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1.
Kai Zhang Huijuan Su Muhan Yang Jing Ge Guiyao Li Jun Yi Yang Wang 《Biotechnology letters》2017,39(6):905-909
Objectives
To establish a positive cloning system with a zero background for high-throughput DNA cloning purpose.Results
The cloning vector, pRI857, and the genomic-library construction vector, pRI857-BAC, were constructed based on the mechanism of expression of the thermo-sensitive cI857 repressor gene that can stringently repress the PR promoter and kanamycin resistance gene (PR-kan R ) at 30 °C, but have no effect on PR-kan R gene at 37 °C or at higher temperatures. When the pRI857 vectors were transformed into E. coli with or without a target foreign DNA fragment inserted at the BfrBI site of the cI857 gene, only colonies with the foreign DNA fragment survive. We extended this method to construct a pRI857-BAC vector for genomic library cloning which displays an efficiency of ~107 cfu per µg of genomic DNA, with no empty vectors detected.Conclusions
Cloning by indirect activation of resistance marker gene represents a novel DNA-capturing system, which can be widely applied for high-throughput DNA cloning.2.
Mengmeng Zhang Mengjia Wang Xiaocui Zhu Wengong Yu Qianhong Gong 《Biotechnology letters》2018,40(5):865-870
Objective
To screen for the quorum-sensing (QS) inhibitors from marine-derived fungi and evaluate their anti-QS properties in Pseudomonas aeruginosa.Results
QS inhibitory activity was found in secondary metabolites of a marine fungus Fusarium sp. Z10 using P. aeruginosa QSIS-lasI biosensor. The major active compound of this fungus was isolated by HPLC and identified as equisetin. Subinhibitory concentration of equisetin could inhibit the formation of biofilm, swarming motility, and the production of virulence factors in P. aeruginosa. The inhibition of las, PQS, and rhl system by equisetin were determined using Escherichia coli MG4/pKDT17, E.coli pEAL08-2, and E.coli pDSY, respectively. Real–time RT-PCR assays showed that equisetin could downregulate the mRNA expression of QS-related genes.Conclusions
Equisetin proved its potential as an inhibitor against P. aeruginosa QS system and might also serve as precursor compound in development of novel therapeutics for infectious diseases by optimal design of structures.3.
Pei Han Yong Huang Yumin Xie Wu Yang Wenying Xiang Peter J. Hylands Cristina Legido-Quigley 《Metabolomics : Official journal of the Metabolomic Society》2018,14(7):91
Introduction
In spite of advances in antibiotics, urinary tract infection (UTI) is still among the most common reasons for antibiotic medication worldwide. Persicaria capitata (Buch.-Ham. ex D. Don) H.Gross (P. capitata) is a herbal medicine used by the Miao people in China to treat UTI. However studies of its mechanism are challenging, owing to the complexity of P. capitata with multiple constituents acting on multiple metabolic pathways.Objective
The objective of this study was to explore the working mechanism of P. capitata on urinary tract infection.Methods
Relinqing® granule, which is solely made from aqueous extracts of the whole P. capitata plant, was used in this study. Urine metabolomics based on gas chromatography-mass spectroscopy was employed to assess the metabolic changes caused by administration of Relinqing® granule in a UTI mouse model. Female specific-pathogen-free Kunming mice were divided into control group (mock infection, saline treatment), model group (E.coli infection, saline treatment), Relinqing® group (E.coli infection, Relinqing® granule treatment), ciprofloxacin group (E.coli infection, ciprofloxacin treatment), and sham-Relinqing® group (no surgery, Relinqing® granule treatment).Results
The results showed that after the treatments, urine levels of itaconic acid in Relinqing® group increased by 4.9 fold and 11.3 fold compared with model and ciprofloxacin groups respectively. Itaconic acid is an endogenous antibacterial metabolite produced by macrophages, which also functions as a checkpoint for metabolic reprogramming of macrophage.Conclusion
Our findings suggest that this herbal medicine can cure urinary tract infection through modulation of immune system.4.
Bing Lin Xu-Zhong Yang Xue-Wei Cao Tao-Zhu Zhang Fu-Jun Wang Jian Zhao 《Biotechnology letters》2017,39(1):71-78
Objective
To evaluate the anti-tumor effects of trichosanthin after fusion with a cell penetrating peptide, heparin-binding peptide (HBP), derived from human heparin-binding EGF-like growth factor (HB-EGF).Results
The fusion protein of trichosanthin-HBP was expressed in Escherichia coli BL21 and purified by Ni–NTA affinity chromatography. The HBP domain had no influence on the topological inactivation activity and N-glycosidase activity of trichosanthin. Trichosanthin-HBP significantly inhibited the growth of tested cancer cells which are impervious to trichosanthin. Tumor cell apoptosis and both the mitochondrial- and death receptor-mediated apoptotic signaling pathways induced by trichosanthin-HBP were more significant than those induced by trichosanthin in HeLa cells.Conclusion
HBP is an efficient intracellular delivery vehicle for trichosanthin and makes trichosanthin-HBP become a promising agent for cancer therapy.5.
Chih-Yueh Liu Chang-Ching Weng Chih-Hsiang Lin Chiou-Ying Yang Kwok-Kong Tony Mong Yaw-Kuen Li 《Biotechnology letters》2017,39(3):407-413
Objectives
A Neissaria bacterial pilus sugar, bacillosamine, was synthesized and, for the first time, used as a probe to screen a single-chain variable fragment (scFv).Results
Four Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca and Neisseria subflava, and two negative controls, Streptococcus pneumoniae and Escherichia coli, were tested through ELISA, immunostaining and gold nanoparticle immunological assay. All results indicated that the selected scFv is feasible for the specific detection of Neisseria species via the recognition of bacillosamine.Conclusions
The recombinant scFv could detect Neisseria strains at 106 CFU/ml.6.
Tianzhen Li Wei Zhou Huiping Bi Yibin Zhuang Tongcun Zhang Tao Liu 《Biotechnology letters》2018,40(7):1057-1065
Objectives
To achieve biosynthesis of caffeoylmalic acid from glucose in engineered Escherichia coli.Results
We constructed the biosynthetic pathway of caffeoylmalic acid in E. coli by co-expression of heterologous genes RgTAL, HpaBC, At4CL2 and HCT2. To enhance the production of caffeoylmalic acid, we optimized the tyrosine metabolic pathway of E. coli to increase the supply of the substrate caffeic acid. Consequently, an E. coli–E. coli co-culture system was used for the efficient production of caffeoylmalic acid. The final titer of caffeoylmalic acid reached 570.1 mg/L.Conclusions
Microbial production of caffeoylmalic acid using glucose has application potential. In addition, microbial co-culture is an efficient tool for producing caffeic acid esters.7.
Objectives
To construct an Escherichia coli strain capable of producing riboflavin with high titer and yield.Results
A low copy number plasmid pLS01 containing a riboflavin operon under the control of a constitutive promoter was constructed and introduced into Escherichia coli MG1655. Subsequently, the pfkA, edd and ead genes were disrupted, and the resulting strain LS02T produced 667 mg riboflavin/l in MSY medium supplied with 10 g glucose/l in flask cultivation. In a fed-batch process, riboflavin production of the strain reached 10.4 g/l with a yield of 56.8 mg riboflavin/g glucose.Conclusion
To our knowledge, this is the first report of engineered E. coli strains that can produce more than 10 g riboflavin/l in fed-batch cultivation, indicating that E. coli has potential for riboflavin production.8.
Sharmila Narayanan Deepanjalee Dutta Neha Arora Lingaraj Sahoo Siddhartha Sankar Ghosh 《Biotechnology letters》2017,39(10):1591-1598
Objectives
To investigate the potential of recombinant phytaspase loaded manganese (Mn) doped zinc sulphide (ZnS) quantum dots embedded chitosan nanoparticles for augmenting cisplatin induced chemotherapy of HeLa cells.Results
The recombinant phytaspase was cloned into bacterial expression vector PGEX-4T-2. The expressed and purified recombinant plant phytaspase protein from Escherichia coli BL21 was immobilized onto the cationic nanocomposite. Confocal microscopy elucidated the delivery of these luminescent nanocomposites inside cervical cancer HeLa cells. A 50% reduction in the viability of HeLa cells was achieved only in the case of phytaspase–nanocomposites–cisplatin combination at a dose of phytaspase (42 nM), nanocomposites (56.3 μg/ml) and cisplatin (0.44 μg/ml).Conclusion
Luminescent cationic nanocomposites were developed for intracellular delivery of recombinant phytaspase, which due to its caspase-like activity assisted in substantiating the chemotherapeutic activity of apoptosis inducing drug-cisplatin.9.
Yibin Zhuang Jingjie Jiang Huiping Bi Hua Yin Shaowei Liu Tao Liu 《Biotechnology letters》2016,38(4):619-627
Objectives
To produce rosmarinic acid analogues in the recombinant Escherichia coli BLRA1, harboring a 4-coumarate: CoA ligase from Arabidopsis thaliana (At4CL) and a rosmarinic acid synthase from Coleus blumei (CbRAS).Results
Incubation of the recombinant E. coli strain BLRA1 with exogenously supplied phenyllactic acid (PL) and analogues as acceptor substrates, and coumaric acid and analogues as donor substrates led to production of 18 compounds, including 13 unnatural RA analogues.Conclusion
This work demonstrates the viability of synthesizing a broad range of rosmarinic acid analogues in E. coli, and sheds new light on the substrate specificity of CbRAS.10.
Objectives
To find new metabolic engineering strategies to improve the yield of acetone in Escherichia coli.Results
Results of flux balance analysis from a modified Escherichia coli genome-scale metabolic network suggested that the introduction of a non-oxidative glycolysis (NOG) pathway would improve the theoretical acetone yield from 1 to 1.5 mol acetone/mol glucose. By inserting the fxpk gene encoding phosphoketolase from Bifidobacterium adolescentis into the genome, we constructed a NOG pathway in E.coli. The resulting strain produced 47 mM acetone from glucose under aerobic conditions in shake-flasks. The yield of acetone was improved from 0.38 to 0.47 mol acetone/mol glucose which is a significant over the parent strain.Conclusions
Guided by computational analysis of metabolic networks, we introduced a NOG pathway into E. coli and increased the yield of acetone, which demonstrates the importance of modeling analysis for the novel metabolic engineering strategies.11.
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 1917.12.
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.13.
Thijs Welle Anna T. Hoekstra Ineke A. J. J. M. Daemen Celia R. Berkers Matheus O. Costa 《Metabolomics : Official journal of the Metabolomic Society》2017,13(7):83
Introduction
Swine dysentery caused by Brachyspira hyodysenteriae is a production limiting disease in pig farming. Currently antimicrobial therapy is the only treatment and control method available.Objective
The aim of this study was to characterize the metabolic response of porcine colon explants to infection by B. hyodysenteriae.Methods
Porcine colon explants exposed to B. hyodysenteriae were analyzed for histopathological, metabolic and pro-inflammatory gene expression changes.Results
Significant epithelial necrosis, increased levels of l-citrulline and IL-1α were observed on explants infected with B. hyodysenteriae.Conclusions
The spirochete induces necrosis in vitro likely through an inflammatory process mediated by IL-1α and NO.14.
Thais Freitas da Silva Renata Estebanez Vollú Joana Montezano Marques Joana Falcão Salles Lucy Seldin 《Plant and Soil》2017,414(1-2):69-79
Background
The fungus Colletotrichum is a plant pathogen that causes the anthracnose disease, resulting in huge losses in various crops including the rose-scented geranium (Pelargonium graveolens). Although the bacterial community associated with plants has an important role in the establishment of plant diseases, little is known about what happens in P. graveolens.Aims
To increase the knowledge about the bacterial community associated with P. graveolens and its relationship with anthracnose disease symptoms.Methods
Quantitative PCR and high-throughput sequencing were combined to determine the presence of the fungus Colletotrichum and to reveal the bacterial communities associated with different plant parts – root, stem and leaf – and in the rhizosphere and bulk soil, and also to determine the respective bacterial communities associated with P. graveolens leaves symptomatic and asymptomatic for anthracnose disease.Results
The fungus Colletotrichum was detected in all plant parts and in the surrounding soil. Bacterial communities varied spatially in plants, and the disease symptoms also influenced the composition of the bacterial community. Abundances of operational taxonomic units (OTUs) assigned to the phylum Actinobacteria and to the genus Streptococcus were greatly increased in asymptomatic leaves.Conclusions
The bacterial community associated to geranium leaves responds to anthracnose symptoms.15.
Objective
A potential thermotolerant l-leucine dehydrogenase from Laceyella sacchari (Ls-LeuDH) was over-expressed in E. coli, purified and characterized.Results
Ls-LeuDH had excellent thermostability with a specific activity of 183 U/mg at pH 10.5 and 25 °C. It retained a high activity in 200 mM carbonate buffer from pH 9.5 to 11. The optimal temperature for Ls-LeuDH was 60 °C.Conclusion
It is the first time that a thermostable and highly active LeuDH originating from L. sacchari has been characterized. It may be useful for medical and pharmaceutical applications.16.
Man Zhao Liang Gao Li Zhang Yanbin Bai Liang Chen Meilan Yu Feng Cheng Jie Sun Zhao Wang Xiangxian Ying 《Biotechnology letters》2017,39(11):1741-1746
Objectives
To characterize a recombinant carbonyl reductase from Saccharomyces cerevisiae (SceCPR1) and explore its use in asymmetric synthesis of (R)-pantolactone [(R)-PL].Results
The NADPH-dependent SceCPR1 exhibited strict (R)-enantioselectivity and high activity in the asymmetric reduction of ketopantolactone (KPL) to (R)-PL. Escherichia coli, coexpressing SceCPR1 and glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), was constructed to fulfill efficient NADPH regeneration. During the whole-cell catalyzed asymmetric reduction of KPL, the spontaneous hydrolysis of KPL significantly affected the yield of (R)-PL, which was effectively alleviated by the employment of the substrate constant-feeding strategy. The established whole-cell bioreduction for 6 h afforded 458 mM (R)-PL with the enantiomeric excess value of >99.9% and the yield of 91.6%.Conclusions
Escherichia coli coexpressing SceCPR1 and EsGDH efficiently catalyzed the asymmetric synthesis of (R)-PL through the substrate constant-feeding strategy.17.
Objectives
To characterize a novel feruloyl esterase from Escherichia coli BL21 DE3.Results
The gene encoding BioH was cloned and overexpressed in E. coli. The protein was purified and its catalytic activity was assessed. BioH exhibited feruloyl esterase activity toward a broad range of substrates, and the corresponding kinetic constants for the methyl ferulate, ethyl ferulate, and methyl p-coumarate substrates were: K m values of 0.48, 6.3, and 1.9 mM, respectively, and k cat /K m values of 9.3, 3.8, and 3.8 mM?1 s?1, respectively.Conclusions
Feruloyl esterase from E. coli was expressed for the first time. BioH was confirmed to be a feruloyl esterase.18.
Keke Zhang Mei Huang Jiangshan Ma Zeyi Liu Jiarui Zeng Xuanming Liu Ting Xu Xiang Wang Ying Liu Zhigang Bu Yonghua Zhu 《Biotechnology letters》2018,40(5):871-880
Objective
To identify and characterize a novel bacterial pyranose 2-oxidase (P2Ox) and investigate its potential use in lignin degradation applications.Results
A new bacterial P2Ox (PaP2Ox) enzyme was identified in the lignocellulolytic bacterium Pantoea ananatis Sd-1. The PaP2Ox open reading frame was cloned, and the encoded protein was heterologously expressed in an Escherichia coli expression system. Unlike another reported bacterial P2Ox enzyme, the purified PaP2Ox exhibits a homotetrameric spatial conformation that is similar to fungal P2Oxs, with each subunit having a molecular mass of 65 kDa. The recombinant PaP2Ox exhibits maximum activity at 50 °C and pH 6.5 with d-glucose as its preferred substrate. In addition, this enzyme was shown to work in combination with bacterial laccase in lignin degradation.Conclusions
The bacterial enzyme PaP2Ox has potential use in ligninolytic systems and shows promising value in industrial biotechnological applications.19.
20.
Ying-ge Wang Jin-mei Cheng Hai-bo Ding Xi Lin Guo-hao Chen Mei Zhou Sheng-nan Ye 《Mycopathologia》2018,183(3):551-558