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Applied Microbiology and Biotechnology (v.73, #5)

PHA synthase engineering toward superbiocatalysts for custom-made biopolymers by Christopher T. Nomura; Seiichi Taguchi (pp. 969-979).

Poly-3-hydroxyalkanoates [P(3HA)s] are biologically produced polyesters that have attracted much attention as biodegradable polymers that can be produced from biorenewable resources. These polymers have many attractive properties for use as bulk commodity plastics, fishing lines, and medical uses that are dependent on the repeating unit structures. Despite the readily apparent benefits of using P(3HA)s as replacements for petrochemical-derived plastics, the use and distribution of P(3HA)s have been limited by their cost of production. This problem is currently being addressed by the engineering of enzymes involved in the production of P(3HA)s. Polyhydroxyalkanoate (PHA) synthase (PhaC) enzymes, which catalyze the polymerization of 3-hydroxyacyl-CoA monomers to P(3HA)s, were subjected to various forms of protein engineering to improve the enzyme activity or substrate specificity. This review covers the recent history of PHA synthase engineering and also summarizes studies that have utilized engineered PHA synthases.

Keywords: Polyhydroxyalkanoates; PHA synthase; Genetic engineering; In vitro evolution; Escherichia coli ; Ralstonia eutropha ; Arabidopsis thaliana ; PHA monomer-supplying enzymes

Biosynthesis and engineering of isoprenoid small molecules by Sydnor T. Withers; Jay D. Keasling (pp. 980-990).

Isoprenoid secondary metabolites are a rich source of commercial products that have not been fully explored. At present, there are isoprenoid products used in cancer therapy, the treatment of infectious diseases, and crop protection. All isoprenoids share universal prenyl diphosphate precursors synthesized via two distinct pathways. From these universal precursors, the biosynthetic pathways to specific isoprenoids diverge resulting in a staggering array of products. Taking advantage of this diversity has been the focus of much effort in metabolic engineering heterologous hosts. In addition, the engineering of the mevalonate pathway has increased levels of the universal precursors available for heterologous production. Finally, we will describe the efforts to produce to commercial terpenoids, paclitaxel and artemisinin.

Keywords: Isoprenoids; Terpenes; Synthetic biology; Metabolic engineering; Artemisinin

Modeling of enzyme production kinetics by Mani Thilakavathi; Tanmay Basak; Tapobrata Panda (pp. 991-1007).

Models of single cells, cell populations, and cultures can be most useful in organizing information in a comprehensive system description, as well as in optimizing and controlling actual production operations. Models discussed in this article are of various degrees of biological structure and mathematical complexity. The models are developed based on the biomass formation, substrate consumption, and product formation. The potentials and the limitations of all the models have been reported. The parameter estimation by different methods has been discussed in this communication. These parameters will be helpful to explore the areas where future-modeling studies may be especially valuable.

Keywords: Enzyme kinetics; Modeling; Parameters estimation; Sensitivity function; Structured model

Optimisation and comparison of liquid and dry formulations of the biocontrol yeast Pichia anomala J121 by Petter Melin; Sebastian Håkansson; Johan Schnürer (pp. 1008-1016).

The biocontrol yeast Pichia anomala J121 can effectively reduce mould growth on moist cereal grains during airtight storage. Practical use of microorganisms requires formulated products that meet a number of criteria. In this study we compared different formulations of P. anomala. The best way to formulate P. anomala was freeze-drying. The initial viability was as high as 80%, with trehalose previously added to the yeast. Freeze-dried products could be stored at temperatures as high as 30 °C for a year, with only a minor decrease in viability. Vacuum-drying also resulted in products with high storage potential, but the products were not as easily rehydrated as freeze-dried samples. Upon desiccating the cells using fluidised-bed drying or as liquid formulations, a storage temperature of 10 °C was required to maintain viability. Dependent on the type of formulation, harvesting of cells at different nutritional stresses affected the initial viabilities, e.g. the initial viability for fluidised-bed-dried cells was higher when the culture was fed with excess glucose, but for freeze-drying it was superior when cells were harvested after depletion of carbon. Using micro-silos we found that the biocontrol activity remained intact after drying, storage and rehydration for all formulations.

Keywords: Pichia anomala; Biocontrol; Formulation; Shelf-life; Trehalose

Fermentation of 1,3-propanediol by a lactate deficient mutant of Klebsiella oxytoca under microaerobic conditions by Guang Yang; Jiesheng Tian; Jilun Li (pp. 1017-1024).

Klebsiella oxytoca M5al is an excellent 1,3-propanediol (1,3-PD) producer, but too much lactic acid yielded greatly lessened the fermentation efficiency for 1,3-PD. To counteract the disadvantage, four lactate deficient mutants were obtained by knocking out the ldhA gene of lactate dehydrogenase (LDH) of K. oxytoca M5al. The LDH activities of the four mutants were from 3.85 to 6.92% of the parental strain. The fed-batch fermentation of 1,3-PD by mutant LDH3, whose LDH activity is the lowest, was studied. The results showed that higher 1,3-PD concentration, productivity, and molar conversion rate from glycerol to 1,3-PD can be gained than those of the wild type strain and no lactic acid is produced under both anaerobic and microaerobic conditions. Sucrose fed during the fermentation increased the conversion and sucrose added at the beginning increased the productivity. In fed-batch fermentation with sucrose as cosubstrate under microaerobic conditions, the 1,3-PD concentration, conversion, and productivity were improved significantly to 83.56 g l⁻¹, 0.62 mol mol⁻¹, and 1.61 g l⁻¹ h⁻¹, respectively. Furthermore, 60.11 g l⁻¹ 2,3-butanediol was also formed as major byproduct in the broth.

Biotransformation of isoeugenol to vanillin by Pseudomonas putida IE27 cells by Mamoru Yamada; Yukiyoshi Okada; Toyokazu Yoshida; Toru Nagasawa (pp. 1025-1030).

The ability to produce vanillin and/or vanillic acid from isoeugenol was screened using resting cells of various bacteria. The vanillin- and/or vanillic-acid-producing activities were observed in strains belonging to the genera Achromobacter, Aeromonas, Agrobacerium, Alcaligenes, Arthrobacter, Bacillus, Micrococcus, Pseudomonas, Rhodobacter, and Rhodococcus. Strain IE27, a soil isolate showing the highest vanillin-producing activity, was identified as Pseudomonas putida. We optimized the culture and reaction conditions for vanillin production from isoeugenol using P. putida IE27 cells. The vanillin-producing activity was induced by adding isoeugenol to the culture medium but not vanillin or eugenol. Under the optimized reaction conditions, P. putida IE27 cells produced 16.1 g/l vanillin from 150 mM isoeugenol, with a molar conversion yield of 71% at 20 °C after a 24-h incubation in the presence of 10% (v/v) dimethyl sulfoxide.

Keywords: Isoeugenol; Vanillin; Pseudomonas putida IE27

Regulation of avilamycin biosynthesis in Streptomyces viridochromogenes: effects of glucose, ammonium ion, and inorganic phosphate by Chuan-he Zhu; Fu-ping Lu; Ya-nan He; Zhen-lin Han; Lian-xiang Du (pp. 1031-1038).

Effects of glucose, ammonium ions and phosphate on avilamycin biosynthesis in Streptomyces viridochromogenes AS4.126 were investigated. Twenty grams per liter of glucose, 10 mmol/L ammonium ions, and 10 mmol/L phosphate in the basal medium stimulated avilamycin biosynthesis. When the concentrations of glucose, ammonium ions, and phosphate in the basal medium exceeded 20 g/L, 10 mmol/L, and 10 mmol/L, respectively, avilamycin biosynthesis greatly decreased. When 20 g/L glucose was added at 32 h, avilamycin yield decreased by 70.2%. Avilamycin biosynthesis hardly continued when 2-deoxy-glucose was added into the basal medium at 32 h. There was little influence on avilamycin biosynthesis with the addition of the 3-methyl-glucose (20 g/L) at 32 h. In the presence of excess (NH₄)₂SO₄ (20 mmol/L), the activities of valine dehydrogenase and glucose-6-phosphate dehydrogenase were depressed 47.7 and 58.3%, respectively, of that of the control at 48 h. The activity of succinate dehydrogenase increased 49.5% compared to the control at 48 h. The intracellular adenosine triphosphate level and 6-phosphate glucose content of S. viridochromogenes were 128 and 129%, respectively, of that of the control at 48 h, with the addition of the 40 mmol/L of KH₂PO₄. As a result, high concentrations of glucose, ammonium ions, and inorganic phosphate all led to the absence of the precursors for avilamycin biosynthesis and affected antibiotic synthesis.

Keywords: Regulation; Biosynthesis; Avilamycin; Streptomyces viridochromogenes AS4.126

High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant Saccharomyces cerevisiae by Kaisa Karhumaa; Romain Fromanger; Bärbel Hahn-Hägerdal; Marie-F. Gorwa-Grauslund (pp. 1039-1046).

Xylose fermentation performance was studied of a previously developed Saccharomyces cerevisiae strain TMB 3057, carrying high xylose reductase (XR) and xylitol dehydrogenase (XDH) activity, overexpressed non-oxidative pentose phosphate pathway (PPP) and deletion of the aldose reductase gene GRE3. The fermentation performance of TMB 3057 was significantly improved by increased ethanol production and reduced xylitol formation compared with the reference strain TMB 3001. The effects of the individual genetic modifications on xylose fermentation were investigated by comparing five isogenic strains with single or combined modifications. All strains with high activity of both XR and XDH had increased ethanol yields and significantly decreased xylitol yields. The presence of glucose further reduced xylitol formation in all studied strains. High activity of the non-oxidative PPP improved the xylose consumption rate. The results indicate that ethanolic xylose fermentation by recombinant S. cerevisiae expressing XR and XDH is governed by the efficiency by which xylose is introduced in the central metabolism.

Heat-inducible production of β-glucuronidase in tobacco hairy root cultures by Kung-Ta Lee; Shih-Cheng Chen; Bor-Luen Chiang; Takashi Yamakawa (pp. 1047-1053).

The production of β-glucuronidase (GUS) driven by the Arabidopsis small heat shock protein 18.2 promoter in liquid cultures of transgenic tobacco (Nicotiana tabacum) hairy roots is reported. Clone GD-3, showing high GUS heat induction and a moderate growth rate, was selected from 436 clones for study. Treatment of GD-3 with heat shock at 36–42°C for 2 h then recovery at 27°C resulted in an increase in GUS specific activity, while higher heat-shock temperatures led to a decline. These results were in accordance with the change in esterase activity, a measure of tissue viability. Using 2 h of 42°C heat shock and a recovery phase at 27°C, GUS specific activity increased rapidly and reached a maximum of 267.6 nmol 4-methylumbelliferyl β-D-glucuronic acid (MU) min⁻¹ mg⁻¹ protein at 24 h of recovery. When tissues were continuously heated at 42°C and tested without a recovery period, GUS mRNA was detectable at 2 h and peaked at 5 h, but GUS activity was not seen until 10 h and did not peak until 28 h; in addition, the maximum activity was lower than that seen after heat shock for only 30 min or 2 h, followed by recovery. This shows that recovery at normal temperature is crucial for the heat-inducible heterogeneous expression system of transgenic hairy roots. Multiple heat-shock treatments showed that this system was heat reinducible, although a gradual decline in GUS specific activity was seen in the second and third cycles.

Keywords: sHSP18.2 promoter; GUS; Transgenic hairy roots

Optimization of nutrient parameters for lovastatin production by Monascus purpureus MTCC 369 under submerged fermentation using response surface methodology by Sadik Ali Sayyad; Bibhu Prasad Panda; Saleem Javed; Mohd Ali (pp. 1054-1058).

Lovastatin, an inhibitor of HMG-CoA reductase, was produced by submerged fermentation using Monascus purpureus MTCC 369. Five nutritional parameters screened using Plackett–Burman experimental design were optimized by Box–Behnken factorial design of response surface methodology for lovastatin production in shake flask cultures. Maximum lovastatin production of 351 mg/l were predicted in medium containing 29.59 g/l dextrose, 3.86 g/l NH₄Cl, 1.73 g/l KH₂PO₄, 0.86 g/l MgSO₄·7H₂O, and 0.19 g/l MnSO₄·H₂O using response surface plots and point prediction tool of DESIGN EXPERT 7.0 (Statease, USA) software.

Keywords: Lovastatin; Monascus purpureus ; Optimization; Response surface methodology

Immobilization of bacteria in silica matrices using citric acid in the sol–gel process by Gisela S. Alvarez; Martín F. Desimone; Luis E. Diaz (pp. 1059-1064).

The aim of this work was to use citric acid in the sol–gel process to generate an inorganic polymer that allows bacterial survival for long periods of time and to study the influence of different storage temperatures. We compared gram-negative Escherichia coli and gram-positive Staphylococcus aureus, immobilized and preserved at different storage temperatures in silica matrices prepared by the method proposed. Immobilized E. coli and S. aureus in silica matrices were stored in sealed tubes at 20, 4, −20, and −70°C for 4 months during which the number of viable cells was analyzed. Results show that the immobilization in silica matrices using citric acid, to neutralize the alkalinity of the silica precursors, makes the technique not only biocompatible but also easier to perform since polymerization does not occur immediately as it does when hydrochloric acid is utilized.

Keywords: Citric acid; Escherichia coli ; Immobilization; Preservation; Sol–gel; Staphylococcus aureus

Cloning, expression, and characterization of a Baeyer–Villiger monooxygenase from Pseudomonas fluorescens DSM 50106 in E. coli by Anett Kirschner; Josef Altenbuchner; Uwe T. Bornscheuer (pp. 1065-1072).

A gene encoding a Baeyer–Villiger monooxygenase (BVMO) identified in Pseudomonas fluorescens DSM 50106 was cloned and functionally expressed in Escherichia coli JM109. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis showed an estimated 56 kDa-size protein band corresponding to the recombinant enzyme. Expression in BL21 (DE3) resulted mainly in the formation of inclusion bodies. This could be overcome by coexpression of molecular chaperones, especially the DnaK/DnaJ/GrpE complex, leading to increased production of soluble BVMO enzyme in recombinant E. coli. Examination of the substrate spectra using whole-cell biocatalysis revealed a high specificity of the BVMO for aliphatic open-chain ketones. Thus, octyl acetate, heptyl propionate, and hexyl butyrate were quantitatively formed from the corresponding ketone substrates. Several other esters were obtained in conversion >68%. Selected esters were also produced on preparative scale.

Keywords: Baeyer–Villiger monooxygenase; Aliphatic ketones; Pseudomonas fluorescens ; Cloning

Purification, characterization and cloning of aldehyde dehydrogenase from Rhodococcus erythropolis UPV-1 by Arrate Jaureguibeitia; Laura Saá; María J. Llama; Juan L. Serra (pp. 1073-1086).

The enzyme responsible for formaldehyde removal in industrial wastewaters by cells of Rhodococcus erythropolis UPV-1 was identified as a broad-specific aldehyde dehydrogenase (EC 1.2.1.3). The enzyme was purified to electrophoretic homogeneity from ethanol-grown cells with a specific activity of 19.5 U mg⁻¹ protein and an activity recovery of 56%. The enzyme showed an isoelectric point (pI) of 5.3 and was a trimer of 162 kDa consisting of three identical 54-kDa subunits. It was specific for NAD⁺ and showed hyperbolic kinetics for this coenzyme (K _m=90 μM), but sigmoidal kinetics for the aliphatic aldehydes used as substrates. The enzyme affinity for aldehydes increased with their hydrocarbon chain length, ranging from 333 μM for formaldehyde to 85 nM for n-octanal. The corresponding calculated Hill coefficients were in the 1.55–2.77 range. With n-propanal as substrate, the optimum pH and temperature for activity were 9.5–10.0 and 47.5°C, respectively, with an E _a for catalysis of 28.6 kJ mol⁻¹. NAD⁺ protected the enzyme against thermal inactivation, but aldehydes were ineffective. The activity was severely inhibited by p-hydroxymercuribenzoate, indicating that a thiol was essential for catalysis. The 1,524-bp aldhR gene encoding a 507-amino-acid protein was expressed in cells of Escherichia coli M15 as a hexahistidine-tagged protein.

Monoterpenes as novel substrates for oxidation and halo-hydroxylation with chloroperoxidase from Caldariomyces fumago by Bjoern-Arne Kaup; Umberto Piantini; Matthias Wüst; Jens Schrader (pp. 1087-1096).

Chloroperoxidase (CPO) from Caldariomyces fumago was analysed for its ability to oxidize ten different monoterpenes with hydrogen peroxide as oxidant. In the absence of halide ions geraniol and, to a lesser extent, citronellol and nerol were converted into the corresponding aldehydes, whereas terpene hydrocarbons did not serve as substrates under these conditions. In the presence of chloride, bromide and iodide ions, every terpene tested was converted into one or more products. (1S)-(+)-3-carene was chosen as a model substrate for the CPO-catalysed conversion of terpenes in the presence of sodium halides. With chloride, bromide and iodide, the reaction products were the respective (1S,3R,4R,6R)-4-halo-3,7,7-trimethyl-bicyclo[4.1.0]-heptane-3-ols, as identified by ¹H and ¹³C nuclear magnetic resonance. These product formations turned out to be strictly regio- and stereoselective and proceeded very rapidly and almost quantitatively. Initial specific activities of halohydrin formation increased from 4.22 U mg⁻¹ with chloride to 12.22 U mg⁻¹ with bromide and 37.11 U mg⁻¹ with iodide as the respective halide ion. These results represent the first examples of the application of CPO as a highly efficient biocatalyst for monoterpene functionalization. This is a promising strategy for ‘green’ terpene chemistry overcoming drawbacks usually associated with cofactor-dependent oxygenases, whole-cell biocatalysts and conventional chemical methods used for terpene conversions.

Keywords: Chloroperoxidase; Caldariomyces fumago ; Terpenes; Oxidation; Stereoselective; Halohydrin

A gene linB2 responsible for the conversion of β-HCH and 2,3,4,5,6-pentachlorocyclohexanol in Sphingomonas sp. BHC-A by Jun Wu; Qing Hong; Peng Han; Jian He; Shunpeng Li (pp. 1097-1105).

Commercial formulations of hexachlorocyclohexane (HCH) consist of a mixture of four isomers: α, β, γ, and δ. All four isomers are toxic and recalcitrant pollutants. β-HCH is more problematic due to its longer persistence in the environment. Sphingomonas sp. BHC-A was able to degrade not only α-, γ-, and δ-HCH but also β-HCH. To clone a gene responsible for the degradation of β-HCH, a Tn5 mutation was introduced into BHC-A, and one mutant BHC-A45 defective in β-HCH degradation was selected. Sequencing analysis showed this mutant had a Tn5 insertion at the site of one haloalkane dehalogenase gene, designated linB2. linB2 was overexpressed in Escherichia coli and the 32-kDa product LinB2 showed the conversion activity of not only β-HCH to β-2,3,4,5,6-pentachlorocyclohexanol (β-PCHL) but also β-PCHL to β-2,3,5,6-tetrachloro-1,4-cyclohexanediol.

Characterization of the third chitinase Chi18C of Clostridium paraputrificum M-21 by Kenji Morimoto; Michiko Yoshimoto; Shuichi Karita; Tetsuya Kimura; Kunio Ohmiya; Kazuo Sakka (pp. 1106-1113).

A novel chitinase gene chiC of Clostridium paraputrificum M-21, a chitinolytic and hydrogen-gas-producing bacterium, was characterized along with its translated product. The chi18C gene encodes 683 amino acids (signal peptide included) with a deduced molecular weight of 74,651. Chi18C is a modular enzyme composed of a family-18 catalytic module of glycoside hydrolases, two reiterated modules of unknown function, and a family-12 carbohydrate-binding module. Recombinant Chi18C was active toward soluble and insoluble chitin preparations, and synthetic substrates such as 4-methylumbelliferyl-β-d-N-N′-N″-triacetylchitotriose, but not active toward 4-MU-N-acetylglucosamine or 4-MU-β-d-N-N′-diacetylchitobioside. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunological analyses suggested that the expression of chi18C was inducible with chitinous substrates and that Chi18C was secreted into the culture medium. A possible role of Chi18C in the chitinolytic system of C. paraputrificum M-21 is discussed.

Keywords: Chitinase; Clostridium; Paraputrificum

The construction and characterization of a bifunctional EGFP/sAPRIL fusion protein by Zhengbing Guan; Wenjuan Yao; Jilin Ye; Wenbing Dan; Jiayin Shen; Shuangquan Zhang (pp. 1114-1122).

A fusion protein of enhanced green fluorescent protein (EGFP) and soluble domain of human a proliferation-inducing ligand (sAPRIL) was efficiently expressed in Escherichia coli BL 21 (DE3). The soluble EGFP/sAPRIL, around 43 kDa, was purified in milligram amounts using metal chellate affinity chromatography and detected with anti-His₆ and anti-hsAPRIL monoclonal antibody. The chimeric protein exhibited similar fluorescence spectra with free EGFP. In vitro, purified EGFP/sAPRIL specifically bound receptor B cell maturation antigen (BCMA) detected by enzyme linked immunosorbent assay (ELISA) and receptors [including heparan sulfate proteoglycan (HSPGs)]-positive cell lines analyzed by fluorescence-activated cell sorting (FACS). Confocal laser microscopy images visibly showed the HSPGs’-dependent binding of EGFP/sAPRIL to NIH-3T3 cell. In addition, the chimera retained the bioactivity to stimulate/co-stimulate proliferation of NIH-3T3 and Jurkat cell/human B cell in vitro. Therefore, the fusion protein shows a readily obtainable source of biologically active sAPRIL which has considerable potential for single-step fluorescence detection assay in the study of APRIL and its receptors.

Keywords: Fusion protein; EGFP; APRIL; In vitro expression; LSCM

High-level heterologous gene expression in Ochrobactrum anthropi using an A-rich UP element by Mohamed Seleem; Mohammed Ali; M. W. Abd Al-Azeem; Stephen M. Boyle; Nammalwar Sriranganathan (pp. 1123-1127).

DNA regions that flank a gene’s promoter play an important role in determining transcription efficiency by interacting with the carboxy-terminal domain of RNA polymerase α-subunit. We placed an adenine-rich upstream element (UP) between −38 and −59 of the core trc promoter to enhance gene expression in Ochrobactrum anthropi up to 66-fold. The high level of expression achieved by the UP element and the N-terminus fusion of a 6xHis epitope tag facilitated detection and purification of heterologous proteins directly from O. anthropi.

RNAi-based tuning of cell cycling in Drosophila S2 cells—effects on recombinant protein yield by John C. March; William E. Bentley (pp. 1128-1135).

We have demonstrated the RNA interference-based interruption of cellular controllers to increase recombinant protein yield in Drosophila Schneider 2 (S2) cell culture. Double-stranded RNA (dsRNA) was enzymatically synthesized in vitro and transfected into stable cell lines expressing green fluorescent protein (GFP) under an inducible promoter. Components of cell cycling (CycE and ago) were silenced with dsRNA homologous to a 700-nucleotide section of their respective mRNA transcripts. Silencing ago and CycE resulted in increases in product yield of up to 1.8-fold and 4-fold, respectively, relative to a control transfected with nuclease-free water. It is surprising to note that nearly complete silencing of CycE resulted in no significant change in GFP fluorescence after 24 h, and a decrease in fluorescence after 72 h. By partially silencing CycE, however, we were able to retain 80% of the cells in G1 (48-h sample) and increase GFP synthesis by fourfold. Implications for protein synthesis processing are discussed.

Keywords: Drosophila ; S2; Sl-2; Cell culture; RNAi; CycE ; Cyclin E; Archipelago; Ago ; Cell cycle; GFP; Apoptosis

Multiplex PCR for the detection of toxigenic cyanobacteria in dietary supplements produced for human consumption by Martin L. Saker; Martin Welker; Vitor M. Vasconcelos (pp. 1136-1142).

The production of food supplements containing cyanobacteria is a growing worldwide industry. While there have been several reports of health benefits that can be gained from the consumption of these supplements, there have also been a growing number of studies showing the presence of toxins some of which (for example microcystins) are known to affect human health. In this paper, we report a multiplex polymerase chain reaction (PCR) technique that can be used to identify microcystin contamination in dietary supplements produced for human consumption. This method involves a PCR reaction containing three primer pairs, the first of which is used to amplify a 220-bp fragment of 16s rDNA specific to Microcystis, the most common microcystin-producing cyanobacterium. The second primer pair is used to amplify a 300-bp fragment of the mcyA gene, linked to microcystin biosynthesis in Anabaena, Microcystis, and Planktothrix. A third primer pair, used as a positive control, results in the amplification of a 650-bp fragment from the phycocyanin operon common to all cyanobacteria. This technique was found to be useful for detecting the presence of toxigenic Microcystis in all dietary supplements produced from the nontoxic cyanobacterium Aphanizomenon flos-aquae.

One-pot synthesis of genistein from tyrosine by coincubation of genetically engineered Escherichia coli and Saccharomyces cerevisiae cells by Yohei Katsuyama; Ikuo Miyahisa; Nobutaka Funa; Sueharu Horinouchi (pp. 1143-1149).

For production of genistein from N-acetylcysteamine-attached p-coumarate (p-coumaroyl-NAC) supplemented to the medium, a chalcone synthase (CHS) gene from Glycyrrhiza echinata, a chalcone isomerase (CHI) gene from Pueraria lobata, and an isoflavone synthase (IFS) gene from G. echinata were placed under the control of the galactose-inducible GAL promoters in pESC vector and were introduced in Saccharomyces cerevisiae. When the recombinant yeast cells (0.5 g wet weight) were used as “enzyme bags” and incubated at 30°C for 48 h in 100 ml of the buffer containing galactose and 1 mM (265 mg/l) p-coumaroyl-NAC, ca. 340 μg genistein/l was produced. Another system consisting of two enzyme bags was also generated for the purpose of production of genistein from tyrosine. One enzyme bag was an Escherichia coli cell containing a phenylalanine ammonia-lyase gene from a yeast, a 4-coumarate/cinnamate:CoA ligase gene from the actinomycete Streptomyces coelicolor A3(2), the CHS gene, and the CHI gene, in addition to the acetyl-CoA carboxylase gene from Corynebacterium glutamicum, all of which were under the control of the isopropyl-β-d-thiogalactopyranoside-inducible T7 promoter, and thus producing (S)-naringenin from tyrosine. The other enzyme bag was a S. cerevisiae cell containing the IFS gene. Coincubation of the E. coli cells (0.5 g wet weight) and S. cerevisiae cells (0.5 g wet weight) at 26°C for 60 h in 20 ml of the buffer containing 3 mM (543 mg/l) tyrosine as the starting substrate yielded ca. 6 mg genistein/l.

Keywords: Combinatorial biosynthesis; Isoflavone; Saccharomyces cerevisiae ; Genistein; Naringenin

pspA overexpression in Streptomyces lividans improves both Sec- and Tat-dependent protein secretion by Kristof Vrancken; Sophie De Keersmaeker; Nick Geukens; Elke Lammertyn; Jozef Anné; Lieve Van Mellaert (pp. 1150-1157).

Streptomyces is an interesting host for the secretory production of recombinant proteins because of its innate capacity to secrete proteins at high level in the culture medium. In this report, we evaluated the importance of the phage-shock protein A (PspA) homologue on the protein secretion yield in Streptomyces lividans. The PspA protein is supposed to play a role in the maintenance of the proton motive force (PMF). As the PMF is an energy source for both Sec- and Tat-dependent secretion, we evaluated the influence of the PspA protein on both pathways by modulating the pspA expression. Results indicated that pspA overexpression can improve the Tat-dependent protein secretion as illustrated for the Tat-dependent xylanase C and enhanced green fluorescent protein (EGFP). The effect on Sec-dependent secretion was less pronounced and appeared to be protein dependent as evidenced by the increase in subtilisin inhibitor (Sti-1) secretion but the lack of increase in human tumour necrosis factor (hTNFα) secretion in a pspA-overexpressing strain.

Keywords: Streptomyces ; Protein production; Phage-shock protein; pspA; Twin arginine; Secretion

Production of cyclopiazonic acid, aflatrem, and aflatoxin by Aspergillus flavus is regulated by veA, a gene necessary for sclerotial formation by Rocio M. Duran; Jeffrey W. Cary; Ana M. Calvo (pp. 1158-1168).

The plant pathogenic fungus Aspergillus flavus produces several types of mycotoxins. The most well known are the carcinogenic compounds called aflatoxins. In addition, A. flavus produces cyclopiazonic acid and aflatrem mycotoxins, contributing to the toxicity of A. flavus infected crops. Cyclopiazonic acid is a specific inhibitor of calcium-dependent ATPase in the sarcoplasmic reticulum that results in altered cellular Ca⁺⁺ levels. Aflatrem is a potent tremorgenic mycotoxin known to lead to neurological disorders. Previously we showed that a gene called veA controls aflatoxin and sclerotial production in A. parasiticus. In this study in A. flavus, we show that the veA homolog in A. flavus not only is necessary for the production of aflatoxins B1 and B2 and sclerotia, but also regulates the synthesis of the mycotoxins cyclopiazonic acid and aflatrem. The A. flavus ΔveA mutant was completely blocked in the production of aflatrem and showed greater than twofold decrease in cyclopiazonic acid production. The genes involved in the synthesis of cyclopiazonic acid are unknown; however, the aflatrem gene cluster has been characterized. Northern hybridization analysis showed that veA is required for expression of the A. flavus aflatrem genes atmC, atmG, and atmM. This is the first report of a regulatory gene governing the production of cyclopiazonic acid and aflatrem mycotoxins.

Keywords: Aspergillus flavus; Cyclopiazonic acid; Aflatrem; Aflatoxin; Sclerotia; Mycotoxin regulation

Growth of E. coli BL21 in minimal media with different gluconeogenic carbon sources and salt contents by Oleg Paliy; Thusitha S. Gunasekera (pp. 1169-1172).

Escherichia coli strain BL21 is commonly used as a host strain for protein expression and purification. For structural analysis, proteins are frequently isotopically labeled with deuterium (²H), ¹³C, or ¹⁵N by growing E. coli cultures in a medium containing the appropriate isotope. When large quantities of fully deuterated proteins are required, E. coli is often grown in minimal media with deuterated succinate or acetate as the carbon source because these are less expensive. Despite the widespread use of BL21, we found no data on the effect of different minimal media and carbon sources on BL21 growth. In this study, we assessed the growth behavior of E. coli BL21 in minimal media with different gluconeogenic carbon sources. Though BL21 grew reasonably well on glycerol and pyruvate, it had a prolonged lag-phase on succinate (20 h), acetate (10 h), and fumarate (20 h), attributed to the physiological adaptation of E. coli cells. Wild-type strain NCM3722 (K12) grew well on all the substrates. We also examined the growth of E. coli BL21 in minimal media that differed in their salt composition but not in their source of carbon. The commonly used M9 medium did not support the optimum growth of E. coli BL21 in minimal medium. The addition of ferrous sulphate to M9 medium (otherwise lacking it) increased the growth rate of E. coli cultures and significantly increased their cell density in the stationary phase.

Keywords: Escherichia coli ; BL21; Gluconeogenesis; Minimal medium; Acetate; Succinate

In vitro evaluation of the prebiotic activity of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel by G. Mandalari; C. Nueno Palop; K. Tuohy; G. R. Gibson; R. N. Bennett; K. W. Waldron; G. Bisignano; A. Narbad; C. B. Faulds (pp. 1173-1179).

The prebiotic effect of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel was studied using pure and mixed cultures of human faecal bacteria. This was compared to the prebiotic effect of fructo-oligosaccharides (FOS). Individual species of bifidobacteria and lactobacilli responded positively to the addition of the bergamot extract, which contained oligosaccharides in the range of three to seven. Fermentation studies were also carried out in controlled pH batch mixed human faecal cultures and changes in gut bacterial groups were monitored over 24 h by fluorescent in situ hybridisation, a culture-independent microbial assessment. Addition of the bergamot oligosaccharides (BOS) resulted in a high increase in the number of bifidobacteria and lactobacilli, whereas the clostridial population decreased. A prebiotic index (PI) was calculated for both FOS and BOS after 10 and 24 h incubation. Generally, higher PI scores were obtained after 10 h incubation, with BOS showing a greater value (6.90) than FOS (6.12).

Keywords: Fructo-oligosaccharides; Bergamot oligosaccharides; Prebiotics; Pectic; Lactobacilli; Bifidobacteria

Sustained generation of electricity by the spore-forming, Gram-positive, Desulfitobacterium hafniense strain DCB2 by C. E. Milliken; H. D. May (pp. 1180-1189).

Desulfitobacterium hafniense strain DCB2 generates electricity in microbial fuel cells (MFCs) when humic acids or the humate analog anthraquinone-2,6-disulfonate (AQDS) is added as an electron-carrying mediator. When utilizing formate as fuel, the Gram-positive, spore-forming bacterium generated up to 400 mW/m² of cathode surface area in a single-chamber MFC with a platinum-containing air-fed cathode. Hydrogen, lactate, pyruvate, and ethanol supported electricity generation, but acetate, propionate, and butyrate did not. Scanning electron microscopy indicated that strain DCB2 colonized the surface of a current-generating anode but not of an unconnected electrode. The electricity was recovered fully within minutes after the exchange of the medium in the anode chamber and within a week after an exposure of a colonized anode to 90°C for 20 min. Of the six strains of Desulfitobacteria tested, all of which would reduce AQDS, only D. hafniense strain DCB2 continued to reduce AQDS and generate electricity for more than 24 h, indicating that reduction of the humate analog alone is insufficient to sustain electrode reduction.

Interaction effects of lactic acid and acetic acid at different temperatures on ethanol production by Saccharomyces cerevisiae in corn mash by Tara Graves; Neelakantam V. Narendranath; Karl Dawson; Ronan Power (pp. 1190-1196).

The combined effects of lactic acid and acetic acid on ethanol production by S. cerevisiae in corn mash, as influenced by temperature, were examined. Duplicate full factorial experiments (three lactic acid concentrations × three acetic acid concentrations) were performed to evaluate the interaction between lactic and acetic acids on the ethanol production of yeast at each of the three temperatures, 30, 34, and 37°C. Corn mash at 30% dry solids adjusted to pH 4 after lactic and acetic acid addition was used as the substrate. Ethanol production rates and final ethanol concentrations decreased (P<0.001) progressively as the concentration of combined lactic and acetic acids in the corn mash increased and the temperature was raised from 30 to 37°C. At 30°C, essentially no ethanol was produced after 96 h when 0.5% w/v acetic acid was present in the mash (with 0.5, 2, and 4% w/v lactic acid). At 34 and 37°C, the final concentrations of ethanol produced by the yeast were noticeably reduced by the presence of 0.3% w/v acetic acid and ≥2% w/v lactic acid. It can be concluded that, as in previous studies with defined media, lactic acid and acetic acid act synergistically to reduce ethanol production by yeast in corn mash. In addition, the inhibitory effects of combined lactic and acetic acid in corn mash were more apparent at elevated temperatures.

Keywords: Corn mash; Lactic acid; Acetic acid; Temperature; Saccharomyces cerevisiae ; Synergy

Isolation and characterization of dihydromonacolin-MV from Monascus purpureus for antioxidant properties by Mohan A. Dhale; S. Divakar; S. Umesh Kumar; G. Vijayalakshmi (pp. 1197-1202).

The methanolic extract of Monascus purpureus cultivated by solid-state fermentation on rice showed strong 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and better yield as compared to other polarity based extracted fractions. It was selected for further purification of the antioxidant. The activity-guided repeated fractionation of methanolic extract on a silica gel column chromatography yielded a compound that exhibited strong antioxidant activity. Based on the spectroscopic analysis by UV, IR, ¹H NMR, ¹³C NMR, 2D-HSQCT NMR, and MS, the antioxidant isolated was elucidated as a derivative of dihydromonacolin-K, where the ester group is 2-methyl propionate, designated as dihydromonacolin-MV. The DPPH radical was significantly scavenged by the dihydromonacolin-MV (IC₅₀ 20±1 μg ml⁻¹). The dihydromonacolin-MV showed strong inhibition of lipid peroxidation in a liposome model with an IC₅₀ value of 5.71±0.38 μg ml⁻¹ and superoxide radical scavenging activity with an IC₅₀ value of 163.97±2.68 μg ml⁻¹.

Keywords: Monascus purpureus ; Dihydromonacolin-MV; Antioxidant; DPPH lipid peroxidation; Superoxide radical

Methanol utilizing Desulfotomaculum species utilizes hydrogen in a methanol-fed sulfate-reducing bioreactor by Melike Balk; Jan Weijma; Heleen P. Goorissen; Mariska Ronteltap; Theo A. Hansen; Alfons J. M. Stams (pp. 1203-1211).

A sulfate-reducing bacterium, strain WW1, was isolated from a thermophilic bioreactor operated at 65°C with methanol as sole energy source in the presence of sulfate. Growth of strain WW1 on methanol or acetate was inhibited at a sulfide concentration of 200 mg l⁻¹, while on H₂/CO₂, no apparent inhibition occurred up to a concentration of 500 mg l⁻¹. When strain WW1 was co-cultured under the same conditions with the methanol-utilizing, non-sulfate-reducing bacteria, Thermotoga lettingae and Moorella mulderi, both originating from the same bioreactor, growth and sulfide formation were observed up to 430 mg l⁻¹. These results indicated that in the co-cultures, a major part of the electron flow was directed from methanol via H₂/CO₂ to the reduction of sulfate to sulfide. Besides methanol, acetate, and hydrogen, strain WW1 was also able to use formate, malate, fumarate, propionate, succinate, butyrate, ethanol, propanol, butanol, isobutanol, with concomitant reduction of sulfate to sulfide. In the absence of sulfate, strain WW1 grew only on pyruvate and lactate. On the basis of 16S rRNA analysis, strain WW1 was most closely related to Desulfotomaculum thermocisternum and Desulfotomaculum australicum. However, physiological properties of strain WW1 differed in some aspects from those of the two related bacteria.

Keywords: Methanol; Thermophile; Sulfate-reducing bacterium; Desulfotomaculum

Ring test assessment of the mKir2.1 growth based assay in Saccharomyces cerevisiae using parametric models and model-free fits by Guido Hasenbrink; Lucie Kolacna; Jost Ludwig; Hana Sychrova; Maik Kschischo; Hella Lichtenberg-Fraté (pp. 1212-1221).

Inward rectifying K⁺ (Kir) channels are a subfamily of the potassium channel superfamily. They mediate potassium influx into the cells, a process responding to the polarization state, a variety of intracellular messengers and specific auxiliary proteins, thereby they are involved in important physiological processes such as the pacemaker activity in the heart, insulin release, and potassium uptake in glial cells. The Saccharomyces cerevisiae mKir2.1 in vitro assay was subjected to a ring test assessment. Compound-associated mKir2.1 modulating effects were detected by growth determination of functionally complemented S. cerevisiae cells in a 96-well format within 15 h. Dose–response diagrams and EC₅₀ value calculations were determined by parametric model and model-free fits using cubic spline interpolation. These characteristics were evaluated by statistical methods to determine reproducibility among working groups. Nonparametric bootstrap simulations of the variability of the data revealed that EC₅₀ values of the mKir2.1 indicator strain were well-matched (81–92 μM), enabling unambiguous quantitative statements about inhibitory effects and no significant influence of the different laboratory conditions. Limitations of the assay include compounds/samples that are either insoluble under the conditions of the test or strongly cytotoxic to yeast. Thus, the described test is a sensitive and reliable tool that can be used in different laboratories and is applicable in drug discovery and development as simple and reliable prescreening procedure.

Keywords: Short-term in vitro test; Parametric models; Model-free fits; Saccharomyces cerevisiae

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Applied Microbiology and Biotechnology (v.73, #5)