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Applied Microbiology and Biotechnology (v.73, #4)

Ecological and biotechnological aspects of lichens by Ilona Oksanen (pp. 723-734).

Lichens and the partners from three different kingdoms are both taxonomically and physiologically a very diverse group, which makes them interesting from both ecological and biotechnological points of view. A lichen is a mutual ecophysiological innovation in many extreme environments in which symbiosis seems to protect the partners. Lichen’s ability to grow in harsh environments can be advantageous, resulting in important ecological niches, or disadvantageous when lichens occupy and cause biodeterioration of cultural monuments. Recently, new candidate compounds for drugs, UVB protection, and antifreeze proteins for frozen foods were discovered. Lichens were also found to have potential in bioplastic degradation and prevention of desertification. Nevertheless, there is still large potential for further industrial screening and research on lichen products. Due to improved culture techniques of isolated symbionts, increased knowledge of their secondary metabolism and improved methods for solubilizing lichen metabolites, the screening and activity tests can be implemented more easily today than in the past.

Keywords: Lichen; Green algae; Cyanobacteria; Fungi; Symbiosis; Natural product

The promise of synthetic biology by Jürgen Pleiss (pp. 735-739).

DNA synthesis has become one of the technological bases of a new concept in biology: synthetic biology. The vision of synthetic biology is a systematic, hierarchical design of artificial, biology-inspired systems using robust, standardized, and well-characterized building blocks. The design concept and examples from four fields of application (genetic circuits, protein design, platform technologies, and pathway engineering) are discussed, which demonstrate the usefulness and the promises of synthetic biology. The vision of synthetic biology is to develop complex systems by simplified solutions using available material and knowledge. Synthetic biology also opens a door toward new biomaterials that do not occur in nature.

Keywords: Modular design; Genetic circuits; Protein design; DNA synthesis

In vivo engineering of proteins with nitrogen-containing tryptophan analogs by Sandra Lepthien; Birgit Wiltschi; Bojana Bolic; Nediljko Budisa (pp. 740-754).

Recently, it has become possible to reprogram the protein synthesis machinery such that numerous noncanonical amino acids can be translated into target sequences yielding tailor-made proteins. The canonical amino acid tryptophan (Trp) encoded by a single nucleotide triplet (UGG) is a particularly interesting target for protein engineering and design. Trp-residues can be substituted with a variety of analogs and surrogates generated biosynthetically or by organic chemistry. Among them, nitrogen-containing tryptophan analogs occupy a central position, as they have distinct chemical properties in comparison with aliphatic amines and imines. They resemble purine bases of DNA and share their capacity for pH-sensitive intramolecular charge transfer. These special properties of the analogs can be directly transmitted into related protein structures via in vivo ribosome-mediated translation. Proteins expressed in this way are further endowed with unique properties like new spectral, altered redox and titration features or might serve as useful biomaterials. We present and discuss current works and future developments in protein engineering with nitrogen-containing tryptophan analogs and related compounds as well as their relevance for academic and applicative research.

Keywords: Amino- and azatryptophan; Genetic code; Nucleobase fluorescence; Protein design and engineering; DNA mimicry

Application of kaolin to improve citric acid production by a thermophilic Aspergillus niger by Sikander Ali (pp. 755-762).

Citric acid production by a thermophilic strain of the filamentous fungus Aspergillus niger IIB-6 in a medium containing blackstrap cane molasses was improved by the addition of kaolin to the fermentation medium. The fermentation was run in a 7.5-l stirred bioreactor (60% working volume). The optimal sugar concentration was found to be 150 g/l. Kaolin (1.0 ml) was added to the fermentation medium to enhance volumetric production. The best results in terms of product formation were observed when 15 parts per million (ppm) kaolin was added 24 h after inoculation. With added kaolin, citric acid production was enhanced 2.34-fold, compared to a control fermentation without added kaolin. The length of incubation to attain this product yield was shortened from 168 to 96 h. The comparison of kinetic parameters showed improved citrate synthase activity of the culture (Y _p/x=7.046 g/g). When the culture grown at various kaolin concentrations was monitored for Q _p, Q _s, and q _p, there was significant improvement in these variables over the control. Specific production by the culture (q _p=0.073 g/g cells/h) was improved several fold. The addition of kaolin substantially improved the enthalpy (ΔH _D=74.5 kJ/mol) and entropy of activation (ΔS=−174 J/mol/K) for citric acid production, free energies for transition state formation, and substrate binding for sucrose hydrolysis. The performance of fuzzy logic control of the bioreactor was found to be very promising for an improvement (≈4.2-fold) in the production of citric acid (96.88 g/l), which is of value in commercial applications.

Keywords: Citric acid; Aspergillus niger ; Kaolin; Fermentation; Molasses; Fuzzy logic control

Production of hydrolysate with antioxidative activity by enzymatic hydrolysis of extruded corn gluten by Xi-qun Zheng; Li-te Li; Xiao-lan Liu; Xiao-jie Wang; Jie Lin; Di Li (pp. 763-770).

Hydrolysate of extruded corn gluten with higher solubility and antioxidative property was prepared. Extrusion and starch removal of corn gluten were applied as pretreatment before enzymatic hydrolysis by Alcalase. The amylase hydrolysis of starch at 70°C for 3 h resulted in the removal of the starch from the extruded corn gluten. The best hydrolysis results can be obtained by conducting the hydrolysis at 60°C with water addition 20 g/g protein, enzyme addition 0.048 Ansen units/g protein, pH 8.5, and 120 min. Degree of hydrolysis of extruded and nonextruded corn gluten reached 39.54 and 31.16%, respectively, under the optimal condition. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the optimal hydrolysate revealed that proteolysis of extruded corn gluten was more extensive than proteolysis of its counterpart which was not subjected to extrusion. The molecular weight of the peptides in the optimal hydrolysate was mainly over 3,710–660 Da as determined by gel filtration chromatography. The hydrolysates displayed good solubility and antioxidative activity. The separation profile of the hydrolysate on an ion exchange chromatography of Q-Sepharose Fast Flow showed that many kinds of peptides had antioxidative effect. A new peptide with antioxidative activity was purified, and its amino acid sequence was Phe-Pro-Leu-Glu-Met-Met-Pro-Phe, which was identified by Q-TOF2 mass spectrometry.

Keywords: Corn gluten; Enzyme; Extrusion; Hydrolysate; Molecular weight distribution; Solubility; Antioxidative activity

Metabolism of isoeugenol via isoeugenol-diol by a newly isolated strain of Bacillus subtilis HS8 by Yongmei Zhang; Ping Xu; Shuai Han; Haiqin Yan; Cuiqing Ma (pp. 771-779).

A bacterium designated as HS8 was newly isolated from soil based on its ability to degrade isoeugenol. The strain was identified as Bacillus subtilis according to its 16S rDNA sequence analysis and biochemical characteristics. The metabolic pathway for the degradation of isoeugenol was examined. Isoeugenol-diol, for the first time, was detected as an intermediate from isoeugenol to vanillin by a bacterial strain. Isoeugenol was converted to vanillin via isoeugenol-diol, and vanillin was then metabolized via vanillic acid to guaiacol by strain HS8. These metabolites, vanillin, vanillic acid, and guaiacol, are all valuable aromatic compounds in flavor production. At the same time, the bipolymerization of isoeugenol was observed, which produced dehydrodiisoeugenol and decreased the vanillin yield. High level of vanillic acid decarboxylase activity was detected in cell-free extract. These findings provided a detailed profile of isoeugenol metabolism by a B. subtilis strain for the first time, which would improve the production of valuable aromatic compounds by biotechnology.

Keywords: Isoeugenol; Isoeugenol-diol; Vanillin; Metabolic pathway; Bacillus subtilis

Stability of plasmid and expression of a recombinant gonadotropin-releasing hormone (GnRH) vaccine in Escherichia coli by Jinshu Xu; Wenjia Li; Jie Wu; Yin Zhang; Zheng Zhu; Jingjing Liu; Zhuoyi Hu (pp. 780-788).

In our previous studies, the recombinant gonadotropin-releasing hormone (GnRH) peptide was constructed into a T7 RNA polymerase-based expression system. The recombinant gene encoding GnRH3-hinge-MVP, which contained three repeated GnRH units, a fragment of hinge region (225-232/225′-232′), and a T cell epitope of measles virus protein, was cloned into Escherichia coli BL21 harboring pED-GnRH3. The high activity of T7 RNA polymerase could make the expression system very powerful for high-level expression of the recombinant protein. However, during the large-scale production of recombinant protein, the productivity of the fermentation process was directly affected by many factors, such as plasmid stability, protein production, and culture conditions. In this study, we studied the effects of various culture conditions on the plasmid stability and the target protein yield including selective pressure, the time of induction by lactose, and the number of successive cultures. The results indicate that the plasmid instability may be caused by a loss of plasmid rather than structural change. However, to go down to future generations, engineered bacteria have the stability of plasmid and protein yield to a large extent. The amount of the fusion protein was also up to 40% of the total cell protein after the 50th generation. These data would be useful for the industrial production of the recombinant GnRH vaccine.

Keywords: The recombinant GnRH peptide; Plasmid stability; High-level expression; Anti-GnRH antibody responses

Growth characteristics of Rhodopseudomonas palustris cultured outdoors, in an underwater tubular photobioreactor, and investigation on photosynthetic efficiency by Pietro Carlozzi; Benjamin Pushparaj; Alessandro Degl’Innocenti; Antonella Capperucci (pp. 789-795).

The underwater tubular photobioreactor is a fully controlled outdoor system to study photosynthetic bacteria. Before growing bacteria cells outdoors, two modified van Niel medium (vN-A, vN-B) were tested under artificial light. During exponential growth, the specific growth rates were 0.0416 and 0.0434 h⁻¹, respectively; vN-B was chosen for outdoor experiments. The growth behavior of Rhodopseudomonas palustris was investigated under a natural light–dark cycle (sunrise–sunset, 15L/9D) and a forced light–dark cycle (9:00–19:00, 10L/14D). Relationships between solar radiations, daily growth rates, and biomass output rates were also investigated. After determining the elemental biomass molar composition and its combustion heat, some trends of photosynthetic efficiency (PE) were obtained over daylight. The PE trends were always of the oscillatory type, with the exception of that achieved at low biomass concentration. Under a natural light/dark cycle, the maximum PE (11.2%) was attained at sunset, while under a forced light/dark cycle, the highest PE (8.5%) was achieved in the morning. Three initial biomass concentrations were investigated (0.65, 1.01, and 1.54 g l⁻¹). The stoichiometric equation for bacteria cells indicated that 87.7% of the carbon of acetic acid was converted to biomass and only 12.3% was lost as CO₂.

Keywords: Photobioreactors; Rhodopseudomonas palustris ; Photosynthetic efficiency

Biochemical characterization of the decaprenyl diphosphate synthase of Rhodobacter sphaeroides for coenzyme Q₁₀ production by Hossein Shahbani Zahiri; Kambiz Akbari Noghabi; Yong Chul Shin (pp. 796-806).

Coenzyme Q₁₀ (CoQ₁₀), like other CoQs of various organisms, plays indispensable roles not only in energy generation but also in several other processes required for cells’ survival. In this study, a gene encoding for a decaprenyl diphosphate synthase (Rsdds) was cloned from Rhodobacter sphaeroides in Escherichia coli. The in vivo catalytic activity and product specificity of Rsdds were compared with those of a counterpart enzyme from Agrobacterium tumefaciens (Atdds) in E. coli as a heterologous host. In contrast with Atdds, Rsdds showed lower catalytic activity but higher product specificity for CoQ₁₀ production, as indicated by the amount of CoQ₉ formation. The higher product specificity of Rsdds was also confirmed by utilizing both Rsdds and Atdds for in vitro synthesis of polyprenyl diphosphates. Thin layer chromatography indicated that the Rsdds enzyme resulted in relatively much less solanesyl diphosphate formation. The purified Rsdds catalyzed the addition of isopentenyl diphosphate to dimethyl allyl diphosphate, geranyl diphosphate, ω,E,E-farnesyl diphosphate (FPP), and ω,E,E,E-geranylgeranyl diphosphate as priming substrates. The kinetic parameters of V _max (pmol/min), K _M (μM), k _cat (1/min), and k _cat /K _M of the enzyme using FPP as the most appropriate substrate were determined to be 264.6, 13.1, 8.8, and 0.67, respectively.

Molecular cloning and characterization of two intracellular β-glucosidases belonging to glycoside hydrolase family 1 from the basidiomycete Phanerochaete chrysosporium by Takeshi Tsukada; Kiyohiko Igarashi; Makoto Yoshida; Masahiro Samejima (pp. 807-814).

cDNAs encoding two glycoside hydrolase family 1 β-glucosidases (BGL1A and BGL1B) were cloned from the basidiomycete Phanerochaete chrysosporium, and the substrate specificities of the recombinant enzymes and the expression patterns of the two genes were investigated in relation to cellobiose metabolism by the fungus. The cDNA sequences contained open reading frames of 1,389 base pairs (bp) (bgl1A) and 1,623 bp (bgl1B), encoding 462 and 530 amino acids, respectively. Although high sequence identity (65%) was observed between the deduced amino acid sequences of the two enzymes, an apparent difference was observed at the C-terminal region: BGL1B has a 63-amino acid extension, which has no similarity with any known protein. Both recombinant enzymes expressed in Escherichia coli showed hydrolytic activity towards several β-glycosidic compounds. However, the substrate recognition patterns of the two enzymes were quite different from each other. In particular, cellobiose was hydrolyzed more effectively by BGL1B than by BGL1A. The expression of the two genes in the fungus was monitored by reverse transcription-PCR, which showed that bgl1A was expressed constitutively in both glucose- and cellobiose-containing culture, whereas bgl1B was expressed in cellobiose culture but was repressed in glucose culture, possibly because of carbon catabolite repression. We conclude that BGL1B contributes to cellobiose metabolism during cellulose degradation by P. chrysosporium.

Keywords: Phanerochaete chrysosporium ; Beta-glucosidase; Glycoside hydrolase; Cellulose degradation

Identification and characterization of IS1 transposition in plasmid amplification mutants of E. coli clones producing DNA vaccines by Kristala L. Jones Prather; M. Celina Edmonds; Jerrell W. Herod (pp. 815-826).

Merck Research Laboratories has developed a highly productive Escherichia coli fermentation process to produce plasmid DNA for use as vaccines. The process consists of a fed-batch fermentation in a chemically defined medium. Initiation of the feed stream precedes a growth-limited phase in which plasmid DNA is amplified. The fermentation is only maximally productive for a small fraction of E. coli transformants designated as high-producers, while the predominant low-producer population does not amplify plasmid DNA. In experiments undertaken to probe this phenomenon, transposition of the 768-bp E. coli insertion sequence IS1 into an HIV DNA vaccine vector was observed in several low-producer clones. IS1 was found to insert in or near the neomycin resistance gene in nearly a dozen unique sites from within a single population of plasmid molecules. The fraction of IS1-containing plasmids within several clones was determined by quantitative polymerase chain reaction and was found to increase with increasing cultivation time in the chemically defined medium. Because transposition into an antibiotic-resistance gene is unlikely to affect plasmid amplification, the genomes of high- and low-producers of three different HIV DNA vaccine vectors were subsequently profiled by restriction fragment length polymorphism analysis. In all three cases, IS1 insertional mutations were found in the genomes of the predominant low-producers, while the genomes of the high-producers were indistinguishable from untransformed cells. The insertions reside on similarly sized fragments for two of the low-producer clones, and the fragment size is smaller for the third clone. The third clone also produces much less plasmid DNA than a typical low-producer. The results suggest the presence of an IS1 insertional mutation that affects plasmid replication and amplification, possibly in a position-dependent manner.

Keywords: DNA vaccine; IS1 transposition; Q-PCR; RFLP

Orthric Rieske dioxygenases for degrading mixtures of 2,4-dinitrotoluene/naphthalene and 2-amino-4,6-dinitrotoluene/4-amino-2,6-dinitrotoluene by Brendan G. Keenan; Thomas K. Wood (pp. 827-838).

Pollutants are frequently found as mixtures yet it is difficult to engineer enzymes with broad substrate ranges on aromatics. Inspired by the archetypal nitroarene dioxygenase, which shares its electron transport with a salicylate monooxygenase, we have created an innovative and general approach to expand the substrate range of dioxygenase enzymes in a single cell. We have developed here a series of novel, hybrid dioxygenase enzymes that function with a single ferredoxin reductase and ferredoxin that are used to transport two electrons from nicotinamide adenine dinucleotide to the two independent terminal oxygenases. Each independent alpha-oxygenase may then be used simultaneously to create orthric enzymes that degrade mixtures of environmental pollutants. Specifically, we created a hybrid dioxygenase system consisting of naphthalene dioxygenase/dinitrotoluene dioxygenase to simultaneously degrade 2,4-dinitrotoluene and naphthalene (neither enzyme alone had significant activity on both compounds) and dinitrotoluene dioxygenase/nitrobenzene dioxygenase to simultaneously degrade the frequently encountered 2,4,6-trinitrotoluene reduction products 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene.

Keywords: Dioxygenases; Dinitrotoluene; Naphthalene; Hybrid enzyme

Differential regulation of manganese peroxidases and characterization of two variable MnP encoding genes in the white-rot fungus Physisporinus rivulosus by Terhi K. Hakala; Kristiina Hildén; Pekka Maijala; Cia Olsson; Annele Hatakka (pp. 839-849).

Manganese peroxidase (MnP) production in the white-rot basidiomycete Physisporinus rivulosus T241i was studied. Separate MnP isoforms were produced in carbon-limited liquid media supplemented with Mn²⁺, veratryl alcohol, or sawdust. The isoforms had different pH ranges for the oxidation of Mn²⁺ and 2,6-dimethoxyphenol. Although lignin degradation by white-rot fungi is often triggered by nitrogen depletion, MnPs of P. rivulosus were efficiently produced also in the presence of high-nutrient nitrogen, especially in cultures supplemented with veratryl alcohol. Two MnP encoding genes, mnpA and mnpB, were identified, and their corresponding cDNAs were characterized. Structurally, the genes showed marked dissimilarity, and the expression of the two genes implicated quantitative variation and differential regulation in response to manganese, veratryl alcohol, or sawdust. The variability in regulation and properties of the isoforms may widen the operating range for efficient lignin degradation by P. rivulosus.

Keywords: Manganese peroxidase; White-rot fungi; Physisporinus rivulosus ; Gene expression; Mn²⁺ oxidation; Lignin degradation

A novel GH43 α-l-arabinofuranosidase from Humicola insolens: mode of action and synergy with GH51 α-l-arabinofuranosidases on wheat arabinoxylan by Hanne R. Sørensen; Christel T. Jørgensen; Carsten H. Hansen; Christian I. Jørgensen; Sven Pedersen; Anne S. Meyer (pp. 850-861).

A novel α-l-arabinofuranosidase (α-AraF) belonging to glycoside hydrolase (GH) family 43 was cloned from Humicola insolens and expressed in Aspergillus oryzae. ¹H-NMR analysis revealed that the novel GH43 enzyme selectively hydrolysed (1→3)-α-l-arabinofuranosyl residues of doubly substituted xylopyranosyl residues in arabinoxylan and in arabinoxylan-derived oligosaccharides. The optimal activity of the cloned enzyme was at pH 6.7 and 53 °C. Two other novel α-l-arabinofuranosidases (α-AraFs), both belonging to GH family 51, were cloned from H. insolens and from the white-rot basidiomycete Meripilus giganteus. Both GH51 enzymes catalysed removal of (1→2) and (1→3)-α-l-arabinofuranosyl residues from singly substituted xylopyranosyls in arabinoxylan; the highest arabinose yields were obtained with the M. giganteus enzyme. Combinations (50:50) of the GH43 α-AraF from H. insolens and the GH51 α-AraFs from either M. giganteus or H. insolens resulted in a synergistic increase in arabinose release from water-soluble wheat arabinoxylan in extended reactions at pH 6 and 40 °C. This synergistic interaction between GH43 and GH51 α-AraFs was also evident when a GH43 α-AraF from a Bifidobacterium sp. was supplemented in combination with either of the GH51 enzymes. The synergistic effect is presumed to be a result of the GH51 α-AraFs being able to catalyse the removal of single-sitting (1→2)–α-l-arabinofuranosyls that resulted after the GH43 enzyme had catalysed the removal of (1→3)–α-l-arabinofuranosyl residues on doubly substituted xylopyranosyls in the wheat arabinoxylan.

Keywords: Arabinoxylan hydrolysis; GH43 α-l-arabinofuranosidase; GH51 α-l-arabinofuranosidase; Synergy; Meripilus giganteus ; Humicola insolens

Complete decolorization of the anthraquinone dye Reactive blue 5 by the concerted action of two peroxidases from Thanatephorus cucumeris Dec 1 by Yasushi Sugano; Yuko Matsushima; Makoto Shoda (pp. 862-871).

It is useful to identify and examine organisms that may prove useful for the treatment of dye-contaminated wastewater. Here, we report the purification and characterization of a new versatile peroxidase (VP) from the decolorizing microbe, Thanatephorus cucumeris Dec 1 (TcVP1). The purified TcVP1 after Mono P column chromatography showed a single band at 43 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Amino acid sequencing revealed that the N terminus of TcVP1 had the highest homology to Trametes versicolor MPG1, lignin peroxidase G (LiPG) IV, Bjerkandera adusta manganese peroxidase 1 (MnP1), and Bjerkandera sp. RBP (12 out of 14 amino acid residues, 86% identity). Mn²⁺ oxidizing assay revealed that TcVP1 acted like a classical MnP at pH ∼5, while dye-decolorizing and oxidation assays of aromatic compounds revealed that the enzyme acted like a LiP at pH ∼3. TcVP1 showed particularly high decolorizing activity toward azo dyes. Furthermore, coapplication of TcVP1 and the dye-decolorizing peroxidase (DyP) from T. cucumeris Dec 1 was able to completely decolorize a representative anthraquinone dye, Reactive blue 5, in vitro. This decolorization proceeded sequentially; DyP decolorized Reactive blue 5 to light red-brown compounds, and then TcVP1 decolorized these colored intermediates to colorless. Following extended reactions, the absorbance corresponding to the conjugated double bond from phenyl (250–300 nm) decreased, indicating that aromatic rings were also degraded. These findings provide important new insights into microbial decolorizing mechanisms and may facilitate the future development of treatment strategies for dye wastewater.

Keywords: Decolorization; DyP; Versatile peroxidase; Anthraquinone dye

Production of a chimeric enzyme tool associating the Trichoderma reesei swollenin with the Aspergillus niger feruloyl esterase A for release of ferulic acid by Anthony Levasseur; Markku Saloheimo; David Navarro; Martina Andberg; Frédéric Monot; Tiina Nakari-Setälä; Marcel Asther; Eric Record (pp. 872-880).

The main goals of this work were to produce the fusion protein of the Trichoderma reesei swollenin I (SWOI) and Aspergillus niger feruloyl esterase A (FAEA) and to study the effect of the physical association of the fusion partners on the efficiency of the enzyme. The fusion protein was produced up to 25 mg l⁻¹ in the T. reesei strains Rut-C30 and CL847. In parallel, FAEA alone was produced for use as a control protein in application tests. Recombinant FAEA and SWOI–FAEA were purified to homogeneity and characterized. The biochemical and kinetic characteristics of the two recombinant proteins were found to be similar to those of native FAEA, except for the temperature stability and specific activity of the SWOI–FAEA. Finally, the SWOI–FAEA protein was tested for release of ferulic acid from wheat bran. A period of 24 h of enzymatic hydrolysis with the SWOI–FAEA improved the efficiency of ferulic acid release by 50% compared with the results obtained using the free FAEA and SWOI. Ferulic acid is used as an antioxidant and flavor precursor in the food and pharmaceutical industries. This is the first report of a potential application of the SWOI protein fused with an enzyme of industrial interest.

Keywords: Trichoderma reesei ; Chimeric protein; Swollenin; Feruloyl esterase; Biotechnology; Fungus

Selection and characterisation of a xylitol-derepressed Aspergillus niger mutant that is apparently impaired in xylitol transport by Peter J. I. van de Vondervoort; Marco J. L. de Groot; George J. G. Ruijter; Jaap Visser (pp. 881-886).

Aspergillus niger is known for its biotechnological applications, such as the use of xylanase enzyme for the degradation of hemicellulose. Depending on culture conditions, several polyols may also be accumulated, such as xylitol during D-xylose oxidation. Also during industrial fermentation of xylose for the production of fuel ethanol by recombinant yeast, xylitol is a by-product. We studied xylitol metabolism by isolating mutants that have impaired xylitol-mediated repression. Genetic and biochemical characterisation revealed that one of these mutants was affected not only in xylitol-mediated carbon repression, but also had impaired xylitol transport.

Keywords: Mutant selection; Xylitol transport; Gene regulation; Pentose repression; Xylose fermentation

Genome-scale in silico aided metabolic analysis and flux comparisons of Escherichia coli to improve succinate production by Qingzhao Wang; Xun Chen; Yudi Yang; Xueming Zhao (pp. 887-894).

In the post-genome era, it is one challenge to understand the cellular metabolism at the systematic levels. Mathematical modeling of microorganisms and subsequent computer simulation are effective tools for systems biology. In this paper, based on the genome-scale Escherichia coli stoichiometric model iJR904, through the GAMS linear programming package, the in silico maximal succinate yield was estimated to be 1.714 mol/mol glucose. When another two constraints were added, the maximal succinate yield dropped to 1.60 mol/mol glucose. Further analysis substantiated the uniqueness of the flux distribution under such constraints. After comparisons with the metabolic flux analysis (MFA) results computed from the wet experimental data of the three kinds of E. coli, three potential improvement target sites, the glucose phosphotransferase transport system, the pyruvate carboxylase, and the glyoxylate shunt, were identified and selected for the genetic modifications. All the three genetic modified strains showed increased succinate yield. The final strain TUQ19/pQZ6 had a high yield of 1.29 mol succinate/mol glucose and high productivity. The success of the above experiments proved that this in silico optimal succinate production pathway is reasonable and practical. This method may also be used as a general strategy to help enhance the yields of other favorable metabolites in E. coli.

An acetoin-regulated expression system of Bacillus subtilis by Jörg Silbersack; Britta Jürgen; Michael Hecker; Bernd Schneidinger; Rainer Schmuck; Thomas Schweder (pp. 895-903).

An expression system, which is based on the promoter of the acoABCL operon of Bacillus subtilis was developed and characterized. The acoABCL operon codes for the acetoin dehydrogenase complex, which is the major enzyme system responsible for the catabolism of acetoin in B. subtilis. Besides weak organic acids, the neutral overflow metabolite acetoin is metabolized by the cells in the early stationary phase. Transcription of reporter gene fusions with the acoA promoter of this operon is strongly repressed by glucose but induced by acetoin as soon as the preferred carbon source glucose is exhausted. The co-expression of an additional copy of the regulator gene acoR led to more than twofold higher activity of the acoA promoter. It is demonstrated that the induction of this promoter in growing cells with acetoin is possible with non-phosphotransferase system sugars as carbon and energy source and in a ccpA mutant background. Moreover, it could be shown that the activity of the acoA-directed expression system correlates with the level of acetoin in the medium. During glucose limitation, the utilization of the alternative energy source acetoin keeps the protein synthesis machinery of B. subtilis cells active and thus allows for a long lasting acoA-controlled expression of recombinant genes.

Keywords: Bacillus subtilis ; acoABCL operon; Expression system; Fed-batch fermentation; Acetoin

Heterologous protein secretion in Lactococcus lactis is enhanced by the Bacillus subtilis chaperone-like protein PrsA by Agneta Lindholm; Ulla Ellmén; Marja Tolonen-Martikainen; Airi Palva (pp. 904-914).

The Bacillus subtilis lipoprotein PrsA enhances the yield of several homologous and heterologous exported proteins in B. subtilis by being involved in the posttranslocational stage of the secretion process. In this work, we have studied the effect of B. subtilis PrsA on the secretion of Bacillus amyloliquefaciens α-amylase (AmyQ), a target protein for PrsA, and Bacillus licheniformis penicillinase (PenP) a nontarget protein for PrsA, in Lactococcus lactis. Two compatible plasmids were constructed and introduced into L. lactis strain NZ9000: one high copy plasmid, expressing the AmyQ gene (amyQ) or the PenP gene (penP), and one low copy plasmid, expressing the PrsA encoding gene (prsA). When amyQ and prsA were simultaneously expressed under the nisin-inducible promoter P nisA, Western blotting experiments revealed a 15- to 20-fold increase in the total yield of AmyQ and a sixfold increase in secreted AmyQ activity, compared to a control strain lacking prsA. When expressed under the same induction conditions, PrsA had no effect on the secretion or total yield of PenP. These results show that the secretion yield of some heterologous proteins can be significantly increased in L. lactis when coproduced with the B. subtilis PrsA protein.

Keywords: Protein secretion; Lactococcus lactis ; PrsA; Bacillus subtilis ; α-Amylase; Penicillinase

Effective cellulose production by a coculture of Gluconacetobacter xylinus and Lactobacillus mali by Akira Seto; Yu Saito; Mayumi Matsushige; Hiroki Kobayashi; Yasuyuki Sasaki; Naoto Tonouchi; Takayasu Tsuchida; Fumihiro Yoshinaga; Kenji Ueda; Teruhiko Beppu (pp. 915-921).

A microbial colony that contained a marked amount of cellulose was isolated from vineyard soil. The colony was formed by the associated growth of two bacterial strains: a cellulose-producing acetic acid bacterium (st-60-12) and a lactic acid bacterium (st-20). The 16S rDNA-based taxonomy indicated that st-60-12 belonged to Gluconacetobacter xylinus and st-20 was closely related to Lactobacillus mali. Cocultivation of the two organisms in corn steep liquor/sucrose liquid medium resulted in a threefold higher cellulose yield when compared to the st-60-12 monoculture. A similar enhancement was observed in a coculture with various L. mali strains but not with other Lactobacillus spp. The enhancement of cellulose production was most remarkable when sucrose was supplied as the substrate. L. mali mutants for exocellular polysaccharide (EPS) production were defective in promoting cellulose production, but the addition of EPS to the monoculture of st-60-12 did not affect cellulose productivity. Scanning electron microscopic observation of the coculture revealed frequent association between the st-60-12 and L. mali cells. These results indicate that cell–cell interaction assisted by the EPS-producing L. mali promotes cellulose production in st-60-12.

Structure of microbial communities performing the simultaneous reduction of Fe(II)EDTA.NO²⁻and Fe(III)EDTA⁻ by Rajkumari Kumaraswamy; J. Gijs Kuenen; Robbert Kleerebezem; Mark C. M. van Loosdrecht; Gerard Muyzer (pp. 922-931).

BioDeNOx is a combined physicochemical and biological process for the removal of nitrogen oxides (NOx) from flue gas. In the present study, two anaerobic bioreactors performing BioDeNOx were run consecutively (RUN-1 and RUN-2) at a dilution rate of 0.01 h⁻¹ with Fe(II)EDTA.NO²⁻ and Fe(III)EDTA⁻ as electron acceptors and ethanol as electron donor. The measured protein concentration of the reactor biomass of both runs was 120 mg/l. Different molecular methods were used to determine the identity and abundance of the bacterial populations in both bioreactors. Bacillus azotoformans strain KT-1 was recognized as a key player in Fe(II)EDTA.NO²⁻ reduction. PCR-denaturing gradient gel electrophoresis analysis of the reactor biomass showed a greater diversity in RUN-2 than in RUN-1. Enrichments of Fe(II)EDTA.NO²⁻ and Fe(III)EDTA⁻ reducers and activity assays were conducted using the biomass from RUN-2 as an inoculum. The results on substrate turnover, overall microbial diversity, and enrichments and finally activity assays confirmed that ethanol was used as electron donor for Fe(II)EDTA.NO²⁻ reduction. In addition, the Fe(III)EDTA⁻ reduction rate of the microbial community proved to be feasible enough to run the bioreactors, ruling out the chemical reduction of Fe(III)EDTA⁻ with sulfide as was proposed by other researchers.

Keywords: Nitrogen oxides; BioDeNOx; DGGE; Bacillus azotoformans ; Denitrification

Fungal bioconversion of toxic polychlorinated biphenyls by white-rot fungus, Phlebia brevispora by Ichiro Kamei; Shigenori Sonoki; Koichi Haraguchi; Ryuichiro Kondo (pp. 932-940).

Toxic coplanar polychlorinated biphenyls (Co-PCBs) were used as substrates for a degradation experiment with white-rot fungus, Phlebia brevispora TMIC33929, which is capable of degrading polychlorinated dibenzo-p-dioxins. Eleven PCB congener mixtures (7 mono-ortho- and 4 non-ortho-PCBs) were added to the cultures of P. brevispora and monitored by high resolution gas chromatography and mass spectrometry (HRGC/HRMS). Five PCB congeners, 3,3′,4,4′-tetrachlorobiphenyl, 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl were degraded by P. brevispora. To investigate the fungal metabolism of PCB, each Co-PCB was treated separately by P. brevispora and the metabolites were analyzed by gas chromatography and mass spectrometry (GC/MS) and identified on the basis of the GC/MS comparison with the authentic compound. Meta-methoxylated metabolite was detected from the culture containing each compound. Additionally, para-dechlorinated and -methoxylated metabolite was also detected from the culture with 2,3,3′,4,4′-pentachlorobiphenyl, 2,3′,4,4′,5-pentachlorobiphenyl, and 2,3′,4,4′,5,5′-hexachlorobiphenyl, which are mono-ortho-PCBs. In this paper, we identified the congener specific degradation of coplanar PCBs by P. brevispora, and clearly proved for the first time by identifying the metabolites that the white-rot fungus, P. brevispora, transformed recalcitrant coplanar PCBs.

Keywords: P450; Coplanar PCBs; White rot fungi; Bioremediation; Phlebia brevispora; Hydroxylation

Degradation of carbazole and its derivatives by a Pseudomonas sp. by Li Li; Qinggang Li; Fuli Li; Quan Shi; Bo Yu; Fengrui Liu; Ping Xu (pp. 941-948).

Carbazole, carbazoles with monomethyl or dimethyls substituted on different positions (C₁-carbazoles or C₂-carbazoles), and benzocarbazoles, as toxic and mutagenic components of petroleum and creosote contamination, were biodegradable by an isolated bacterial strain Pseudomonas sp. XLDN4-9. C₁-carbazoles were degraded in preference to carbazole and C₂-carbazoles. The biodegradation of C₁-carbazoles or C₂-carbazoles was influenced by the positions of methyl substitutions. Among C₁-carbazole isomers, 1-methyl carbazole was the most susceptible. C₂-carbazole isomers with substitutions on the same benzo-nucleus were more susceptible at a concentration of less than 3.4 μg g⁻¹ petroleum, especially when harboring one substitution on position 1. In particular, 1,5-dimethyl carbazole was the most recalcitrant dimethyl isomer.

Keywords: Biodegradation; Carbazole; Carbazole derivatives; Pseudomonas sp.; Angular dioxygenase

Oil biodegradation by Bacillus strains isolated from the rock of an oil reservoir located in a deep-water production basin in Brazil by Claudia Duarte da Cunha; Alexandre S. Rosado; Gina V. Sebastián; Lucy Seldin; Irene von der Weid (pp. 949-959).

Sixteen spore forming Gram-positive bacteria were isolated from the rock of an oil reservoir located in a deep-water production basin in Brazil. These strains were identified as belonging to the genus Bacillus using classical biochemical techniques and API 50CH kits, and their identity was confirmed by sequencing of part of the 16S rRNA gene. All strains were tested for oil degradation ability in microplates using Arabian Light and Marlin oils and only seven strains showed positive results in both kinds of oils. They were also able to grow in the presence of carbazole, n-hexadecane and polyalphaolefin (PAO), but not in toluene, as the only carbon sources. The production of key enzymes involved with aromatic hydrocarbons biodegradation process by Bacillus strains (catechol 1,2-dioxygenase and catechol 2,3-dioxygenase) was verified spectrophotometrically by detection of cis,cis-muconic acid and 2-hydroxymuconic semialdehyde, and results indicated that the ortho ring cleavage pathway is preferential. Furthermore, polymerase chain reaction (PCR) products were obtained when the DNA of seven Bacillus strains were screened for the presence of catabolic genes encoding alkane monooxygenase, catechol 1,2-dioxygenase, and/or catechol 2,3-dioxygenase. This is the first study on Bacillus strains isolated from an oil reservoir in Brazil.

Keywords: Oil; Biodegradation; Bacillus strains

Bioaugmenting anaerobic digestion of biosolids with selected strains of Bacillus, Pseudomonas, and Actinomycetes species for increased methanogenesis and odor control by Metin Duran; Nalan Tepe; Deniz Yurtsever; Vito L. Punzi; Charles Bruno; Raj J. Mehta (pp. 960-966).

The objective of this study was to evaluate the effects of bioaugmenting anaerobic biosolids digestion with a commercial product containing selected strains of bacteria from genera Bacillus, Pseudomonas, and Actinomycetes, along with ancillary organic compounds containing various micronutrients. Specifically, the effects of the bioaugment in terms of volatile solids destruction and generation and fate of odor-causing compounds during anaerobic digestion and during storage of the digested biosolids were studied. Two bench-scale anaerobic digesters receiving primary and secondary clarifier biosolids from various full-scale biological wastewater treatment plants were operated. One of the digesters received the bioaugment developed by Organica Biotech, while the other was operated as control. The bioaugmented digester generated 29% more net CH₄ during the 8 weeks of operation. In addition, the average residual propionic acid concentration in the bioaugmented digester was 54% of that in the control. The monitoring of two organic sulfide compounds, methyl mercaptan (CH₃SH) and dimethyl sulfide (CH₃SCH₃), clearly demonstrated the beneficial effects of the bioaugmentation in terms of odor control. The biosolids digested in the bioaugmented digester generated a negligible amount of CH₃SH during 10 days of post-digestion storage, while CH₃SH concentration in the control reached nearly 300 ppm_v during the same period. Similarly, peak CH₃SCH₃ generated by stored biosolids from the bioaugmented digester was only 37% of that from the control.

Keywords: Anaerobic digestion-biosolids; Bioaugmentation; Odor

Biosynthesis of poly(ε-l-lysine)s in two newly isolated strains of Streptomyces sp. by Hideo Hirohara; Munenori Takehara; Masayuki Saimura; Atsushi Ikezaki; Masahiro Miyamoto (pp. 967-967).

Growth of E. coli BL21 in minimal media with different gluconeogenic carbon sources and salt contents by Thusitha S. Gunasekera; Oleg Paliy (pp. 968-968).

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Applied Microbiology and Biotechnology (v.73, #4)