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1.
Shin-Hye Yu So-Hyung Kwak Thi Thanh Hanh Nguyen Ye-Seul Seo Chaeri Song Il Kyoon Mok Doman Kim 《Biotechnology letters》2018,40(2):375-381
Objectives
To develop preventive canine oral health bio-materials consisting of probiotics and glucanase to reduce insoluble glucan and volatile sulfur compound formation.Results
Co-cultivation of Enterococcus faecium T7 with Streptococcus mutans at inoculation ratio of 3:1 (v/v) resulted in 25% reduction in the growth of Streptococcus mutans. Amounts of soluble and insoluble glucans produced by S. mutans were decreased to 70 and 55%, respectively. Insoluble glucan was decreased from 0.6 µg/ml in S. mutans culture to 0.03 µg/ml in S. mutans co-cultivated with E. faecium T7 in the presence of Lipomyces starkeyi glucanase. Volatile sulfur compound, a main component of halitosis produced by Fusobacteria nucleatum, was decreased by co-cultivating F. nucleatum with E. faecium.Conclusion
E. faecium and glucanase can be combined as potentially active ingredients of oral care products for pets by reducing plaque-forming bacteria growth and their by-products that cause cavity and periodontal disease.2.
Objectives
To investigate the mode of action of leucocin K7 against Listeria monocytogenes and to assess its inhibitory effect on Lis. monocytogenes in refrigerated milk.Results
A bacteriocin-producing strain, Leuconostoc mesenteroides K7, was isolated from a fermented pickle. The bacteriocin, leucocin K7, exhibited antagonistic activity against Lis. monocytogenes with an MIC of 28 µg/ml. It was sensitive to proteaseS and displayed good thermal stability and broad active pH range. Leucocin K7 had no effect on the efflux of ATP from Lis. monocytogenes but triggered the efflux of K+ and the intracellular hydrolysis of ATP. It also dissipated the transmembrane electrical potential completely and transmembrane pH gradient partially. It 80 AU/ml inhibited the growth of Lis. monocytogenes by 2.3–3.9 log units in milk; when combined with glycine (5 mg/ml), it completely eliminated viable Lis. monocytogenes over 7 daysConclusion
Leucocin K7 shows different mode of action from nisin and may have potential application in milk preservation.3.
4.
Junhong Wei Jinjin Tian Guoqing Pan Jie Xie Jialing Bao Zeyang Zhou 《Biotechnology letters》2017,39(6):857-864
Objective
To develop a reliable and easy to use expression system for antibiotic production improvement of Streptomyces.Results
A two-compound T7 RNA polymerase-dependent gene expression system was developed to fulfill this demand. In this system, the T7 RNA polymerase coding sequence was optimized based on the codon usage of Streptomyces coelicolor. To evaluate the functionality of this system, we constructed an activator gene overexpression strain for enhancement of actinorhodin production. By overexpression of the positive regulator actII-ORF4 with this system, the maximum actinorhodin yield of engineered strain was 15-fold higher and the fermentation time was decreased by 48 h.Conclusion
The modified two-compound T7 expression system improves both antibiotic production and accelerates the fermentation process in Streptomyces. This provides a general and useful strategy for strain improvement of important antibiotic producing Streptomyces strains.5.
Objectives
To enhance acid tolerance of Candida glabrata for pyruvate production by engineering AMP metabolism.Results
The physiological function of AMP deaminase in AMP metabolism from C. glabrata was investigated by deleting or overexpresseing the corresponding gene, CgAMD1. At pH 4, CgAMD1 overexpression resulted in 59 and 51% increases in biomass and cell viability compared to those of wild type strain, respectively. In addition, the intracellular ATP level of strain Cgamd1Δ/CgAMD1 was down-regulated by 22%, which led to a 94% increase in pyruvate production. Further, various strengths of CgAMD1 expression cassettes were designed, thus resulting in a 59% increase in pyruvate production at pH 4. Strain Cgamd1Δ/CgAMD1 (H) was grown in a 30 l batch bioreactor at pH 4, and pyruvate reached 46.1 g/l.Conclusion
CgAMD1 overexpression plays an active role in improving acid tolerance and pyruvate fermentation performance of C. glabrata at pH 4.6.
Carla Ceoloni Paola Forte Ljiljana Kuzmanović Silvio Tundo Ilaria Moscetti Pasquale De Vita Maria Elena Virili Renato D’Ovidio 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2017,130(10):2005-2024
Key message
A major locus for resistance to different Fusarium diseases was mapped to the most distal end of Th. elongatum 7EL and pyramided with Th. ponticum beneficial genes onto wheat 7DL.Abstract
Perennial Triticeae species of the Thinopyrum genus are among the richest sources of valuable genes/QTL for wheat improvement. One notable and yet unexploited attribute is the exceptionally effective resistance to a major wheat disease worldwide, Fusarium head blight, associated with the long arm of Thinopyrum elongatum chromosome 7E (7EL). We targeted the transfer of the temporarily designated Fhb-7EL locus into bread wheat, pyramiding it with a Th. ponticum 7el1L segment stably inserted into the 7DL arm of wheat line T4. Desirable genes/QTL mapped along the T4 7el1L segment determine resistance to wheat rusts (Lr19, Sr25) and enhancement of yield-related traits. Mapping of the Fhb-7EL QTL, prerequisite for successful pyramiding, was established here on the basis of a bioassay with Fusarium graminearum of different 7EL-7el1L bread wheat recombinant lines. These were obtained without resorting to any genetic pairing promotion, but relying on the close 7EL-7el1L homoeology, resulting in 20% pairing frequency between the two arms. Fhb-7EL resided in the telomeric portion and resistant recombinants could be isolated with useful combinations of more proximally located 7el1L genes/QTL. The transferred Fhb-7EL locus was shown to reduce disease severity and fungal biomass in grains of infected recombinants by over 95%. The same Fhb-7EL was, for the first time, proved to be effective also against F. culmorum and F. pseudograminearum, predominant agents of crown rot. Prebreeding lines possessing a suitable 7EL-7el1L gene/QTL assembly showed very promising yield performance in preliminary field tests.7.
Grietje Kuipers Alexandros Karyolaimos Zhe Zhang Nurzian Ismail Gianluca Trinco David Vikström Dirk Jan Slotboom Jan-Willem de Gier 《Microbial cell factories》2017,16(1):226
Background
To optimize the production of membrane and secretory proteins in Escherichia coli, it is critical to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein production strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-production of its natural inhibitor T7 lysozyme. This setup enables to precisely tune target gene expression rates in Lemo21(DE3). The t7lys gene is expressed from the pLemo plasmid using the titratable rhamnose promoter. A disadvantage of the Lemo21(DE3) setup is that the system is based on two plasmids, a T7 expression vector and pLemo. The aim of this study was to simplify the Lemo21(DE3) setup by incorporating the key elements of pLemo in a standard T7-based expression vector.Results
By incorporating the gene encoding the T7 lysozyme under control of the rhamnose promoter in a standard T7-based expression vector, pReX was created (ReX stands for Regulated gene eXpression). For two model membrane proteins and a model secretory protein we show that the optimized production yields obtained with the pReX expression vector in BL21(DE3) are similar to the ones obtained with Lemo21(DE3) using a standard T7 expression vector. For another secretory protein, a c-type cytochrome, we show that pReX, in contrast to Lemo21(DE3), enables the use of a helper plasmid that is required for the maturation and hence the production of this heme c protein.Conclusions
Here, we created pReX, a T7-based expression vector that contains the gene encoding the T7 lysozyme under control of the rhamnose promoter. pReX enables regulated T7-based target gene expression using only one plasmid. We show that with pReX the production of membrane and secretory proteins can be readily optimized. Importantly, pReX facilitates the use of helper plasmids. Furthermore, the use of pReX is not restricted to BL21(DE3), but it can in principle be used in any T7 RNAP-based strain. Thus, pReX is a versatile alternative to Lemo21(DE3).8.
Real-time metabolomic analysis of lactic acid bacteria as monitored by in vitro NMR and chemometrics
Parvaneh Ebrahimi Flemming Hofmann Larsen Henrik Max Jensen Finn Kvist Vogensen Søren Balling Engelsen 《Metabolomics : Official journal of the Metabolomic Society》2016,12(4):77
Introduction
Lactic acid bacteria (LAB) play an important role in the food industry as starter cultures to manufacture fermented food, and as probiotics. In recent years, there has been an increasing interest in using LAB cultures for biopreservation of food products. It is therefore of great interest to study the detailed metabolism of these bacteria.Objectives
This study aimed at developing an efficient analytical protocol for real-time in vitro NMR measurements of LAB fermentations, from sample preparation, over data acquisition and preprocessing, to the extraction of the kinetic metabolic profiles.Method
The developed analytical protocol is applied to an experimental design with two LAB strains (Lactobacillus rhamnosus DSM 20021 and Lactobacillus plantarum subsp. plantarum DSM 20174), two initial pH levels (pHi 6.5 and 5.5), two levels of glucose concentration (2.5 and 0.25 g/l), and two batch fermentation replicates.Results
The design factors proved to be strongly significant and led to interesting biological information. The protocol allowed for detailed real-time kinetic analysis of 11 major metabolites involved in the glycolysis, pyruvate catabolism, amino acid catabolism and cell energy metabolism. New biological knowledge was obtained about the different patterns of glutamine and aspartic acid consumption by the two strains. It was observed that L. plantarum consumes more glutamine at low pH (pH 5.5) whereas the opposite applies to L. rhamnosus. Regarding aspartic acid, both of the strains consume it higher at low pH, and overall L. plantarum consumes it more. L. rhamnosus did not consume aspartic acid at pH 6.5.Conclusion
The developed analytical protocol for real-time in vitro NMR measurements of bacterial metabolism allows a relatively easy investigation of different fermentation factors such as new strains, new substrates, cohabitations, temperature, and pH and has a great potential in biopreservation studies to discover new efficient bioprotective cultures.9.
Objectives
To identify a novel nitrilase with S-selectivity toward mandelonitrile that can produce (S)-mandelic acid in one step.Results
A novel nitrilase PpL19 from Pseudomonas psychrotolerans L19 was discovered by genome mining. It showed S-selectivity with an enantiomeric excess of 52.7 % when used to hydrolyse (R, S)-mandelonitrile. No byproduct was observed. PpL19 was overexpressed in Escherichia coli BL21 (DE3) and formed inclusion bodies that were active toward mandelonitrile and stable across a broad range of temperature and pH. In addition, PpL19 hydrolysed nitriles with diverse structures; arylacetonitriles were the optimal substrates. Homology modelling and docking studies of both enantiomers of mandelonitrile in the active site of nitrilase PpL19 shed light on the enantioselectivity.Conclusions
A novel nitrilase PpL19 from P. psychrotolerans L19 was mined and distinguished from other nitrilases as it was expressed as an active inclusion body and showed S-selectivity toward mandelonitrile.10.
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.11.
Ningning Sun Yuanchao Qian Weiwei Wang Yaohua Zhong Meixue Dai 《Biotechnology letters》2018,40(7):1119-1126
Objective
Improve the hydrolysis efficiency of the Trichoderma reesei cellulase system by heterologously expressing cellobiohydrolase Cel7A (Te-Cel7A) from the thermophilic fungus Talaromyces emersonii.Results
Te-Cel7A was expressed in T. reesei under control of the cdna1 promoter and the generated transformant QTC14 could successfully secrete Te-Cel7A into the supernatant using glucose as carbon source. The recombinant Te-Cel7A had a temperature optimum at 65 °C and an optimal pH of 5, which were similar to those from the native host. The culture supernatant of QTC14 exhibited a 28.8% enhancement in cellobiohydrolase activity and a 65.2% increase in filter paper activity relative to that of the parental strain QP4. Moreover, the QTC14 cellulase system showed higher thermal stability than that of the parental strain QP4. In the saccharification of delignified corncob residue, the cellulose conversion of QTC14 showed 13.9% higher than that of QP4 at the end of reaction.Conclusions
The thermophilic fungus-derived cellulases could be efficiently expressed by T. reesei and the recombinant cellulases had potential applications for biomass conversion.12.
Objective
To ascertain the effect of chitin-binding domain (ChBD) and fibronectin type III domain (FN3) on the characterization of the intact chitinase from Bacillus thuringiensis.Results
An intact chitinase gene (chi74) from B. thuringiensis HZP7 and its truncated genes (chi54, chi63 and chi66) were expressed in Escherichia coli BL21. The expression products were analyzed after purification. All chitinases were active from pH 4–7.5 and from 20 to 80 °C with identical optimal: pH 5.5 and 60 °C. The activity of colloid chitin degradation for Chi74 was the highest, followed by Chi66, Chi63 and Chi54. Ag+ reduced the activity of Chi74, Chi54, Chi63 and Chi66, but Mg2+ enhanced them. The effect of Ag+ and Mg2+ was more significant on the activity of Chi54 than on the activities of Chi63, Chi66 and Chi74.Conclusion
ChBDChi74 and FN3Chi74 domains play a role in exerting enzymatic activity and can improve the stability of chitinase.13.
Objectives
To characterize a novel ene-reductase from Meyerozyma guilliermondii and achieve the ene-reductase-mediated reduction of activated C=C bonds.Results
The gene encoding an ene-reductase was cloned from M. guilliermondii. Sequence homology analysis showed that MgER shared the maximal amino acid sequence identity of 57 % with OYE2.6 from Scheffersomyces stipitis. MgER showed the highest specific activity at 30 °C and pH 7 (100 mM sodium phosphate buffer), and excellent stereoselectivities were achieved for the reduction of (R)-carvone and ketoisophorone. Under the reaction conditions (30 °C and pH 7.0), 150 mM (R)-carvone could be completely converted to (2R,5R)-dihydrocarvone within 22 h employing purified MgER as catalyst, resulting in a yield of 98.9 % and an optical purity of >99 % d.e.Conclusion
MgER was characterized as a novel ene-reductase from yeast and showed great potential for the asymmetric reduction of activated C=C bonds of α,β-unsaturated compounds.14.
Objectives
To clone and express a neopullulanase gene from Lactobacillus mucosae LM1 in Escherichia coli and characterise the resulting recombinant neopullulanase.Results
An ORF in L. mucosae corresponding to a neopullulanase was cloned and expressed in E. coli. The predicted amino acid sequence of the neopullulanase contained catalytic sites and conserved motifs that are present in members of the neopullulanase subfamily. The resulting recombinant neopullulanase was efficiently purified by Ni–NTA affinity chromatography. The purified enzyme optimally hydrolyses pullulan at 37 °C and pH 6.0, producing panose as the major reaction product.Conclusions
To the best of our knowledge, this is the first report of the cloning, expression and characterisation of a neopullulanase gene from a lactic acid bacterium.15.
Rongguang Zhang Chen Wang Wenbin Cheng Guangcai Duan Qingfeng Shi Shuaiyin Chen Qingtang Fan 《Biotechnology letters》2018,40(3):585-590
Objective
To develop a safe and effective oral vaccine against Helicobacter pylori using its HpaA protein expressed in Lactococcus lactis.Results
The gene encoding HpaA was obtained by PCR and ligated to pNZ8110-lysM following digestion with NaeI + SphI. The recombinant plasmid was transferred into E. coli for multiplication, and then into L. lactis. The recombinant L. lactis was induced to express HpaA, resulting in two products of 29 and 25 kDa, both of which yielded positive immunoreaction with mouse antisera against H. pylori, as confirmed by immunoblot assays. The 29 kDa product constituted 12% of the cell lysates. Oral inoculation with the engineered L. lactis evoked significantly elevated serum IgG level in mice (P < 0.05).Conclusions
A novel engineered L. lactis strain was developed that efficiently produces whole HpaA protein with desired antigenicity and potent immunogenicity. It provides a basis for approaches to L. lactis-delivered anti-H. pylori vaccination.16.
Aiqin Zhang Wei Zang Xiyang Zhang Yangyang Ma Xiufeng Yan Qiuying Pang 《Plant and Soil》2016,409(1-2):175-202
Background and aims
Soil salinization with high pH condition is a major abiotic stress to plant growth and crop productivity. Helianthus tuberosus L. is an important stress tolerant plant and can survive in the saline-alkali soil and semiarid areas. The aim of this study is to identify the effect of alkali stress on H. tuberosus through global proteomics analysis and improve understanding of the alkalinity resistance of plants.Methods
H. tuberosus seedlings were exposed to different level alkali stress for 7 days. Protein profiling was quantified by conducting MS-based comparative proteomics analysis. RT-PCR study was carried out to analyze the mRNA expression levels of candidate alkali stress response proteins.Results
The response of H. tuberosus to alkali stress was detected at both physiological and molecular levels. 104 differentially expressed proteins from H. tuberosus leaves response to Na2CO3 treatment were successfully identified. Functional categorization of these identified proteins showed that the accumulation level of proteins involved in glycolysis, TCA cycle, PSI system, ROS scavenging and signal transduction increased under alkali stress.Conclusions
Based on the observation of plant growth and the investigation of molecular regulation, H.tuberosus could resist certain alkali stress by modulating carbohydrate metabolism and redox homeostasis. These findings provide a new sight into the underlying molecular mechanisms of alkali resistance in plant.17.
Objectives
To develop orally administrated anti-Helicobacter pylori vaccination, a Lactococcus lactis strain was genetically constructed for fusion expression of H. pylori protective antigens HpaA and Omp22.Results
The fusion gene of omp22 and hpaA with an adapter encoding three glycines was cloned from a plasmid pMAL-c2x-omp22-hpaA into Escherichia coli MC1061 and L. lactis NZ3900 successively using a shutter vector pNZ8110. Expression of the fusion gene in L. lactis was induced with nisin resulting in production of proteins with molecular weights of 50 and 28 kDa. Both of them were immunoreactive with mouse anti-H. pylori sera as determined via western blotting. Oral vaccination of BALB/c mice using the L. lactis strain carrying pNZ8110-omp22-hpaA elicited significant systematic humoral immune response (P < 0.05).Conclusions
This is the first report showing that a fusion protein of two H. pylori antigens was efficiently expressed in L. lactis with immunogenicity. This is a considerable step towards H. pylori vaccines.18.
19.
Background
Dogs are the most common pet animals worldwide. They may harbour a wide range of parasites with zoonotic potential, thus causing a health risk to humans. In Nigeria, epidemiological knowledge on these parasites is limited.Methods
In a community-based study, we examined 396 dogs in urban and rural areas of Ilorin (Kwara State, Central Nigeria) for ectoparasites and intestinal helminths. In addition, a questionnaire regarding knowledge and practices was applied to pet owners.Results
Nine ectoparasite species belonging to four taxa and six intestinal helminth species were identified: fleas (Ctenocephalides canis, Pulex irritans, Tunga penetrans), mites (Demodex canis, Otodectes sp., Sarcoptes scabiei var. canis), ticks (Rhipicephalus sanguineus, Ixodes sp.), and lice (Trichodectes canis); and Toxocara canis, Ancylostoma sp., Trichuris vulpis, Dipylidium caninum, Taenidae and Strongyloides sp. Overall prevalence of ectoparasites was 60.4% and of intestinal helminths 68.4%. The occurrence of C. canis, R. sanguineus, T. canis, Ancylostoma sp. and T. vulpis was most common (prevalence 14.4% to 41.7%). Prevalence patterns in helminths were age-dependent, with T. canis showing a decreasing prevalence with age of host, and a reverse trend in other parasite species. Knowledge regarding zoonoses was very limited and the diseases not considered a major health problem. Treatment with antiparasitic drugs was more frequent in urban areas.Conclusion
Parasites of importance for human health were highly prevalent in Nigerian dogs. Interventions should include health education provided to dog owners and the establishment of a program focusing on zoonotic diseases.20.
Akihiro Ito Tadashi Ishida Yasuyoshi Washio Akio Yamazaki Hiromasa Tachibana 《BMC pulmonary medicine》2017,17(1):211