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1.
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.2.
Objectives
To improve the stability and sweetness of the sweet-tasting protein, monellin, by using site-directed mutagenesis and a Pichia pastoris expression system with a GAPDH constitutive promoter.Results
Both wild-type and E2 N mutant of single-chain monellin gene were cloned into the PGAPZαA vector and expressed in Pichia pastoris. The majority of the secreted recombinant protein, at 0.15 g/l supernatant, was monellin. This was purified by Sephadex G50 chromatography. The sweetness threshold of wild-type and E2 N were 30 μg/ml and 20 μg/ml, respectively. Compared with the proteins expressed in Escherichia coli, the thermostability of both proteins was improved. The N-terminal sequence is determinative for the sweetness of the proteins expressed in yeast strains.Conclusions
Site-directed mutagenesis, modification of the N-terminus of monellin, and without the need of methanol induction in P. pastoris expression system, indicate the possibility for large-scale production of this sweet-tasting protein.3.
4.
Xiaojie Duan Mingming Zheng Yu Liu Zhengqiang Jiang Shaoqing Yang 《Biotechnology letters》2016,38(12):2127-2135
Objectives
To identify novel cold-active lipases from fungal sources and improve their production by heterologous expression in Pichia pastoris.Results
A novel cold-active lipase gene (ReLipB) from Rhizomucor endophyticus was cloned. ReLipB was expressed at a high level in Pichia pastoris using high cell-density fermentation in a 5-l fermentor with the highest lipase activity of 1395 U/ml. The recombinant lipase (RelipB) was purified and biochemically characterized. ReLipB was most active at pH 7.5 and 25 °C. It was stable from pH 4.5–9.0. It exhibited broad substrate specificity towards p-nitrophenyl (pNP) esters (C2–C16) and triacylglycerols (C2–C12), showing the highest specific activities towards pNP laurate (231 U/mg) and tricaprylin (1840 U/mg), respectively. In addition, the enzyme displayed excellent stability with high concentrations of organic solvents including cyclohexane, n-hexane, n-heptane, isooctane and petroleum ester and surfactants.Conclusions
A novel cold-active lipase from Rhizomucor endophyticus was identified, expressed at a high level and biochemically characterized. The high yield and unique enzymatic properties make this lipase of some potential for industrial applications.5.
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.6.
Xuechang Wu Lijie Zhang Xinna Jin Yahong Fang Ke Zhang Lei Qi Daoqiong Zheng 《Biotechnology letters》2016,38(7):1097-1106
7.
Jiwei Mao Quanli Liu Xiaofei Song Hesuiyuan Wang Hui Feng Haijin Xu Mingqiang Qiao 《Biotechnology letters》2017,39(7):977-982
Objective
To identify new enzymatic bottlenecks of l-tyrosine pathway for further improving the production of l-tyrosine and its derivatives.Result
When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l?1, respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l?1) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain.Conclusion
Combinatorial metabolic engineering provides a new strategy for further improvement of l-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.8.
Deqiang Zhu Jianrong Wu Xiaobei Zhan Li Zhu Zhiyong Zheng Minjie Gao 《Biotechnology letters》2017,39(2):227-234
Objectives
N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).Results
PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l?1, compared with 3.6 ± 0.15 g l?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l?1 in a 1 l fermenter.Conclusions
The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.9.
Adones Sales Luana Ferreira Afonso Juliana Alves Americo Mauro de Freitas Rebelo Glaucia Maria Pastore Juliano Lemos Bicas 《Biotechnology letters》2018,40(3):561-567
Objective
To investigate the biocatalytic potential of Colletotrichum acutatum and Colletotrichum nymphaeae for monoterpene biotransformation.Results
C. acutatum and C. nymphaeae used limonene, α-pinene, β-pinene, farnesene, citronellol, linalool, geraniol, perillyl alcohol, and carveol as sole carbon and energy sources. Both species biotransformed limonene and linalool, accumulating limonene-1,2-diol and linalool oxides, respectively. α-Pinene was only biotransformed by C. nymphaeae producing campholenic aldehyde, pinanone and verbenone. The biotransformation of limonene by C. nymphaeae yielded 3.34–4.01 g limonene-1,2-diol l?1, depending on the substrate (R-(+)-limonene, S-(?)-limonene or citrus terpene (an agro-industrial by-product). This is among the highest concentrations already reported for this product.Conclusions
This is the first report on the biotransformation of these terpenes by Colletotrichum spp. and the biotransformation of limonene to limonene-1,2-diol possibly involves enzymes similar to those found in Grosmannia clavigera.10.
Background
Species of Paris Sect. Marmorata are valuable medicinal plants to synthesize steroidal saponins with effective pharmacological therapy. However, the wild resources of the species are threatened by plundering exploitation before the molecular genetics studies uncover the genomes and evolutionary significance. Thus, the availability of complete chloroplast genome sequences of Sect. Marmorata is necessary and crucial to the understanding the plastome evolution of this section and facilitating future population genetics studies. Here, we determined chloroplast genomes of Sect. Marmorata, and conducted the whole chloroplast genome comparison.Results
This study presented detailed sequences and structural variations of chloroplast genomes of Sect. Marmorata. Over 40 large repeats and approximately 130 simple sequence repeats as well as a group of genomic hotspots were detected. Inverted repeat contraction of this section was inferred via comparing the chloroplast genomes with the one of P. verticillata. Additionally, almost all the plastid protein coding genes were found to prefer ending with A/U. Mutation bias and selection pressure predominately shaped the codon bias of most genes. And most of the genes underwent purifying selection, whereas photosynthetic genes experienced a relatively relaxed purifying selection.Conclusions
Repeat sequences and hotspot regions can be scanned to detect the intraspecific and interspecific variability, and selected to infer the phylogenetic relationships of Sect. Marmorata and other species in subgenus Daiswa. Mutation and natural selection were the main forces to drive the codon bias pattern of most plastid protein coding genes. Therefore, this study enhances the understanding about evolution of Sect. Marmorata from the chloroplast genome, and provide genomic insights into genetic analyses of Sect. Marmorata.11.
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.12.
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 4.13.
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.14.
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.15.
Xiaohong Chen Junjun Chen Yuanxing Zhang Ping Zhu Yong Deng Qin Liu 《Biotechnology letters》2016,38(6):959-967
Objective
To achieve secreted expression of the truncated capsid protein from porcine circovirus type 2 (PCV2) in Pichia pastoris.Results
A truncated cap gene (tcap) with a deleted N-terminal nuclear localization signal was optimized and synthesized. Effective secreted expression was achieved in P. pastoris GS115. The high-productive recombinant strain for tCap was grown in a 5 l bioreactor and the productivity of tCap in supernatant reached 250 μg/ml. Furthermore, serum antibody test demonstrated that adjuvant-assisting tCap induced a significant increase of specific PCV2-Cap antibody over time in mice and a similar antibody level in pigs compared with a commercial Cap-based subunit vaccine.Conclusion
This work establishes a secreted expression strategy in P. pastoris for the production of PCV2 Cap with superior bioactivity, and this strategy might provide potential uses in developing Cap-based subunit vaccine in the future.16.
Korey J. Brownstein Mahmoud Gargouri William R. Folk David R. Gang 《Metabolomics : Official journal of the Metabolomic Society》2017,13(11):133
Introduction
Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.Objectives
We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.Methods
Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.Results
Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.Conclusions
Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.17.
Ryosuke Fujiwara Shuhei Noda Yoshifumi Kawai Tsutomu Tanaka Akihiko Kondo 《Biotechnology letters》2016,38(9):1543-1549
Objectives
To find a novel host for the production of 4-vinylphenol (4VPh) by screening Streptomyces species.Results
The conversion of p-coumaric acid (pHCA) to 4VPh in Streptomyces mobaraense was evaluated using a medium containing pHCA. S. mobaraense readily assimilated pHCA after 24 h of cultivation to produce 4VPh. A phenolic acid decarboxylase, derived from S. mobaraense (SmPAD), was purified following heterologous expression in Escherichia coli. SmPAD was evaluated under various conditions, and the enzyme’s kcat/Km value was 0.54 mM ?1 s?1. Using intergenetic conjugation, a gene from Rhodobacter sphaeroides encoding a tyrosine ammonia lyase, which catalyzes the conversion of l-tyrosine to p-coumaric acid, was introduced into S. mobaraense. The resulting S. mobaraense transformant produced 273 mg 4VPh l?1 from 10 g glucose l?1.Conclusion
A novel strain suitable for the production of 4VPh and potentially other aromatic compounds was isolated.18.
Ziv Dadon Assaf Cohen Yael M. Szterenlicht Marc V. Assous Yair Barzilay David Raveh-Brawer Amos M. Yinnon Gabriel Munter 《Annals of clinical microbiology and antimicrobials》2017,16(1):68
Background
Streptococcus gordonii is an infrequent cause of infective endocarditis (IE); associated spondylodiskitis has not yet been described in the literature.Purpose
We describe 2 patients who presented with new-onset, severe back pain; blood cultures revealed S. gordonii bacteremia, which led to the diagnosis of spondylodiskitis and IE. We review our 2-decade experience with S. gordonii bacteremia to describe the clinical and epidemiological characteristics of these patients.Results
In our hospital over the last 20 years (1998–2017), a total of 15 patients with S. gordonii bacteremia were diagnosed, including 11 men and 4 women, and the mean age was 65 ± 22 (range 23–95). The most common diagnosis was IE (9 patients), spondylodiskitis (the presented 2 patients, who in addition were diagnosed with endocarditis), necrotizing fasciitis (1), sternitis (1), septic arthritis (1) and pneumonia (1). The 11 patients with IE were treated with penicillin ± gentamicin, or ceftriaxone for 6 weeks, 5 required valve surgery and 10/11 (91%) attained complete cure. The 2 patients with diskitis required 2–3 months of intravenous antibiotics to achieve complete cure.Conclusion
Spondylodiskitis was the presenting symptom of 2/11 (18%) patients with S. gordonii endocarditis. Spondylodiskitis should probably be looked for in patients diagnosed with S. gordonii endocarditis and back pain as duration of antibiotic treatment to achieve complete cure may be considerably longer.19.