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  • Correction to: A pH-responsive genetic sensor for the dynamic regulation of D-xylonic acid accumulation in Escherichia coli
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-16
    Angelo B. Bañares, Kris Niño G. Valdehuesa, Kristine Rose M. Ramos, Grace M. Nisola, Won-Keun Lee, Wook-Jin Chung

    In the published version, the y-axis data of Fig. 3c was incorrectly inserted (OD600 instead of D-xylonate (g L-1) and the x-axes of Figs. 3b, 3d, 3e and 3f ended at 48 h instead of 72 h. See the correct Fig. 3 below.

    更新日期:2020-02-10
  • Yeast bioprospecting versus synthetic biology—which is better for innovative beverage fermentation?
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-18
    Lucien Alperstein, Jennifer M Gardner, Joanna F Sundstrom, Krista M Sumby, Vladimir Jiranek

    Abstract Producers often utilise some of the many available yeast species and strains in the making of fermented alcoholic beverages in order to augment flavours, aromas, acids and textural properties. But still, the demand remains for more yeasts with novel phenotypes that not only impact sensory characteristics but also offer process and engineering advantages. Two strategies for finding such yeasts are (i) bioprospecting for novel strains and species and (ii) genetic modification of known yeasts. The latter enjoys the promise of the emerging field of synthetic biology, which, in principle, would enable scientists to create yeasts with the exact phenotype desired for a given fermentation. In this mini review, we compare and contrast advances in bioprospecting and in synthetic biology as they relate to alcoholic fermentation in brewing and wine making. We explore recent advances in fermentation-relevant recombinant technologies and synthetic biology including the Yeast 2.0 Consortium, use of environmental yeasts, challenges, constraints of law and consumer acceptance.

    更新日期:2020-02-10
  • Combination of the CRP mutation and ptsG deletion in Escherichia coli to efficiently synthesize xylitol from corncob hydrolysates
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-16
    Xinsong Yuan, Shuai Tu, Jianping Lin, Lirong Yang, Huahao Shen, Mianbin Wu

    Abstract The biotechnology-based production of xylitol has received widespread attention because it can use cheap and renewable lignocellulose as a raw material, thereby decreasing costs and pollution. The simultaneous use of various sugars in lignocellulose hydrolysates is a primary prerequisite for efficient xylitol production. In this study, a ΔptsG and crp* combinatorial strategy was used to generate Escherichia coli W3110 strain IS5-dI, which completely eliminated glucose repression and simultaneously used glucose and xylose. This strain produced 164 g/L xylitol from detoxified corncob hydrolysates during a fed-batch fermentation in a 15-L bioreactor, which was 14.7% higher than the xylitol produced by the starting strain, IS5-d (143 g/L), and the xylitol productivity was 3.04 g/L/h. These results represent the highest xylitol concentration and productivity reported to date for bacteria and hemicellulosic sugars. Additionally, strain IS5-dG, which differs from IS5-dI at CRP amino acid residue 127 (I127G), was tolerant to the toxins in corncob hydrolysates. In a fed-batch fermentation experiment involving a 15-L bioreactor, IS5-dG produced 137 g/L xylitol from non-detoxified corncob hydrolysates, with a productivity of 1.76 g/L/h. On the basis of these results, we believe that IS5-dI and IS5-dG may be useful host strains for the industrial-scale production of xylitol from detoxified or non-detoxified corncob hydrolysates.

    更新日期:2020-02-10
  • A novel multifunctional GH9 enzyme from Paenibacillus curdlanolyticus B-6 exhibiting endo/exo functions of cellulase, mannanase and xylanase activities
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-24
    Vanarat Phakeenuya, Khanok Ratanakhanokchai, Akihiko Kosugi, Chakrit Tachaapaikoon

    Abstract PcMulGH9, a novel glycoside hydrolase family 9 (GH9) from Paenibacillus curdlanolyticus B-6, was successfully expressed in Escherichia coli. It is composed of a catalytic domain of GH9, two domains of carbohydrate-binding module family 3 (CBM3) and two domains of fibronectin type 3 (Fn3). The PcMulGH9 enzyme showed broad activity towards the β-1,4 glycosidic linkages of cellulose, mannan and xylan, including cellulose and xylan contained in lignocellulosic biomass, which is rarely found in GH9. The enzyme hydrolysed substrates with bifunctional endo-/exotypes cellulase, mannanase and xylanase activities, but predominantly exhibited exo-activities. This enzyme released cellobiose as a major product from cellohexaose, while mannotriose and xylotriose were major hydrolysis products from mannohexaose and xylohexaose, respectively. Moreover, PcMulGH9 could hydrolyse untreated corn hull and rice straw into xylo- and cello-oligosaccharides. Enzyme kinetics, site-directed mutagenesis and molecular docking revealed that Met394, located at the binding subsite + 2, was involved in broad substrate specificity of PcMulGH9 enzyme. This study offers new knowledge of the multifunctional cellulase/mannanase/xylanase in GH9. The PcMulGH9 enzyme showed a novel function of GH9, which increases its potential for saccharification of lignocellulosic biomass into value-added products, especially oligosaccharides.

    更新日期:2020-02-10
  • Microbially induced calcium carbonate precipitation to design a new type of bio self-healing dental composite
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-15
    Mostafa Seifan, Zahra Sarabadani, Aydin Berenjian

    Abstract Crack propagation is one of the issues associating with dental composites which can significantly affect their performance. Current solutions for preventing and stopping the cracks include maximizing the filler to matrix ratio as well as fiber reinforcing of composites which are not always reliable. The precipitation of calcium carbonate (CaCO3) minerals by the generally recognized as safe (GRAS) bacteria can be seen as a novel approach to address this shortcoming. In the present study, the effect of microbially induced calcium carbonate precipitation (MICP) on filling dental composites’ cracks and cavities was studied. In this first step, the capability of different GRAS bacteria to induce CaCO3 precipitation was investigated. In the next step, the capability of potent bacteria to initiate MCIP in solid matrix was evaluated. For this purpose, the CaCO3-bacteria along with necessary nutrients were introduced into different dental composites in two ways, namely, powder and paste form. The light-cured composites were analyzed using optical microscopy, scanning electron microscopy (SEM), and energy-dispersive X-ray (EDS) to identify and characterize the precipitated CaCO3 crystals. It was shown that the incorporation of powder healing compound in two composites resulted in precipitation of CaCO3, while no crystals were formed when a paste form of healing compound was mixed with composites. The results evidently show that MICP can be a feasible alternative to current inefficient approaches to address microcracking issues in dental composites.

    更新日期:2020-02-10
  • Enrichment of novel Actinomycetales and the detection of monooxygenases during aerobic 1,4-dioxane biodegradation with uncontaminated and contaminated inocula
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-20
    Vidhya Ramalingam, Alison M. Cupples

    Abstract 1,4-Dioxane, a co-contaminant at many chlorinated solvent sites, is a problematic groundwater pollutant because of risks to human health and characteristics which make remediation challenging. In situ 1,4-dioxane bioremediation has recently been shown to be an effective remediation strategy. However, the presence/abundance of 1,4-dioxane degrading species across different environmental samples is generally unknown. Here, the objectives were to identify which 1,4-dioxane degrading functional genes are present and which genera may be using 1,4-dioxane and/or metabolites to support growth across different microbial communities. For this, laboratory sample microcosms and abiotic control microcosms (containing media) were inoculated with four uncontaminated soils and sediments from two contaminated sites. Live control microcosms were treated in the same manner, except 1,4-dioxane was not added. 1,4-Dioxane decreased in live microcosms with all six inocula, but not in the abiotic controls, suggesting biodegradation occurred. A comparison of live sample microcosms and live controls (no 1,4-dioxane) indicated nineteen genera were enriched following exposure to 1,4-dioxane, suggesting a growth benefit for 1,4-dioxane biodegradation. The three most enriched were Mycobacterium, Nocardioides, and Kribbella (classifying as Actinomycetales). There was also a higher level of enrichment for Arthrobacter, Nocardia, and Gordonia (all three classifying as Actinomycetales) in one soil, Hyphomicrobium (Rhizobiales) in another soil, Clavibacter (Actinomycetales) and Bartonella (Rhizobiales) in another soil, and Chelativorans (Rhizobiales) in another soil. Although Arthrobacter, Mycobacterium, and Nocardia have previously been linked to 1,4-dioxane degradation, Nocardioides, Gordonia, and Kribbella are potentially novel degraders. The analysis of the functional genes associated with 1,4-dioxane demonstrated three genes were present at higher relative abundance values, including Rhodococcus sp. RR1 prmA, Rhodococcus jostii RHA1 prmA, and Burkholderia cepacia G4 tomA3. Overall, this study provides novel insights into the identity of the multiple genera and functional genes associated with aerobic degradation of 1,4-dioxane in mixed communities.

    更新日期:2020-02-10
  • Novel approaches for efficient in vivo fermentation production of noncoding RNAs
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-17
    Ai-Ming Yu, Neelu Batra, Mei-Juan Tu, Colleen Sweeney

    Abstract Genome-derived noncoding RNAs (ncRNAs), including microRNAs (miRNAs), small interfering RNAs (siRNAs), and long noncoding RNAs (lncRNAs), play an essential role in the control of target gene expression underlying various cellular processes, and dysregulation of ncRNAs is involved in the pathogenesis and progression of various diseases in virtually all species including humans. Understanding ncRNA biology has opened new avenues to develop novel RNA-based therapeutics. Presently, ncRNA research and drug development is dominated by the use of ncRNA mimics that are synthesized chemically in vitro and supplemented with extensive and various types of artificial modifications and thus may not necessarily recapitulate the properties of natural RNAs generated and folded in living cells in vivo. Therefore, there are growing interests in developing novel technologies for in vivo production of RNA molecules. The two most recent major breakthroughs in achieving an efficient, large-scale, and cost-effective fermentation production of recombinant or bioengineered RNAs (e.g., tens of milligrams from 1 L of bacterial culture) are (1) using stable RNA carriers and (2) direct overexpression in RNase III-deficient bacteria, while other approaches offer a low yield (e.g., nano- to microgram scales per liter). In this article, we highlight these novel microbial fermentation-based technologies that have shifted the paradigm to the production of true biological ncRNA molecules for research and development.

    更新日期:2020-02-10
  • Alternative strategies for the application of aminoglycoside antibiotics against the biofilm-forming human pathogenic bacteria
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-22
    Fazlurrahman Khan, Dung Thuy Nguyen Pham, Young-Mog Kim

    Abstract Aminoglycosides are one of the common classes of antibiotics that have been widely used for treating infections caused by pathogenic bacteria. The mechanism of bactericidal action by aminoglycosides is well-known, by which it terminates the cytoplasmic protein synthesis. However, the potentials of aminoglycosides become hindered when facing the evolution of bacterial resistance mechanisms. Among multiple resistance mechanisms displayed by bacteria against antibiotics, the formation of biofilm is the mechanism that provides a barrier for antibiotics to reach the cellular level. Bacteria present in the biofilm also get protection against the impact of host immune responses, harsh environmental conditions, and other antimicrobial treatments. Hence, with the multifaceted resistance developed by biofilm-forming pathogenic bacteria, antibiotics are therefore discontinued for further applications. However, the recent research developed several alternative strategies such as optimization of the active concentration, modification of the environmental conditions, modification of the chemical structure, combinatorial application with other active agents, and formulation with biocompatible carrier materials to revitalize and exploit the new potential of aminoglycosides. The present review article describes the above mentioned multiple approaches and possible mechanisms for the application of aminoglycosides to treat biofilm-associated infections.

    更新日期:2020-02-10
  • Nanosecond electric pulses rapidly enhance the inactivation of Gram-negative bacteria using Gram-positive antibiotics
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-22
    Ram Anand Vadlamani, Agni Dhanabal, David A. Detwiler, Rusha Pal, James McCarthy, Mohamed N. Seleem, Allen L. Garner

    Abstract Physically disrupting microorganism membranes to enable antibiotics to overcome resistance mechanisms that inhibit or excrete antibiotics has great potential for reducing antibiotic doses and rendering resistance mechanisms inert. We demonstrate the synergistic inactivation of a Gram-positive (Staphylococcus aureus) and two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria by combining 222 30 kV/cm electric pulses (EPs) or 500 20 kV/cm EPs with 300-ns EP duration with various antibiotics with different mechanisms of action is demonstrated. Doses of antibiotics that produced no inactivation in 10 min of exposure in solution with bacteria induced several log reductions under the influence of nanosecond EPs. Combining 2 μg/L or 20 μg/mL of rifampicin with the 30 kV/cm EPs enhanced Staphylococcus aureus inactivation compared with EPs alone, while only a few of the other combinations demonstrated improvement. Combining 2 μg/L or 20 μg/mL of mupirocin or rifampicin with either EP train enhanced E. coli inactivation compared with EPs alone. Combining 2 μg/L or 20 μg/mL of erythromycin or vancomycin with the 30 kV/cm EPs enhanced E. coli inactivation compared with EPs alone. These results indicate that EPs can make Gram-positive antibiotics efficient for inactivating Gram-negative bacteria with future studies required to optimize EP parameters for other antibiotics and Gram-negative bacteria.

    更新日期:2020-02-10
  • l -Alanine specifically potentiates fluoroquinolone efficacy against Mycobacterium persisters via increased intracellular reactive oxygen species
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-15
    Junfeng Zhen, Shuangquan Yan, Yuzhu Li, Cao Ruan, Yue Li, Xue Li, Xiaokang Zhao, Xi Lv, Yan Ge, Ulrich Aymard Ekomi Moure, Jianping Xie

    Abstract Tuberculosis caused by Mycobacterium tuberculosis remains a major global health concern; M. tuberculosis drug resistance and persistence further fueled the situation. Nutrient supportive therapy was intensively pursued to complement the conventional treatment, as well as their synergy with current antibiotics. To explore whether l-alanine can synergize with fluoroquinolones against M. tuberculosis, M. smegmatis was used as a surrogate in this study. We found that l-alanine can boost the bactericidal efficacy of fluoroquinolones, increasing the production of intracellular reactive oxygen species. This effect is very significant for persisters. Accelerated tricarboxylic acid cycle and/or nucleotide metabolism were observed after the addition of l-alanine. M. smegmatis MSMEG2660 is a homolog of the alanine dehydrogenase (Rv2780, MSMEG2659) negative regulator Rv2779c and involved in the l-alanine potentiation of fluoroquinolone via funneling more alanine into tricarboxylic acid. Deletion mutant of the MSMEG2660 (∆Ms2660) became more susceptible, and more readily revived from persistence. We firstly found that l-alanine can synergize with fluoroquinolones against Mycobacterium, especially the persisters via promoting metabolism. This will inspire new avenue to eliminate Mycobacterium persisters.

    更新日期:2020-02-10
  • Restricting mycotoxins without killing the producers: a new paradigm in nano-fungal interactions
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-02-05
    Rubaiya Jesmin, Anindya Chanda

    Abstract Over the past several years, numerous studies have demonstrated the feasibility of using engineered nanoparticles as antifungals, especially against those fungal pathogens that produce mycotoxins and infect plants, animals, and humans. The high dosage of nanoparticles has been a concern in such antifungal applications due to the potential toxicological and ecotoxicological impacts. To address such concerns, we have recently introduced the idea of inhibiting mycotoxin biosynthesis using low doses of engineered nanoparticles. At such low doses these particles are minimally toxic to humans and the environment. From our studies we realize that for the effective use of nanotechnology to intervene in the biology of fungal pathogens and for an accurate evaluation of the impacts of the increasingly growing nanomaterials in the environment on fungi and their interacting biotic partners, there is a pressing need for a rigorous understanding of nano-fungal interactions, which is currently far from complete. In this minireview, we build on the available evidence from nano-bio interaction research and our recent interaction studies with Aspergillus cells and engineered silver nanoparticles to introduce a potential theoretical model for nano-fungal interactions. The aim of the proposed model is to provide an initial insight on how nanoparticle uptake and their transformation inside fungal cells, possibly influence the production of mycotoxins and other secondary metabolites of filamentous fungi .

    更新日期:2020-02-06
  • Identification and characterization of two fatty acid elongases in Lipomyces starkeyi
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-02-05
    Tomohiko Matsuzawa, Yasushi Kamisaka, Tomoko Maehara, Hiroaki Takaku, Katsuro Yaoi

    Abstract The oleaginous yeast Lipomyces starkeyi is a potential cost-effective source for the production of microbial lipids. Fatty acid elongases have vital roles in the syntheses of long-chain fatty acids. In this study, two genes encoding fatty acid elongases of L. starkeyi, LsELO1, and LsELO2 were identified and characterized. Heterologous expression of these genes in Saccharomyces cerevisiae revealed that LsElo1 is involved in the production of saturated long-chain fatty acids with 24 carbon atoms (C24:0) and that LsElo2 is involved in the conversion of C16 fatty acids to C18 fatty acids. In addition, both LsElo1 and LsElo2 were able to elongate polyunsaturated fatty acids. LsElo1 elongated linoleic acid (C18:2) to eicosadienoic acid (C20:2), and LsElo2 elongated α-linolenic acid (C18:3) to eicosatrienoic acid (C20:3). Overexpression of LsElo2 in L. starkeyi caused a reduction in C16 fatty acids, such as palmitic and palmitoleic acids, and an accumulation of C18 fatty acids such as oleic and linoleic acids. Our findings have the potential to contribute to the remodeling of fatty acid composition and the production of polyunsaturated long-chain fatty acids in oleaginous yeasts.

    更新日期:2020-02-06
  • Synthetic antibody discovery against native antigens by CRISPR/Cas9-library generation and endoplasmic reticulum screening
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-02-04
    Joana H. Ministro, Soraia S. Oliveira, Joana G. Oliveira, Miguel Cardoso, Frederico Aires-da-Silva, Sofia Corte-Real, Joao Goncalves

    Abstract Despite the significant advances of antibodies as therapeutic agents, there is still much room for improvement concerning the discovery of these macromolecules. Here, we present a new synthetic cell-based strategy that takes advantage of eukaryotic cell biology to produce highly diverse antibody libraries and, simultaneously, link them to a high-throughput selection mechanism, replicating B cell diversification mechanisms. The interference of site-specific recognition by CRISPR/Cas9 with error-prone DNA repair mechanisms was explored for the generation of diversity, in a cell population containing a gene for a light chain antibody fragment. We achieved up to 93% of cells containing a mutated antibody gene after diversification mechanisms, specifically inside one of the antigen-binding sites. This targeted variability strategy was then integrated into an intracellular selection mechanism. By fusing the antibody with a KDEL retention signal, the interaction of antibodies and native membrane antigens occurs inside the endoplasmic reticulum during the process of protein secretion, enabling the detection of high-quality leads for expression and affinity by flow cytometry. We successfully obtained antibody lead candidates against CD3 as proof of concept. In summary, we developed a novel antibody discovery platform against native antigens by endoplasmic synthetic library generation using CRISPR/Cas9, which will contribute to a faster discovery of new biotherapeutic molecules, reducing the time-to-market.

    更新日期:2020-02-04
  • Identification of the genes involved in growth characters of medicinal fungus Ophiocordyceps sinensis based on Agrobacterium tumefaciens –mediated transformation
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-02-04
    Guiqing Liu, Li Cao, Zhongchen Rao, Xuehong Qiu, Richou Han

    Abstract Ophiocordyceps sinensis, one of the well-known and precious fungal species in the world, parasitizes soil-dwelling larvae of ghost moths on the Tibetan Plateau. The genetic intractability of this extremely psychrophilic and slow-growing O. sinensis fungus is a major limitation on the molecular study. In this study, an Agrobacterium tumefaciens–mediated genetic transformation (ATMT) system for this fungus was established. ATMT procedure was optimized based on the fungal recipient, Agrobacterium strains, and different co-cultivation conditions. Blastospores were ideal recipients for this system. Acetosyringone (AS) was not essential for the transformation of O. sinensis. Sixty to 100 hygromycin B–resistant transformants per 1 × 106 blastospores were obtained. Southern blot analysis indicated the presence of a random single-copy integration of T-DNA into the O. sinensis genome. The insertional transformants with altered growth characters such as colony, blastospore, and fruiting body production were selected to identify the T-DNA flanking sequences by modified hiTAIL-PCR and FPNI-PCR techniques. Eight genes, including genes for an MFS transporter, a 2-oxoglutarate dehydrogenase, a DNA-directed RNA polymerase III complex subunit Rpc37, a cytochrome oxidase subunit I, a mitochondrial import inner membrane translocase subunit tim54, a cytidine deaminase, a phosphoribosylaminoimidazole carboxylase, and a histone H3-like centromeric protein cse-4 were identified. This ATMT system provides a useful tool for gene discovery and characterization of O. sinensis and contributes to the better understanding of the mysterious life cycle of O. sinensis and the molecular interaction between this fungus and its host insects.

    更新日期:2020-02-04
  • Fungi from the extremes of life: an untapped treasure for bioactive compounds
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-02-04
    Muhammad Ibrar, Muhammad Wajid Ullah, Sehrish Manan, Umer Farooq, Muhammad Rafiq, Fariha Hasan

    Abstract More than 80% of the Earth surface is consisted of hostile and harsh environments, classified as extreme from an anthropogenic perspective. Microorganisms with acclimatized nature dominate these extreme ecosystems of the biosphere. Survivals in such environments initiate an inductive force leading to the production of noteworthy metabolites having peculiar biochemistry. Recent investigations on extremophilic fungi for unprecedented bioactive compounds emphasize their remarkable potential as sources of new therapeutics. The present review covers the literature published in the last 15 years and highlights the biological activities and structure of compounds isolated from the extremophilic fungi. The compounds are grouped based on their biological functions such as cytotoxicity, lipid-lowering ability, and antimicrobial, antioxidant, nematocidal, anti-inflammatory, anti-malarial, and antifouling activities. A total of 155 compounds isolated from 25 Penicillium species, 16 Aspergillus species, and 23 other species are presented, which include 105 new and 50 known bioactive compounds. Out of these, 77 have known cytotoxic activity and 46 are antimicrobial in nature, while there are 32 other compounds with different activities including nematocidal, anti-allergic, antioxidant, and anti-inflammatory. Key Points • A broad compilation of bioactive compounds from extremophilic fungi. • Classification of bioactive compounds based on their biological functions. • Production of cytotoxic compounds is common among all kind of extremophilic fungi. • Bioactive compounds have no direct role in adaptation process of extremophiles.

    更新日期:2020-02-04
  • Engineering a Pichia pastoris nitrilase whole cell catalyst through the increased nitrilase gene copy number and co-expressing of ER oxidoreductin 1
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-02-04
    Qi Shen, Zhuang Yu, Pei-jin Lv, Qian Li, Shu-Ping Zou, Neng Xiong, Zhi-Qiang Liu, Ya-Ping Xue, Yu-Guo Zheng

    Abstract 1-Cyanocyclohexaneacetic acid (1-CHAA) is a critical intermediate for the synthesis of the antiepileptic agent gabapentin. Previously, our group has established a novel manufacturing route for 1-CHAA through bioconversion catalyzed by an Escherichia coli (E. coli) nitrilase whole cell catalyst. However, the nitrilase expressed in E. coli has several drawbacks such as a low level of reusability, which hampered its industrial application. Herein, we investigated the potential of using the methylotrophic yeast Pichia pastoris (P. pastoris) for producing the nitrilase whole cell catalyst. To achieve strains with high catalytic activities, we investigated the effects of the promoter choice, expressing cassette copy number, and co-expression of chaperone on the production of nitrilase. Our results demonstrated that the strain harboring the multicopy integrations of nitrilase gene under the control of the alcohol oxidase 1 (AOX1) promoter and co-expressing of ER oxidoreductin 1 (ERO1) exhibited an 18-fold enhancement in the nitrilase activity compared with the strain containing a single integration of nitrilase gene under the control of glyceraldehyde-3-phosphate (GAP) dehydrogenase promoter. This optimized P. pastoris strain, compared with the E. coli nitrilase whole cell catalyst, shows greatly improved levels of reusability and thermostability while has a similar high-substrate tolerance.

    更新日期:2020-02-04
  • Correction to: Lipid accumulation in prokaryotic microorganisms from arid habitats
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-02-03
    Philippa Hauschild, Annika Röttig, Mohamed H. Madkour, Ahmed M. Al-Ansari, Naief H. Almakishah, Alexander Steinbüchel

    There is an error in the Original Publication of this paper for “Acknowledgements” section was missing.

    更新日期:2020-02-03
  • Comparative genomics of the aconidial Aspergillus niger strain LDM3 predicts genes associated with its high protein secretion capacity
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-02-03
    Yu-Fei Sui, Li-Ming Ouyang, Tabea Schütze, Shu Cheng, Vera Meyer, Ying-Ping Zhuang

    Abstract Aspergillus niger is widely used as a cell factory for homologous and heterologous protein production. As previous studies reported that reduced sporulation favors protein secretion in A. niger, in this study, we conducted a comparative genomic analysis of the non-sporulating industrially exploited A. niger strain LDM3 in China and the reference protein secretion strain CBS 513.88 to predict the key genes that might define the genetic basis of LDM3’s high protein-producing potential in silico. After sequencing using a hybrid approach combining Illumina and PacBio sequencing platforms, a high-quality genome sequence of LDM3 was obtained which harbors 11,209 open reading frames (ORFs). LDM3 exhibits large chromosomal rearrangements in comparison to CBS 513.88. An alignment of the two genome sequences revealed that the majority of the 457 ORFs uniquely present in LDM3 possessed predicted functions in redox pathways, protein transport, and protein modification processes. In addition, bioinformatic analyses revealed the presence of 656 ORFs in LDM3 with non-synonymous mutations encoding for proteins related to protein translation, protein modification, protein secretion, metabolism, and energy production. We studied the impact of two of these on protein production in the established lab strain N402. Both tupA and prpA genes were selected because available literature suggested their involvement in asexual sporulation of A. niger. Our co-expression network analysis supportively predicted the role of tupA in protein secretion and the role of prpA in energy generation, respectively. By knockout experiments, we showed that the ΔtupA mutant displayed reduced sporulation (35%) accompanied by higher total protein secretion (65%) compared to its parental strain. Such an effect was, however, not observed in the ΔprpA mutant.

    更新日期:2020-02-03
  • The (p)ppGpp-mediated stringent response regulatory system globally inhibits primary metabolism and activates secondary metabolism in Pseudomonas protegens H78
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-02-03
    Lingyu Wu, Zheng Wang, Yejun Guan, Xianqing Huang, Huimin Shi, Yujie Liu, Xuehong Zhang

    Abstract Pseudomonas protegens H78 produces multiple secondary metabolites, including antibiotics and iron carriers. The guanosine pentaphosphate or tetraphosphate ((p)ppGpp)-mediated stringent response is utilized by bacteria to survive during nutritional starvation and other stresses. RelA/SpoT homologues are responsible for the biosynthesis and degradation of the alarmone (p)ppGpp. Here, we investigated the global effect of relA/spoT dual deletion on the transcriptomic profiles, physiology, and metabolism of P. protegens H78 grown to mid- to late log phase. Transcriptomic profiling revealed that relA/spoT deletion globally upregulated the expression of genes involved in DNA replication, transcription, and translation; amino acid metabolism; carbohydrate and energy metabolism; ion transport and metabolism; and secretion systems. Bacterial growth was partially increased, while the cell survival rate was significantly reduced by relA/spoT deletion in H78. The utilization of some nutritional elements (C, P, S, and N) was downregulated due to relA/spoT deletion. In contrast, relA/spoT mutation globally inhibited the expression of secondary metabolic gene clusters (plt, phl, prn, ofa, fit, pch, pvd, and has). Correspondingly, antibiotic and iron carrier biosynthesis, iron utilization, and antibiotic resistance were significantly downregulated by the relA/spoT mutation. This work highlights that the (p)ppGpp-mediated stringent response regulatory system plays an important role in inhibiting primary metabolism and activating secondary metabolism in P. protegens.

    更新日期:2020-02-03
  • Novel caffeine degradation gene cluster is mega-plasmid encoded in Paraburkholderia caffeinilytica CF1
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-02-03
    Di Sun, Xueying Yang, Chao Zeng, Bo Li, Yi Wang, Chunzhi Zhang, Wenzhong Hu, Xianzhen Li, Ziqing Gao

    Abstract The widespread use of caffeine in food and drug industries has caused great environmental pollution. Herein, an efficient caffeine-degrading strain Paraburkholderia caffeinilytica CF1 isolated from a tea garden in China can utilize caffeine as its sole carbon and nitrogen source. Combination of chromatographic and spectrophotometric techniques confirmed that strain CF1 adopts N-demethylation pathway for caffeine degradation. Whole genome sequencing of strain CF1 reveals that it has two chromosomes with sizes 3.62 Mb and 4.53 Mb, and a 174-kb mega-plasmid. The plasmid P1 specifically harbors the genes essential for caffeine metabolism. By analyzing the sequence alignment and quantitative real-time PCR data, the redundant gene cluster of caffeine degradation was elucidated. Genes related to catalyzing the N1-demethylation of caffeine to theobromine, the first step of caffeine degradation were heterologously expressed, and methylxanthine N1-demethylase was purified and characterized. Above all, this study systematically unravels the molecular mechanism of caffeine degradation by Paraburkholderia. Key Points • Caffeine degradation cluster in Paraburkholderia caffeinilytica CF1 was located in mega-plasmid P1. • The whole genome and the caffeine degrading pathway of P. caffeinilytica CF1 were sequenced and elucidated, respectively. • This study succeeded in heterologous expression of methylxanthine N1-demethylase (CdnA) and Rieske oxygenase reductase (CdnD) and illuminated the roles of CdnA and CdnD in caffeine degradation of P. caffeinilytica CF1.

    更新日期:2020-02-03
  • Valorization of cheese whey using microbial fermentations
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-02-03
    Teresa Zotta, Lisa Solieri, Lucilla Iacumin, Claudia Picozzi, Maria Gullo

    Abstract Cheese whey (CW), the liquid resulting from the precipitation and removal of milk casein during cheese-making, and the second cheese whey (SCW) derived from the production of cottage and ricotta cheeses are the main byproducts of dairy industry. The major constituent of CW and SCW is lactose, contributing to the high BOD and COD content. Because of this, CW and SCW are high-polluting agents and their disposal is still a problem for the dairy sector. CW and SCW, however, also consist of lipids, proteins, and minerals, making them useful for production of various compounds. In this paper, microbial processes useful to promote the bioremediation of CW and SCW are discussed, and an overview on the main whey-derived products is provided. Special focus was paid to the production of health-promoting whey drinks, vinegar, and biopolymers, which may be exploited as value-added products in different segments of food and pharmaceutical industries.

    更新日期:2020-02-03
  • Cis and trans isomers of the vitamin menaquinone-7: which one is biologically significant?
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-02-03
    Neha Lal, Aydin Berenjian

    Abstract Recently, several studies have indicated that an adequate intake of menaquinone-7 (MK-7) offers numerous health benefits. However, the low availability of MK-7 in the diet necessitates the development of dietary supplements or functional food products to complement natural food sources and meet the daily intake requirements. Like most biological molecules, MK-7 can exist as geometric isomers that can occur in the cis, trans, and cis/trans forms; however, only the all-trans form is biologically significant. MK-7 is traditionally produced through bacterial fermentation, but various synthetic preparations have lately become available. The isomer composition in the final product is influenced by numerous factors, including the methods of production and purification, as well as particular environmental and storage conditions. The MK-7 profile obtained from the various production methods has not yet been elucidated, and the ideal method for the synthesis of the all-trans form of the vitamin is also debatable. Consequently, the quantification of the MK-7 profile of various products is necessary to develop an understanding of the factors that influence the proportion of isomers that are obtained in different preparations. Several possible methods exist for the quantification of MK-7 isomers, and of these, liquid chromatography in conjunction with mass spectrometry techniques appears to be the most promising. Evaluation of the isomer composition is an important consideration, as only the all-trans form sustains biological activity. Furthermore, knowledge of the prominent factors that influence the MK-7 composition may also enable their manipulation to obtain a more favorable MK-7 profile in the final product.

    更新日期:2020-02-03
  • CRISPR-Cas9 assisted functional gene editing in the mushroom Ganoderma lucidum
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-21
    Ping-An Wang, Han Xiao, Jian-Jiang Zhong

    Abstract The genetic manipulation of basidiomycete mushrooms is notoriously difficult and immature, and there is a lack of research reports on clustered regularly interspaced short palindromic repeat (CRISPR) based gene editing of functional genes in mushrooms. In this work, Ganoderma lucidum, a famous traditional medicinal basidiomycete mushroom, which produces a type of unique triterpenoid-anti-tumor ganoderic acids (GAs), was used, and a CRISPR/CRISPR-associated protein-9 nuclease (Cas9) editing system for functional genes of GA biosynthesis was constructed in the mushroom. As proof of concept, the effect of different gRNA constructs with endogenous u6 promoter and self-cleaving ribozyme HDV on ura3 disruption efficiency was investigated at first. The established system was applied to edit a cytochrome P450 monooxygenase (CYP450) gene cyp5150l8, which is responsible for a three-step biotransformation of lanosterol at C-26 to ganoderic acid 3-hydroxy-lanosta-8, 24-dien-26 oic acid. As a result, precisely edited cyp5150l8 disruptants were obtained after sequencing confirmation. The fermentation products of the wild type (WT) and cyp5150l8 disruptant were analyzed, and a significant decrease in the titer of four identified GAs was found in the mutant compared to WT. Another CYP gene involved in the biosynthesis of squalene-type triterpenoid 2, 3; 22, 23-squalene dioxide, cyp505d13, was also disrupted using the established CRISPR-Cas9 based gene editing platform of G. lucidum. The work will be helpful to strain molecular breeding and biotechnological applications of G. lucidum and other basidiomycete mushrooms.

    更新日期:2020-01-27
  • Characterization of an 17β-estradiol-degrading bacterium Stenotrophomonas maltophilia SJTL3 tolerant to adverse environmental factors
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-13
    Weiliang Xiong, Chong Yin, Wanli Peng, Zixin Deng, Shuangjun Lin, Rubing Liang

    Abstract Bioremediation of environmental estrogens requires microorganisms with stable degradation efficiency and great stress tolerance in complex environments. In this work, Stenotrophomonas maltophilia SJTL3 isolated from wastewater was found to be able to degrade over 90% of 10 μg/mL 17β-estradiol (E2) in a week and the degradation dynamic was fitted by the first-order kinetic equations. Estrone was the first and major intermediate of E2 biodegradation. Strain SJTL3 exhibited strong tolerance to several adverse conditions like extreme pH (3.0–11.0), high osmolality (2%), co-existing heavy metals (6.25 μg/mL of Cu2+) and surfactants (5 CMC of Tween 80), and retained normal cell vitality and stable E2-degradaing efficiency. In solid soil, strain SJTL3 could remove nearly 100% of 1 μg/mL of E2 after the bacteria inoculation and 8-day culture. As to the contamination of 10 μg/mL E2 in soil, the biodegradation efficiency was about 90%. The further obtainment of the whole genome of strain SJTL3 and genome analysis revealed that this strain contained not only the potential genes responsible for estrogen degradation, but also the genes encoding proteins involved in stress tolerance. This work could promote the estrogen-biodegrading mechanism study and provide insights into the bioremediation application.

    更新日期:2020-01-15
  • Fungal P 450 monooxygenases - the diversity in catalysis and their promising roles in biocontrol activity
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-19
    Razak Hussain, Mushtaq Ahmed, Tabreiz Ahmad Khan, Yusuf Akhter

    Abstract The fungal P450s catalyze vital monooxygenation reactions in primary and secondary metabolism, which may lead to the production of diverse secondary metabolites. Many of these, such as from the family of trichothecenes, involve in biocontrol activities. The diversified nature of fungal P450 monooxygenases makes their host organisms adoptable to various ecological niches. The available genome data analysis provided an insight into the activity and mechanisms of the fungal P450s. However, still more structural and functional studies are needed to elucidate the details of its catalytic mechanism, and the advance studies are also required to decipher further about their dynamic role in various aspects of trichothecene oxygenations. This mini review will provide updated information on different fungal P450 monooxygenases, their genetic diversity, and their role in catalyzing various biochemical reactions leading to the production of plant growth promoting secondary metabolites.

    更新日期:2020-01-15
  • Engineered bacterial biofloc formation enhancing phenol removal and cell tolerance
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-13
    Xiao Jia, Shun Zhang, Jiawei Li, Juan Xia, Ruilian Yao, Xinqing Zhao, Bing Wu, Fengwu Bai, Yi Xiao

    Abstract A microbial floc consisting of a community of microbes embedded in extracellular polymeric substances matrix can provide microbial resistances to toxic chemicals and harsh environments. Phenol is a toxic environmental pollutant and a typical lignin-derived phenolic inhibitor. In this study, we genetically engineered Escherichia coli cells by expressions of diguanylate cyclases (DGCs) to promote proteinaceous and aliphatic biofloc formation. Compared with the planktonic E. coli cells, the biofloc-forming cells improved phenol removal rate by up to 2.2-folds, due to their substantially improved tolerance (up to 149%) to phenol and slightly enhanced cellular activity (20%) of phenol hydroxylase (PheH). The engineered bioflocs also improved E. coli tolerance to other toxic compounds such as furfural, 5-hydroxymethylfurfural, and guaiacol. Additionally, the strategy of the engineered biofloc formation was applicable to Pseudomonas putida and enhanced its tolerance to phenol. This study highlights a strategy to form engineered bioflocs for improved cell tolerance and removal of toxic compounds, enabling their universality of use in bioproduction and bioremediation.

    更新日期:2020-01-15
  • Diversity of volatile organic compound production from leucine and citrate in Enterococcus faecium
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-11
    Matilde D’Angelo, Gabriela P. Martino, Victor S. Blancato, Martín Espariz, Axel Hartke, Nicolas Sauvageot, Abdellah Benachour, Sergio H. Alarcón, Christian Magni

    Abstract Enterococcus faecium is frequently isolated from fermented food; in particular, they positively contribute to the aroma compound generation in traditional cheese. Citrate fermentation is a desirable property in these bacteria, but this feature is not uniformly distributed among E. faecium strains. In the present study, three selected E. faecium strains, IQ110 (cit−), GM70 (cit+ type I), and Com12 (cit+ type II), were analyzed in their production of aroma compounds in milk. End products and volatile organic compounds (VOCs) were determined by solid-phase micro-extraction combined with gas chromatography mass spectrometry (SPME-GC-MS). Principal component analysis (PCA) of aroma compound profiles revealed a different VOC composition for the three strains. In addition, resting cell experiments of E. faecium performed in the presence of leucine, citrate, or pyruvate as aroma compound precursors allowed us to determine metabolic differences between the studied strains. GM70 (cit+ type I) showed an active citrate metabolism, with increased levels of diacetyl and acetoin generation relative to Com12 or to citrate defective IQ110 strains. In addition, in the experimental conditions tested, a defective citrate-fermenting phenotype for the Com12 strain was found, while its leucine degradation and pyruvate metabolism were conserved. In conclusion, rational selection of E. faecium strains could be performed based on genotypic and phenotypic analyses. This would result in a performing strain, such as GM70, that could positively contribute to flavor, with typical notes of diacetyl, acetoin, 3-methyl butanal, and 3-methyl butanol in an adjuvant culture.

    更新日期:2020-01-15
  • Zinc supplementation improves the harvest purity of β-glucuronidase from CHO cell culture by suppressing apoptosis
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-19
    Ryan J. Graham, Stephanie Ketcham, Adil Mohammad, Bandaranayake M. B. Bandaranayake, Ty Cao, Bidesh Ghosh, James Weaver, Seongkyu Yoon, Patrick J. Faustino, Muhammad Ashraf, Celia N. Cruz, Chikkathur N. Madhavarao

    The variability of trace metals in cell culture media is a potential manufacturing concern because it may significantly affect the production and quality of therapeutic proteins. Variability in trace metals in CHO cell culture has been shown to impact critical production metrics such as cell growth, viability, nutrient consumption, and production of recombinant proteins. To better understand the influence of excess supplementation, zinc and copper were initially supplemented with 50-μM concentrations to determine the impact on the production and quality of β-glucuronidase, a lysosomal enzyme, in a parallel bioreactor system. Ethylenediaminetetraacetic acid (EDTA), a metal chelator, was included as another treatment to induce a depletion of trace metal bioavailability to examine deficiency. Samples were drawn daily to monitor cell growth and viability, nutrient levels, β-glucuronidase activity, and trace zinc flux. Cell cycle analysis revealed the inhibition of sub-G0/G1 species in zinc supplemented cultures, maintaining higher viability compared to the control, EDTA-, and copper-supplemented cultures. Enzyme activity analysis in the harvests revealed higher specific activity of β-glucuronidase in reactors supplemented with zinc. A confirmation run was conducted with supplementations of zinc at concentrations of 50, 100, and 150 μM. Further cell cycle analysis and caspase-3 analysis demonstrated the role of zinc as an apoptosis suppressor responsible for the enhanced harvest purity of β-glucuronidase from zinc-supplemented bioreactors.

    更新日期:2020-01-15
  • Quantitative multi-omics analysis of the effects of mitochondrial dysfunction on lipid metabolism in Saccharomyces cerevisiae
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-12
    Xiaopeng Guo, Miaomiao Zhang, Yue Gao, Guozhen Cao, Dong Lu, Wenjian Li

    In this study, combined genome, transcriptome, and metabolome analysis was performed for eight Saccharomyces cerevisiae mitochondrial respiration-deficient mutants. Each mutant exhibited a unique nuclear genome mutation pattern; the nuclear genome mutations, and thus potentially affected genes and metabolic pathways, showed a co-occurrence frequency of ≤ 3 among the eight mutants. For example, only a lipid metabolism-related pathway was likely to be affected by the nuclear genome mutations in one of the mutants. However, large deletions in the mitochondrial genome were the shared characteristic among the eight mutants. At the transcriptomic level, lipid metabolism was the most significantly enriched Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway for differentially expressed genes (DEGs) co-occurring in both ≥ 4 and ≥ 5 mutants. Any identified DEG enriched in lipid metabolism showed the same up-/down-regulated pattern among nearly all eight mutants. Further, 126 differentially expressed lipid species (DELS) were identified, which also showed the same up-/down-regulated pattern among nearly all investigated mutants. It was conservatively demonstrated that the similar change pattern of lipid metabolism in the entire investigated mutant population was attributed to mitochondrial dysfunction. The change spectrum of lipid species was presented, suggesting that the number and change degree of up-regulated lipid species were higher than those of down-regulated lipid species. Additionally, energy storage lipids increased in content and plasma-membrane phospholipid compositions varied in the relative proposition. The results for the genome, transcriptome, and lipidome were mutually validated, which provides quantitative data revealing the roles of mitochondria from a global cellular perspective.

    更新日期:2020-01-15
  • Antimicrobial secondary metabolites from agriculturally important bacteria as next-generation pesticides
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-19
    Chetan Keswani, Harikesh B. Singh, Carlos García-Estrada, John Caradus, Ya-Wen He, Samia Mezaache-Aichour, Travis R. Glare, Rainer Borriss, Estibaliz Sansinenea

    Abstract The whole organisms can be packaged as biopesticides, but secondary metabolites secreted by microorganisms can also have a wide range of biological activities that either protect the plant against pests and pathogens or act as plant growth promotors which can be beneficial for the agricultural crops. In this review, we have compiled information about the most important secondary metabolites of three important bacterial genera currently used in agriculture pest and disease management.

    更新日期:2020-01-15
  • Advances in acrylamide bioproduction catalyzed with Rhodococcus cells harboring nitrile hydratase
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-19
    Song Jiao, Fulong Li, Huimin Yu, Zhongyao Shen

    Acrylamide is an important bulk chemical used for producing polyacrylamide, which is widely applied in diverse fields, such as enhanced oil recovery and water treatment. Acrylamide production with a superior biocatalyst, free-resting Rhodococcus cells containing nitrile hydratase (NHase), has been proven to be simple but effective, thereby becoming the main method adopted in industry to date. Under the harsh industrial conditions, however, NHase-containing Rhodococcus cells in a natural state are prone to deactivation. Thus, multiple genetic strategies able to evolve recombinant Rhodococcus biocatalysts at either the enzyme or cell level have been reported. While most of the methods on enzyme engineering concentrate on NHase stability enhancement by strengthening the flexible sites, Rhodococcus cell engineering with various methods can enhance both the NHase activity and stability as well. Developing some new types of reactors, especially the microreactor, is also an effective way to improve the hydration process efficiency. Compared with the conventional stirred tank reactor, the membrane dispersion microreactor can enhance the heat and mass transfer in the hydration process with Rhodococcus cells as biocatalysts, thereby significantly improving the productivity of the acrylamide bioproduction process.

    更新日期:2020-01-15
  • Metabolic engineering for the production of fat-soluble vitamins: advances and perspectives
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-14
    Panhong Yuan, Shixiu Cui, Yanfeng Liu, Jianghua Li, Guocheng Du, Long Liu

    Fat-soluble vitamins are vitamins that are insoluble in water, soluble in fat, and organic solvents; they are found in minute amount in various foods. Fat-soluble vitamins, including vitamins A, D, E, and K, have been widely used in food, cosmetics, health care products, and pharmaceutical industries. Fat-soluble vitamins are currently produced via biological and chemical synthesis. In recent years, fat-soluble vitamin production by biotechnological routes has been regarded as a very promising approach. Based on biosynthetic pathways, considerable advances of α-tocopherol and β-carotenes have been achieved in transgenic plants and microalgae. Microbial fermentation, as an alternative method for the production of vitamin K and β-carotenes, is attracting considerable attention because it is an environment friendly process. In this review, we address the function and applications of fat-soluble vitamins, and an overview of current developments in the production of fat-soluble vitamins in transgenic plants, microalgae, and microorganisms. We focus on the metabolic and process engineering strategies for improving production of fat-soluble vitamins, and we hope this review can be useful for the people who are interested in the production of fat-soluble vitamins by biotechnological routes.

    更新日期:2020-01-15
  • Enzymatic characterization and regulation of gene expression of PhoK alkaline phosphatase in Sphingobium sp. strain TCM1
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-12
    Shouji Takahashi, Yuka Morooka, Takahito Kumakura, Katsumasa Abe, Yoshio Kera

    Abstract Sphingobium sp. strain TCM1 can significantly degrade chlorinated organophosphorus flame retardants, such as tris(2-chloroethyl) phosphate. The PhoK of strain TCM1 (Sb-PhoK) is the main alkaline phosphatase (APase) that catalyzes the last step in the degradation pathway. Here, we purified and characterized Sb-PhoK produced in E. coli, and analyzed the regulation of Sb-phoK gene expression in strain TCM1. The recombinant Sb-PhoK was produced in the mature form, lacking a putative signal peptide, and formed a homodimer. Purified Sb-PhoK exhibited 384 U/mg of specific activity at 37 °C. The optimum temperature was 50 °C, and Sb-PhoK was completely inactivated when incubated at 60 °C for 10 min. The optimum pH was 10, with stability observed at pH 6.0–10.5. Sb-PhoK was suggested to contain two Ca2+ and one Zn2+ per subunit, but excess addition of Zn2+ into the reaction mixture markedly inhibited the enzyme activity. Sb-PhoK showed phosphatase activity against various phosphorylated compounds, except for bis(p-nitrophenyl) phosphate, indicating that it is a phosphomonoesterase with broad substrate specificity. The Km and kcat for p-nitrophenyl phosphate were 2.31 mM and 1270 s−1, respectively, under optimal conditions. The enzyme was strongly inhibited by vanadate, dithiothreitol, and SDS, but was highly resistant to urea and Triton X-100. Sb-phoK gene expression was regulated by the inorganic phosphate concentration in culture medium, and was induced at a low inorganic phosphate concentration. The deletion of Sb-phoB gene resulted in no induction of Sb-phoK gene even at a low inorganic phosphate concentration, confirming that Sb-PhoK is a member of Pho regulon.

    更新日期:2020-01-15
  • Novel strategy to improve the colonizing ability of Irpex lacteus in non-sterile wheat straw for enhanced rumen and enzymatic digestibility
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-19
    Dongze Niu, Sasa Zuo, Jianjun Ren, Huhetaoli, Mingli Zheng, Di Jiang, Chuncheng Xu

    Pretreatment with white rot fungi is a promising method to enhance the digestibility of lignocelluloses; however, sterilization of feedstocks prior to inoculation is one of the costliest steps. To improve the colonizing ability of white rot fungi under non-sterile condition, Irpex lacteus, Pleurotus ostreatus, and Phanerochaete chrysosporium were inoculated in the wheat straw ensiled for 28 days and incubated for 56 days to determine the changes in microbe counts, organic acid content, chemical composition, and rumen and enzymatic digestibility. Results showed that ensiling produced abundant organic acids and suppressed most microbes in wheat straw. Significant growth of I. lacteus was observed after 3 days of incubation, and molds were only detectable at day 7 in the group. At the end of incubation, aerobic bacteria and lactic acid bacteria decreased by 18% and 38% in the wheat straw treated with I. lacteus, but molds, aerobic bacteria, and lactic acid bacteria thrived in those treated with P. ostreatus and P. chrysosporium. Even more, P. ostreatus and P. chrysosporium increased the lignin content of the ensiled wheat straw by 34% and 65%. However, I. lacteus selectively degraded lignin by 28% and improved the rumen and enzymatic digestibility by 18% and 34%. The finding indicates that ensiling prior to fermentation with I. lacteus is an effective method to control spoilage microbes and to enhance the rumen and enzymatic digestibility of wheat straw.

    更新日期:2020-01-15
  • A plasmid-based genomic screening system for transcriptional regulators of non-adjacent xenobiotic catabolism genes
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-10
    Minggen Cheng, Ziyu Xing, Luyao Lu, Feng Chen, Jian He, Xing Huang

    Bacteria play an important role in the catabolism of environmental xenobiotics. The study of transcriptional regulation has greatly enhanced our understanding of the molecular mechanisms associated with these processes. However, genes encoding transcription factors (TFs) for xenobiotic catabolism are usually not located in the immediate vicinity of the catabolic genes that they regulate; therefore, functional identification of these TFs is difficult. Significantly modified from a metagenome screening method substrate-induced gene expression (SIGEX), here we propose a synthetic pSRGFP-18 plasmid-based tool as a TF reporter system. In short, two multiple cloning sites (MCS) were designed; one in front of an egfp reporter gene was constructed for promoter insertion, and the other MCS was used for shotgun cloning of genomic DNA. Based on the regulatory relationship between TFs and the promoter of their associated catabolic genes, this approach allowed us to screen for TF genes using a genome shotgun approach. This system performed well when testing the known operons. Following statistical analysis of known catabolic operons in Escherichia coli and Bacillus subtilis, the suggested region of the target promoter for this system was from − 250 to + 150. Furthermore, to broaden the applicability of this plasmid, a series of pSRGFP-18 and pBBR1-based pSRGFP-X plasmids were constructed, which had broad host ranges and contained different antibiotic markers. This study outlines a powerful tool to enable functional identification of TFs for bacterial xenobiotic catabolism.

    更新日期:2020-01-15
  • OxyR-controlled surface polysaccharide production and biofilm formation in Acinetobacter oleivorans DR1
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-20
    Bora Shin, Chulwoo Park, Woojun Park

    Abstract The genomes of several Acinetobacter species possess three distinct polysaccharide-producing operons [two poly-N-acetyl glucosamine (PNAG) and one K-locus]. Using a microfluidic device, an increased amount of polysaccharides and enhanced biofilm formation were observed following continuous exposure to H2O2 and removal of the H2O2-sensing key regulator, OxyR, in Acinetobacter oleivorans DR1 cells. Gene expression analysis revealed that genes located in PNAG1, but not those in PNAG2, were induced and that genes in the K-locus were expressed in the presence of H2O2. Interestingly, the expression of the K-locus gene was enhanced in the PNAG1 mutant and vice versa. The absence of either OxyR or PNAG1 resulted in enhanced biofilm formation, higher surface hydrophobicity, and increased motility, implying that K-locus-driven polysaccharide production in both the oxyR and PNAG1 deletion mutants may be related to these phenotypes. Both the oxyR and K-locus deletion mutants were more sensitive to H2O2 compared with the wildtype and PNAG1 mutant strains. Purified OxyR binds to the promoter regions of both polysaccharide operons with a higher affinity toward the K-locus promoter. Although oxidized OxyR could bind to both promoter regions, the addition of dithiothreitol further enhanced the binding efficiency of OxyR, suggesting that OxyR might function as a repressor for controlling these polysaccharide operons.

    更新日期:2020-01-15
  • Production and characterisation of a marine Halomonas surface-active exopolymer
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-07
    Tony Gutierrez, Gordon Morris, Dave Ellis, Barbara Mulloy, Michael D. Aitken

    Abstract During screening for novel emulsifiers and surfactants, a marine gammaproteobacterium, Halomonas sp. MCTG39a, was isolated and selected for its production of an extracellular emulsifying agent, P39a. This polymer was produced by the new isolate during growth in a modified Zobell’s 2216 medium amended with 1% glucose, and was extractable by cold ethanol precipitation. Chemical, chromatographic and nuclear magnetic resonance spectroscopic analysis confirmed P39a to be a high-molecular-weight (~ 261,000 g/mol) glycoprotein composed of carbohydrate (17.2%) and protein (36.4%). The polymer exhibited high emulsifying activities against a range of oil substrates that included straight-chain aliphatics, mono- and alkyl- aromatics and cycloparaffins. In general, higher emulsification values were measured under low (0.1 M PBS) compared to high (synthetic seawater) ionic strength conditions, indicating that low ionic strength is more favourable for emulsification by the P39a polymer. However, as observed with other bacterial emulsifying agents, the polymer emulsified some aromatic hydrocarbon species, as well as refined and crude oils, more effectively under high ionic strength conditions, which we posit could be due to steric adsorption to these substrates as may be conferred by the protein fraction of the polymer. Furthermore, the polymer effected a positive influence on the degradation of phenanthrene by other marine bacteria, such as the specialist PAH-degrader Polycyclovorans algicola. Collectively, based on the ability of this Halomonas high-molecular-weight glycoprotein to emulsify a range of pure hydrocarbon species, as well as refined and crude oils, it shows promise for the bioremediation of contaminated sites.

    更新日期:2020-01-15
  • Genome mining integrating semi-rational protein engineering and nanoreactor design: roadmap for a robust biocatalyst for industrial resolution of Vince lactam
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-11
    Hongxia Li, Shuaihua Gao, Yan Qiu, Chaoqun Liang, Shaozhou Zhu, Guojun Zheng

    Biomanufacturing of chemicals using biocatalysts is an attractive strategy for the production of valuable pharmaceuticals since it is usually more economical and has a much-reduced environmental impact. However, there are often challenges such as their thermal instability that should be overcome before a newly discovered enzyme is eventually translated into industrial processes. In this work, we describe a roadmap for the development of a robust catalyst for industrial resolution of Vince lactam, a key intermediate for the synthesis of carbocyclic-nucleoside-related pharmaceuticals. By a genome mining strategy, a new (+)-γ-lactamase (MiteL) from Microbacterium testaceum was successfully discovered and biochemically characterized. In vitro studies showed that the enzyme exhibited high activity but poor enantioselectivity (E = 6.3 ± 0.2) toward racemic Vince lactam, and thus, it is not suitable for industrial applications. Based on structural modeling and docking studies, a semi-rational engineering strategy combined with an efficient screening method was then applied to improve the enantioselectivity of MiteL. Several mutants with significant shifting stereoselectivity toward (−)-γ-lactam were obtained by site-saturation mutagenesis. Synergy effects led to the final mutant F14D/Q114R/M117L, which enabled efficient acquisition of (−)-γ-lactam with a high E value (> 200). The mutant was biochemically characterized, and the docking studies suggested a plausible mechanism for its improved selectivity. Finally, a sunflower-like nanoreactor was successfully constructed to improve the mutant’s robustness via protein supramolecular self-assembly. Thus, the synergism between semi-rational protein engineering and self-assembling immobilization enabled construction of a nanoreactor with superior properties, which can be used for resolution of Vince lactam in large scale.

    更新日期:2020-01-15
  • Evaluation of the protective immunity of Riemerella anatipestifer OmpA
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-21
    Xinxin Xu, Yaohui Xu, Shuang Miao, Pan Jiang, Junsheng Cui, Yanshan Gong, Panpan Tan, Xiaoli Du, Nazrul Islam, Qinghai Hu

    Riemerella anatipestifer is responsible for an economically important disease of commercially raised ducks. No or only few cross-protection was observed between different serotypes of R. anatipestifer strains, and so far no protective antigen in this bacterium has been identified. OmpA is a predominant immunogenic protein of R. anatipestifer, and within the 1467 bp ompA ORF (ompA1467), there is another 1164 bp ORF (ompA1164) with the same C-terminal. In this study, our results showed that the full sequence of ompA1467 from some R. anatipestifer strains with different serotypes shared the same amino acid sequence. Animal experiments showed that the soluble recombinant protein rOmpA1164, but not rOmpA1467, could provide partial protective immunity against challenge. Moreover, there was no significant difference in protective immunity between ducklings immunized with Th4△ompA bacterin and those immunized with Th4 bacterin. In addition, OmpA1467 was the main existing form of OmpA in R. anatipestifer cells by gel electrophoresis and western blot analyses. The results suggested that OmpA1467 was not a protective antigen of R. anatipestifer, and antibodies against proteins other than OmpA play a critical role in the process of anti-R. anatipestifer infection.

    更新日期:2020-01-15
  • Characterization of three GH35 β-galactosidases, enzymes able to shave galactosyl residues linked to rhamnogalacturonan in pectin, from Penicillium chrysogenum 31B
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-18
    Tatsuya Kondo, Yuichi Nishimura, Kaori Matsuyama, Megumi Ishimaru, Masami Nakazawa, Mitsuhiro Ueda, Tatsuji Sakamoto

    Abstract Three recombinant β-galactosidases (BGALs; PcBGAL35A, PcBGAL35B, and PcGALX35C) belonging to the glycoside hydrolase (GH) family 35 derived from Penicillium chrysogenum 31B were expressed using Pichia pastoris and characterized. PcBGAL35A showed a unique substrate specificity that has not been reported so far. Based on the results of enzymological tests and 1H-nuclear magnetic resonance, PcBGAL35A was found to hydrolyze β-1,4-galactosyl residues linked to l-rhamnose in rhamnogalacturonan-I (RG-I) of pectin, as well as p-nitrophenyl-β-d-galactopyranoside and β-d-galactosyl oligosaccharides. PcBGAL35B was determined to be a common BGAL through molecular phylogenetic tree and substrate specificity analysis. PcGALX35C was found to have similar catalytic capacities for the β-1,4-galactosyl oligomer and polymer. Furthermore, PcGALX35C hydrolyzed RG-I-linked β-1,4-galactosyl oligosaccharide side chains with a degree of polymerization of 2 or higher in pectin. The amino acid sequence similarity of PcBGAL35A was approximately 30% with most GH35 BGALs, whose enzymatic properties have been characterized. The amino acid sequence of PcBGAL35B was approximately 80% identical to those of BGALs from Penicillium sp. The amino acid sequence of PcGALX35C was classified into the same phylogenetic group as PcBGAL35A. Pfam analysis revealed that the three BGALs had five domains including a catalytic domain. Our findings suggest that PcBGAL35A and PcGALX35C are enzymes involved in the degradation of galactosylated RG-I in pectin. The enzymes characterized in this study may be applied for products that require pectin processing and for the structural analysis of pectin.

    更新日期:2020-01-15
  • Modulation effect of tea consumption on gut microbiota
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-18
    Yu-Chuan Liu, Xin-Yu Li, Liang Shen

    Tea is one of the most popular beverages in the world and possesses a wide range of beneficial effects for human health. The modulation of tea on gut microbiota has gained much interest in recent years. The present study discussed the modulation effect of various types of tea on gut microbiota, which plays crucial roles in human health, as investigated by in vitro animal and human studies. The currently available findings from a total of 23 studies support the modulation effects of tea liquid, tea extract, and its major active components, including polyphenols, polysaccharides, and teasaponin, on gut microbiota. Overall, tea possesses prebiotic-like effect and can alleviate the gut microbiota dysbiosis induced by high-fat diet in gut microbiota, despite the detailed bacterial taxa may alter depending on the types of tea supplemented. Current evidence implies that the modulation effect on gut microbiota may be an important action mechanism underlying the beneficial effect of tea consumption in daily life and also the great potential of strategically chosen tea extract to develop functional foods.

    更新日期:2020-01-15
  • Chemoenzymatic synthesis of the pH responsive surfactant octyl β-D-glucopyranoside uronic acid
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2019-12-07
    Ngoc T. N. Ngo, Carl Grey, Patrick Adlercreutz

    Methodology was developed to expand the range of benign alkyl glycoside surfactants to include also anionic types. This was demonstrated possible through conversion of the glycoside to its carboxyl derivative. Specifically, octyl β-D-glucopyranoside (OG) was oxidised to the corresponding uronic acid (octyl β-D-glucopyranoside uronic acid, OG-COOH) using the catalyst system T. versicolor laccase/2,2,6,6-tetramethylpiperidinyloxy (TEMPO) and oxygen from air as oxidant. The effects of oxygen supply methodology, concentrations of laccase, TEMPO and OG as well as reaction temperature were evaluated. At 10 mM substrate concentration, the substrate was almost quantitatively converted into product, and even at a substrate concentration of 60 mM, 85% conversion was reached within 24 h. The surfactant properties of OG-COOH were markedly dependent on pH. Foaming was only observed at low pH, while no foam was formed at pH values above 5.0. Thus, OG-COOH can be an attractive low-foaming surfactant, for example for cleaning applications and emulsification, in a wide pH range (pH 1.5–10.0).

    更新日期:2020-01-15
  • Correction to: Heterologous production of chlortetracycline in an industrial grade Streptomyces rimosus host
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-14
    Xuefeng Wang, Shouliang Yin, Jing Bai, Yang Liu, Keqiang Fan, Huizhuan Wang, Fang Yuan, Baohua Zhao, Zilong Li, Weishan Wang

    The original version of this article contains an error.

    更新日期:2020-01-14
  • Transgene suppression in plants by foliar application of in vitro-synthesized small interfering RNAs
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-14
    Alexandra S. Dubrovina, Olga A. Aleynova, Andrey R. Suprun, Zlata V. Ogneva, Konstantin V. Kiselev

    Abstract Recent research has shown that plants can uptake long dsRNAs and dsRNA-derived siRNAs that target important genes of infecting fungi or viruses when applied on the surface of plant leaves. The external RNAs were capable of local and systemic movement inducing plant resistance against the pathogens. Few studies have been made for plant gene regulation by foliar application of RNAs. In this study, several types of ssRNA and siRNA duplexes targeting the neomycin phosphotransferase II (NPTII) transgene were in vitro-synthesized and externally applied to the leaf surface of 4-week-old transgenic Arabidopsis thaliana plants. External application of the synthetic NPTII-encoding siRNAs down-regulated NPTII transcript levels in transgenic A. thaliana 1 and 7 days post-treatment with a higher and more consistent effect being observed for siRNAs methylated at 3′ ends. We also analyzed the effects of external NPTII-encoding dsRNA precursors and a dsRNA-derived heterogenous siRNA mix. Digestion of the NPTII-dsRNA to the heterogeneous siRNAs did not improve efficiency of the transgene suppression effect. Key Points• Foliar application of siRNAs down-regulated a commonly used transgene in Arabidopsis. • A more consistent effect was observed for methylated siRNAs. • The findings are important for development of plant gene regulation approaches.

    更新日期:2020-01-14
  • Significant improvement of the enantioselectivity of a halohydrin dehalogenase for asymmetric epoxide ring opening reactions by protein engineering
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-14
    Feng Xue, Li-Hui Zhang, Qing Xu

    Abstract Halohydrin dehalogenases (HHDHs) have attracted much attention due to their ability to synthesize enantiomerically enriched epoxides and β-haloalcohols. However, most of the HHDHs exhibit low enantioselectivity. Here, a HHDH from the alphaproteobacteria isolate 46_93_T64 (AbHHDH), which shows only poor enantioselectivity in the catalytic resolution of rac-PGE (E = 9.9), has been subjected to protein engineering to enhance its enantioselectivity. Eight mutants (R89K, R89Y, V137I, P178A, N179Q, N179L, F187L, F187A) showed better enantioselectivity than the wild type. The best single mutant N179L (E = 93.0) showed a remarkable 9.4-fold increase in the enantioselectivity. Then, the single mutations were combined to produce the double, triple, quadruple, and quintuple mutants. Among the combinational mutants, the best variant (R89Y/N179L) showed an increased E value of up to 48. The E values of the variants N179L and R89Y/N179L for other epoxides 2–7 were 12.2 to > 200, which showed great improvement compared to 1.2 to 10.5 for the wild type. Using the variant N179L, enantiopure (R)-PGE with > 99% ee could be readily prepared, affording a high yield and a high concentration.

    更新日期:2020-01-14
  • Colletotrichum: species complexes, lifestyle, and peculiarities of some sources of genetic variability
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-14
    Leandro Lopes da Silva, Hanna Lorena Alvarado Moreno, Hilberty Lucas Nunes Correia, Mateus Ferreira Santana, Marisa Vieira de Queiroz

    Abstract The genus Colletotrichum comprises species with different lifestyles but is mainly known for phytopathogenic species that infect crops of agronomic relevance causing considerable losses. The fungi of the genus Colletotrichum are distributed in species complexes and within each complex some species have particularities regarding their lifestyle. The most commonly found and described lifestyles in Colletotrichum are endophytic and hemibiotrophic phytopathogenic. Several of these phytopathogenic species show wide genetic variability, which makes long-term maintenance of resistance in plants difficult. Different mechanisms may play an important role in the emergence of genetic variants but are not yet fully understood in this genus. These mechanisms include heterokaryosis, a parasexual cycle, sexual cycle, transposable element activity, and repeat-induced point mutations. This review provides an overview of the genus Colletotrichum, the species complexes described so far and the most common lifestyles in the genus, with a special emphasis on the mechanisms that may be responsible, at least in part, for the emergence of new genotypes under field conditions.

    更新日期:2020-01-14
  • Discovery and mechanism of intestinal bacteria in enzymatic cleavage of C–C glycosidic bonds
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-13
    Bin Wei, Ya-Kun Wang, Wen-Hui Qiu, Si-Jia Wang, Yue-Hong Wu, Xue-Wei Xu, Hong Wang

    C-Glycosides, a special type of glycoside, are frequently distributed in many kinds of medicinal plants, such as puerarin and mangiferin, showing various and significant bioactivities. C-Glycosides are usually characterized by the C–C bond that forms between the anomeric carbon of sugar moieties and the carbon atom of aglycon, which is usually resistant against acidic hydrolysis and enzymatic treatments. Interestingly, C-glycosides could be cleaved by several intestinal bacteria, but whether the enzymatic cleavage of C–C glycosidic bond is reduction or hydrolysis has been controversial; furthermore, whether existence of a “C-glycosidase” directly catalyzing the cleavage is not clear. Here we review research advances about the discovery and mechanism of intestinal bacteria in enzymatic cleavage of C–C glycosidic bond with an emphasis on the identification of enzymes manipulation the deglycosylation. Finally, we give a brief conclusion about the mechanism of C-glycoside deglycosylation and perspectives for future study in this field.

    更新日期:2020-01-13
  • Two new ene-reductases from photosynthetic extremophiles enlarge the panel of old yellow enzymes: Ct OYE and Gs OYE
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-13
    Marina Simona Robescu, Mattia Niero, Mélanie Hall, Laura Cendron, Elisabetta Bergantino

    Looking for new ene-reductases with uncovered features beneficial for biotechnological applications, by mining genomes of photosynthetic extremophile organisms, we identified two new Old Yellow Enzyme homologues: CtOYE, deriving from the cyanobacterium Chroococcidiopsis thermalis, and GsOYE, from the alga Galdieria sulphuraria. Both enzymes were produced and purified with very good yields and displayed catalytic activity on a broad substrate spectrum by reducing α,β-unsaturated ketones, aldehydes, maleimides and nitroalkenes with good to excellent stereoselectivity. Both enzymes prefer NADPH but demonstrate a good acceptance of NADH as cofactor. CtOYE and GsOYE represent robust biocatalysts showing high thermostability, a wide range of pH optimum and good co-solvent tolerance. High resolution X-ray crystal structures of both enzymes have been determined, revealing conserved features of the classical OYE subfamily as well as unique properties, such as a very long loop entering the active site or an additional C-terminal alpha helix in GsOYE. Not surprisingly, the active site of CtOYE and GsOYE structures revealed high affinity toward anions caught from the mother liquor and trapped in the anion hole where electron-withdrawing groups such as carbonyl group are engaged. Ligands (para-hydroxybenzaldehyde and 2-methyl-cyclopenten-1-one) added on purpose to study complexes of GsOYE were detected in the enzyme catalytic cavity, stacking on top of the FMN cofactor, and support the key role of conserved residues and FMN cofactor in the catalysis.

    更新日期:2020-01-13
  • Production, characterization, and application of a monoclonal antibody specific for the extracellular domain of human P2X7R
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-13
    Mingxuan Li, Shuping Luo, Yunfang Zhang, Lina Jia, Chuanyu Yang, Xiaoxiang Peng, Ronglan Zhao

    This paper focuses on the production of a high-affinity monoclonal antibody (mAb) that can efficiently detect and block purinergic ligand-gated ion channel 7 receptor (P2X7R). To achieve this goal, the extracellular domain of human P2X7R, P2X7R-ECD, was used as an immunogen for BALB/c mice, inducing them to produce spleen lymphocytes that were subsequently fused with myeloma cells. Screening of the resultant hybridoma clones resulted in the selection of one stable positive clone that produced a qualified mAb, named 4B3A4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the purity of the purified 4B3A4 mAb was above 85%, with prominent bands corresponding to molecular weights of 55 kDa (heavy chain) and 25 kDa (light chain), and the BCA assay showed that the concentration of the purified 4B3A4 mAb was 0.3 mg/mL. Western blot analysis revealed that the 4B3A4 mAb could specifically recognize and bind both P2X7R-ECD and the full-length P2X7R protein. Laser scanning confocal microscopy (LSCM) revealed that the 4B3A4 mAb specifically bound to P2X7R on the membrane of human peripheral blood mononuclear cells (PBMCs). P2X7R expression was significantly different between healthy individuals and people with certain cancers as determined by flow cytometry (FCM). In addition, the 4B3A4 mAb significantly reduced ATP-stimulated Ca2+ entry and YO-PRO-1 uptake, which indicated that the 4B3A4 mAb effectively blocked P2X7R activity. These data indicate that the 4B3A4 mAb can be further used as not only an antibody to detect cell surface P2X7R but also as a therapeutic antibody to target P2X7R-related signaling pathways.

    更新日期:2020-01-13
  • Identification of Zur boxes and determination of their roles in the differential regulation of the Zur regulon in Agrobacterium tumefaciens C58
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-11
    Puttamas Nuonming, Sasimaporn Khemthong, Rojana Sukchawalit, Skorn Mongkolsuk

    Zinc uptake regulator (Zur) is a transcriptional regulator that represses zinc acquisition genes under high zinc conditions. The aim of this study was to identify and investigate the role of Zur-binding motifs (Zur boxes) in the differential regulation of Zur target genes, including the zinT, znuA, znuCB-zur operon, the troCBA operon, and yciC, in Agrobacterium tumefaciens. DNase I footprinting and gel shift assays were performed, confirming that Zur directly binds to 18-bp inverted repeat motifs found in the promoter of these Zur-regulated genes. Furthermore, promoter-lacZ fusions and mutagenesis of the identified Zur boxes were performed to assess the role of each Zur box. A Zur box found in the zinT promoter was required for zinc-dependent repression by Zur. The intergenic region between the znuA gene and the znuCB-zur operon contains two Zur boxes, named A and C, which immediately precede the genes znuA and znuC, respectively. Zur box A, but not Zur box C, was essential for the repression of the znuA promoter. Both Zur boxes A and C were implicated in the repression of the znuC promoter, in which mutation of either box alone was sufficient for full derepression of the znuC promoter. Three Zur boxes named T, M, and Y were identified in the intergenic region between the troCBA operon and the yciC gene. Zur box Y, which immediately precedes yciC, was shown to be responsible for Zur repression of the yciC promoter. In contrast, two Zur boxes, T and M, were essential for the complete repression of the troCBA operon, and full derepression of the troC promoter was exhibited when both Zur boxes were mutated simultaneously. Sequence analysis of the identified Zur boxes revealed a correlation between deviation from the core recognition sequence of the Zur box and the requirement of two Zur boxes for Zur regulation of distinctive promoters.

    更新日期:2020-01-13
  • Prevention of biofilm formation by quorum quenching
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-11
    E. Paluch, J. Rewak-Soroczyńska, I. Jędrusik, E. Mazurkiewicz, K. Jermakow

    Quorum sensing (QS) is a mechanism that enables microbial communication. It is based on the constant secretion of signaling molecules to the environment. The main role of QS is the regulation of vital processes in the cell such as virulence factor production or biofilm formation. Due to still growing bacterial resistance to antibiotics that have been overused, it is necessary to search for alternative antimicrobial therapies. One of them is quorum quenching (QQ) that disrupts microbial communication. QQ-driving molecules can decrease or even completely inhibit the production of virulence factors (including biofilm formation). There are few QQ strategies that comprise the use of the structural analogues of QS receptor autoinductors (AI). They may be found in nature or be designed and synthesized via chemical engineering. Many of the characterized QQ molecules are enzymes with the ability to degrade signaling molecules. They can also impede cellular signaling cascades. There are different techniques used for testing QS/QQ, including chromatography-mass spectroscopy, bioluminescence, chemiluminescence, fluorescence, electrochemistry, and colorimetry. They all enable qualitative and quantitative measurements of QS/QQ molecules. This article gathers the information about the mechanisms of QS and QQ, and their effect on microbial biofilm formation. Basic methods used to study QS/QQ, as well as the medical and biotechnological applications of QQ, are also described. Basis research methods are also described as well as medical and biotechnological application.

    更新日期:2020-01-13
  • Phenotypic and genomic analysis of multiple heavy metal–resistant Micrococcus luteus strain AS2 isolated from industrial waste water and its potential use in arsenic bioremediation
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-11
    Shahid Sher, Syed Zajif Hussain, Abdul Rehman

    Multiple heavy metal–resistant bacterium, Micrococcus luteus strain AS2, was isolated from industrial waste water of District Sheikhupura, Pakistan. The isolated bacterium showed minimum inhibitory concentrations of 55 and 275 mM against arsenite and arsenate. The bacterial strain also showed resistance against other heavy metal ions, i.e., lead, cadmium, chromium, mercury, nickel, and zinc, apart from arsenic. The optimum temperature and pH were 37 °C and 7, respectively. The antioxidant enzymes such as catalase were significantly increased under arsenite stress. The increase in 43.9% of GSH/GSSG and 72.72% of non-protein thiol was determined under15 mM arsenite stress. Bacterial genome was sequenced through Illumina and Nanopore and genes related to arsenic and other heavy metals were identified and blast (tblastx) on NCBI. Through scanning electron microscopy, no morphological changes were observed in bacterial cells under arsenite stress. The peaks appeared in EDX showed that there is surface adsorption of arsenite in bacterial cell while it was confirmed from Fourier transformed infrared spectroscopy analysis that there is some interaction between arsenite and functional groups present on the surface of bacterial cell. The SDS-PAGE analysis of whole-cell proteins under 15 mM arsenite stress clearly revealed that there is upregulation of some proteins in ranged of 60 to 34 kDa. The bioremediation efficiency (E) of bacterial biomass was 72% after 2 h and 99% after 10 h. The bioremediation efficiency of bacterial biomass is an indicator for the isolated bacterium to employ as a potential candidate for the amelioration of sites contaminated with arsenic.

    更新日期:2020-01-13
  • Effects of pH, temperature and salinity on P3HB synthesis culturing the marine Rhodovulum sulfidophilum DSM-1374
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-11
    Pietro Carlozzi, Tiziana Di Lorenzo, Demetrios F. Ghanotakis, Eleftherios Touloupakis

    Rhodovulum sulfidophilum DSM-1374 is a potential producer of polyester when growing in phototrophic conditions. The present study investigated on a polyester product (P3HB) by culturing Rhodovulum sulfidophilum DSM-1374 in two different photobioreactors (PBR-1 and PBR-2) both with 4-L working volumes. PBR-1 is equipped with an internal rotor having 4 paddles to mix the bacterial culture while PBR-2 has an internal coil-shaped rotor. After selecting PBR-1, which best performed in the preliminary experiment, the effect of different stressing growth conditions as pH (7.0, 8.0, and 9.0), temperature (25, 30, and 35 °C), and medium salinity (1.5, 2.5, 3.5, and 4.5%) were tested. When the pH of the culture was set to 8.0, the capability of the bacterium to synthetize the polyester increased significantly reaching a concentration of 412 mg (P3HB)/L; the increase of the pH at 9.0 caused a reduction of the P3HB concentration in the culture. The medium salinity of 4.5% was the best stress-growth condition to reach the highest concentration of polyester in the culture (820 ± 50 mg (P3HB)/L) with a P3HB mass fraction in the dry biomass of 33 ± 1.5%. Stresses caused by culture temperature are another potential parameter that could increase the synthesis of P3HB.

    更新日期:2020-01-13
  • A tandem GGDEF-EAL domain protein-regulated c-di-GMP signal contributes to spoilage-related activities of Shewanella baltica OS155
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-11
    Feifei Wang, Yongzheng Wang, Congnan Cen, Linglin Fu, Yanbo Wang

    Cyclic diguanylate (c-di-GMP) is a second messenger involved in the regulation of various physiological processes in bacteria. However, its function in spoilage bacteria has not yet been addressed. Here, we studied the function of a tandem GGDEF-EAL domain protein, Sbal_3235, in the spoilage bacterium Shewanella baltica OS155. The deletion of sbal_3235 significantly reduced the c-di-GMP level, biofilm formation, and exopolysaccharide, trimethylamine (TMA), and putrescine production; sbal_3235 deletion also downregulated the expression of the torS and speF genes and affected membrane fatty acid composition. Site-directed mutagenesis in conserved GGDEF and EAL motifs abolished diguanylate cyclase (DGC) and phosphodiesterase (PDE) activity, respectively. These data indicate that Sbal_3235 is an essential contributor to the c-di-GMP pool with bifunctional DGC and PDE activity, which is involved in the biofilm formation and spoilage activity of S. baltica OS155. Our findings expand the biochemical role of c-di-GMP and uncover its link to spoilage activities, providing novel targets for food quality and safety controlling.

    更新日期:2020-01-13
  • Naproxen in the environment: its occurrence, toxicity to nontarget organisms and biodegradation
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-10
    Danuta Wojcieszyńska, Urszula Guzik

    Abstract This article summarizes the current knowledge about the presence of naproxen in the environment, its toxicity to nontarget organisms and the microbial degradation of this drug. Currently, naproxen has been detected in all types of water, including drinking water and groundwater. The concentrations that have been observed ranged from ng/L to μg/L. These concentrations, although low, may have a negative effect of long-term exposure on nontarget organisms, especially when naproxen is mixed with other drugs. The biological decomposition of naproxen is performed by fungi, algae and bacteria, but the only well-described pathway for its complete degradation is the degradation of naproxen by Bacillus thuringiensis B1(2015b). The key intermediates that appear during the degradation of naproxen by this strain are O-desmethylnaproxen and salicylate. This latter is then cleaved by 1,2-salicylate dioxygenase or is hydroxylated to gentisate or catechol. These intermediates can be cleaved by the appropriate dioxygenases, and the resulting products are incorporated into the central metabolism. Key points •High consumption of naproxen is reflected in its presence in the environment. •Prolonged exposure of nontargeted organisms to naproxen can cause adverse effects. •Naproxen biodegradation occurs mainly through desmethylnaproxen as a key intermediate.

    更新日期:2020-01-11
  • Bionanomining: biotechnological synthesis of metal nanoparticles from mining waste—opportunity for sustainable management of mining environmental liabilities
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-10
    Liey-si Wong-Pinto, Andrew Menzies, Javier I. Ordóñez

    Abstract Mining is an important activity for many countries, especially some in development, such as Chile, where it is a pillar of its economy. However, it generates large impacts that are undesirable for the population such as the generation of polluting solid and effluents with a high content of heavy metals and metalloids, which are traditionally accumulated in deposits. In recent years, bionanomining emerged as a cutting-edge scientific-technological development associated with the application of micro- and macro-organisms to generate nanotechnological products by using mining and industrial wastes and wastewaters. Biomass of many species of bacteria, plants, algae and fungi have the ability to reduce or oxidise cations, which can physically be deposited as nanometric materials such as the nanoparticles. Nanoparticles are materials that are increasingly used, and therefore, their demand increase, based on the high surface area characteristics to improve thermal, electrical and optical properties of materials, and metallic ones have also antimicrobial activity. This review addresses the biosynthesis of metal nanoparticles, focusing on mining waste recovery strategies, which is an emerging reality in mining countries. Transformation of potentially hazardous waste into a valuable product through techniques that are eco-friendly is an opportunity to develop sustainably depressed or polluted sites.

    更新日期:2020-01-11
  • Diverse and common features of trehalases and their contributions to microbial trehalose metabolism
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-10
    Masayoshi Sakaguchi

    Trehalose is a stable disaccharide that consists of two glucose units linked primarily by an α,α-(1 → 1)-linkage, and it has been found in a wide variety of organisms. In these organisms, trehalose functions not only as a source of carbon energy but also as a protector against various stress conditions. In addition, this disaccharide is attractive for use in a wide range of applications due to its bioactivities. In trehalose metabolism, direct trehalose-hydrolyzing enzymes are known as trehalases, which have been reported for bacteria, archaea, and eukaryotes, and are classified into glycoside hydrolase 37 (GH37), GH65, and GH15 families according to the Carbohydrate-Active enZyme (CAZy) database. The catalytic domains (CDs) of these enzymes commonly share (α/α)6-barrel structures and have two amino acid residues, Asp and/or Glu, that function as catalytic residues in an inverting mechanism. In this review, I focus on diverse and common features of trehalases within different GH families and their contributions to microbial trehalose metabolism.

    更新日期:2020-01-11
  • A putative mechanism underlying secondary metabolite overproduction by Streptomyces strains with a 23S rRNA mutation conferring erythromycin resistance
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-10
    Kanata Hoshino, Yu Imai, Keiichiro Mukai, Ryoko Hamauzu, Kozo Ochi, Takeshi Hosaka

    Mutations in rrn encoding ribosomal RNA (rRNA) and rRNA modification often confer resistance to ribosome-targeting antibiotics by altering the site of their interaction with the small (30S) and large (50S) subunits of the bacterial ribosome. The highly conserved central loop of domain V of 23S rRNA (nucleotides 2042–2628 in Escherichia coli; the exact position varies by species) of the 50S subunit, which is implicated in peptidyl transferase activity, is known to be important in macrolide interactions and resistance. In this study, we identified an A2302T mutation in the rrnA-23S rRNA gene and an A2281G mutation in the rrnC-23S rRNA gene that were responsible for resistance to erythromycin in the model actinomycete Streptomyces coelicolor A3(2) and its close relative Streptomyces lividans 66, respectively. Interestingly, genetic and phenotypic characterization of the erythromycin-resistant mutants indicated a possibility that under coexistence of the 23S rRNA mutation and mutations in other genes, S. coelicolor A3(2) and S. lividans 66 can produce abundant amounts of the pigmented antibiotics actinorhodin and undecylprodigiosin depending on the combinations of mutations. Herein, we report the unique phenomenon occurring by unexpected characteristics of the 23S rRNA mutations that can affect the emergence of additional mutations probably with an upswing in spontaneous mutations and enrichment in their variations in Streptomyces strains. Further, we discuss a putative mechanism underlying secondary metabolite overproduction by Streptomyces strains with a 23S rRNA mutation conferring erythromycin resistance.

    更新日期:2020-01-11
  • Poly(lactic acid)/carvacrol-based materials: preparation, physicochemical properties, and antimicrobial activity
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-10
    Roberto Scaffaro, Andrea Maio, Antonia Nostro

    Abstract The current demand for new antimicrobial systems has stimulated research for the development of poly(lactic acid)/carvacrol (PLA/CAR)-based materials able to hinder the growth and spread of microorganisms. The eco-friendly characteristics of PLA and cytocompatibility make it very promising in the perspective of green chemistry applications as material for food and biomedical employments. The broad-spectrum biological and pharmacological properties of CAR, including antimicrobial activity, make it an interesting bioactive molecule that can be easily compounded with PLA by adopting the same techniques as those commonly used for PLA manufacturing. This review critically discusses the most common methods to incorporate CAR into a PLA matrix and their interference on the morphomechanical properties, release behavior, and antimicrobial activity of systems. The high potential of PLA/CAR materials in terms of chemical-physical and antimicrobial properties can be exploited for the future development of food packaging, coated medical devices, or drug delivery systems.

    更新日期:2020-01-11
  • Novel mutagenesis and screening technologies for food microorganisms: advances and prospects
    Appl. Microbiol. Biotechnol. (IF 3.670) Pub Date : 2020-01-09
    Qinghui Yu, Yuncheng Li, Bo Wu, Wei Hu, Mingxiong He, Guoquan Hu

    Abstract Microorganisms are indispensable in the food industry, but wild-type strains hardly meet the current industrial demands due to several undesirable traits. Therefore, microbial strain improvement offers a critical solution to enhance the food industry. Traditional techniques for food microbial improvement, such as the use of chemical mutagens and manual isolation/purification, are inefficient, time-consuming, and laborious, restricting further progress in the area of food fermentation. In this review, the applications of novel mutagenesis and screening technologies used for the improvement of food microbes were summarized, including random mutagenesis based on physical irradiation, microbial screening facilitated by a microtiter plate, fluorescence-activated cell or droplet sorting, and microscaled fermentation in a microtiter plate or microbioreactor. In comparison with conventional methods, these new tools have the potential in accelerating microbial strain improvement and their combined applications could create a new trend for strain development. However, several problems that could affect its potential application may include the following: the lack of specific mutagenesis devices and biosensing systems, the insufficient improvement of the mixed culture system, the low efficiency when using filamentous fungi and flocculating bacteria, and the insufficient safety assessment on harnessing genome-editing technology. Therefore, future works on strain improvement remain challenging for the food industry.

    更新日期:2020-01-09
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