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Applied Microbiology and Biotechnology (v.81, #1)

Artificial enzymes, “Chemzymes”: current state and perspectives by Jeannette Bjerre; Cyril Rousseau; Lavinia Marinescu; Mikael Bols (pp. 1-11).

Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models that successfully perform Michaelis–Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called “Chemzymes”, based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well as chemzymes that catalyze conjugate additions, cycloadditions, and self-replicating processes. The focus will be mainly on cyclodextrin-based chemzymes since they have shown to be good candidate structures to base an enzyme model skeleton on. In addition hereto, other molecules that encompass binding properties will also be presented.

Keywords: Supramolecular; Cyclodextrin; Biomimetic; Enzyme model; Catalysis

Synthesis and application of dipeptides; current status and perspectives by Makoto Yagasaki; Shin-ichi Hashimoto (pp. 13-22).

The functions and applications of l-α-dipeptides (dipeptides) have been poorly studied compared with proteins or amino acids. Only a few dipeptides, such as aspartame (l-aspartyl-l-phenylalanine methyl ester) and l-alanyl-l-glutamine (Ala-Gln), are commercially used. This can be attributed to the lack of an efficient process for dipeptide production though various chemical or chemoenzymatic method have been reported. Recently, however, novel methods have arisen for dipeptide synthesis including a nonribosomal peptide-synthetase-based method and an l-amino acid α-ligase-based method, both of which enable dipeptides to be produced through fermentative processes. Since it has been revealed that some dipeptides have unique physiological functions, the progress in production methods will undoubtedly accelerate the applications of dipeptides in many fields. In this review, the functions and applications of dipeptides, mainly in commercial use, and methods for dipeptide production including already proven processes as well as newly developed ones are summarized. As aspartame and Ala-Gln are produced using different industrial processes, the manufacturing processes of these two dipeptides are compared to clarify the characteristics of each procedure.

Keywords: Dipeptide; l-Amino acid α-ligase; NRPS; Aspartame; l-Alanyl-l-glutamine

In situ removal and purification of biosurfactants by automated surface enrichment by Julia Glazyrina; Stefan Junne; Peter Thiesen; Klaus Lunkenheimer; Peter Goetz (pp. 23-31).

A new method is described to remove and separate biosurfactants from complex mixtures by compressing and harvesting the liquid surface layer. This method was applied to Bacillus subtilis cultures, in which the lipopeptide antibiotic fengycin as well as the polyketide antibiotic bacillaene were produced. The automated harvesting and collection in a custom-built glass body called ‘flounder’ was repeated several hundred times. The fengycin concentration in the fractions was found to be four times higher than in the culture centrifugate. Of the overall fengycin, 50% (w/w) were recovered after 300 cycles, 95% (w/w) after 800 harvesting cycles. A separation of fengycin from the less surface-active bacillaene could be achieved due to stronger surface activity of fengycin. The ratio of partition coefficients of fengycin and bacillaene was nine times higher compared to foam fractionation. A stepwise increase of the equilibrium surface tension in the centrifugate from 29 to 33 mN/m indicated a fractionated separation of different surface-active substances. The utilization of cell containing culture broth instead of centrifugate had only slight effects on separation efficiency. These results demonstrate the possibility to separate biosurfactants directly from cultivation without the use of extraction solvents or foam formation.

Keywords: Biosurfactant; Separation; Bacillus subtilis ; Antibiotic; Fengycin; Bacillaene

High-level expression of a functional humanized single-chain variable fragment antibody against CD25 in Pichia pastoris by Lin Wan; Huawei Cai; Hao Yang; Yanrong Lu; Yingying Li; Xiaowei Li; Shengfu Li; Jie Zhang; Youping Li; Jingqiu Cheng; Xiaofeng Lu (pp. 33-41).

CD25 is a marker molecule expressed on many T and B cell neoplasms and on activated T cells that mediate organ transplant rejection and many autoimmune diseases. Single-chain variable fragments (scFvs) have many advantages over whole antibodies for use in antibody-targeted immunotherapy due to their small size. Daclizumab is an Food and Drug Administration-approved humanized anti-CD25 antibody. We attempted to produce a daclizumab-derived scFv, designated as Dmab(scFv), in Pichia pastoris. The Dmab(scFv) gene was designed based on the variable regions of the heavy and light chains of daclizumab, cloned into the yeast vector pPIC9K, and expressed in P. pastoris strain GS115. Under optimized conditions (culture medium pH, 6.0–6.5; methanol concentration added daily, 3.0%; inoculum density, OD₆₀₀ = 60; induction time point, 72–96 h), the yield of soluble recombinant Dmab(scFv) was approximately 80 mg l⁻¹. Flow cytometry, immunohistochemistry, and Western blotting revealed that the purified Dmab(scFv) could bind strongly to the membrane of CD25-positive cells, including SNT-8 cells, ConA-stimulated human peripheral blood mononuclear cells (hPBMCs), and rat splenocytes, but not to CD25-negative cells, including Raji cells, unstimulated hPBMCs, and rat splenocytes. These results suggest that Dmab(scFv) produced in P. pastoris is active and specific toward CD25-positive cells and has potential for use in CD25-targeted immunotherapy.

Keywords: Single-chain variable fragment; Immunotherapy; CD25; Pichia pastoris

Expression of serotonin derivative synthetic genes on a single self-processing polypeptide and the production of serotonin derivatives in microbes by Munyoung Park; Kiyoon Kang; Sangkyu Park; Young Soon Kim; Sun-Hwa Ha; Shin Woo Lee; Mi-Jeong Ahn; Jung-Myung Bae; Kyoungwhan Back (pp. 43-49).

The plant-specific serotonin derivatives feruloylserotonin (FS) and 4-coumaroylserotonin (CS) are synthesized by the enzymes 4-coumarate:coenzyme A ligase (4CL) and serotonin N-hydroxycinnamoyltransferase (SHT). To express these genes coordinately, SHT was fused in-frame with the self-processing FDMV 2A sequence followed by 4CL in a single open reading frame and introduced into Escherichia coli or Saccharomyces cerevisiae. The transgenes were abundantly expressed in both recombinant microbes, but functional expression was achieved only in yeast, with cleavage at the 2A sequence yielding monomeric SHT-2A and 4CL as judged by immunoblot and product analyses. In the presence of an exogenous supply of precursors such as serotonin and ferulic acid, the recombinant yeast synthesized 4.5 mg l⁻¹ FS in the medium while 0.02 mg l⁻¹ FS was produced in the cells. Time-course analysis indicated peak accumulation of FS at 24 h after induction, and this level was maintained until 96 h. The optimum precursor concentration was 2 mM. A series of serotonin derivatives was produced by adding various cinnamate derivative precursors with serotonin; 2.5 mg l⁻¹ caffeoylserotonin (CaS) and 1.4 mg l⁻¹ CS were produced, whereas no sinapoylserotonin or cinnamoylserotonin was yielded.

Keywords: Serotonin derivatives; 2A sequence of foot-and-mouth disease virus; Co-ordinate expression; Feruloylserotonin; Escherichia coli ; Saccharomyces cerevisiae

Cloning, expression, and characterization of an aldehyde dehydrogenase from Escherichia coli K-12 that utilizes 3-Hydroxypropionaldehyde as a substrate by Ji-Eun Jo; Subramanian Mohan Raj; Chelladurai Rathnasingh; Edwardraja Selvakumar; Woo-Chel Jung; Sunghoon Park (pp. 51-60).

3-Hydroxypropionaldehyde (3-HPA), an intermediary compound of glycerol metabolism in bacteria, serves as a precursor to 3-Hydroxypropionic acid (3-HP), a commercially valuable platform chemical. To achieve the effective conversion of 3-HPA to 3-HP, an aldH gene encoding an aldehyde dehydrogenase in Escherichia coli K-12 (AldH) was cloned, expressed, and characterized for its properties. The recombinant AldH exhibited broad substrate specificity for various aliphatic and aromatic aldehydes. AldH preferred NAD⁺ over NADP⁺ as a cofactor for the oxidation of most aliphatic aldehydes tested. Among the aldehydes used, the specific activity was highest (38.1 U mg⁻¹ protein) for 3-HPA at pH 8.0 and 37 °C. The catalytic efficiency (k _cat) and the specificity constant (k _cat/K _m) for 3-HPA in the presence of NAD⁺ were 28.5 s⁻¹ and 58.6 × 10³ M⁻¹ s⁻¹, respectively. The AldH activity was enhanced in the presence of disulfide reductants such as dithiothreitol (DTT) or 2-mercaptoethanol, while several metal ions, particularly Hg²⁺, Ag⁺, Cu²⁺, and Zn²⁺, inhibited AldH activity. This study illustrates that AldH is a potentially useful enzyme in converting 3-HPA to 3-HP.

Keywords: Aldehyde dehydrogenase; aldH ; 3-Hydroxypropionaldehyde dehydrogenase; 3-Hydroxypropionic acid; Enzyme characterization; Escherichia coli

Cloning, purification, and characterization of a thermostable α-l-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari by Sabriye Canakci; Murat Kacagan; Kadriye Inan; Ali Osman Belduz; Badal C. Saha (pp. 61-68).

The gene, AbfAC26Sari, encoding an α-l-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari, was isolated, cloned, sequenced, and characterizated. On the basis of amino acid sequence similarities, this 57-kDa enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. Characterization of the purified recombinant α-l-arabinofuranosidase produced in Escherichia coli BL21 revealed that it is active at a broad pH range (pH 4.5 to 9.0) and at a broad temperature range (45–85°C) and it has an optimum pH of 5.5 and an optimum temperature of 65°C. Kinetic experiment at 65°C with p-nitrophenyl α-l-arabinofuranoside as a substrate gave a V _max and K _m values of 1,019 U/mg and 0.139 mM, respectively. The enzyme had no apparent requirement of metal ions for activity, and its activity was strongly inhibited by 1 mM Cu²⁺ and Hg²⁺. The recombinant arabinofuranosidase released l-arabinose from arabinan, arabinoxylan, oat spelt xylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. Endoarabinanase activity was not detected. These findings suggest that AbfAC26Sari is an exo-acting enzyme.

Keywords: α-l-Arabinofuranosidase; Anoxybacillus kestanbolensis ; Arabinan; Arabinoxylan; Arabinooligosaccharides

A cyanophycin synthetase from Thermosynechococcus elongatus BP-1 catalyzes primer-independent cyanophycin synthesis by Toshinobu Arai; Kuniki Kino (pp. 69-78).

Cyanophycin synthesis is catalyzed by cyanophycin synthetase (CphA). It was believed that CphA requires l-aspartic acid (Asp), l-arginine (Arg), ATP, Mg²⁺, and a primer (low-molecular mass cyanophycin) for cyanophycin synthesis and catalyzes the elongation of a low-molecular mass cyanophycin. Despite extensive studies of cyanophycin, the mechanism of primer supply is still unclear, and already-known CphAs were primer-dependent enzymes. In the present study, we found that recombinant CphA from Thermosynechococcus elongatus BP-1 (Tlr2170 protein) catalyzed in vitro cyanophycin synthesis in the absence of a primer. The Tlr2170 protein showed strict substrate specificity toward Asp and Arg. The optimum pH was 9.0, and Mg²⁺ or Mn²⁺ was essential for cyanophycin synthesis. KCl enhanced the cyanophycin synthesis activity of the Tlr2170 protein; in contrast, dithiothreitol did not. The Tlr2170 protein appeared to be a 400 ± 9 kDa homo-tetramer. The Tlr2170 protein showed thermal stability and retained its 80% activity after a 60-min incubation at 50°C. In addition, we examined cyanophycin synthesis at 30°C, 40°C, 50°C, and 60°C. SDS-PAGE analysis showed that the molecular mass of cyanophycin increased with increased reaction temperature.

Keywords: Cyanophycin synthetase; Primer independent; Thermosynechococcus elongatus BP-1

Heterologous expression of lipase in Escherichia coli is limited by folding and disulfide bond formation by Yali Xu; Amrita Yasin; Raymond Tang; Jeno M. Scharer; Murray Moo-Young; C. Perry Chou (pp. 79-87).

Functional expression of lipase B from Pseudozyma antarctica (PalB) in the cytoplasm of Escherichia coli BL21(DE3) and its mutant derivative Origami B(DE3) was explored. Coexpression of DsbA was found to be effective in enhancing PalB expression. The improvement was particularly pronounced with Origami B(DE3) as a host, suggesting that both folding and disulfide bond formation may be major factors limiting PalB expression. Fusion tag technique was also explored by constructing several PalB fusions for the evaluation of their expression performance. While the solubility was enhanced for most PalB fusions, only the DsbA tag was effective in boosting PalB activity, possibly by both enhanced solubility and correct disulfide bond formation. Our results suggest that PalB activity is closely associated with correct disulfide bond formation, and increased solubilization by PalB fusions does not necessarily result in activity enhancement.

Keywords: Disulfide bond formation; Fusion tag; Escherichia coli ; Lipase; Recombinant protein production

Production of 2-methyl-1-butanol in engineered Escherichia coli by Anthony F. Cann; James C. Liao (pp. 89-98).

Recent progress has been made in the production of higher alcohols by harnessing the power of natural amino acid biosynthetic pathways. Here, we describe the first strain of Escherichia coli developed to produce the higher alcohol and potential new biofuel 2-methyl-1-butanol (2MB). To accomplish this, we explored the biodiversity of enzymes catalyzing key parts of the isoleucine biosynthetic pathway, finding that AHAS II (ilvGM) from Salmonella typhimurium and threonine deaminase (ilvA) from Corynebacterium glutamicum improve 2MB production the most. Overexpression of the native threonine biosynthetic operon (thrABC) on plasmid without the native transcription regulation also improved 2MB production in E. coli. Finally, we knocked out competing pathways upstream of threonine production (ΔmetA, Δtdh) to increase its availability for further improvement of 2MB production. This work led to a strain of E. coli that produces 1.25 g/L 2MB in 24 h, a total alcohol content of 3 g/L, and with yields of up to 0.17 g 2MB/g glucose.

Keywords: Metabolic engineering; Biofuel; E. coli

Induction of ortho- and meta-cleavage pathways in Pseudomonas in biodegradation of high benzoate concentration: MS identification of catabolic enzymes by Bin Cao; Anli Geng; Kai-Chee Loh (pp. 99-107).

The degradation pathways of benzoate at high concentration in Pseudomonas putida P8 were directly elucidated through mass spectrometric identification of some key catabolic enzymes. Proteins from P. putida P8 grown on benzoate or succinate were separated using two-dimensional gel electrophoresis. For cells grown on benzoate, eight distinct proteins, which were absent in the reference gel patterns from succinate-grown cells, were found. All the eight proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as catabolic enzymes involved in benzoate degradation. Among them, CatB (EC5.5.1.1), PcaI (EC2.8.3.6), and PcaF (EC2.3.1.174) were the enzymes involved in the ortho-cleavage pathway; DmpC (EC1.2.1.32), DmpD (EC3.1.1.-), DmpE (EC4.2.1.80), DmpF (EC1.2.1.10), and DmpG (EC4.1.3.-) were the meta-cleavage pathway enzymes. In addition, enzyme activity assays showed that the activities of both catechol 1,2-dioxygenase (C12D; EC1.13.11.1) and catechol 2,3-dioxygenase (C23D; EC1.13.11.2) were detected in benzoate-grown P. putida cells, undoubtedly suggesting the simultaneous expression of both the ortho- and the meta-cleavage pathways in P. putida P8 during the biodegradation of benzoate at high concentration.

Keywords: Benzoate biodegradation; Ortho-cleavage pathway; Meta-cleavage pathway; Pseudomonas putida ; 2-DE; MALDI-TOF MS

Heterologous production of epothilones B and D in Streptomyces venezuelae by Sung Ryeol Park; Je Won Park; Won Seok Jung; Ah Reum Han; Yeon-Hee Ban; Eun Ji Kim; Jae Kyung Sohng; Sang Jun Sim; Yeo Joon Yoon (pp. 109-117).

Epothilones, produced from the myxobacterium Sorangium cellulosum, are potential anticancer agents that stabilize microtubules in a similar manner to paclitaxel. The entire epothilone biosynthetic gene cluster was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin polyketide synthase gene cluster. The resulting strains produced approximately 0.1 μg/l of epothilone B as a sole product after 4 days cultivation. Deletion of an epoF encoding the cytochrome P450 epoxidase gave rise to a mutant that selectively produces 0.4 μg/l of epothilone D. To increase the production level of epothilones B and D, an additional copy of the positive regulatory gene pikD was introduced into the chromosome of both S. venezuleae mutant strains. The resulting strains showed enhanced production of corresponding compounds (approximately 2-fold). However, deletion of putative transport genes, orf3 and orf14 in the epothilone D producing S. venezuelae mutant strain, led to an approximately 3-fold reduction in epothilone D production. These results introduce S. venezuelae as an alternative heterologous host for the production of these valuable anticancer agents and demonstrate the possibility of engineering this strain as a generic heterologous host for the production of polyketides and hybrid polyketide-nonribosomal peptides.

Keywords: Epothilone; Heterologous production; Streptomyces venezuelae

Antagonistic effect of divalent cations Ca²⁺ and Mg²⁺ on the morphological development of Streptomyces hygroscopicus var. geldanus by Lynne F. Dobson; Daniel G. O’Shea (pp. 119-126).

Supplementation of the divalent cations calcium and magnesium to submerged cultures of Streptomyces hygroscopicus var. geldanus greatly influenced morphological development and secondary metabolite synthesis. The disparate response could be explained in terms of the differential effects of Ca²⁺ and Mg²⁺ ions on cell surface hydrophobicity. Cultures supplemented with calcium ions were found to be hydrophobic, which resulted in cell concentration-dependent aggregation. In contrast, those grown in a magnesium-rich medium were found to be hydrophilic with the organism growing as freely dispersed filaments that synthesised geldanamycin at an optimal rate in comparison to hydrophobic pellets.

Keywords: Calcium; Geldanamycin; Hydrophobicity; Magnesium; Morphology; Streptomyces hygroscopicus

Characterization of developmental colony formation in Corynebacterium glutamicum by Hideaki Takano; Akiko Shimizu; Rina Shibosawa; Rie Sasaki; Shino Iwagaki; Osamu Minagawa; Kazuki Yamanaka; Kiyoshi Miwa; Teruhiko Beppu; Kenji Ueda (pp. 127-134).

We report that Corynebacterium glutamicum colonies exhibit a developmental transition in culture. When cultured on a routinely used complete medium (CM2B), this bacterium first formed a flat translucent colony. Subsequently, some parts of this colony expanded to form small spherical yellow colonies that finally developed into a single large yellow colony. The small flat colony consisted of long thick cells, which were occasionally V or Y shaped, while the large yellow colony consisted of short small rods. A similar colony development pattern was observed in Corynebacterium ammoniagenes and Corynebacterium callunae. Analysis following shotgun cloning revealed that the introduction of a multi-copy-number plasmid carrying amtR, a global transcriptional regulator for nitrogen metabolism, into C. glutamicum cells induced precocious colony development. An amtR-null C. glutamicum mutant exhibited delayed development. Detailed observations of C. glutamicum cells cultured on CM2B medium containing buffers at various pH values revealed that the colony growth was rapid at a pH value of 6.4 or higher and slow but distinct at a pH of less than 6.4. This pH threshold increased to 6.8 following the addition of 0.1% glucose into the medium.

Keywords: Corynebacterium glutamicum ; Morphology; Development; Ammonia; amtR

Modeling of growth kinetics for Pseudomonas putida during toluene degradation by N.-C. Choi; J.-W. Choi; S.-B. Kim; D.-J. Kim (pp. 135-141).

Glucose has been often used as a secondary substrate to enhance the degradation of primary substrate as well as the increase of biomass, especially for the inhibitory range of substrate concentration. In this study, we investigated the effect of glucose concentration on growth kinetics of Pseudomonas putida during toluene degradation for a wide concentration range (60–250 mg/l). Batch microcosm studies were conducted in order to monitor bacterial growth for three different initial concentrations (2, 5, 10 mg/ml) of glucose for a given toluene concentration. Modeling of growth kinetics was also performed for each growth curve of glucose dose using both Monod and Haldane kinetics. Batch studies revealed that bacterial growth showed a distinct inhibitory phase above some limit (∼170 mg/l) for the lowest (2 mg/ml) glucose dose, but the degree of inhibition decreased as the glucose dose increased, leading to three different growth patterns. The bacterial growth followed each of the modified Wayman and Tseng, Wayman and Tseng, and Luong model as the glucose dose increased from 2 to 10 mg/ml. This indicates that glucose has a prominent influence on bacterial growth during toluene degradation and that different kinetics should be adopted for each broth condition.

Keywords: Growth kinetics; Toluene degradation; Glucose; Inhibition

Coffee pulp koji of Aspergillus sojae as stable immobilized catalyst of chlorogenate hydrolase by Osao Adachi; Yoshitaka Ano; Yoshihiko Akakabe; Emiko Shinagawa; Kazunobu Matsushita (pp. 143-151).

Chlorogenate hydrolase (EC 3.1.1.42, CHase) was highly induced in mycelia of Aspergillus sojae AKU 3312 grown in Czapek medium containing either instant coffee powder or coffee pulp as inducer. No CHase formation was observed in the mycelia when cultivated without the inducer. CHase was purified readily from CHase-induced mycelia to high homogeneity, and the purified CHase revealed the molecular weight of 180,000 consisting of two identical subunits of 88 kDa. Equimolar quinate (QA) and caffeate (CA) were confirmed on hydrolysis of chlorogenate (CGA). The purified CHase was only useful for a laboratory scale hydrolysis of CGA. For practical QA and CA production using scaled up hydrolysis of vegetable extracts of natural CGA resources, the enzyme activity of purified CHase decreased and denatured irreversibly. Preparation of coffee pulp koji and its application to QA and CA production were proposed instead of purified CHase. When coffee pulp koji was heated at 60°C for 30 min, CHase survived without any appreciable loss of enzyme activity while vegetative mycelial growth and spore germination were terminated. The heated coffee pulp koji thus prepared was effective itself as stable immobilized catalyst of CHase for QA and CA production from vegetable CGA resources such as coffee powders, coffee pulp, and others.

Keywords: Aspergillus sojae ; Caffeate; Chlorogenate hydrolase; Coffee pulp koji (molded coffee pulp); Quinate

Effect of bulk liquid BOD concentration on activity and microbial community structure of a nitrifying, membrane-aerated biofilm by Leon S. Downing; Robert Nerenberg (pp. 153-162).

Membrane-aerated biofilms (MABs) are an effective means to achieve nitrification and denitrification of wastewater. In this research, microsensors, fluorescence in situ hybridization (FISH), and modeling were used to assess the impact of bulk liquid biological oxygen demand (BOD) concentrations on the activity and microbial community structure of nitrifying MABs. With 1 g m⁻³ BOD in the bulk liquid, the nitrification rate was 1.3 g N m⁻² day⁻¹, slightly lower than the 1.5 g N m⁻² day⁻¹ reported for no bulk liquid BOD. With bulk liquid BOD concentrations of 3 and 10 g m⁻³, the rates decreased to 1 and 0.4 g N m⁻² day⁻¹, respectively. The percent denitrification increased from 20% to 100% when the BOD increased from 1 to 10 g m⁻³ BOD. FISH results indicated increasing abundance of heterotrophs with increasing bulk liquid BOD, consistent with the increased denitrification rates. Modeling was used to assess the effect of BOD on nitrification rates and to compare an MAB to a conventional biofilm. The model-predicted nitrification rates were consistent with the experimental results. Also, nitrification in the MAB was much less sensitive to BOD inhibition than the conventional biofilm. The MAB achieved concurrent nitrification and denitrification, whereas little denitrification occurred in the conventional biofilm.

Keywords: Nitrification; Denitrification; Wastewater; BOD inhibition; Membrane-aerated biofilm reactor; Membrane biofilm reactor

Diversity of the resident microbiota in a thermophilic municipal biogas plant by Agnes Weiss; Valérie Jérôme; Ruth Freitag; Helmut K. Mayer (pp. 163-173).

Biogas plants continuously convert biological wastes mainly into a mixture of methane, CO₂ and H₂O—a conversion that is carried out by a consortium of bacteria and archaea. Especially in the municipal plants dedicated towards waste treatment, the reactor feed may vary considerably, exposing the resident microbiota to a changing variety of substrates. To evaluate how and if such changes influence the microbiology, an established biogas plant (6,600 m³, up to 600 m³ biogas per h) was followed over the course of more than 2 years via polymerase chain reaction–denaturing gradient gel electrophoresis of 16S rRNA genes and subsequent sequencing. Both the bacterial and the archaeal community remained stable over the investigation. Of the bacterial consortium, about half of the sequences were in decreasing order of occurrence: Thermoacetogenium sp., Anaerobaculum mobile, Clostridium ultunense, Petrotoga sp., Lactobacillus hammesii, Butyrivibrio sp., Syntrophococcus sucromutans, Olsenella sp., Tepidanaerobacter sp., Sporanaerobacter acetigenes, Pseudoramibacter alactolyticus, Lactobacillus fuchuensis or Lactobacillus sakei, Lactobacillus parabrevis or Lactobacillus spicheri and Enterococcus faecalis. The other half matched closely to ones from similar habitats (thermophilic anaerobic methanogenic digestion). The archaea consisted of Methanobrevibacter sp., Methanoculleus bourgensis, Methanosphaera stadtmanae, Methanimicrococcus blatticola and uncultured Methanomicrobiales. The role of these species in methane production is discussed.

Keywords: Biogas plant; Cloning; DGGE; Methanogenic consortium; Microbiota

Quantitative analysis of isoprenoid diphosphate intermediates in recombinant and wild-type Escherichia coli strains by T. Vallon; S. Ghanegaonkar; O. Vielhauer; A. Müller; C. Albermann; G. Sprenger; M. Reuss; K. Lemuth (pp. 175-182).

In biotechnology, the heterologous biosynthesis of isoprenoid compounds in Escherichia coli is a field of great interest and growth. In order to achieve higher isoprenoid yields in heterologous E. coli strains, it is necessary to quantify the pathway intermediates and adjust gene expression. In this study, we developed a precise and sensitive nonradioactive method for the simultaneous quantification of the isoprenoid precursors farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) in recombinant and wild-type E. coli cells. The method is based on the dephosphorylation of FPP and GGPP into the respective alcohols and involves their in situ extraction followed by separation and detection using gas chromatography–mass spectrometry. The integration of a geranylgeranyl diphosphate synthase gene into the E. coli chromosome leads to the accumulation of GGPP, generating quantities as high as those achieved with a multicopy expression vector.

Keywords: Isoprenoid diphosphate; Quantification; Geranylgeranyl diphosphate synthase; Heterologous expression; Chromosomal integration

Application of sandwich ELISA for detecting tag fusion proteins in high throughput by Zhu-wei Xu; Tao Zhang; Chao-jun Song; Qi Li; Ran Zhuang; Kun Yang; An-gang Yang; Bo-quan Jin (pp. 183-189).

Based on a series of mAbs against four frequently used tags—the human Ig Fc fragment, GST, maltose-binding protein, and thioredoxin—we developed corresponding sandwich enzyme-linked immunosorbent assay (ELISA) to detect these tag fusion proteins. As a supplement for Western blot, the successfully established ELISA was specific, sensitive, quantitative, easy to perform, time-saving, and last but not least, suitable for high-throughput screening of tag fusion proteins. Determination of soluble tag fusion proteins expressed by various systems with the sandwich ELISA developed in the present study could be a valuable and promising tool for the wide application of tag-protein fusion systems in the rapidly growing field of proteomics research.

Keywords: ELISA; Fusion tag; Tag fusion protein

Phage Mu-driven two-plasmid system for integration of recombinant DNA in the Methylophilus methylotrophus genome by Elena G. Abalakina; Irina L. Tokmakova; Natalya V. Gorshkova; Evgueni R. Gak; Valerii Z. Akhverdyan; Sergey V. Mashko; Yurgis A. V. Yomantas (pp. 191-200).

A phage Mu-driven two-plasmid system for DNA integration in Escherichia coli genome has been adjusted for Methylophilus methylotrophus. Constructed helper plasmids with broad-host-range replicons carry thermo-inducible genes for transposition factors MuA and MuB. Integrative plasmids that are only replicated in E. coli could be mobilized to M. methylotrophus and contained mini-Mu unit with a short terminus of Mu DNA, Mu-attL/R. Mini-Mu unit was integrated in the M. methylotrophus genome via mobilization of the integrative plasmid to the cells carrying the helper in conditions of thermo-induced expression of MuA and MuB. In this system, mini-Mu unit was mainly integrated due to replicative transposition, and the integrated copy could be amplified in the M. methylotrophus chromosome in the presence of helper plasmid. A kan-gene flanked by FRT sites was inserted in one of the mini-Mu units, and it could be readily excised by yeast FLP recombinase that is encoded by the designed plasmid. The multiple Mu-driven gene insertion was carried out by integration of the Bacillus amyloliquefaciens α-amylase gene followed by curing the Km^R marker before integration of the second mini-Mu unit with Pseudomonas putida xylE gene encoding catechol 2,3-dioxygenase (C23O).

Keywords: Methylotrophs; Mini-Mu unit; Replicative transposition; Amplification; Excisable marker

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Applied Microbiology and Biotechnology (v.81, #1)

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Applied Microbiology and Biotechnology (v.81, #1)