ligand prediction, and revealed the presence of six ligands binding sites and two ligands (2 Mn2+ and dAMP).9.
Marie GB Hansen Mette Christoffersen Line R Thuesen Morten R Petersen Anders M Bojesen 《Acta veterinaria Scandinavica》2010,52(1):3
Background
Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are able to infect horses. However, the extend to which Danish horses are infected and seroconvert due to these two bacteria is unknown. The aim of the present study was to evaluate the seroprevalence of B. burgdorferi sensu lato and A. phagocytophilum in Danish horses.Methods
A total of 390 blood samples collected from all major regions of Denmark and with a geographical distribution corresponding to the density of the Danish horse population were analyzed. All samples were examined for the presence of antibodies against B. burgdorferi sensu lato and A. phagocytophilum by the use of the SNAP®4DX ® ELISA test.Results
Overall, 29.0% of the horses were seropositive for B. burgdorferi sensu lato whereas 22.3% were seropositive for A. phagocytophilum.Conclusions
Antibodies against B burgdorferi sensu lato and A. phagocytophilum are commonly found among Danish horses thus showing that Danish horses are frequently infected by these organisms.10.
Pradeep K Chatterjee Leighcraft A Shakes Naima Stennett Vanessa L Richardson Tennison L Malcolm Ken R Harewood 《BMC research notes》2010,3(1):38
Background
Chromatin adjoining the site of integration of a transgene affects expression and renders comparisons of closely related transgenes, such as those derived from a BAC deletion series retrofitted with enhancer-traps, unreliable. Gene targeting to a pre-determined site on the chromosome is likely to alleviate the problem.Findings
A general procedure to replace the loxP site located at one end of genomic DNA inserts in BACs with lox66 is described. Truncating insert DNA from the loxP end with a Tn10 transposon carrying a lox66 site simultaneously substitutes the loxP with a lox66 sequence. The replacement occurs with high stringency, and the procedure should be applicable to all BACs in the public domain. Cre recombination of loxP with lox66 or lox71 was found to be as efficient as another loxP site during phage P1 transduction of small plasmids containing those sites. However the end-deletion of insert DNA in BACs using a lox66 transposon occurred at no more than 20% the efficiency observed with a loxP transposon. Differences in the ability of Cre protein available at different stages of the P1 life cycle to recombine identical versus non-identical lox-sites is likely responsible for this discrepancy. A possible mechanism to explain these findings is discussed.Conclusions
The loxP/lox66 replacement procedure should allow targeting BACs to a pre-positioned lox71 site in zebrafish chromosomes; a system where homologous recombination-mediated "knock-in" technology is unavailable.11.
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14.
Elaheh Sajadi Valiollah Babaipour Ali Asghar Deldar Bagher Yakhchali Seyed Safa-Ali Fatemi 《Biotechnology letters》2017,39(9):1395-1401
Objectives
To evaluate the crystallinity index of the cellulose produced by Escherichia coli Nissle 1917 after heterologous expression of the cellulose synthase subunit D (bcsD) gene of Gluconacetobacter xylinus BPR2001.Results
The bcsD gene of G. xylinus BPR2001 was expressed in E. coli and its protein product was visualized using SDS-PAGE. FTIR analysis showed that the crystallinity index of the cellulose produced by the recombinants was 0.84, which is 17% more than that of the wild type strain. The increased crystallinity index was also confirmed by X-ray diffraction analysis. The cellulose content was not changed significantly after over-expressing the bcsD.Conclusion
The bcsD gene can improve the crystalline structure of the bacterial cellulose but there is not any significant difference between the amounts of cellulose produced by the recombinant and wild type E. coli Nissle 1917.15.
Mattis Kupferschmid Moyira Osny Aquino-Gil Hosam Shams-Eldin Jörg Schmidt Nao Yamakawa Frédéric Krzewinski Ralph T. Schwarz Tony Lefebvre 《Malaria journal》2017,16(1):485
Background
Post-translational modifications (PTMs) constitute a huge group of chemical modifications increasing the complexity of the proteomes of living beings. PTMs have been discussed as potential anti-malarial drug targets due to their involvement in many cell processes. O-GlcNAcylation is a widespread PTM found in different organisms including Plasmodium falciparum. The aim of this study was to identify O-GlcNAcylated proteins of P. falciparum, to learn more about the modification process and to understand its eventual functions in the Apicomplexans.Methods
The P. falciparum strain 3D7 was amplified in erythrocytes and purified. The proteome was checked for O-GlcNAcylation using different methods. The level of UDP-GlcNAc, the donor of the sugar moiety for O-GlcNAcylation processes, was measured using high-pH anion exchange chromatography. O-GlcNAcylated proteins were enriched and purified utilizing either click chemistry labelling or adsorption on succinyl-wheat germ agglutinin beads. Proteins were then identified by mass-spectrometry (nano-LC MS/MS).Results
While low when compared to MRC5 control cells, P. falciparum disposes of its own pool of UDP-GlcNAc. By using proteomics methods, 13 O-GlcNAcylated proteins were unambiguously identified (11 by click-chemistry and 6 by sWGA-beads enrichment; 4 being identified by the 2 approaches) in late trophozoites. These proteins are all part of pathways, functions and structures important for the parasite survival. By probing clicked-proteins with specific antibodies, Hsp70 and α-tubulin were identified as P. falciparum O-GlcNAc-bearing proteins.Conclusions
This study is the first report on the identity of P. falciparum O-GlcNAcylated proteins. While the parasite O-GlcNAcome seems close to those of other species, the structural differences exhibited by the proteomes provides a glimpse of innovative therapeutic paths to fight malaria. Blocking biosynthesis of UDP-GlcNAc in the parasites is another promising option to reduce Plasmodium life cycle.16.
Background
The ability to respond rapidly to fluctuations in environmental changes is decisive for cell survival. Under these conditions trehalose has an essential protective function and its concentration increases in response to enhanced expression of trehalose synthase genes, TPS1, TPS2, TPS3 and TSL1. Intriguingly, the NTH1 gene, which encodes neutral trehalase, is highly expressed at the same time. We have previously shown that trehalase remains in its inactive non-phosphorylated form by the action of an endogenous inhibitor. Recently, a comprehensive two-hybrid analysis revealed a 41-kDa protein encoded by the YLR270w ORF, which interacts with NTH1p.Results
In this work we investigate the correlation of this Trehalase Associated Protein, in trehalase activity regulation. The neutral trehalase activity in the ylr270w mutant strain was about 4-fold higher than in the control strain. After in vitro activation by PKA the ylr270w mutant total trehalase activity increased 3-fold when compared to a control strain. The expression of the NTH1 gene promoter fused to the heterologous reporter lacZ gene was evaluated. The mutant strain lacking YLR270w exhibited a 2-fold increase in the NTH1-lacZ basal expression when compared to the wild type strain.Conclusions
These results strongly indicate a central role for Ylr270p in inhibiting trehalase activity, as well as in the regulation of its expression preventing a wasteful futile cycle of synthesis-degradation of trehalose.17.
Camila Gazolla Volpiano Bruno Brito Lisboa Jackson Freitas Brilhante São José Andreia Mara Rotta de Oliveira Anelise Beneduzi Luciane Maria Pereira Passaglia Luciano Kayser Vargas 《Plant and Soil》2018,432(1-2):229-243
Aims
To identify Rhizobium strains’ ability to biocontrol Sclerotium rolfsii, a fungus that causes serious damage to the common bean and other important crops, 78 previously isolated rhizobia from common bean were assessed.Methods
Dual cultures, volatiles, indole-acetic acid (IAA), siderophore production and 16S rRNA sequencing were employed to select strains for pot and field experiments.Results
Thirty-three antagonistic strains were detected in dual cultures, 16 of which were able to inhibit ≥84% fungus mycelial growth. Antagonistic strains produced up to 36.5 μg mL?1 of IAA, and a direct correlation was verified between IAA production and mycelium inhibition. SEMIA 460 inhibited 45% of mycelial growth through volatile compounds. 16S rRNA sequences confirmed strains as Rhizobium species. In pot condition, common bean plants grown on S. rolfsii-infested soil and inoculated with SEMIA 4032, 4077, 4088, 4080, 4085, or 439 presented less or no disease symptoms. The most efficient strains under field conditions, SEMIA 439 and 4088, decreased disease incidence by 18.3 and 14.5% of the S. rolfsii-infested control.Conclusions
Rhizobium strains could be strong antagonists towards S. rolfsii growth. SEMIA 4032, 4077, 4088, 4080, 4085, and 439 are effective in the biological control of the collar rot of the common bean.18.
Objectives
To evaluate transient expression of RNA interference (RNAi) effectors in Nicotiana benthamiana plants by using recombinant virus vectors and also oral delivery of the effectors for silencing of Mythimna separata endogenous gene expression.Results
Mythimna separata is a serious pest of corn production in China. To evaluate RNAi approaches to target specific RNAs in M. separate, we cloned fragments of the M. separata chitinase sequences into a virus vector in order to produce RNAi effectors during virus infection and replication in plants. When the infected plants were fed to M. separata, expression levels of target MseChi1 and MseChi2 genes were down-regulated by 76 and 45 %, respectively, and sequence-specific siRNAs were detected in recipient insects. RNAi-based silencing of chitinase genes also led to body weight decreases by 43 %.Conclusion
Our research demonstrates target mRNA knockdown and suggests a promising application for controlling of M. separata by in planta expression of RNAi effectors using a recombinant plant virus.19.
Han Bin Pek Pei Yu Lim Chengcheng Liu Dong-Yup Lee Xuezhi Bi Fong Tian Wong Dave Siak-Wei Ow 《Biotechnology letters》2017,39(5):759-765
Objectives
To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis.Results
The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively.Conclusions
Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.20.