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Applied Microbiology and Biotechnology (v.94, #5)

The microbiology of metalworking fluids by Ratul Saha; Robert S. Donofrio (pp. 1119-1130).

Metalworking fluids (MWFs) are complex mixtures of chemicals and are indispensable materials in industry. They are used as cooling and lubricating agents in different machining process such as grinding, milling, and cutting. The quality of MWFs is affected by physical, chemical, and microbial contaminates. In particular, MWFs are highly vulnerable to microbial contamination, which may act both as potential pathogens and deteriorgens. Microbial contamination is of major concern due to potential health hazards such as skin dermatitis and hypersensitivity pneumonitis. The contaminated MWFs can exhibit high degrees of microbial loading, ranging from 10⁴ to 10¹⁰ colony-forming units (CFU)/ml. Wide varieties of microorganisms are reported to colonize MWFs. Traditional culturing techniques are not only laborious and time consuming but also underestimate the actual distribution of the microorganisms present in the contaminated MWFs. Therefore, rapid molecular methods such as real-time PCR and fluorescent in situ hybridization are implemented to monitor the microbial load. In industry, biocides are presently used to control microbial contamination. However, it has its own disadvantages and therefore, in recent years, alternative methods such as UV irradiation were evaluated to reduce microbial contamination in MWFs. Microbes inhabiting the MWF are also capable of forming biofilm which is detrimental to the MWF system. Biofilm is resistant to common disinfectant methods, and thus further research and development is required to effectively control its formation within MWF systems. This review is intended to discuss the overall microbiological aspects of MWF.

Keywords: Metalworking fluids; Real-time PCR; FISH; Biocides; Biofilm

Two-component signal transduction in Corynebacterium glutamicum and other corynebacteria: on the way towards stimuli and targets by Michael Bott; Melanie Brocker (pp. 1131-1150).

In bacteria, adaptation to changing environmental conditions is often mediated by two-component signal transduction systems. In the prototypical case, a specific stimulus is sensed by a membrane-bound histidine kinase and triggers autophosphorylation of a histidine residue. Subsequently, the phosphoryl group is transferred to an aspartate residue of the cognate response regulator, which then becomes active and mediates a specific response, usually by activating and/or repressing a set of target genes. In this review, we summarize the current knowledge on two-component signal transduction in Corynebacterium glutamicum. This Gram-positive soil bacterium is used for the large-scale biotechnological production of amino acids and can also be applied for the synthesis of a wide variety of other products, such as organic acids, biofuels, or proteins. Therefore, C. glutamicum has become an important model organism in industrial biotechnology and in systems biology. The type strain ATCC 13032 possesses 13 two-component systems and the role of five has been elucidated in recent years. They are involved in citrate utilization (CitAB), osmoregulation and cell wall homeostasis (MtrAB), adaptation to phosphate starvation (PhoSR), adaptation to copper stress (CopSR), and heme homeostasis (HrrSA). As C. glutamicum does not only face changing conditions in its natural environment, but also during cultivation in industrial bioreactors of up to 500 m³ volume, adaptability can also be crucial for good performance in biotechnological production processes. Detailed knowledge on two-component signal transduction and regulatory networks therefore will contribute to both the application and the systemic understanding of C. glutamicum and related species.

Keywords: Histidine kinase; Response regulator; Sensors; Regulation; C. diphtheriae

Two-component signal transduction in Corynebacterium glutamicum and other corynebacteria: on the way towards stimuli and targets by Michael Bott; Melanie Brocker (pp. 1131-1150).

Keywords: Histidine kinase; Response regulator; Sensors; Regulation; C. diphtheriae

Benefit of Monascus-fermented products for hypertension prevention: a review by Bao-Hong Lee; Tzu-Ming Pan (pp. 1151-1161).

γ-Aminobutyric acid (GABA) has been reported to play a neurotransmitter in the central nervous system thereby exerting an inhibition in nerve impulse, in turn ameliorating depression; in addition, recent study also reveals the anti-hypertensive effect of GABA in vivo. Edible fungi of the Monascus species have been used as traditional Chinese medicine in eastern Asia for several centuries. Monascus-fermented products possess a number of functional secondary metabolites, including anti-inflammatory pigments (such as monascin and ankaflavin), monacolins, dimerumic acid, and GABA. Several scientific studies have shown that these secondary metabolites have anti-inflammatory, anti-oxidative, and anti-tumor activities. Moreover, many published reports have shown the efficacy of Monascus-fermented products in the prevention or amelioration of some diseases, including hypercholesterolemia, hyperlipidemia, hypertension, diabetes, obesity, Alzheimer’s disease, and numerous types of cancer in recent studies. The current article discusses and provides evidence to elucidate the anti-hypertensive benefit of Monascus-fermented metabolites, including anti-inflammatory pigments and GABA.

Keywords: γ-Aminobutyric acid (GABA); Monascus ; Monascin; Ankaflavin; Anti-hypertensive

Features and technical applications of ω-transaminases by M. Shaheer Malik; Eul-Soo Park; Jong-Shik Shin (pp. 1163-1171).

Chiral amines in enantiopure forms are important chemical building blocks, which are most well recognized in the pharmaceutical industries for imparting desirable biological activity to chemical entities. A number of synthetic strategies to produce chiral amines via biocatalytic as well as chemical transformation have been developed. Recently, ω-transaminase (ω-TA) has attracted growing attention as a promising catalyst which provides an environment-friendly access to production of chiral amines with exquisite stereoselectivity and excellent catalytic turnover. To obtain enantiopure amines using ω-TAs, either kinetic resolution of racemic amines or asymmetric amination of achiral ketones is employed. The latter is usually preferred because of twofold higher yield and no requirement of conversion of a ketone product back to racemic amine. However, the choice of a production process depends on several factors such as reaction equilibrium, substrate reactivity, enzyme inhibition, and commercial availability of substrates. This review summarizes the biochemical features of ω-TA, including reaction chemistry, substrate specificity, and active site structure, and then introduces recent advances in expanding the scope of ω-TA reaction by protein engineering and public database searching. We also address crucial factors to be considered for the development of efficient ω-TA processes.

Keywords: ω-transaminase; Chiral amines; Kinetic resolution; Asymmetric synthesis; Product inhibition

MDCK cell-cultured influenza virus vaccine protects mice from lethal challenge with different influenza viruses by Kun Liu; Zhidong Yao; Liangyan Zhang; Junli Li; Li Xing; Xiliang Wang (pp. 1173-1179).

Influenza epidemics are major health concern worldwide. Vaccination is the major strategy to protect the general population from a pandemic. Currently, most influenza vaccines are manufactured using chicken embroynated eggs, but this manufacturing method has potential limitations, and cell-based vaccines offer a number of advantages over the traditional method. We reported here using the scalable bioreactor to produce pandemic influenza virus vaccine in a Madin–Darby canine kidney cell culture system. In the 7.5-L bioreactor, the cell concentration reached to 3.2 × 10⁶ cells/mL and the highest virus titers of 256 HAU/50 μL and 1 × 10⁷ TCID50/mL. The HA concentration was found to be 11.2 μg/mL. The vaccines produced by the cell-cultured system induced neutralization antibodies, cross-reactive T-cell responses, and were protective in a mouse model against different lethal influenza virus challenge. These data indicate that microcarrier-based cell-cultured influenza virus vaccine manufacture system in scalable bioreactor could be used to produce effective pandemic influenza virus vaccines.

Keywords: MDCK; Bioreactor; Influenza virus; Vaccine

A process for high-efficiency isoflavone deglycosylation using Bacillus subtilis natto NTU-18 by Lun-Cheng Kuo; Ren-Yu Wu; Kung-Ta Lee (pp. 1181-1188).

In order to produce isoflavone aglycosides effectively, a process of isoflavone hydrolysis by Bacillus subtilis natto NTU-18 (BCRC 80390) was established. This process integrates the three stages for the production of isoflavone aglycosides in one single fermenter, including the growth of B. subtilis natto, production of β-glucosidase, deglycosylation of fed isoflavone glycosides. After 8 h of batch culture of B. subtilis natto NTU-18 in 2 L of soy medium, a total of 3 L of soy isoflavone glucoside solution containing 3.0 mg/mL of daidzin and 1.0 mg/mL of genistin was fed continuously over 34 h. The percentage deglycosylation of daidzin and genistin was 97.7% and 94.6%, respectively. The concentration of daidzein and genistein in the broth reached 1,066.8 μg/mL (4.2 mM) and 351 μg/mL (1.3 mM), respectively, and no residual daidzin or genistin was detected. The productivity of the bioconversion of daidzein and genistein over the 42 h of culture was 25.6 mg/L/h and 8.5 mg/L/h, respectively. This showed that this is an efficient bioconversion process for selective estrogen receptor modulator production.

Keywords: Isoflavones; Bacillus subtilis natto; β-glucosidase; Isoflavone deglycosylation; Fed-batch bioconversion

High-yield enzymatic bioconversion of hydroquinone to α-arbutin, a powerful skin lightening agent, by amylosucrase by Dong-Ho Seo; Jong-Hyun Jung; Suk-Jin Ha; Hyun-Kug Cho; Dong-Hyun Jung; Tae-Jip Kim; Nam-In Baek; Sang-Ho Yoo; Cheon-Seok Park (pp. 1189-1197).

α-Arbutin (α-Ab) is a powerful skin whitening agent that blocks epidermal melanin biosynthesis by inhibiting the enzymatic oxidation of tyrosine and l-3,4-dihydroxyphenylalanine (l-DOPA). α-Ab was effectively synthesized from hydroquinone (HQ) by enzymatic biotransformation using amylosucrase (ASase). The ASase gene from Deinococcus geothermalis (DGAS) was expressed and efficiently purified from Escherichia coli using a constitutive expression system. The expressed DGAS was functional and performed a glycosyltransferase reaction using sucrose as a donor and HQ as an acceptor. The presence of a single HQ bioconversion product was confirmed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The HQ bioconversion product was isolated by silica gel open column chromatography and its chemical structure determined by ¹H and ¹³ C nuclear magnetic resonance (NMR). The product was determined to be hydroquinone-O-α-d-glucopyranoside with a glucose molecule linked to HQ through an α-glycosidic bond. However, the production yield of the transfer reaction was significantly low (1.3%) due to the instability of HQ in the reaction mixture. The instability of HQ was considerably improved by antioxidant agents, particularly ascorbic acid, implying that HQ is labile to oxidation. A maximum yield of HQ transfer product of 90% was obtained at a 10:1 molar ratio of donor (sucrose) and acceptor (HQ) molecules in the presence of 0.2 mM ascorbic acid.

Keywords: Amylosucrase; α-arbutin; Ascorbic acid; Deinococcus geothermalis ; Hydroquinone

Photothermal effects of folate-conjugated Au nanorods on HepG2 cells by Hua Jin; Peihui Yang; Jiye Cai; Jinhui Wang; Mei Liu (pp. 1199-1208).

Photothermal cancer therapy, as a prospective approach for local cancer treatment, is attracting increasing interests. In this paper, gold nanorods were conjugated with folate (folate/AuNRs), and their photothermal effects on hepatocellular carcinoma cell line (HepG2) using MTT assay, flow cytometry, as well as on the cellular morphology, cytoskeleton, cell surface adhesion, and stiffness detected at subcellular level by an atomic force microscope (AFM) were investigated. The results indicated that near-infrared laser-induced hyperthermia of folate/AuNRs could break the cell membrane integrity and homeostasis and then lead to the depolymerization of cytoskeleton and influx of intracellular Ca²⁺. Thus, folate/AuNRs can be as effective and promising nanomaterials for photothermal therapy of folate receptor bearing tumor.

Keywords: Photothermal therapy; Folate-conjugated gold nanorods; HepG2 cells; Atomic force microscopy

CysK from Lactobacillus casei encodes a protein with O-acetylserine sulfhydrylase and cysteine desulfurization activity by Biljana Bogicevic; Hélène Berthoud; Reto Portmann; Leo Meile; Stefan Irmler (pp. 1209-1220).

A gene encoding an O-acetyl-l-serine sulfhydrylase (cysK) was cloned from Lactobacillus casei FAM18110 and expressed in Escherichia coli. The purified recombinant enzyme synthesized cysteine from sulfide and O-acetyl-l-serine at pH 5.5 and pH 7.4. At pH 7.4, the apparent K _M for O-acetyl-l-serine (OAS) and sulfide were 0.6 and 6.7 mM, respectively. Furthermore, the enzyme showed cysteine desulfurization activity in the presence of dithiothreitol at pH 7.5, but not at pH 5.5. The apparent K _M for l-cysteine was 0.7 mM. The synthesis of cystathionine from homocysteine and serine or OAS was not observed. When expressed in a cysMK mutant of Escherichia coli, the cloned gene complemented the cysteine auxotrophy of the mutant. These findings suggested that the gene product is mainly involved in cysteine biosynthesis in L. casei. Quantitative real-time PCR and a mass spectrometric assay based on selected reaction monitoring demonstrated that L. casei FAM18110 is constitutively overexpressing cysK.

Keywords: Lactobacillus casei ; Cysteine; Hydrogen sulfide; O-acetylserine sulfhydrylase; Desulfurization

Stereoselective hydrolysis of aryl-substituted dihydropyrimidines by hydantoinases by U. Engel; C. Syldatk; J. Rudat (pp. 1221-1231).

In this study, we investigated the possibility of using a modified hydantoinase process for the production of optically pure β-amino acids. Two aryl-substituted dihydropyrimidines d,l-6-phenyl-5,6-dihydrouracil (PheDU) and para-chloro-d,l-6-phenyl-5,6-dihydrouracil (pClPheDU) were synthesized. Hydrolysis of these novel substrates to the corresponding N-carbamoyl-β-amino acids by three recombinant d-hydantoinases and several bacterial strains was tested. All applied recombinant d-hydantoinases and eight bacterial isolates catalyzed the conversion of PheDU to N-carbamoyl-β-phenylalanine (NCβPhe). Some of these biocatalysts showed an enantioselectivity for either the d- or the l-PheDU enantiomer. The second dihydropyrimidinase substrate pClPheDU was hydrolyzed by all three recombinant d-hydantoinases and six of the wild-type strains. To our knowledge, this is the first dihydropyrimidinase activity reported with this aryl-substituted dihydropyrimidine. For selected biocatalysts, hydantoinase activity towards aryl-substituted hydantoins was demonstrated as well. However, none of the bacterial strains tested so far exhibited any carbamoylase activity towards NCβPhe.

Keywords: Beta-amino acid; Hydantoinase; Dihydropyrimidinase; Carbamoylase

Nanotube-supported bioproduction of 4-hydroxy-2-butanone via in situ cofactor regeneration by Liang Wang; Hongfang Zhang; Chi-Bun Ching; Yuan Chen; Rongrong Jiang (pp. 1233-1241).

Nicotinamide cofactor-dependent oxidoreductases have been widely employed during the bioproduction of varieties of useful compounds. Efficient cofactor regeneration is often required for these biotransformation reactions. Herein, we report the synthesis of an important pharmaceutical intermediate 4-hydroxy-2-butanone (4H2B) via an immobilized in situ cofactor regeneration system composed of NAD⁺-dependent glycerol dehydrogenase (GlyDH) and NAD⁺-regenerating NADH oxidase (nox). Both enzymes were immobilized on functionalized single-walled carbon nanotubes (SWCNTs) through the specific interaction between the His-tagged enzymes and the modified SWCNTs. GlyDH demonstrated ca. 100% native enzyme activity after immobilization. The GlyDH/nox ratio, pH, and amount of nicotinamide cofactor were examined to establish the optimum reaction conditions for 4H2B production. The nanoparticle-supported cofactor regeneration system become more stable and the yield of 4H2B turned out to be almost twice (37%) that of the free enzyme system after a 12-h reaction. Thus, we believe that this non-covalent specific immobilization procedure can be applied to cofactor regeneration system for bioconversions.

Keywords: Glycerol dehydrogenase; Cofactor regeneration; 4-hydroxy-2-butanone; NADH oxidase

Understanding the mechanistic roles of 30Kc6 gene in apoptosis and specific productivity in antibody-producing Chinese hamster ovary cells by Zesong Wang; Xuhui Ma; Li Fan; Won Jong Rhee; Tai Hyun Park; Liang Zhao; Wen-Song Tan (pp. 1243-1253).

Previously, we reported that the expression of Bombyx mori 30Kc6 gene in Chinese hamster ovary (CHO) cells increases recombinant protein production by both inhibiting apoptosis and enhancing specific productivity. In this study, in order to gain a thorough understanding of the roles of 30Kc6 gene in antibody production, the mechanisms modulating cell apoptosis and specific productivity were investigated. 30Kc6 gene was introduced into a CHO cell line producing a chimeric anti-human CD20 monoclonal antibody. The stable expression of 30Kc6 increased cell viability and productivity by 46.7% and 3.4-folds, respectively. It was observed that the Bax translocation from cytosol to mitochondria and the cytochrome c (cyt c) release from mitochondrial intermembrane space to cytosol were repressed, which resulted in a decrease in the activation of apoptosis executioner, caspase-3. On the other hand, 30Kc6 expression increased the specific productivity by 2.3-folds. However, at the transcription level, the relative levels of heavy and light chain mRNAs increased only by 8.3% and 8.7%, respectively, which was not accountable for the observed increment in the specific productivity. Instead, the mitochondrial membrane potential was maintained and the ATP generation was stimulated. A higher ATP level could activate the mammalian target of rapamycin (mTOR), which drives the translation initiation and elongation by phosphorylating eukaryotic initiation factor 4E binding protein 1 (4EBP1) and S6 kinase 1 (S6K1). In the 30Kc6-expressing cells, both the 4EBP1 and S6K1 were phosphorylated at higher levels, which indicated that the increased specific productivity primarily resulted from the boost of translation process. Furthermore, it was also found that the specific uptake rates of glucose and glutamine were not affected by 30Kc6 expression, demonstrating that the enhanced ATP generation and consequently maintained mTOR activity were due to 30Kc6 expression but not the different metabolic uptake rates. In conclusion, 30Kc6 expression inhibited apoptosis by repressing the Bax translocation, which down-regulated the downstream cascade responses including cyt c release and caspase-3 activation. Also, 30Kc6 expression increased the specific productivity by enhancing the translation process.

Keywords: CHO cells; mAb; 30Kc6 ; Apoptosis; Specific productivity

Injection of duck recombinant follistatin fusion protein into duck muscle tissues stimulates satellite cell proliferation and muscle fiber hypertrophy by He-he Liu; Ji-wen Wang; Hai-yue Yu; Rong-ping Zhang; Xi Chen; Hai-bo Jin; Fei Dai; Liang Li; Feng Xu (pp. 1255-1263).

Follistatin (FST) can inhibit the expression of myostatin, which is a predominant inhibitor of muscle development. The potential application of myostatin-based technology has been prompted in different ways in agriculture. We previously constructed an expression vector of duck FST and isolated the FST fusion protein. After the protein was purified and refolded, it was added to the medium of duck myoblasts cultured in vitro. The results show that the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide value of the myoblasts in the duck FST treatment group is higher than that in the control group, which indicates that the duck FST fusion protein exhibits the biological activities that can accelerate myoblast proliferation. To further investigate the roles of duck FST on muscle development, we injected the protein into the duck muscle tissues in vivo. The results show that both the duck muscle fiber cross-sectional area and the satellite cell activation frequency are influenced more in the FST treatment group than they are in the control group. In addition to these phenomena, expression of MyoD and Myf5 were increased, and the expression of myostatin was decreased. Together, these results suggest the potential for using duck FST fusion protein to inhibit myostatin activity and subsequently to enhance muscle growth in vivo. The mechanism by which FST regulates muscle development in the duck is similar to that in mammals and fishes.

Keywords: Prokaryotic expression; Follistatin ; Satellite cell; Activation; Hypertrophy

Flagellin gene (fliC) of Thermus thermophilus HB8: characterization of its product and involvement to flagella assembly and microbial motility by Christos P. Papaneophytou; Rigini M. Papi; Anastasia A. Pantazaki; Dimitrios A. Kyriakidis (pp. 1265-1277).

Thermus thermophilus HB8 flagellin protein (FliC) is encoded by the TTHC004 (fliC) gene, which is located in the pTT8 plasmid of the bacterium. Flagellin monomer and flagella fibres were isolated from a culture of T. thermophilus grown in rich medium, or in mineral salt medium with sodium gluconate as the carbon source. Western blot immunodetection with anti-FliC revealed a stable complex (FliC)₁(FliS)₂ of flagellin (FliC, 27.7 kDa) with a homodimer of FliS (FliS, 18.2 kDa) that are encoded by TTHC004 and TTHC003 genes, respectively. The complex is dissociable at low pHs and/or by heat treatment. Glycan staining of purified flagella and treatment with N-glycosidase F suggested that flagellin of T. thermophilus is a glycosylated protein. Size exclusion chromatography revealed that flagellar filaments (FliC) have a molecular mass higher than 200 kDa. The formation of flagella is enhanced after prolonged cultivation time where phosphate and other nutrient were depleted, giving in the bacterium considerable swimming motility in low viscosity media.

Keywords: T. thermophilus HB8; fliC gene; Flagellin; Flagella assembly; Motility

Characterization of Lactobacillus salivarius CECT 5713, a strain isolated from human milk: from genotype to phenotype by Susana Langa; Antonio Maldonado-Barragán; Susana Delgado; Rebeca Martín; Virginia Martín; Esther Jiménez; José L. Ruíz-Barba; Baltasar Mayo; Ruth I. Connor; Juan Evaristo Suárez; Juan M. Rodríguez (pp. 1279-1287).

Lactobacillus salivarius CECT 5713, isolated from human milk, has immunomodulatory, anti-inflammatory and antiinfectious properties, as revealed by several in vitro and in vivo assays, which suggests a strong potential as a probiotic strain. In this work, the relationships between several genetic features of L. salivarius CECT 5713 and the corresponding phenotypes were evaluated. Although it contains a plasmid-encoded bacteriocin cluster, no bacteriocin biosynthesis was observed, possibly due to a 4-bp deletion at the beginning of the histidine kinase determinant abpK. The genome of L. salivarius CECT 5713 harbours two apparently complete prophages of 39.6 and 48 kbp. Upon induction, the 48-kbp prophage became liberated from the bacterial genome, but no DNA replication took place, which resulted in lysis of the cultures but not in phage progeny generation. The strain was sensitive to most antibiotics tested and no transmissible genes potentially involved in antibiotic resistance were detected. Finally, the genome of L. salivarius CECT 5713 contained four ORFs potentially involved in human molecular mimetism. Among them, protein 1230 was considered of particular relevance because of its similarity with dendritic cell-related proteins. Subsequently, in vitro assays revealed the ability of L. salivarius CECT 5713 to stimulate the maturation of immature dendritic cells and to inhibit the in vitro infectivity of HIV-1.

Keywords: Lactobacillus salivarius ; Probiotic; Genome; Phage; Antibiotic resistance; Bacteriocin; Dendritic cell; HIV

Differential proteomic analysis of an engineered Streptomyces coelicolor strain reveals metabolic pathways supporting growth on n-hexadecane by Giuseppe Gallo; Luca Lo Piccolo; Giovanni Renzone; Ruggero La Rosa; Andrea Scaloni; Paola Quatrini; Anna Maria Puglia (pp. 1289-1301).

The alkB gene, encoding an alkane monooxygenase in the actinomycete Gordonia sp. SoCg, was expressed in the non-alkane-degrading actinomycete Streptomyces coelicolor M145. The resulting engineered strain, M145-AH, can grow on n-hexadecane as sole carbon source. To unravel proteins associated with growth on n-alkanes, proteome of M145-AH after 6, 24, and 48 h of incubation in the Bushnell-Haas (BH) mineral medium containing n-hexadecane as sole carbon source (H condition) and in BH without any carbon source (0 condition) were compared using 2D-differential gel electrophoresis. Proteome analysis revealed significant changes only at 48 h, showing 48 differentially abundant proteins identified by mass spectrometry procedures. To asses if these proteins were specifically related to n-hexadecane metabolism, their expression was investigated, comparing H proteome with that of M145-AH incubated in BH with glucose as sole carbon source (G condition). Thus, protein expression profiles at 6, 24, and 48 h under H, 0, and G conditions were combined, revealing that M145-AH regulates in a temporally- and carbon source-dependent manner the expression of proteins involved in regulatory events, central carbon metabolism, respiration, β-oxidation, membrane transport, and amino acid and protein metabolism. Interestingly, 21 % of them, mostly involved in membrane transport and protein metabolism, showed a n-hexadecane-dependent regulation with regulatory proteins such as CRP likely to have a key role in M145-AH n-hexadecane growth. These results, expanding the knowledge on n-alkane utilization in Gram-positive bacteria, reveal genes to be targeted to develop an efficient S. coelicolor M145-AH-based bioremediation system.

Keywords: n-hexadecane utilization; n-alkane monoxygenase; Streptomyces ; Engineered strain; 2D-DIGE analysis; Proteomics

Mode of coniferous wood decay by the white rot fungus Phanerochaete carnosa as elucidated by FTIR and ToF-SIMS by Sonam Mahajan; Dragica Jeremic; Robyn E. Goacher; Emma R. Master (pp. 1303-1311).

The softwood degrading white-rot fungus, Phanerochaete carnosa, was investigated for its ability to degrade two coniferous woods: balsam fir and lodgepole pine. P. carnosa grew similarly on these wood species, and like the hardwood-degrading white-rot fungus Ceriporiopsis subvermispora, P. carnosa demonstrated selective degradation of lignin, as observed by Fourier transform infrared spectroscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Lignin degradation across cell walls of decayed pine samples was also evaluated by ToF-SIMS and was shown to be uniform. This study illustrates softwood lignin utilization by a white-rot fungus and reveals the industrial potential of the lignocellulolytic activity elicited by this fungus.

Keywords: Coniferous softwood; Basidiomycota ; White-rot decay; Fourier transform infrared spectroscopy; Time-of-flight secondary ion mass spectrometry; Principal component analysis; Selective degradation

Improvement of glutathione production by metabolic engineering the sulfate assimilation pathway of Saccharomyces cerevisiae by Kiyotaka Y. Hara; Kentaro Kiriyama; Akiko Inagaki; Hideki Nakayama; Akihiko Kondo (pp. 1313-1319).

Glutathione (GSH) is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae. In this study, we demonstrated that engineering in sulfate assimilation metabolism can significantly improve GSH production. The intracellular GSH content of MET14 and MET16 over-expressing strains increased up to 1.2 and 1.4-fold higher than that of the parental strain, respectively, whereas those of APA1 and MET3 over-expressing strains decreased. Especially, in the MET16 over-expressing strain, the volumetric GSH concentration was up to 1.7-fold higher than that of the parental strain as a result of the synergetic effect of the increases in the cell concentration and the intracellular GSH content. Additionally, combinatorial mutant strains that had been engineered to contain both the sulfur and the GSH synthetic metabolism synergistically increased the GSH production. External addition of cysteine to S. cerevisiae is well known as a way to increase the intracellular GSH content; however, it results a decrease in cell growth. This study showed that the engineering of sulfur metabolism in S. cerevisiae proves more valuable than addition of cysteine as a way to boost GSH production due to the increases in both the intracellular GSH content and the cell growth.

Keywords: Glutathione; Yeast; Saccharomyces cerevisiae ; Cysteine; Sulfate

The rhizospheres of traditional medicinal plants in Panxi, China, host a diverse selection of actinobacteria with antimicrobial properties by Ke Zhao; Petri Penttinen; Qiang Chen; Tongwei Guan; Kristina Lindström; Xiaoling Ao; Lili Zhang; Xiaoping Zhang (pp. 1321-1335).

Actinobacteria are a prolific source of antibiotics. Since the rate of discovery of novel antibiotics is decreasing, actinobacteria from unique environments need to be explored. In particular, actinobacterial biocontrol strains from medicinal plants need to be studied as they can be a source of potent antibiotics. We combined culture-dependent and culture-independent methods in analyzing the actinobacterial diversity in the rhizosphere of seven traditional medicinal plant species from Panxi, China, and assessed the antimicrobial activity of the isolates. Each of the plant species hosted a unique set of actinobacterial strains. Out of the 64 morphologically distinct isolates, half were Streptomyces sp., eight were Micromonospora sp., and the rest were members of 18 actinobacterial genera. In particular, Ainsliaea henryi Diels. hosted a diverse selection of actinobacteria, although the 16S ribosomal RNA (rRNA) sequence identity ranges of the isolates and of the 16S rRNA gene clone library were not congruent. In the clone library, 40% of the sequences were related to uncultured actinobacteria, emphasizing the need to develop isolation methods to assess the full potential of the actinobacteria. All Streptomyces isolates showed antimicrobial activity. While the antimicrobial activities of the rare actinobacteria were limited, the growth of Escherichia coli, Verticillium dahliae, and Fusarium oxysporum were inhibited only by rare actinobacteria, and strains related to Saccharopolyspora shandongensis and Streptosporangium roseum showed broad antimicrobial activity.

Keywords: Panxi plateau; Rhizospheric actinobacteria; Diversity; Antimicrobial activity

Expression of the soybean allergenic protein P34 in Escherichia coli and its indirect ELISA detection method by Bin Liu; Da Teng; Xiumin Wang; Yalin Yang; Jianhua Wang (pp. 1337-1345).

To detect the soybean allergen P34 (Gly m Bd 30K) from soybean products, the full-length cDNA sequence of P34 was synthesized and inserted into the prokaryotic expression vector pET-28a. The P34 protein was expressed in Escherichia coli BL21 (DE3) as an inclusion body under the induction of 0.8 mmol/L isopropyl β-d-1-thiogalactopyranoside. After purification with His-Bind affinity chromatography, the purity quotient of the recombinant protein was over 92 %, and its molecular weight (approximately 33 kDa) was very close to that of the native soybean P34. The polyclonal antibody (pAB) against P34 was prepared with the purified recombinant P34. The generated pAB, named as pAB-P34, exhibited high specificity to the P34 protein of the soybean meal. The indirect enzyme-linked immunosorbent assay (iELISA) based on pAB-P34 was established to determine the P34 content of soybean products. The CVs of the recovery tests of P34 were less than 7.77 %, which indicated that iELISA had high reproducibility and accuracy. Therefore, the recombinant P34 produced in the E. coli expression system, the prepared pAB-P34, and the developed iELISA could provide a valuable tool for sensitive detection of P34 in various soybean products and for future studies on allergies related to soybean P34.

Keywords: Soybean allergen; P34/Gly m Bd 30K; Prokaryotic expression; pAB; Indirect ELISA

Mining of unexplored habitats for novel chitinases—chiA as a helper gene proxy in metagenomics by Mariana Silvia Cretoiu; Anna Maria Kielak; Waleed Abu Al-Soud; Søren J. Sørensen; Jan Dirk van Elsas (pp. 1347-1358).

The main objective of this study was to assess the abundance and diversity of chitin-degrading microbial communities in ten terrestrial and aquatic habitats in order to provide guidance to the subsequent exploration of such environments for novel chitinolytic enzymes. A combined protocol which encompassed (1) classical overall enzymatic assays, (2) chiA gene abundance measurement by qPCR, (3) chiA gene pyrosequencing, and (4) chiA gene-based PCR-DGGE was used. The chiA gene pyrosequencing is unprecedented, as it is the first massive parallel sequencing of this gene. The data obtained showed the existence across habitats of core bacterial communities responsible for chitin assimilation irrespective of ecosystem origin. Conversely, there were habitat-specific differences. In addition, a suite of sequences were obtained that are as yet unregistered in the chitinase database. In terms of chiA gene abundance and diversity, typical low-abundance/diversity versus high-abundance/diversity habitats was distinguished. From the combined data, we selected chitin-amended agricultural soil, the rhizosphere of the Arctic plant Oxyria digyna and the freshwater sponge Ephydatia fluviatilis as the most promising habitats for subsequent bioexploration. Thus, the screening strategy used is proposed as a guide for further metagenomics-based exploration of the selected habitats.

Keywords: chiA ; Bacterial community; Environment; Functional screening; chiA pyrosequencing

Determination of low concentration of Paracoccus denitrificans encapsulated in polyvinyl alcohol LentiKat’s pellets by Lenka Vacková; Radek Stloukal; Jiří Wanner (pp. 1359-1364).

The aim of this work was to compare three methods to determinate low concentrations of Paracoccus denitrificans encapsulated in polyvinyl alcohol pellets, which is important for evaluation and optimization of pellet production as well as for monitoring of biomass growth. Pellets with different and well-defined biomass concentrations were used for experiments. The following fast and simple methods were tested: (1) dissolution of polyvinyl alcohol in hot water followed by dry weight estimation, (2) dissolution of polyvinyl alcohol in hot water followed by optical density measurement, (3) and extraction and quantification of proteins. Dry weight estimation proved to be problematic as it was difficult to separate biomass from polymeric carrier. Optical density measurement showed good linearity of dependence of optical density on biomass content, but determined limits of detection and limits of quantification were not within the range necessary for intended application. The only tested method meeting the requirements for sensitivity was determination of protein concentration after protein extraction.

Keywords: Biomass determination; Immobilization; Encapsulation; Paracoccus denitrificans ; Polyvinyl alcohol

Reactivation characteristics of stored aerobic granular sludge using different operational strategies by Xiangjuan Yuan; Dawen Gao; Hong Liang (pp. 1365-1374).

Aerobic granules after 6 months storage were employed in identical sequencing batch reactors (SBRs) using synthetic wastewater to investigate the impacts of different operational strategies on granules’ reactivation process. The SBRs were operated under three operational strategies for reactivation of (a) different organic loading rate (OLR); (b) different ammonia concentration; and (c) different shear force (a superficial upflow air velocity). The results indicated that granules after long-term storage could be successfully recovered after 7 days of operation, and the excellent granule reactivation performance was closely related to the operational strategies, since inappropriate operational strategies could cause the outgrowth of filamentous bacteria and granule disintegration. Based on comprehensive comparison of reactivation performance under different operational strategies, the optimal operation strategy for granule reactivation was suggested at OLR of 0.8 kg COD/m³/day, ammonia concentration of 15–20 mg/L, and a superficial upflow air velocity of 2.6 cm/s. After 7 days operation under the optimal strategy, the dark brown granules (12 months storage) restored their bioactivities to previous state, in terms of COD removal efficiency (97.44%) and specific oxygen uptake rate (40.63 mg O₂/g SS h⁻¹). The results shed light on the future practical application of stored aerobic granules as bioseed for reactor fast start-up.

Keywords: Aerobic granules; Organic loading rate (OLR); Ammonia concentration; Shear force; Specific oxygen utilization rate (SOUR)

Adaptation mechanisms of bacteria during the degradation of polychlorinated biphenyls in the presence of natural and synthetic terpenes as potential degradation inducers by Slavomíra Zorádová-Murínová; Hana Dudášová; Lucia Lukáčová; Milan Čertík; Katarína Šilharová; Branislav Vrana; Katarína Dercová (pp. 1375-1385).

In this study, we examined the effect of polychlorinated biphenyls (PCBs) in the presence of natural and synthetic terpenes and biphenyl on biomass production, lipid accumulation, and membrane adaptation mechanisms of two PCB-degrading bacterial strains Pseudomonas stutzeri and Burkholderia xenovorans LB400. According to the results obtained, it could be concluded that natural terpenes, mainly those contained in ivy leaves and pine needles, decreased adaptation responses induced by PCBs in these strains. The adaptation processes under investigation included growth inhibition, lipid accumulation, composition of fatty acids, cis/trans isomerization, and membrane saturation. Growth inhibition effect decreased upon addition of these natural compounds to the medium. The amount of unsaturated fatty acids that can lead to elevated membrane fluidity increased in both strains after the addition of the two natural terpene sources. The cells adaptation changes were more prominent in the presence of carvone, limonene, and biphenyl than in the presence of natural terpenes, as indicated by growth inhibition, lipid accumulation, and cis/trans isomerization. Addition of biphenyl and carvone simultaneously with PCBs increased the trans/cis ratio of fatty acids in membrane fractions probably as a result of fluidizing effects of PCBs. This stimulation is more pronounced in the presence of PCBs as a sole carbon source. This suggests that PCBs alone have a stronger effect on bacterial membrane adaptation mechanisms than when added together with biphenyl or natural or synthetic terpenes.

Keywords: Membrane lipids; Polychlorinated biphenyls; Adaptation; Bacteria; Environmental stress; Terpenes; Stimulators

Metabolic pathway analysis of Scheffersomyces (Pichia) stipitis: effect of oxygen availability on ethanol synthesis and flux distributions by Pornkamol Unrean; Nhung H. A. Nguyen (pp. 1387-1398).

Elementary mode analysis (EMA) identifies all possible metabolic states of the cell metabolic network. Investigation of these states can provide a detailed insight into the underlying metabolism in the cell. In this study, the flux states of Scheffersomyces (Pichia) stipitis metabolism were examined. It was shown that increasing oxygen levels led to a decrease of ethanol synthesis. This trend was confirmed by experimental evaluation of S. stipitis in glucose–xylose fermentation. The oxygen transfer rate for an optimal ethanol production was 1.8 mmol/l/h, which gave the ethanol yield of 0.40 g/g and the ethanol productivity of 0.25 g/l/h. For a better understanding of the cell’s regulatory mechanism in response to oxygenation levels, EMA was used to examine metabolic flux patterns under different oxygen levels. Up- and downregulation of enzymes in the network during the change of culturing condition from oxygen limitation to oxygen sufficiency were identified. The results indicated the flexibility of S. stipitis metabolism to cope with oxygen availability. In addition, relevant genetic targets towards improved ethanol-producing strains under all oxygenation levels were identified. These targeted genes limited the metabolic functionality of the cell to function according to the most efficient ethanol synthesis pathways. The presented approach is promising and can contribute to the development of culture optimization and strain engineers for improved lignocellulosic ethanol production by S. stipitis.

Keywords: Scheffersomyces (Pichia) stipitis metabolism; Oxygenation; Elementary mode analysis; Metabolic network analysis; Ethanol production

Erratum to: Biotechnology and the improvement of silage (tropical and temperate) rumen digestion: a mini-review by D. A. Flores (pp. 1399-1399).

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Applied Microbiology and Biotechnology (v.94, #5)

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Applied Microbiology and Biotechnology (v.94, #5)