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Applied Microbiology and Biotechnology (v.93, #3)

Beneficial effects of Lactobacillus paracasei subsp. paracasei NTU 101 and its fermented products by Shen-Shih Chiang; Tzu-Ming Pan (pp. 903-916).

It is well-known that probiotics have a number of beneficial health effects in humans and animals, including the reduction of symptoms in lactose intolerance and enhancement of the bioavailability of nutrients. Probiotics have showed to possess antimutagenic, anticarcinogenic and hypocholesterolemic properties. Further, they were also observed to have antagonistic actions against intestinal and food-borne pathogens, to decrease the prevalence of allergies in susceptible individuals and to have immunomodulatory effects. Typically, the bacteria colonise the intestinal tract first and then reinforce the host defence systems by inducing a generalised mucosal immune response, balanced T-helper cell response, self-limited inflammatory response and secretion of polymeric IgA. Scientific reports showed that the Taiwan native lactic acid bacterium from newborn infant faeces identified as Lactobacillus paracasei subsp. paracasei NTU 101 and its fermented products proved to be effective for the management of blood cholesterol and pressure, prevention of gastric mucosal lesion development, immunomodulation and alleviation of allergies, anti-osteoporosis and inhibition the fat tissue accumulation. This review article describes that the beneficial effects of this Lactobacillus strains and derivative products may be suitable for human and animals.

Keywords: Lactobacillus paracasei subsp. paracasei NTU 101; Hyperlipidemia; Hypertension; Gastric mucosal lesion; Osteoporosis; Obesity

CHO cells in biotechnology for production of recombinant proteins: current state and further potential by Jee Yon Kim; Yeon-Gu Kim; Gyun Min Lee (pp. 917-930).

Recombinant Chinese hamster ovary cells (rCHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins. Recent advances in cell culture technology for rCHO cells have achieved significant improvement in protein production leading to titer of more than 10 g/L to meet the huge demand from market needs. This achievement is associated with progression in the establishment of high and stable producer and the optimization of culture process including media development. In this review article, we focus on current strategies and achievements in cell line development, mainly in vector engineering and cell engineering, for high and stable protein production in rCHO cells. The approaches that manipulate various DNA elements for gene targeting by site-specific integration and cis-acting elements to augment and stabilize gene expression are reviewed here. The genetic modulation strategy by “direct” cell engineering with growth-promoting and/or productivity-enhancing factors and omics-based approaches involved in transcriptomics, proteomics, and metabolomics to pursue cell engineering are also presented.

Keywords: CHO cells; Cell line development; Vector engineering; Cell engineering; Omics-based approaches

Synthesis and assembly of fungal melanin by Helene C. Eisenman; Arturo Casadevall (pp. 931-940).

Melanin is a unique pigment with myriad functions that is found in all biological kingdoms. It is multifunctional, providing defense against environmental stresses such as ultraviolet (UV) light, oxidizing agents and ionizing radiation. Melanin contributes to the ability of fungi to survive in harsh environments. In addition, it plays a role in fungal pathogenesis. Melanin is an amorphous polymer that is produced by one of two synthetic pathways. Fungi may synthesize melanin from endogenous substrate via a 1,8-dihydroxynaphthalene (DHN) intermediate. Alternatively, some fungi produce melanin from l-3,4-dihydroxyphenylalanine (l-dopa). The detailed chemical structure of melanin is not known. However, microscopic studies show that it has an overall granular structure. In fungi, melanin granules are localized to the cell wall where they are likely cross-linked to polysaccharides. Recent studies suggest the fungal melanin may be synthesized in internal vesicles akin to mammalian melanosomes and transported to the cell wall. Potential applications of melanin take advantage of melanin's radioprotective properties and propensity to bind to a variety of substances.

Keywords: Fungi; Melanin; Cell wall; Vesicle; Chitin; Radioprotection

Applied modern biotechnology for cultivation of Ganoderma and development of their products by Xuan-Wei Zhou; Kai-Qi Su; Yong-Ming Zhang (pp. 941-963).

A white-rot basidiomycete Ganoderma spp. has long been used as a medicinal mushroom in Asia, and it has an array of pharmacological properties for immunomodulatory activity. There have been many reports about the bioactive components and their pharmacological properties. In order to analyze the current status of Ganoderma products, the detailed process of cultivation of Ganoderma spp. and development of their products are restated in this review article. These include the breeding, cultivating, extracting bioactive component, and processing Ganoderma products, etc. This article will expand people’s common knowledge on Ganoderma, and provide a beneficial reference for research and industrial production.

Keywords: Ganoderma spp. biotechnology; Breeding and cultivation; Development and utilizations; Ganoderma-based products; Quality control

MALDI-TOF MS in microbiological diagnostics—identification of microorganisms and beyond (mini review) by Andreas Wieser; Lukas Schneider; Jette Jung; Sören Schubert (pp. 965-974).

Few developments in microbiological diagnostics have had such a rapid impact on species level identification of microorganisms as matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Conventional differentiation methods rely on biochemical criteria and require additional pre-testing and lengthy incubation procedures. In comparison, MALDI-TOF MS can identify bacteria and yeast within minutes directly from colonies grown on culture plates. This radically new, methodically simple approach profoundly reduces the cost of consumables and time spent on diagnostics. The reliability and accuracy of the method have been demonstrated in numerous studies and different systems are already commercially available. Novel applications of the system besides microbial species level identification are also being explored. This includes identification of pathogens from positive blood cultures or directly from patient samples, such as urine. Currently, intriguing MALDI-TOF MS developments are being made regarding the phenotypic detection of certain antibiotic resistance mechanisms, e.g., β-lactamases and carbapenemases. This mini review provides an overview of the literature in the field and also includes our own data and experiences gathered from over 4 years of routine MALDI-TOF MS use in a university hospital’s microbiological diagnostics facility.

Keywords: MALDI-TOF; Microbiological diagnostic; Pathogen identification; Blood cultures; Antibiotic resistance; Strain typing

Best practices for fungal germplasm repositories and perspectives on their implementation by Aric Wiest; Robert Schnittker; Mike Plamann; Kevin McCluskey (pp. 975-982).

In over 50 years, the Fungal Genetics Stock Center has grown to become a world-recognized biological resource center. Along with this growth comes the development and implementation of myriad practices for the management and curation of a diverse collection of filamentous fungi, yeast, and molecular genetic tools for working with the fungi. These practices include techniques for the testing, manipulation, and preservation of individual fungal isolates as well as for processing of thousands of isolates in parallel. In addition to providing accurate record keeping, an electronic managements system allows the observation of trends in strain distribution and in sample characteristics. Because many ex situ fungal germplasm repositories around the world share similar objectives, best-practice guidelines have been developed by a number of organizations such as the Organization for Economic Cooperation and Development or the International Society for Biological and Environmental Repositories. These best-practice guidelines provide a framework for the successful operation of collections and promote the development and interactions of biological resource centers around the world.

Keywords: Biological resource center; Neurospora; Cryopreservation; Lyophilization; L-drying; High-throughput screening

Structure, function and selective inhibition of bacterial acetyl-coa carboxylase by S. W. Polyak; A. D. Abell; M. C. J. Wilce; L. Zhang; G. W. Booker (pp. 983-992).

Acetyl-CoA carboxylase (ACC) catalyses the first committed step in fatty acid biosynthesis: a metabolic pathway required for several important biological processes including the synthesis and maintenance of cellular membranes. ACC employs a covalently attached biotin moiety to bind a carboxyl anion and then transfer it to acetyl-CoA, yielding malonyl-CoA. These activities occur at two different subsites: the biotin carboxylase (BC) and carboxyltransferase (CT). Structural biology, together with small molecule inhibitor studies, has provided new insights into the molecular mechanisms that govern ACC catalysis, specifically the BC and CT subunits. Here, we review these recent findings and highlight key differences between the bacterial and eukaryotic isozymes with a view to establish those features that provide an opportunity for selective inhibition. Especially important are examples of highly selective small molecule inhibitors capable of differentiating between ACCs from different phyla. The implications for early stage antibiotic discovery projects, stemming from these studies, are discussed.

Keywords: Acetyl-CoA carboxylase; Enzyme; Inhibition; Antibiotics; Fatty acid biosynthesis

Culture-independent methods for studying environmental microorganisms: methods, application, and perspective by Can Su; Liping Lei; Yanqing Duan; Ke-Qin Zhang; Jinkui Yang (pp. 993-1003).

Since the application of molecular methods, culture-independent methods (CIMs) have been developed to study microbial communities from various environments. In the past 20 years, several methods based on the direct amplification and analyses of the small subunit ribosomal RNA gene have been developed to directly study environmental microorganisms. These methods include denaturing/temperature gradient gel electrophoresis, single-strand-conformation polymorphism, restriction fragment length polymorphism, terminal restriction fragment length polymorphism, and quantitative polymerase chain reaction (PCR). Similarly, non-PCR-based molecular techniques, such as microarray and fluorescence in situ hybridization have also been adopted. In recent years, several novel fields of investigation such as metagenomics, metatranscriptomics, metaproteomics, and single-cell genomics were developed, largely propelled by the innovation and application of next-generation sequencing methods. Several single-cell-based technologies such as Raman microspectroscopy and nano-scale secondary ion mass spectrometry are also increasingly used in the fields of microbial ecology and environmental microbiology. The application of these methods has revolutionized microbiology by allowing scientists to directly analyze natural microbial communities in situ, including their genes, transcripts, proteins, and metabolites and how their interactions impact their distribution patterns. In this review, we present an up-to-date review on different CIMs and their applications, our focuses are on the comparison of different CIMs and their application in the analyses of microbial diversities and communities.

Keywords: Culture-independent methods; Microbial community; Small subunit ribosomal RNA gene; Molecular analysis

The great screen anomaly—a new frontier in product discovery through functional metagenomics by David Matthias Ekkers; Mariana Silvia Cretoiu; Anna Maria Kielak; Jan Dirk van Elsas (pp. 1005-1020).

Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product.

Keywords: Metagenomics; Functional screening; Natural product discovery; Gene expression

Controlling the oxidoreduction potential of the culture of Clostridium acetobutylicum leads to an earlier initiation of solventogenesis, thus increasing solvent productivity by Shaohua Wang; Yan Zhu; Yanping Zhang; Yin Li (pp. 1021-1030).

Fermentative production of solvents (acetone, butanol, and ethanol) by Clostridium acetobutylicum is generally a biphasic process consisting of acidogenesis and solventogenesis. We report that the biphasic metabolism of C. acetobutylicum could be changed by oxidoreduction potential (ORP) regulation. When using air to control the ORP of the fermentation broth at −290 mV, an earlier initiation of solventogenesis was achieved. Solvent production reached 25.6 g l⁻¹ (2.8 g acetone l⁻¹, 16.8 g butanol l⁻¹, 6.0 g ethanol l⁻¹), a 35% increase compared with the ORP uncontrolled process. Metabolic flux analysis revealed that there was a general increase of the central carbon flux in the first 24 h of fermentation when ORP was controlled at −290 mV, compared with the control. Specifically, the solvent ratio (acetone:butanol:ethanol) was changed from 25:64:11 to 11:66:23 at ORP level of −290 mV, which might have resulted from the rigidity at acetyl-CoA node and the flexibility at acetoacetyl-CoA and butyryl-CoA nodes in response to ORP regulation.

Keywords: Clostridium acetobutylicum ; Oxidoreduction potential (redox potential); Solvent productivity; Earlier initiation of solventogenesis

Development of a measles vaccine production process in MRC-5 cells grown on Cytodex1 microcarriers and in a stirred bioreactor by Khaled Trabelsi; Samy Majoul; Samia Rourou; Héla Kallel (pp. 1031-1040).

Measles vaccination remains the most efficient way to control the spread of the virus. This work focuses on the production of a measles vaccine using stirred conditions as an advanced option for process scale up. Non-porous Cytodex 1 microcarriers were used to support MRC-5 cell growth in suspension cultures. Virus replication was first optimized in spinner flasks, and the effects of various operational parameters were investigated. Cell infection with AIK-C measles strain at an MOI (multiplicity of infection) of 0.005, without glucose regulation and in M199 medium, resulted in a virus titer of 10^6.25 TCID₅₀ (median tissue culture infective dose)/ml. To optimize the production process in a 7-l bioreactor, we carried out various perfused cultures using minimum essential medium (MEM) + 5% FCS diluted with phosphate-buffered saline (PBS). We achieved a high cell density level (4.1 × 10⁶ cells/ml) with an efficient use of the medium when MEM + 5% FCS diluted with PBS at 25% was used during the cell amplification step. Optimization of measles production in MRC-5 cells grown on Cytodex 1 beads in a 7-l bioreactor showed that perfusion was the most efficient when compared to repeated-batch culture. Perfusion at a rate of 0.25 V (reactor volume)/day showed the highest specific productivity (1.6 IVP [infectious virus particle] cell⁻¹ day⁻¹). Testing of several stabilizers containing pharmaceutically improved components such as sugars, amino acids, and charged ions showed that the formulation composed of sucrose and MgCl₂, led to the maintenance of the infectivity of the AIK-C measles virus strain to a significant level, when stored at +28 °C, +4 °C and −60 °C.

Keywords: MRC-5 cells; Cytodex 1 microcarriers; Stirred bioreactor; AIK-C measles strain; Measles vaccine

Utilizing pigment-producing fungi to add commercial value to American beech (Fagus grandifolia) by Sara C. Robinson; Daniela Tudor; Paul A. Cooper (pp. 1041-1048).

American beech (Fagus grandifolia) is an abundant, underutilized tree in certain areas of North America, and methods to increase its market value are of considerable interest. This research utilized pigment-producing fungi to induce color in American beech to potentially establish its use as a decorative wood. Wood samples were inoculated with Trametes versicolor, Xylaria polymorpha, Inonotus hispidus, and Arthrographis cuboidea to induce fungal pigmentation. Black pigmentation (T. versicolor, X. polymorpha, I. hispidus) was sporadic, occurred primarily on the surfaces of the heartwood, but not internally. Pink pigmentation (A. cuboidea) occurred throughout all of the tested beech samples, but was difficult to see in the heartwood due to the darker color of the wood. To increase the visibility of the pink stain, beech blocks were pretreated with T. versicolor for 4 weeks before being inoculated with A. cuboidea. This method significantly increased the saturation of the pink stain on both beech heartwood and sapwood, creating coloration similar to that found on sugar maple. This value-adding process should be particularly effective for small-scale wood pigmentation, and should help establish a market for this currently underutilized wood species.

Keywords: Arthrographis cuboidea ; Fagus grandifolia ; Inonotus hispidus ; Spalting; Trametes ; versicolor ; Xylaria polymorpha

An improved screening method for microorganisms able to convert crude glycerol to 1,3-propanediol and to tolerate high product concentrations by Anne Katrin Ringel; Erik Wilkens; Diana Hortig; Thomas Willke; Klaus-Dieter Vorlop (pp. 1049-1056).

A new screening method was developed and established to find high-performance bacteria for the conversion of crude glycerol to 1,3-propanediol. Three soil samples from palm oil-rich habitats were investigated using crude glycerol of a German biodiesel plant. Nine promising 1,3-propanediol producers could be found. Because of a special pH buffer system, a fast evaluation on microscale and high 1,3-propanediol concentrations up to 40 g L⁻¹ could be achieved. Three strains demonstrated very high product tolerance and were identified as Clostridium butyricum. Two strains, AKR91b and AKR102a, grew and produced 1,3-propanediol in the presence of 60 g L⁻¹ initial 1,3-propanediol, the strain AKR92a even in the presence of 77 g L⁻¹ 1,3-propanediol. The strains AKR91b and AKR102a tolerated up to 150 g L⁻¹ crude glycerol and produced 80% of the 1,3-propanediol attained from pure glycerol of the same concentration. Further criteria for the choice of a production strain were the pathogenicity (risk class), ability to grow on low-cost media, e.g., with less yeast extract, and robustness, e.g., process stability after several bioconversions. Overall, the strain C. butyricum AKR102a was chosen for further process optimization and scale-up due to its high productivity and high final concentration in a pH-regulated bioreactor.

Keywords: Screening; 1,3-Propanediol; Crude glycerol; Microscale; Product tolerance

High-level production of 1,3-propanediol from crude glycerol by Clostridium butyricum AKR102a by Erik Wilkens; Anne Katrin Ringel; Diana Hortig; Thomas Willke; Klaus-Dieter Vorlop (pp. 1057-1063).

The aim of this study was to optimize a biotechnological process for the production of 1,3-propanediol (1,3-PD) based on low-quality crude glycerol derived from biodiesel production. Clostridium butyricum AKR102a was used in fed-batch fermentations in 1-L and 200-L scale. The newly discovered strain is characterized by rapid growth, high product tolerance, and the ability to use crude glycerol at the lowest purity directly gained from a biodiesel plant side stream. Using pure glycerol, the strain AKR102 reached 93.7 g/L 1,3-PD with an overall productivity of 3.3 g/(L*h). With crude glycerol under the same conditions, 76.2 g/L 1,3-PD was produced with a productivity of 2.3 g/(L*h). These are among the best results published so far for natural producers. The scale up to 200 L was possible. Due to the simpler process design, only 61.5 g/L 1,3-PD could be reached with a productivity of 2.1 g/(L*h).

Keywords: Fermentation; Crude glycerol; 1,3-Propanediol; Clostridium butyricum

Coexpression of chaperonin GroEL/GroES markedly enhanced soluble and functional expression of recombinant human interferon-gamma in Escherichia coli by Xiao Yan; Sheng Hu; Yi-Xin Guan; Shan-Jing Yao (pp. 1065-1074).

Recombinant human interferon-gamma (rhIFN-γ) is a protein of great potential for clinical therapy due to its multiple biological activities. However, overexpressing rhIFN-γ in Escherichia coli was found to accumulate as cytoplasmic inclusion bodies. In this work, a system for soluble and active expression of rhIFN-γ was constructed by coexpressing chaperonin GroEL/GroES in E. coli. The rhIFN-γ gene was fused to a pET-28a expression vector, and rhIFN-γ was partially expressed as the soluble form following coexpression with a second vector producing chaperonin GroEL/GroES. The fermentation of recombinant E. coli harboring rhIFN-γ and GroEL/GroES plasmids was investigated, and the optimized conditions were as follows: culture temperature of 25°C, incubation time of 8 h, isopropyl-β-d-thio-galactoside concentration of 0.2 mM, and l-arabinose concentration of 0.5 g/L. As a result, the expression level of rhIFN-γ was improved accordingly by 2.2-fold than the control, while a significantly positive correlation was also found between the ratio of supernatant to precipitate of rhIFN-γ and the amount of chaperonin. Circular dichroism spectra, fluorescence spectra, size exclusion chromatography, and chemical cross-linking method were applied to characterize rhIFN-γ, indicating that the three-dimensional structure of rhIFN-γ was identical to that of the native rhIFN-γ. The enzyme-linked immunosorbent assay for active rhIFN-γ quantification showed that coexpression yielded 72.91 mg rhIFN-γ per liter fermentation broth. Finally, protein–protein interactions between rhIFN-γ and chaperonin were analyzed using the yeast two-hybrid system, which provided the direct evidence that chaperonin GroEL/GroES interacted with rhIFN-γ to increase the soluble expression and presented the potential in producing efficiently recombinant proteins.

Keywords: Recombinant human interferon-gamma; GroEL/GroES coexpression system; Soluble expression; Protein folding; Protein–protein interactions

Efficient synthesis of optically pure alcohols by asymmetric hydrogen-transfer biocatalysis: application of engineered enzymes in a 2-propanol–water medium by Nobuya Itoh; Kentaro Isotani; Masatoshi Nakamura; Kousuke Inoue; Yasuhiro Isogai; Yoshihide Makino (pp. 1075-1085).

We describe an efficient method for producing both enantiomers of chiral alcohols by asymmetric hydrogen-transfer bioreduction of ketones in a 2-propanol (IPA)–water medium with E. coli biocatalysts expressing phenylacetaldehyde reductase (PAR: wild-type and mutant enzymes) from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749 (LSADH). We also describe the detailed properties of mutant PARs, Sar268, and HAR1, which were engineered to have high activity and productivity in media composed of polar organic solvent and water, and the construction of three-dimensional structure of PAR by homology modeling. The K _m and V _max values for some substrates and the substrate specificity of mutant PARs were quite different from those of wild-type PAR. The results well explained the increased productivity of engineered PARs in IPA–water medium.

Keywords: Phenylacetaldehyde reductase (PAR); Rhodococcus sp.; Alcohol dehydrogenase (LSADH); Leifsonia sp.; Optically pure alcohols; Hydrogen-transfer biocatalysis; Engineered enzymes; Polar organic solvent–water medium; 2-Propanol

Biochemical characterization and gene expression of two endo-arabinanases from Penicillium chrysogenum 31B by Tatsuji Sakamoto; Misako Inui; Kana Yasui; Sayaka Tokuda; Mika Akiyoshi; Yohei Kobori; Tetsuko Nakaniwa; Toshiji Tada (pp. 1087-1096).

We previously described five arabinanolytic enzymes secreted by Penicillium chrysogenum 31B into the culture medium. Here, we describe a sixth such enzyme, termed AbnS1. Analysis of the reaction products of debranched arabinan revealed that AbnS1 cleaved the substrate in an endo manner. The optimum temperature of AbnS1 was 60°C, which was much higher than that of a cold-adapted endo-arabinanase (Abnc) produced by this strain. The abns1 cDNA gene encoding AbnS1 was isolated by in vitro cloning. The deduced amino acid sequence of AbnS1 had 70% identity with that of Abnc. Pfam analysis revealed a Glyco_hydro_43 domain at positions 28 to 318 of AbnS1. Semi-quantitative reverse transcription-polymerase chain reaction analysis indicated that the abns1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was only slightly induced with arabinose and arabinan. In contrast, expression of the abnc gene encoding Abnc was strongly induced by arabinose, arabinitol, and arabinan. Using debranched arabinan as substrate, recombinant AbnS1 (rAbnS1) accumulated arabinobiose and arabinotriose as the major products. Recombinant Abnc (rAbnc) released mainly arabinotriose and lesser amounts of arabinose and arabinobiose than did rAbnS1. Branched arabinan was completely degraded to arabinose by the action of rAbnS1 or rAbnc in combination with α-l-arabinofuranosidase.

Keywords: Penicillium chrysogenum ; Endo-arabinanase; Mesophilic enzyme; Nucleotide sequence; Expression

A novel anti-lymphoma protein RE26 from Rozites emodensis (Berk.) Moser by Meng Gong; Minjie Xia; Jing Zhang; Jiao Gu; Xuyang Liu; Jingqiu Cheng (pp. 1097-1108).

A novel antitumor protein, designated RE26, with anti-lymphoma activity was purified from a Tris–HCl buffer extract of Rozites emodensis (Berk.) Moser by three successive steps of ion exchange chromatography. SDS-PAGE and gel filtration chromatography revealed that RE26 is a monomeric protein of 26 kDa, and isoelectrofocusing assay indicated its isoelectric point of 4.3–4.4. RE26 has high stability over a wide pH range (pH 3–11) but is sensitive to temperature and only stable under 40 °C. Partial amino acid sequences of two RE26 peptide fragments were determined by Edman degradation as GLEEEETLLLLFFPP and GTEQE. The half-maximal inhibitory concentration (IC₅₀) of RE26 against tested lymphoma cell lines was around 4 μg/ml. In vitro experiments showed that RE26 could specifically bind to lymphoma cells; activate the caspases, including caspases 3, 8, and 9 in host cells; and induce apoptosis. Experiments in nude mice indicated local RE26 injection adjacent to tumor site could inhibit lymphoma formation.

Keywords: Rozites emodensis (Berk.) Moser; Anti-lymphoma protein; Purification; Character analysis; Apoptosis; Nude mice model

One-step purification and immobilization of thermophilic polyphosphate glucokinase from Thermobifida fusca YX: glucose-6-phosphate generation without ATP by Hehuan Liao; Suwan Myung; Y.-H. Percival Zhang (pp. 1109-1117).

The discovery of stable and active polyphosphate glucokinase (PPGK, EC 2.7.1.63) would be vital to cascade enzyme biocatalysis that does not require a costly ATP input. An open reading frame Tfu_1811 from Thermobifida fusca YX encoding a putative PPGK was cloned and the recombinant protein fused with a family 3 cellulose-binding module (CBM-PPGK) was overexpressed in Escherichia coli. Mg²⁺ was an indispensible activator. This enzyme exhibited the highest activity in the presence of 4 mM Mg²⁺ at 55°C and pH 9.0. Under its suboptimal conditions (pH 7.5), the k _cat and K _m values of CBM-PPGK on glucose were 96.9 and 39.7 s⁻¹ as well as 0.77 and 0.45 mM at 37°C and 50°C respectively. The thermoinactivation of CBM-PPGK was independent of its mass concentration. Through one-step enzyme purification and immobilization on a high-capacity regenerated amorphous cellulose, immobilized CBM-PPGK had an approximately eightfold half lifetime enhancement (i.e., t _1/2 = 120 min) as compared to free enzyme at 50°C. To our limited knowledge, this enzyme was the first thermostable PPGK reported. Free PPGK and immobilized CBM-PPGK had total turnover number values of 126,000 and 961,000 mol product per mol enzyme, respectively, suggesting their great potential in glucose-6-phosphate generation based on low-cost polyphosphate.

Keywords: Enzymatic building block; Cascade enzyme reaction; One-step purification and immobilization; Polyphosphate glucokinase; Synthetic pathway biotransformation (SyPaB); Thermobifida fusca

Pseudomonas putida esterase contains a GGG(A)X-motif confering activity for the kinetic resolution of tertiary alcohols by Jessica Rehdorf; Geoffrey A. Behrens; Giang-Son Nguyen; Robert Kourist; Uwe T. Bornscheuer (pp. 1119-1126).

An esterase from Pseudomonas putida JD1 (PPE) was successfully cloned, actively expressed in Escherichia coli, and characterized. It was discovered that PPE is more active towards short-chain esters, hydrolyzed δ-valerolactone, and ε-caprolactone and was most active at 37°C and pH 8. After purification to homogeneity by Ni–NTA-assisted affinity chromatography, the kinetic parameters K _M and k _cat were determined for p-nitrophenyl acetate and butyrate, respectively, showing better catalytic efficiency for hydrolysis of the acetate residue. Investigation of the protein sequence revealed not only the classical catalytic triad for carboxylesterases, additionally the interesting GGG(A)X-motif, which is associated to activity towards tertiary alcohols, was found. Indeed, enzymatic activity was shown for a set of different tertiary alcohols with enantioselectivities up to E = 20, suggesting PPE to be a promising biocatalyst. In addition, PPE also hydrolyzed 4-hydroxyphenyl acetate, the product of a Baeyer–Villiger monooxygenase-catalyzed oxidation of 4-hydroxyacetophenone with a specific activity of 34.36 U/mg suggesting a physiological role in P. putida JD1.

Keywords: Carboxylesterases; Tertiary alcohols; Biocatalysis; GGG(A)X-motif; Kinetic resolution

α-l-Rhamnosidase of Aspergillus terreus immobilized on ferromagnetic supports by Fernando Soria; Guillermo Ellenrieder; Givanildo Bezerra Oliveira; Mariana Cabrera; Luiz Bezerra Carvalho Jr (pp. 1127-1134).

α-l-Rhamnosidase from Aspergillus terreus was covalently immobilized on the following ferromagnetic supports: polyethylene terephthalate (Dacron-hydrazide), polysiloxane/polyvinyl alcohol (POS/PVA), and chitosan. The powdered supports were magnetized by thermal coprecipitation method using ferric and ferrous chlorides, and the immobilization was carried out via glutaraldehyde. The activity of the Dacron-hydrazide (0.53 nkat/μg of protein) and POS/PVA (0.59 nkat/μg of protein) immobilized enzyme was significantly higher than that found for the chitosan derivative (0.06 nkat/μg of protein). The activity–pH and activity–temperature profiles for all immobilized enzymes did not show difference compared to the free enzyme, except the chitosan derivative that presented higher maximum temperature at 65 °C. The Dacron-hydrazide derivative thermal stability showed a similar behavior of the free enzyme in the temperature range of 40–70 °C. The POS/PVA and chitosan derivatives were stable up to 60 °C, but were completely inactivated at 70 °C. The activity of the preparations did not appreciably decrease after ten successive reuses. Apparent K _m of α-l-rhamnosidase immobilized on magnetized Dacron-hydrazide (1.05 ± 0.22 mM), POS/PVA (0.57 ± 0.09 mM), and chitosan (1.78 ± 0.24 mM) were higher than that estimated for the soluble enzyme (0.30 ± 0.03 mM). The Dacron-hydrazide enzyme derivative showed better performance than the free enzyme to hydrolyze 0.3% narigin (91% and 73% after 1 h, respectively) and synthesize rhamnosides (0.116 and 0.014 mg narirutin after 1 h, respectively).

Keywords: α-l-Rhamnosidase; Aspergillus terreus ; Dacron; POS/PVA; Chitosan; Immobilization

A new Bacillus megaterium whole-cell catalyst for the hydroxylation of the pentacyclic triterpene 11-keto-β-boswellic acid (KBA) based on a recombinant cytochrome P450 system by Sabrina Bleif; Frank Hannemann; Josef Zapp; David Hartmann; Johann Jauch; Rita Bernhardt (pp. 1135-1146).

The use of cytochromes P450 for the regio- and stereoselective hydroxylation of non-activated carbon atoms in biotechnological applications reflects an efficient and cost-effective alternative in comparison to classical organic chemistry. The prokaryotic cytochrome P450 CYP106A2 from Bacillus megaterium ATCC 13368 hydroxylates a variety of 3-oxo-Δ⁴ steroids and recently it was identified to carry out a one-step regioselective allylic hydroxylation of the diterpene abietic acid. The anti-inflammatory pentacyclic triterpene 11-Keto-β-boswellic acid (KBA) was found to be a further substrate of CYP106A2, being the first report of a pentacyclic triterpene conversion by a prokaryotic P450. The reaction products were analyzed by HPLC and the corresponding kinetic parameters were investigated. Structure determination of the main product by NMR revealed a 15α-hydroxylation of this substrate. In order to overcome the inability of a recombinant P450 whole-cell system in E. coli for the uptake of acids with terpene structure, we developed for the first time an expression system for cytochromes P450 in B. megaterium (strains MS941 and ATCC 13368). Interestingly, CYP106A2 was only successfully expressed in the plasmid-less B. megaterium strain MS941 but not in ATCC13368. This recombinant system, with the co-expressed heterologous redox chain of the P450, bovine adrenodoxin reductase (AdR), and bovine adrenodoxin (Adx), was applied for the whole-cell conversion of KBA. The formation of 15α-hydroxy-KBA was increased 15-fold in comparison with the naturally CYP106A2-expressing B. megaterium strain ATCC 13368.

Keywords: CYP106A2; Cytochromes P450; 11-Keto-β-boswellic acid; Whole-cell conversion; Bacillus megaterium

Engineered biosynthesis of glycosylated derivatives of narbomycin and evaluation of their antibacterial activities by Ah Reum Han; Pramod B. Shinde; Je Won Park; Jaeyong Cho; So Ra Lee; Yeon Hee Ban; Young Ji Yoo; Eun Ji Kim; Eunji Kim; Sung Ryeol Park; Byung-Gee Kim; Dong Gun Lee; Yeo Joon Yoon (pp. 1147-1156).

A 14-membered macrolide antibiotic narbomycin produced from Streptomyces venezuelae ATCC 15439 is composed of polyketide macrolactone ring and D-desosamine as a deoxysugar moiety, which acts as an important determinant of its antibacterial activity. In order to generate diverse glycosylated derivatives of narbomycin, expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into S. venezuelae YJ003 mutant strain bearing a deletion of thymidine-5′-diphospho-D-desosamine biosynthetic gene cluster. The resulting recombinants of S. venezuelae produced a range of new analogs of narbomycin, which possess unnatural sugar moieties instead of native deoxysugar D-desosamine. The structures of narbomycin derivatives were determined through nuclear magnetic resonance spectroscopy and mass spectrometry analyses and their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with L-rhamnose or 3-O-demethyl-D-chalcose was demonstrated to exhibit greater antibacterial activity than narbomycin and the clinically relevant erythromycin. This work provides new insight into the functions of deoxysugar biosynthetic enzymes and structure–activity relationships of the sugar moieties attached to the macrolides and demonstrate the potential of combinatorial biosynthesis for the generation of new macrolides carrying diverse sugars with increased antibacterial activities.

Keywords: Streptomyces venezuelae ; Macrolide; Narbomycin; Glycosylation; Antibacterial activity

Heterologous expression and biochemical characterization of novel pyranose 2-oxidases from the ascomycetes Aspergillus nidulans and Aspergillus oryzae by Ines Pisanelli; Petra Wührer; Yazmid Reyes-Dominguez; Oliver Spadiut; Dietmar Haltrich; Clemens Peterbauer (pp. 1157-1166).

A gene encoding a pyranose 2-oxidase (POx; pyranose/oxygen 2-oxidoreductase; glucose 2-oxidase; EC 1.1.3.10) was identified in the genome of the ascomycete Aspergillus nidulans. Attempts to isolate POx directly from A. nidulans cultures or to homologously overexpress the native POx (under control of the constitutive gpdA promoter) in A. nidulans were unsuccessful. cDNA encoding POx was synthesized from mRNA and expressed in Escherichia coli, and the enzyme was subsequently purified and characterized. A putative pyranose 2-oxidase-encoding gene was also identified in the genome of Aspergillus oryzae. The coding sequence was synthetically produced and was also expressed in E. coli. Both purified enzymes were shown to be flavoproteins consisting of subunits of 65 kDa. The A. nidulans enzyme was biochemically similar to POx reported in literature. From all substrates, the highest catalytic efficiency was found with D-glucose. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones and 2,6-dichloroindophenol. As judged by the catalytic efficiencies (k _cat/k _m), some of these quinone electron acceptors are better substrates for pyranose oxidase than oxygen. The enzyme from A. oryzae was physically similar but showed lower kinetic constants compared to the enzyme from A. nidulans. Distinct differences in the stability of the two enzymes may be attributed to a deletion and an insertion in the sequence, respectively.

Keywords: Pyranose oxidase; Aspergillus; Carbohydrate oxidoreductases; Hydrogen peroxide; Electron acceptors

Production of cyanophycin in Rhizopus oryzae through the expression of a cyanophycin synthetase encoding gene by Bas J. Meussen; Ruud A. Weusthuis; Johan P. M. Sanders; Leo H. de Graaff (pp. 1167-1174).

Cyanophycin or cyanophycin granule peptide is a protein that results from non-ribosomal protein synthesis in microorganisms such as cyanobacteria. The amino acids in cyanophycin can be used as a feedstock in the production of a wide range of chemicals such as acrylonitrile, polyacrylic acid, 1,4-butanediamine, and urea. In this study, an auxotrophic mutant (Rhizopus oryzae M16) of the filamentous fungus R. oryzae 99-880 was selected to express cyanophycin synthetase encoding genes. These genes originated from Synechocystis sp. strain PCC6803, Anabaena sp. strain PCC7120, and a codon optimized version of latter gene. The genes were under control of the pyruvate decarboxylase promoter and terminator elements of R. oryzae. Transformants were generated by the biolistic transformation method. In only two transformants both expressing the cyanophycin synthetase encoding gene from Synechocystis sp. strain PCC6803 was a specific enzyme activity detected of 1.5 mU/mg protein. In one of these transformants was both water-soluble and insoluble cyanophycin detected. The water-soluble fraction formed the major fraction and accounted for 0.5% of the dry weight. The water-insoluble CGP was produced in trace amounts. The amino acid composition of the water-soluble form was determined and constitutes of equimolar amounts of arginine and aspartic acid.

Keywords: Cyanophycin; Rhizopus oryzae ; Transformation; Heterologous protein expression

Adaptive evolution of Saccharomyces cerevisiae to generate strains with enhanced glycerol production by D. R. Kutyna; C. Varela; G. A. Stanley; A. R. Borneman; P. A. Henschke; P. J. Chambers (pp. 1175-1184).

The development of new wine yeast strains with improved characteristics is critical in the highly competitive wine market, which faces the demand of ever-changing consumer preferences. Although new strains can be constructed using recombinant DNA technologies, consumer concerns about genetically modified (GM) organisms strongly limit their use in food and beverage production. We have applied a non-GM approach, adaptive evolution with sulfite at alkaline pH as a selective agent, to create a stable yeast strain with enhanced glycerol production; a desirable characteristic for wine palate. A mutant isolated using this approach produced 41% more glycerol than the parental strain it was derived from, and had enhanced sulfite tolerance. Backcrossing to produce heterozygous diploids revealed that the high-glycerol phenotype is recessive, while tolerance to sulfite was partially dominant, and these traits, at least in part, segregated from each other. This work demonstrates the potential of adaptive evolution for development of novel non-GM yeast strains, and highlights the complexity of adaptive responses to sulfite selection.

Keywords: Adaptive evolution; Fermentation; Wine yeast; Glycerol production

The actinobacterium Microbacterium sp. 16SH accepts pBBR1-based pPROBE vectors, forms biofilms, invades roots, and fixes N₂ associated with micropropagated sugarcane plants by Li Lin; Wei Guo; Yongxiu Xing; Xincheng Zhang; Zhengyi Li; Chunjin Hu; Song Li; Yangrui Li; Qianli An (pp. 1185-1195).

Members of the genus Microbacterium lineage of Gram-positive actinobacteria are increasingly being reported to display significant traits associated with environmental biotechnology and bioengineering. 16SH is a nitrogen-fixing bacterial strain isolated from a surface-sterilized stem of sugarcane grown in Guangxi, China. Analysis of 16S rRNA gene sequences revealed that 16SH belonged to the genus Microbacterium. pPROBE-pTet^r plasmids were constructed by cloning the promoter region of the Tet r gene into the promoterless pPROBE-AT, -OT, and -TT vectors derived from the pBBR1 plasmid that has a broad host range of Gram-negative bacteria and sequence similarities to plasmids from Gram-positive bacteria. The pPROBE-pTet^r plasmids expressed the gfp reporter gene and were stably maintained in 16SH cells without antibiotic selection in free-living state and in planta. Confocal microscopy on intact roots of micropropagated sugarcane plantlets showed that gfp-tagged 16SH cells formed biofilms on root maturation and elongation zones but not on root meristem zones and root caps, and colonized in intercellular spaces of root cortices. Inoculation of 16SH significantly increased biomass and nitrogen content of micropropagated sugarcane seedlings grown with a nitrogen fertilization of 6.3 mg N/kg soil. ¹⁵ N isotope dilution assays demonstrated that biological nitrogen fixation contributed to this plant growth promotion. This study for the first time demonstrated that the pBBR1-based pPROBE plasmids provided an efficient genetic transfer system for a Gram-positive Microbacterium strain, and that a nitrogen-fixing Microbacterium endophyte colonized in intact host plants and fixed N₂ associated with the host plants.

Keywords: Microbacterium; pBBR1 plasmid; Biofilm; Endophyte; Nitrogen fixation; Sugarcane

Identification and immunological characteristics of chaperonin GroEL in Riemerella anatipestifer by Xiangan Han; Qinghai Hu; Siyu Ding; Wenjing Chen; Chan Ding; Liang He; Xiaolan Wang; Jiabo Ding; Shengqing Yu (pp. 1197-1205).

Riemerella anatipestifer (RA) infections cause major economic losses in the duck industry. In this study, an immunogenic protein, chaperonin GroEL (GroEL), was identified from the outer membrane of RA strain WJ4 by immunoproteomic assay based on matrix-assisted laser desorption/ionization time of flight mass spectrometry. The complete sequence of the encoding gene, chaperonin groEL (groEL) was amplified and determined to be 1,629 base pairs in length. groEL was then cloned into expression vector pGEX-6P-1, and the expression of the recombinant GroEL (rGroEL) in Escherichia coli strain BL21 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting analysis. Immunization assay showed that ducklings or rabbits immunized with purified rGroEL generated 53- or 160-fold more anti-GroEL antibodies than those with no immunization. Importantly, bactericidal assay showed that rabbit anti-GroEL serum killed 30.0–57.3% of bacteria representing different serotypes, while rabbit anti-bacterin serum killing activity exhibits large serotype-dependent variations between 0.2% and 63.6%. Animal challenge experiment showed that ducklings immunized with rGroEL were 50%, 37.5%, and 37.5% protected from the challenge with RA strains WJ4 (serotype 1), Th4 (serotype 2), and YXb-2 (serotype 10), respectively. In addition, groEL from 34 additional RA strains was amplified by polymerase chain reaction (PCR), and products from nine were sequenced. groEL is highly conserved among RA strains, as the DNA sequence identity was over 97.5% between WJ4 and the nine additional strains. Our results suggest that GroEL may be a good candidate for new RA vaccine development.

Keywords: Immunological characteristics; Chaperonin GroEL; Riemerella anatipestifer

The effect of scale on gene expression: commercial versus laboratory wine fermentations by Debra Rossouw; Neil Jolly; Dan Jacobson; Florian F. Bauer (pp. 1207-1219).

Molecular and cellular processes that are responsible for industrially relevant phenotypes of fermenting microorganisms are a central focus of biotechnological research. Such research intends to generate insights and solutions for fermentation-based industries with regards to issues such as improving product yield or the quality of the final fermentation product. For logistical reasons, and to ensure data reproducibility, such research is mostly carried out in defined or synthetic media and in small-scale fermentation vessels. Two questions are frequently raised regarding the applicability of this approach to solve problems experienced in industrial fermentations: (1) Is synthetic medium a sufficiently accurate approximation of the generally more complex natural (and frequently highly variable) substrates that are employed in most fermentation-based industries, and (2) can results obtained in small-scale laboratory fermentations be extrapolated to large-scale industrial environments? Here, we address the second question through a comparative transcriptomic approach by assessing the response of an industrial wine yeast strain fermenting a natural grape juice in small-scale laboratory and large-scale industrial conditions. In yeast, transcriptome analysis is arguably the best available tool to holistically assess the physiological state of a population and its response to changing environmental conditions. The data suggest that scale does indeed impact on some environmental parameters such as oxygen availability. However, the data show that small-scale fermentations nevertheless accurately reflect general molecular processes and adaptations during large-scale fermentation and that extrapolation of laboratory datasets to real industrial processes can be justified.

Keywords: Transcriptomics; Scale; Commercial wine fermentation

Flocculation in ale brewing strains of Saccharomyces cerevisiae: re-evaluation of the role of cell surface charge and hydrophobicity by Ann Van Holle; Manuela D. Machado; Eduardo V. Soares (pp. 1221-1229).

Flocculation is an eco-friendly process of cell separation, which has been traditionally exploited by the brewing industry. Cell surface charge (CSC), cell surface hydrophobicity (CSH) and the presence of active flocculins, during the growth of two (NCYC 1195 and NCYC 1214) ale brewing flocculent strains, belonging to the NewFlo phenotype, were examined. Ale strains, in exponential phase of growth, were not flocculent and did not present active flocculent lectins on the cell surface; in contrast, the same strains, in stationary phase of growth, were highly flocculent (>98%) and presented a hydrophobicity of approximately three to seven times higher than in exponential phase. No relationship between growth phase, flocculation and CSC was observed. For comparative purposes, a constitutively flocculent strain (S646-1B) and its isogenic non-flocculent strain (S646-8D) were also used. The treatment of ale brewing and S646-1B strains with pronase E originated a loss of flocculation and a strong reduction of CSH; S646-1B pronase E-treated cells displayed a similar CSH as the non-treated S646-8D cells. The treatment of the S646-8D strain with protease did not reduce CSH. In conclusion, the increase of CSH observed at the onset of flocculation of ale strains is a consequence of the presence of flocculins on the yeast cell surface and not the cause of yeast flocculation. CSH and CSC play a minor role in the auto-aggregation of the ale strains since the degree of flocculation is defined, primarily, by the presence of active flocculins on the yeast cell wall.

Keywords: Brewing yeast; Cell–cell adhesion; Cell surface charge; Flocculation; Flo proteins; Hydrophobicity

Bioactive metabolites from Phoma species, an endophytic fungus from the Chinese medicinal plant Arisaema erubescens by Li-Wei Wang; Bai-Ge Xu; Jia-Ying Wang; Zhen-Zhu Su; Fu-Cheng Lin; Chu-Long Zhang; Christian P. Kubicek (pp. 1231-1239).

Through bioassay-guided fractionation, the EtOAc extract of a culture broth of the endophytic fungus Phoma species ZJWCF006 in Arisaema erubescens afforded a new α-tetralone derivative, (3S)-3,6,7-trihydroxy-α-tetralone (1), together with cercosporamide (2), β-sitosterol (3), and trichodermin (4). The structures of compounds were established on the basis of spectroscopic analyses. Compounds 1, 2, and 3 were obtained from Phoma species for the first time. Additionally, the compounds were subjected to bioactivity assays, including antimicrobial activity, against four plant pathogenic fungi (Fusarium oxysporium, Rhizoctonia solani, Colletotrichum gloeosporioides, and Magnaporthe oryzae) and two plant pathogenic bacteria (Xanthomonas campestris and Xanthomonas oryzae), as well as in vitro antitumor activities against HT-29, SMMC-772, MCF-7, HL-60, MGC80-3, and P388 cell lines. Compound 1 showed growth inhibition against F. oxysporium and R. solani with EC₅₀ values of 413.22 and 48.5 μg/mL, respectively. Additionally, compound 1 showed no cytotoxicity, whereas compound 2 exhibited cytotoxic activity against the six tumor cell lines tested, with IC₅₀ values of 9.3 ± 2.8, 27.87 ± 1.78, 48.79 ± 2.56, 37.57 ± 1.65, 27.83 ± 0.48, and 30.37 ± 0.28 μM, respectively. We conclude that endophytic Phoma are promising sources of natural bioactive and novel metabolites.

Keywords: Endophytic fungi; Phoma ; (3S)-3,6,7-trihydroxy-α-tetralone; Cercosporamide; Antifungal; Antitumor

ATP-dependent fructose uptake system in Deinococcus radiodurans by Hui-Yu Lee; Minoti Magotra; Tit-Yee Wong; Chiranjib Chakraborty; Jong-Kang Liu (pp. 1241-1248).

The bacterial phosphoenolpyruvate (PEP)-dependent group translocation system (PTS) requires the presence of both membrane-bound and cytoplasmic components to phosphorylate and translocate sugar. Deinococcus radiodurans has a functional fruA gene coding for the membrane-bound components of the fructose-specific PTS. However, fruB gene coding for the fructose-specific cytosolic components of PTS is a pseudogene. Yet, this bacterium metabolized fructose readily. In vitro studies showed that both cell membranes and cytoplasmic fractions of the cells were needed for fructose phosphorylation. Further studies showed that fructose phosphorylation required ATP, not PEP, as the phosphate donor. Unlike most PEP-dependent PTS systems, fructose phosphorylation is sensitive to sodium fluoride, a kinase inhibitor. Fructose phosphorylation was also inhibited in the presence of antiserum against a kinase phosphorylation site. Rhodobacter capsulatus has a functional fruA–fruB system. Complementation assays by reconstituting the membrane fraction of D. radiodurans to the cytoplasmic fraction of R. capsulatus resulted in a PEP-dependent fructose phosphorylation, whereas mixing the membranes of R. capsulatus and the deinococcal cytosol in vitro resulted in an ATP-dependent fructose phosphorylation.

Keywords: fruAB; Deinococcus radiodurans ; Sugar uptake; PTS

Bacillus thuringiensis Cry3Aa fused to a cellulase-binding peptide shows increased toxicity against the longhorned beetle by Chang-Hua Guo; Shu-Tang Zhao; Yuan Ma; Jian-Jun Hu; Xiao-Jiao Han; Jun Chen; Meng-Zhu Lu (pp. 1249-1256).

Cry3 class toxins are used extensively for biological control of coleopteran larvae. We previously identified a peptide (PCx) from a phage display library that specifically binds Cx-cellulase from the midgut of Anoplophora glabripennis Motschulsky (Asian longhorn beetle) larvae. Here, we added a DNA fragment that encodes the peptide onto either end of the cry3Aa gene and tested the expressed PCx-Cry3Aa and Cry3Aa-PCx proteins for insecticidal activity in the longhorned beetle. An insect bioassay revealed that, compared with native Cry3Aa, the two modified Cry3Aa proteins had significantly higher lethality, with PCx-Cry3Aa exhibiting a mortality rate almost three times that of Cry3Aa. We also proposed that the increased lethality in larvae fed with PCx-Cry3Aa or Cry3Aa-PCx would be attributable to the binding of the toxin with Cx-cellulase, thereby increasing toxin retention in the midgut. The significantly enhanced insecticidal activity of Cry3Aa fused with the Cx-cellulase binding peptide provides a new strategy for increasing toxin efficacy against the longhorned beetle. These uniquely modified Cry3Aa proteins have potential use for pest control.

Keywords: Binding peptide; Cry3Aa; Cx-cellulase; Enhancement; Longhorned beetle

A novel high-throughput B1H-ChIP method for efficiently validating and screening specific regulator–target promoter interactions by Jumei Zeng; Yuqing Li; Shuguang Zhang; Zheng-Guo He (pp. 1257-1269).

Confident protein–DNA interaction (PDI) data could significantly improve our understanding of transcriptional regulation network in both prokaryotes and eukaryotes. New methods need to be established for validating and mining the protein–DNA interaction data produced by bioinformatic tools and large-scale screening assays. In this study, we integrated bacterial one-hybrid technique (B1H) with classical chromatin immunoprecipitation (ChIP) assay to develop an innovative B1H-ChIP method, which has the advantages of being high-throughput, low cost, and easy-to-perform. Using this method, we validated two pairs of previously reported PDIs and further successfully discovered five novel target genes for Mce2R and four novel regulators of the gene dnaA in the human pathogen Mycobacterium tuberculosis. New PDI data suggest that Mce2R may play novel roles in the regulation of multi-drug resistance, cell wall synthesis, and intracellular growth of M. tuberculosis, and there exists a probable selective regulation of dnaA under different host conditions. Our findings provide important new information for understanding unique regulatory mechanisms in the pathogen. The B1H-ChIP approach has wide applications both in validating and discovering PDIs and in unraveling transcriptional regulatory network in prokaryotes and eukaryotes.

Keywords: B1H-ChIP; Protein–DNA interaction; M. tuberculosis ; Mce2R; dnaA

An ELISA based on the repeated foot-and-mouth disease virus 3B epitope peptide can distinguish infected and vaccinated cattle by Mingchun Gao; Runxiang Zhang; Meng Li; Shuang Li; Yongsheng Cao; Bo Ma; Junwei Wang (pp. 1271-1279).

To develop a strategy of differentiating infected from vaccinated animals (DIVA) with foot-and-mouth disease virus (FMDV), a short (27aa) peptide containing three conserved linear B cell epitopes of the FMDV 3B nonstructural protein was designed. This novel BF peptide was synthesized using a gene splicing by overlap extension protocol with preferred codons for Escherichia coli. The resultant eight tandem repeat multimer (1, 2, 4, 6, 8, 16, 24, and 32BF) were expressed as soluble fusion proteins in E. coli. An indirect ELISA was developed based on the recombinant 8BF protein with the aim of specifically distinguishing antibodies induced by FMDV infection but not those induced by vaccination. Using the cut-off value of 0.3, the sensitivity of the assay was 96.8% and the specificities for naive and vaccinated cattle were 99.8 and 99.0%, respectively. The performance of the newly developed epitope-based ELISA was compared with three commercial NSP ELISA kits. The 8BF-ELISA appears to be a promising DIVA test for FMD control and eradication.

Keywords: Foot-and-mouth disease virus; Tandem repeat; Epitope; ELISA; DIVA

Effect of different salt adaptation strategies on the microbial diversity, activity, and settling of nitrifying sludge in sequencing batch reactors by João Paulo Bassin; Robbert Kleerebezem; Gerard Muyzer; Alexandre Soares Rosado; Mark C. M. van Loosdrecht; Marcia Dezotti (pp. 1281-1294).

The effect of salinity on the activity of nitrifying bacteria, floc characteristics, and microbial community structure accessed by fluorescent in situ hybridization and polymerase chain reaction–denaturing gradient gel electrophoresis techniques was investigated. Two sequencing batch reactors (SRB₁ and SBR₂) treating synthetic wastewater were subjected to increasing salt concentrations. In SBR₁, four salt concentrations (5, 10, 15, and 20 g NaCl/L) were tested, while in SBR₂, only two salt concentrations (10 and 20 g NaCl/L) were applied in a more shock-wise manner. The two different salt adaptation strategies caused different changes in microbial community structure, but did not change the nitrification performance, suggesting that regardless of the different nitrifying bacterial community present in the reactor, the nitrification process can be maintained stable within the salt range tested. Specific ammonium oxidation rates were more affected when salt increase was performed more rapidly and dropped 50% and 60% at 20 g NaCl/L for SBR₁ and SBR₂, respectively. A gradual increase in NaCl concentration had a positive effect on the settling properties (i.e., reduction of sludge volume index), although it caused a higher amount of suspended solids in the effluent. Higher organisms (e.g., protozoa, nematodes, and rotifers) as well as filamentous bacteria could not withstand the high salt concentrations.

Keywords: Activated sludge; DGGE; FISH; Microbial diversity; Nitrification; Salt effect; SBR

Kinetics of ferrous iron oxidation by batch and continuous cultures of thermoacidophilic Archaea at extremely low pH of 1.1–1.3 by Paula Gonzalez-Contreras; Jan Weijma; Cees J. N. Buisman (pp. 1295-1303).

The extreme acid conditions required for scorodite (FeAsO₄·2H₂O) biomineralization (pH below 1.3) are suboptimal for growth of most thermoacidophilic Archaea. With the objective to develop a continuous process suitable for biomineral production, this research focuses on growth kinetics of thermoacidophilic Archaea at low pH conditions. Ferrous iron oxidation rates were determined in batch-cultures at pH 1.3 and a temperature of 75°C for Acidianus sulfidivorans, Metallosphaera prunea and a mixed Sulfolobus culture. Ferrous iron and CO₂ in air were added as sole energy and carbon source. The highest growth rate (0.066 h⁻¹) was found with the mixed Sulfolobus culture. Therefore, this culture was selected for further experiments. Growth was not stimulated by increase of the CO₂ concentration or by addition of sulphur as an additional energy source. In a CSTR operated at the suboptimal pH of 1.1, the maximum specific growth rate of the mixed culture was 0.022 h⁻¹, with ferrous iron oxidation rates of 1.5 g L⁻¹ d⁻¹. Compared to pH 1.3, growth rates were strongly reduced but the ferrous iron oxidation rate remained unaffected. Influent ferrous iron concentrations above 6 g L⁻¹ caused instability of Fe²⁺ oxidation, probably due to product (Fe³⁺) inhibition. Ferric-containing, nano-sized precipitates of K-jarosite were found on the cell surface. Continuous cultivation stimulated the formation of an exopolysaccharide-like substance. This indicates that biofilm formation may provide a means of biomass retention. Our findings showed that stable continuous cultivation of a mixed iron-oxidizing culture is feasible at the extreme conditions required for continuous biomineral formation.

Keywords: Iron oxidation; Thermoacidophilic; Archaeon; Sulfolobus; Biomineral; Jarosite; EPS

Characterization of a marine-isolated mercury-resistant Pseudomonas putida strain SP1 and its potential application in marine mercury reduction by Weiwei Zhang; Lingxin Chen; Dongyan Liu (pp. 1305-1314).

The Pseudomonas putida strain SP1 was isolated from marine environment and was found to be resistant to 280 μM HgCl₂. SP1 was also highly resistant to other metals, including CdCl₂, CoCl₂, CrCl₃, CuCl₂, PbCl₂, and ZnSO₄, and the antibiotics ampicillin (Ap), kanamycin (Kn), chloramphenicol (Cm), and tetracycline (Tc). mer operon, possessed by most mercury-resistant bacteria, and other diverse types of resistant determinants were all located on the bacterial chromosome. Cold vapor atomic absorption spectrometry and a volatilization test indicated that the isolated P. putida SP1 was able to volatilize almost 100% of the total mercury it was exposed to and could potentially be used for bioremediation in marine environments. The optimal pH for the growth of P. putida SP1 in the presence of HgCl₂ and the removal of HgCl₂ by P. putida SP1 was between 8.0 and 9.0, whereas the optimal pH for the expression of merA, the mercuric reductase enzyme in mer operon that reduces reactive Hg²⁺ to volatile and relatively inert monoatomic Hg⁰ vapor, was around 5.0. LD₅₀ of P. putida SP1 to flounder and turbot was 1.5 × 10⁹ CFU. Biofilm developed by P. putida SP1 was 1- to 3-fold lower than biofilm developed by an aquatic pathogen Pseudomonas fluorescens TSS. The results of this study indicate that P. putida SP1 is a low virulence strain that can potentially be applied in the bioremediation of HgCl₂ contamination over a broad range of pH.

Keywords: Pseudomonas putida ; Marine environment; mer operon; Bioremediation of HgCl₂ contamination

An unexpected gene cluster for downstream degradation of alkylphenols in Sphingomonas sp. strain TTNP3 by Boris A. Kolvenbach; Hyazinth Dobrowinski; Jan Fousek; Cestmir Vlcek; Andreas Schäffer; Frederic L. P. Gabriel; Hans-Peter E. Kohler; Philippe F. X. Corvini (pp. 1315-1324).

In silico analysis of nucleotide sequences flanking the recently found hydroquinone dioxygenase in Sphingomonas sp. strain TTNP3 revealed a gene cluster that encodes a hydroquinone catabolic pathway. In addition to the two open-reading frames encoding the recently characterized hydroquinone dioxygenase, the cluster consisted of six open-reading frames. We were able to express the three open-reading frames, hqdC, hqdD, and hqdE, and demonstrated that the three gene products, HqdC, HqdD, and HqdE had 4-hydroxymuconic semialdehyde dehydrogenase, maleylacetate reductase, and intradiol dioxygenase activity, respectively. Surprisingly, the gene cluster showed similarities to functionally related clusters found in members of the β- and γ-proteobacteria rather than to those found in other members of the genus Sphingomonas sensu latu.

Keywords: Hydroquinone; Degradation; Sphingomonas; Nonylphenol; Bisphenol A

Impact of impurities in biodiesel-derived crude glycerol on the fermentation by Clostridium pasteurianum ATCC 6013 by Keerthi P. Venkataramanan; Judy J. Boatman; Yogi Kurniawan; Katherine A. Taconi; Geoffrey D. Bothun; Carmen Scholz (pp. 1325-1335).

During the production of biodiesel, crude glycerol is produced as a byproduct at 10% (w/w). Clostridium pasteurianum has the inherent potential to grow on glycerol and produce 1,3-propanediol and butanol as the major products. Growth and product yields on crude glycerol were reported to be slower and lower, respectively, in comparison to the results obtained from pure glycerol. In this study, we analyzed the effect of each impurity present in the biodiesel-derived crude glycerol on the growth and metabolism of glycerol by C. pasteurianum. The crude glycerol contains methanol, salts (in the form of potassium chloride or sulfate), and fatty acids that were not transesterified. Salt and methanol were found to have no negative effects on the growth and metabolism of the bacteria on glycerol. The fatty acid with a higher degree of unsaturation, linoleic acid, was found to have strong inhibitory effect on the utilization of glycerol by the bacteria. The fatty acid with lower or no degrees of unsaturation such as stearic and oleic acid were found to be less detrimental to substrate utilization. The removal of fatty acids from crude glycerol by acid precipitation resulted in a fermentation behavior that is comparable to the one on pure glycerol. These results show that the fatty acids in the crude glycerol have a negative effect by directly affecting the utilization of glycerol as the carbon source, and hence their removal from crude glycerol is an essential step towards the utilization of crude glycerol.

Keywords: Biodiesel-derived crude glycerol; Substrate utilization; C. pasteurianum ; Butanol; Fermentation; Methanol; Fatty acids; Impurities; Inhibition

Cultivation-independent analysis of archaeal and bacterial communities of the formation water in an Indian coal bed to enhance biotransformation of coal into methane by Durgesh Narain Singh; Ashok Kumar; Munish Prasad Sarbhai; Anil Kumar Tripathi (pp. 1337-1350).

Biogenic origin of the significant proportion of coal bed methane has indicated the role of microbial communities in methanogenesis. By using cultivation-independent approach, we have analysed the archaeal and bacterial community present in the formation water of an Indian coal bed at 600–700 m depth to understand their role in methanogenesis. Presence of methanogens in the formation water was inferred by epifluorescence microscopy and PCR amplification of mcrA gene. Archaeal 16S rRNA gene clone library from the formation water metagenome was dominated by methanogens showing similarity to Methanobacterium, Methanothermobacter and Methanolinea whereas the clones of bacterial 16S rRNA gene library were closely related to Azonexus, Azospira, Dechloromonas and Thauera. Thus, microbial community of the formation water consisted of predominantly hydrogenotrophic methanogens and the proteobacteria capable of nitrogen fixation, nitrate reduction and polyaromatic compound degradation. Methanogenic potential of the microbial community present in the formation water was elucidated by the production of methane in the enrichment culture, which contained 16S rRNA gene sequences showing close relatedness to the genus Methanobacterium. Microcosm using formation water as medium as well as a source of inoculum and coal as carbon source produced significant amount of methane which increased considerably by the addition of nitrite. The dominance of Diaphorobacter sp. in nitrite amended microcosm indicated their important role in supporting methanogenesis in the coal bed. This is the first study indicating existence of methanogenic and bacterial community in an Indian coal bed that is capable of in situ biotransformation of coal into methane.

Keywords: Coal bed; Methanogenesis; Methanogenic archaea; Denitrifying bacteria; Microcosm

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Applied Microbiology and Biotechnology (v.93, #3)

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Applied Microbiology and Biotechnology (v.93, #3)