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Applied Microbiology and Biotechnology (v.76, #4)

Prospects for a bio-based succinate industry by James B. McKinlay; C. Vieille; J. Gregory Zeikus (pp. 727-740).

Bio-based succinate is receiving increasing attention as a potential intermediary feedstock for replacing a large petrochemical-based bulk chemical market. The prospective economical and environmental benefits of a bio-based succinate industry have motivated research and development of succinate-producing organisms. Bio-based succinate is still faced with the challenge of becoming cost competitive against petrochemical-based alternatives. High succinate concentrations must be produced at high rates, with little or no by-products to most efficiently use substrates and to simplify purification procedures. Herein are described the current prospects for a bio-based succinate industry, with emphasis on specific bacteria that show the greatest promise for industrial succinate production. The succinate-producing characteristics and the metabolic pathway used by each bacterial species are described, and the advantages and disadvantages of each bacterial system are discussed.

Keywords: Bio-based succinate; 1,4-Butanediol; Metabolic engineering; Escherichia coli ; Actinobacillus succinogenes ; Mannheimia succiniciproducens

Aerobic degradation of lindane (γ-hexachlorocyclohexane) in bacteria and its biochemical and molecular basis by Yuji Nagata; Ryo Endo; Michihiro Ito; Yoshiyuki Ohtsubo; Masataka Tsuda (pp. 741-752).

γ-Hexachlorocyclohexane (γ-HCH, also called γ-BHC and lindane) is a halogenated organic insecticide that causes serious environmental problems. The aerobic degradation pathway of γ-HCH was extensively revealed in bacterial strain Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26. γ-HCH is transformed to 2,5-dichlorohydroquinone through sequential reactions catalyzed by LinA, LinB, and LinC, and then 2,5-dichlorohydroquinone is further metabolized by LinD, LinE, LinF, LinGH, and LinJ to succinyl-CoA and acetyl-CoA, which are metabolized in the citrate/tricarboxylic acid cycle. In addition to these catalytic enzymes, a putative ABC-type transporter system encoded by linKLMN is also essential for the γ-HCH utilization in UT26. Preliminary examination of the complete genome sequence of UT26 clearly demonstrated that lin genes for the γ-HCH utilization are dispersed on three large circular replicons with sizes of 3.5 Mb, 682 kb, and 191 kb. Nearly identical lin genes were also found in other HCH-degrading bacterial strains, and it has been suggested that the distribution of lin genes is mainly mediated by insertion sequence IS6100 and plasmids. Recently, it was revealed that two dehalogenases, LinA and LinB, have variants with small number of amino acid differences, and they showed dramatic functional differences for the degradation of HCH isomers, indicating these enzymes are still evolving at high speed.

Keywords: γ-Hexachlorocyclohexane; Sphingomonads; Dehalogenase

Suppression Subtractive Hybridization (SSH) and its modifications in microbiological research by Xiaowei Huang; Yunxia Li; Qiuhong Niu; Keqin Zhang (pp. 753-760).

Suppression subtractive hybridization (SSH) is an effective approach to identify the genes that vary in expression levels during different biological processes. It is often used in higher eukaryotes to study the molecular regulation in complex pathogenic progress, such as tumorigenesis and other chronic multigene-associated diseases. Because microbes have relatively smaller genomes compared with eukaryotes, aside from the analysis at the mRNA level, SSH as well as its modifications have been further employed to isolate specific chromosomal locus, study genomic diversity related with exceptional bacterial secondary metabolisms or genes with special microbial function. This review introduces the SSH and its associated methods and focus on their applications to detect specific functional genes or DNA markers in microorganisms.

Keywords: SSH; mRNA; Genome; Modified method; Microbiological research

Process optimization of large-scale production of recombinant adeno-associated vectors using dielectric spectroscopy by Alejandro Negrete; Geoffrey Esteban; Robert M. Kotin (pp. 761-772).

A well-characterized manufacturing process for the large-scale production of recombinant adeno-associated vectors (rAAV) for gene therapy applications is required to meet current and future demands for pre-clinical and clinical studies and potential commercialization. Economic considerations argue in favor of suspension culture-based production. Currently, the only feasible method for large-scale rAAV production utilizes baculovirus expression vectors and insect cells in suspension cultures. To maximize yields and achieve reproducibility between batches, online monitoring of various metabolic and physical parameters is useful for characterizing early stages of baculovirus-infected insect cells. In this study, rAAVs were produced at 40-l scale yielding ~1 × 10¹⁵ particles. During the process, dielectric spectroscopy was performed by real time scanning in radio frequencies between 300 kHz and 10 MHz. The corresponding permittivity values were correlated with the rAAV production. Both infected and uninfected reached a maximum value; however, only infected cell cultures permittivity profile reached a second maximum value. This effect was correlated with the optimal harvest time for rAAV production. Analysis of rAAV indicated the harvesting time around 48 h post-infection (hpi), and 72 hpi produced similar quantities of biologically active rAAV. Thus, if operated continuously, the 24-h reduction in the production process of rAAV gives sufficient time for additional 18 runs a year corresponding to an extra production of ~2 × 10¹⁶ particles. As part of large-scale optimization studies, this new finding will facilitate the bioprocessing scale-up of rAAV and other bioproducts.

Keywords: Adeno-associated vectors; Gene therapy; Dielectric spectroscopy; Large-scale; Bioprocessing; Harvesting time

Cell envelope fluidity modification for an effective glutamate excretion in Corynebacterium glutamicum 2262 by Dimitrios Bokas; Davin Uy; Franck Grattepanche; Guy Duportail; Emmanuel Guedon; Stéphane Delaunay; Jean-Louis Goergen (pp. 773-781).

1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was used to assess the cell envelope fluidity of Corynebacterium glutamicum 2262 during a temperature-triggered glutamate producing process. Because the fluorescence lifetime of TMA-DPH was shown to be constant all over the process, fluorescence anisotropy can be considered as a good index of cell envelope fluidity. When the temperature of the fed-batch culture was increased from 33 to 39°C to induce glutamate excretion, the fluorescence anisotropy values decreased from 0.212 ± 0.002 to 0.186 ± 0.002 (corresponding to an increase in the cell fluidity), while the specific glutamate production rate reached its maximal value. The increase in fluidity of the C. glutamicum cell envelope was not due to a physical effect related to the temperature elevation, but rather to an alteration of the composition of the cell envelope. Using a mutant devoid of corynomycolates, significant differences in fluorescence anisotropy values were obtained compared to the wild-type strain, suggesting that TMA-DPH is mainly anchored into the corynomycomembrane. Differences in fluorescence anisotropy were also observed when the bacteria were cultivated at 33, 36, 38, and 39°C in batch cultures, and a linear relationship was obtained between the maximum specific glutamate production rate and the measured fluidity. When using the glutamate non-producing variant of C. glutamicum 2262, the fluorescence anisotropy remained constant at 0.207 ± 0.003 whatever the applied temperature shift. This suggests that the fluidity of the Corynebacteria mycomembrane plays an important role in glutamate excretion during the temperature-triggered process.

Keywords: Corynebacterium; Glutamate; Fluidity; Mycolic acid; TMA-DPH

Optimization of riboflavin production by recombinant Bacillus subtilis RH44 using statistical designs by Qiu-Li Wu; Tao Chen; Yu Gan; Xun Chen; Xue-Ming Zhao (pp. 783-794).

A sequential optimization strategy, based on statistical experimental designs, was used to enhance the production of riboflavin by recombinant Bacillus subtilis RH44. In the first instance, the medium components were optimized in shake flask cultures. After preliminary experiments of nitrogen source selection, the two-level Plackett–Burman (PB) design was implemented to screen medium components that significantly influence riboflavin production. Among the 15 variables tested, glucose, NaNO₃, K₂HPO₄, ZnSO₄, and MnCl₂ were identified as the most significant factors (confidence levels above 95%) for riboflavin production. The optimal values of these five variables were determined by response surface methodology (RSM) based on the central composite design (CCD). The validity of the model developed was verified, and the optimum medium led to a maximum riboflavin concentration of 6.65 g/l, which was 44.3 and 76.4% higher than the improved medium and the basal medium, respectively. A glucose-limited fed-batch culture profile in a 5-l fermentor was consequently designed according to the above optimum medium in shake flasks. A final riboflavin concentration of 16.36 g/l was obtained in 48 h, which further verified the practicability of this optimum strategy.

Keywords: Bacillus subtilis ; Riboflavin production; Plackett–Burman design; Central composite design; Response surface methodology

Identification of Escherichia coli host cell for high plasmid stability and improved production of antihuman ovarian carcinoma × antihuman CD3 single-chain bispecific antibody by Jing-Bo Zhao; Dong-Zhi Wei; Wang-Yu Tong (pp. 795-800).

To improve the plasmid stability during the production of antihuman ovarian carcinoma × antihuman CD3 single-chain bispecific antibody (AhOC×AhCD3), the Escherichia coli BL21(DE3) host cell was optimized serially. Firstly, an isogenic recombination-deficient (recA ⁻) derivative of BL21(DE3), namely BLR(DE3), was used as host instead of BL21(DE3). Although the segregational plasmid stability was greatly improved, AhOC×AhCD3 yield was not improved due to the severe growth inhibition of plasmid-bearing BLR(DE3) cells and the competitive plasmid instability after induction. Secondly, a mutant BLR(DE3), namely BLRM(DE3), was screened by using LB agar plates plus ampicillin and isopropyl-β-d-thiogalactopyranoside. Using this new host, growth inhibition of recombinant cells after induction was eliminated, and plasmids could be stably maintained even after long-time induction in a nonselective medium. At last, about 1.2 g/l AhOC×AhCD3, which was about thrice as much as those of recombinant BL21(DE3) and BLR(DE3) strains, was yielded.

Keywords: Escherichia coli ; Plasmid stability; Antihuman ovarian carcinoma × antihuman CD3 single-chain bispecific antibody

Structural characterization and surface-active properties of a new glycolipid biosurfactant, mono-acylated mannosylerythritol lipid, produced from glucose by Pseudozyma antarctica by Tokuma Fukuoka; Tomotake Morita; Masaaki Konishi; Tomohiro Imura; Hideki Sakai; Dai Kitamoto (pp. 801-810).

Mannosylerythritol lipids (MELs), which are glycolipid biosurfactants produced by Pseudozyma yeasts, show not only excellent interfacial properties but also versatile biochemical actions. In the course of MEL production from glucose as the sole carbon source, P. antarctica was found to produce unknown glycolipids more hydrophilic than conventional “di-acylated MELs,” which have two fatty acyl esters on the mannose moiety. Based on a detailed characterization, the most hydrophilic one was identified as 4-O-(3′-O-alka(e)noyl-β-d-mannopyranosyl)-d-erythritol namely, “mono-acylated MEL.” The mono-acylated MEL reduced the surface tension of water to 33.8 mN/m at a critical micelle concentration (CMC) of 3.6 × 10⁻⁴ M, and its hydrophilic–lipophilic balance was tentatively calculated to be 12.15. The observed CMC was 100-fold higher than that of the MELs hitherto reported. Interestingly, of the yeast strains of the genus Pseudozyma, only P. antarctica and P. parantarctica gave the mono-acylated MEL from glucose, despite a great diversity of di-acylated MEL producers in the genus. These strains produced MELs including the mono-acylated one at a rate of 20–25%. From these results, the new MEL is likely to have great potential for use in oil-in-water-type emulsifiers and washing detergents because of its higher water solubility compared to conventional MELs and will thus contribute to facilitating a broad range of applications for the environmentally advanced surfactants.

Keywords: Biosurfactant; Glycolipid; Mannosylerythritol lipid; Pseudozyma yeasts

Microbial production of R-3-hydroxybutyric acid by recombinant E. coli harboring genes of phbA, phbB, and tesB by Qian Liu; Shao-Ping Ouyang; Ahleum Chung; Qiong Wu; Guo-Qiang Chen (pp. 811-818).

Production of R-3-hydroxybutyric acid (3HB) was observed when genes of β-ketothiolase (PhbA), acetoacetyl CoA reductase (PhbB), and thioesterase II (TesB) were jointly expressed in Escherichia coli. TesB, generally regarded as a medium chain length acyl CoA thioesterase, was found, for the first time, to play an important role for transforming short chain length 3-hydroxybutyrate-CoA to its free fatty acid, namely, 3HB. E. coli BW25113 (pSPB01) harboring phbA, phbB, and tesB genes produced approximately 4 g/l 3HB in shake flask culture within 24 h with glucose used as a carbon source. Under anaerobic growth conditions, 3HB production was found to be more effective, achieving 0.47 g 3HB/g glucose compared with only 0.32 g 3HB/g glucose obtained from aerobic process. When growth was conducted on sodium gluconate, 6 g/l 3HB was obtained. In a 24-h fed-batch growth process conducted in a 6-l fermentor containing 3 l glucose mineral medium, 12 g/l 3HB was produced from 17 g/l cell dry weight (CDW). This was the highest 3HB productivity achieved by a one-stage fermentation process for 3HB production.

Keywords: PHB; 3-hydroxybutyric acid; 3HB; Thioesterase II; TesB; β-ketothiolase; PhbA; Acetoacetyl CoA reductase; PhbB

Alanine production in an H⁺-ATPase- and lactate dehydrogenase-defective mutant of Escherichia coli expressing alanine dehydrogenase by Masaru Wada; Kotomi Narita; Atsushi Yokota (pp. 819-825).

Previously, we reported that pyruvate production was markedly improved in TBLA-1, an H⁺-ATPase-defective Escherichia coli mutant derived from W1485lip2, a pyruvate-producing E. coli K-12 strain. TBLA-1 produced more than 30 g/l pyruvate from 50 g/l glucose by jar fermentation, while W1485lip2 produced only 25 g/l pyruvate (Yokota et al. in Biosci Biotechnol Biochem 58:2164–2167, 1994b). In this study, we tested the ability of TBLA-1 to produce alanine by fermentation. The alanine dehydrogenase (ADH) gene from Bacillus stearothermophilus was introduced into TBLA-1, and direct fermentation of alanine from glucose was carried out. However, a considerable amount of lactate was also produced. To reduce lactate accumulation, we knocked out the lactate dehydrogenase gene (ldhA) in TBLA-1. This alanine dehydrogenase-expressing and lactate dehydrogenase-defective mutant of TBLA-1 produced 20 g/l alanine from 50 g/l glucose after 24 h of fermentation. The molar conversion ratio of glucose to alanine was 41%, which is the highest level of alanine production reported to date. This is the first report to show that an H⁺-ATPase-defective mutant of E. coli can be used for amino acid production. Our results further indicate that H⁺-ATPase-defective mutants may be used for fermentative production of various compounds, including alanine.

Keywords: Alanine production; Escherichia coli ; H⁺-ATPase; Alanine dehydrogenase

Cloning and characterization of a heat-stable CMP-N-acylneuraminic acid synthetase from Clostridium thermocellum by Rahman M. Mizanur; Nicola L. Pohl (pp. 827-834).

In this study, we report the cloning, recombinant expression, and biochemical characterization of a heat-stable CMP-N-acylneuraminic acid (NeuAc) synthetase from Clostridium thermocellum ATCC 27405. A high throughput electrospray ionization mass spectrometry (ESI-MS)-based assay demonstrates that the enzyme has an absolute requirement for a divalent cation for activity and reaches maximum activity in the presence of 10 mM Mn²⁺. The enzyme is active at pH 8–13 in Tris–HCl buffer and at 37–60 °C, and maximum activity is observed at pH 9.5 and 50 °C in the presence of 0.2 mM dithiothreitol. In addition to NeuAc, the enzyme also accepts the analog N-glycolylneuraminic acid (NeuGc) as a substrate. The apparent Michaelis constants for cytidine triphosphate and NeuAc or NeuGc are 240 ± 20, 130 ± 10, and 160 ± 10 μM, respectively, with corresponding turnover numbers of 3.33, 2.25, and 1.66 s⁻¹, respectively. An initial velocity study of the enzymatic reaction indicates an ordered bi–bi catalytic mechanism. In addition to demonstration of a thermostable and substrate-tolerant enzyme, confirmation of the biochemical function of a gene for CMP-NeuAc synthetase in C. thermocellum also opens the question of the biological function of CMP-NeuAc in such nonpathogenic microorganisms.

Production, purification and application-relevant characterisation of an endo-1,3(4)-β-glucanase from Rhizomucor miehei by A. Boyce; G. Walsh (pp. 835-841).

Growth on a wheat bran media induced production of an extracellular β-glucanase by Rhizomucor miehei (DSM 1330). The enzyme was purified to homogeneity. Substrate specificity studies coupled with protein database similarity searching using mass spectrometry-derived sequence data indicate it to be an endo-1,3(4)-β-glucanase (EC 3.2.1.6). The enzyme was characterised in terms of potential suitability for use in animal (poultry) feed. Significant activity was observed over the entire pH range typical of the avian upper digestive tract (pH 2.6–6.5). The enzyme was also found to be more thermostable than current commercialized β-glucanases, particularly when heated at a high enzyme concentration, and retained twice as much residual activity as the latter upon exposure to simulated avian digestive tract conditions. There are no previous reports of the production, purification or characterization of a β-glucanase from a Rhizomucor, and the enzyme’s application-relevant physicochemical characteristics render it potentially suited for use in animal feed.

Keywords: β-glucanase; β-glucan; Rhizomucor miehei ; Animal feed

Characterization of free and immobilized (S)-aminotransferase for acetophenone production by Abraham R. Martin; David Shonnard; Sachin Pannuri; Sanjay Kamat (pp. 843-851).

Enzyme immobilization often improves process economics, but changes in kinetic properties may also occur. The immobilization of a recombinant thermostable (S)-aminotransferase was made by entrapment on calcium alginate—3% (w/v)—and tested with (S)-(−)-(α)-methylbenzylamine for acetophenone production. The best immobilization results were obtained for beads of concentration of 10 mg of spray-dried cells (containing recombinant (S)-aminotransferase) per milliliter of sodium alginate bead. As a result of immobilization, the properties of immobilized spray-dried cells differed from the properties of free spray-dried cells. V _m for the immobilized enzyme was between 0.08 and 0.09 mM/min, while the V _m for free enzyme was 0.06–0.07 mM/min. K _m values differed for immobilized and free spray-dried cells by a factor of between 3 and 5 for (S)-(−)-(α)-methylbenzylamine (6.05 mM for immobilized, 1.78 mM for free) and pyruvate (5.0 mM for immobilized, 1.01 mM for free) at 55°C. Optimum pH values were 7.7 and 8.1 for the free spray-dried cells and the immobilized formulation, respectively. The maximum activity for free spray-dried cells was measured at 55°C, whereas for immobilized ones, it was at 60°C. Activation and deactivation energy values for free spray-dried cells were 15.13 and 41.73 kcal/mol, while those for immobilized spray-dried cells were 8.86 and 48.88 kcal/mol, respectively. Overall, as a result of immobilization, an increase in V _m was measured for the (S)-aminotransferase by 28 to 33% with respect to free enzyme; K _m increased by a factor of three- to fivefold and had a shift of 5°C in optimum temperature, and the activation energy was 41% lower than the activation energy of free (S)-aminotransferase.

Keywords: Immobilization; Aminotransferase; Acetophenone

Identification of pig liver esterase variants by tandem mass spectroscopy analysis and their characterization by E. Brüsehaber; D. Böttcher; A. Musidlowska-Persson; D. Albrecht; M. Hecker; K. Doderer; U. T. Bornscheuer (pp. 853-859).

Pig liver esterase (PLE) is probably the most important carboxyl esterase in organic synthesis and is commercially obtained by extraction of the animal tissue. However, problems occur in its application due to the presence of several isoenzymes (α-, β- and γ-PLE). The functional expression of the γ-isoenzyme was already shown and differences in the enantioselectivity compared to the commercial preparations were confirmed. The amino acid and nucleotide sequences of the α- and β-PLE are still unknown. In this work, putative sequences of the α-isoenzyme were identified from a commercial PLE preparation by 2D gel electrophoresis, digestion with proteases and analysis using Matrix-assisted laser desorption/ionization–time of flight (TOF) and electrospray ionisation quadrupole–TOF mass spectrometry. Based on these results, three amino acid exchanges were introduced into the gene encoding γ-rPLE by site-directed mutagenesis, and the proteins were expressed in E. coli Origami (DE3). The produced PLE mutants were characterised with respect to their substrate specificity and enantioselectivity. No significant differences in the activity towards methyl butyrate were found, but several variants showed substantially enhanced enantioselectivity in the resolution of (R,S)-1-phenyl-2-butyl acetate with E = 100 for the best mutant V236P/A237G.

Keywords: Enantioselectivity; Isoenzymes; MALDI-TOF; Pig liver esterase

Catalytic properties of mycelium-bound lipases from Aspergillus niger MYA 135 by Cintia M. Romero; Mario D. Baigori; Licia M. Pera (pp. 861-866).

A constitutive level of a mycelium-bound lipolytic activity from Aspergillus niger MYA 135 was strongly increased by 97% in medium supplemented with 2% olive oil. The constitutive lipase showed an optimal activity in the pH range of 3.0–6.5, while the mycelium-bound lipase activity produced in the presence of olive oil had two pH optima at pH 4 and 7. Interestingly, both lipolytic sources were cold-active showing high catalytic activities in the temperature range of 4–8°C. These mycelium-bound lipase activities were also very stable in reaction mixtures containing methanol and ethanol. In fact, the constitutive lipase maintained almost 100% of its activity after exposure by 1 h at 37°C in ethanol. A simple methodology to evaluate suitable transesterification activities in organic solvents was also reported.

Keywords: Aspergillus niger ; Mycelium-bound lipase; Solvent tolerance; Enzyme stability; Hydrolytic activity; Transesterification

Enhancing survival of Escherichia coli by increasing the periplasmic expression of Cu,Zn superoxide dismutase from Saccharomyces cerevisiae by Ping Yu (pp. 867-871).

The gene for the Cu,Zn superoxide dismutase (Cu,ZnSOD) from Saccharomyces cerevisiae was cloned and expressed in Escherichia coli LMG194. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the E. coli periplasmic expression vector pBAD/gIIIA, yielding pBAD-1. E. coli was transformed using the constructed plasmid pBAD-1 and induced by adding 0.02% l-arabinose to express Cu,ZnSOD protein. The results indicated that Cu,ZnSOD enzyme activity in the periplasmic space was about fivefold to sixfold higher in the recombinant E. coli strains bearing the sod gene than in the control strains. The yields of Cu,ZnSOD were about threefold higher at 48 h than at 24 h in the recombinant E. coli cells. Significantly higher survival of strains was obtained in cells bearing the sod gene than in the control cells when the cells were treated by heat shock and superoxide-generating agents, such as paraquat and menadione.

Keywords: Cu,Zn superoxide dismutase gene; PCR amplification; Periplasmic expression; Survival

ɛ-Poly-l-lysine producer, Streptomyces albulus, has feedback-inhibition resistant aspartokinase by Y. Hamano; I. Nicchu; T. Shimizu; Y. Onji; J. Hiraki; H. Takagi (pp. 873-882).

Streptomyces albulus NBRC14147 produces ɛ-poly-l-lysine (ɛ-PL), which is an amino acid homopolymer antibiotic. Despite the commercial importance of ɛ-PL, limited information is available regarding its biosynthesis; the l-lysine molecule is directly utilized for ɛ-PL biosynthesis. In most bacteria, l-lysine is biosynthesized by an aspartate pathway. Aspartokinase (Ask), which is the first enzyme in this pathway, is subject to complex regulation such as through feedback inhibition by the end-product amino acids such as l-lysine and/or l-threonine. S. albulus NBRC14147 can produce a large amount of ɛ-PL (1–3 g/l). We therefore suspected that Ask(s) of S. albulus could be resistant to feedback inhibition to provide sufficient l-lysine for ɛ-PL biosynthesis. To address this hypothesis, in this study, we cloned the ask gene from S. albulus and investigated the feedback inhibition of its gene product. As predicted, we revealed the feedback resistance of the Ask; more than 20% relative activity of Ask was detected in the assay mixture even with extremely high concentrations of l-lysine and l-threonine (100 mM each). We further constructed a mutated ask gene for which the gene product Ask (M68V) is almost fully resistant to feedback inhibition. The homologous expression of Ask (M68V) further demonstrated the increase in ɛ-PL productivity.

Keywords: Aspartokinase; Feedback inhibition; ɛ-Poly-l-lysine; Streptomyces albulus

Engineering a regulatory region of jadomycin gene cluster to improve jadomycin B production in Streptomyces venezuelae by Jian-Ting Zheng; Sheng-Lan Wang; Ke-Qian Yang (pp. 883-888).

Streptomyces venezuelae ISP5230 produces a group of jadomycin congeners with cytotoxic activities. To improve jadomycin fermentation process, a genetic engineering strategy was designed to replace a 3.4-kb regulatory region of jad gene cluster that contains four regulatory genes (3′ end 272 bp of jadW2, jadW3, jadR2, and jadR1) and the native promoter upstream of jadJ (P_J) with the ermEp* promoter sequence so that ermEp* drives the expression of the jadomycin biosynthetic genes from jadJ in the engineered strain. As expected, the mutant strain produced jadomycin B without ethanol treatment, and the yield increased to about twofold that of the stressed wild-type. These results indicated that manipulation of the regulation of a biosynthetic gene cluster is an effective strategy to increase product yield.

Keywords: Streptomyces; Jadomycin; Regulation; Promoter replacement

Characterization of Fusarium graminearum inhibitory lipopeptide from Bacillus subtilis IB by J. Wang; J. Liu; H. Chen; J. Yao (pp. 889-894).

Bacillus subtilis strain IB exhibiting inhibitory activity against the Fusarium head blight disease fungus Fusarium graminearum was isolated and identified. The major inhibitory compound was purified from the culture broth through anion exchange, hydrophobic interaction, and reverse phase high-performance liquid chromatography (RP-HPLC) steps. It was a 1,463-Da lipopeptide and had an amino acid composition consisting of Ala, Glx, Ile, Orn, Pro, Thr, and Tyr at a molar ratio of 1:3:1:1:1:1:2. Electrospray ionization mass spectrometry/mass spectrometry (ESI MS/MS) analyses of the natural and the ring-opened peptides showed the antagonist was fengycin, a kind of macrolactone molecule with antifungal activity produced by several Bacillus strains. Fluorescence microscopic analysis indicated this peptide permeabilized and disrupted F. graminearum hyphae.

Keywords: Bacillus subtilis ; Fengycin; Fusarium graminearum ; Fusarium head blight disease

Selective loss of lin genes from hexachlorocyclohexane-degrading Pseudomonas aeruginosa ITRC-5 under different growth conditions by Atul K. Singh; Pankaj Chaudhary; Ankit S. Macwan; Upendra N. Diwedi; Ashwani Kumar (pp. 895-901).

The chlorinated insecticide γ-hexachlorocyclohexane (γ-HCH) is sequentially metabolized by the products of linA, linB, linC, linD, linE, and linF genes to β-ketoadipate, which is subsequently mineralized. Two or more copies of these genes are present in the bacterium Pseudomonas aeruginosa ITRC-5 that was isolated earlier by selective enrichment on technical-HCH. At least one copy of linA, linB, linC, linD, and possibly linE is lost from ITRC-5 upon its growth on γ-HCH. All the lin genes, however, are lost when the bacterium was grown in Luria–Bertani (LB) medium. The loss of lin genes is accompanied with the loss/rearrangement of insertion sequence IS6100 genes. Concomitant to the loss of lin genes, the degradation of HCH-isomers by “γ-HCH grown cells” is slower, when compared with “technical-HCH grown cells”, and is completely lost by “LB-grown cells”. The selective loss of lin genes during different growth conditions has not been reported before and is expected to help in understanding the dynamism of degradative genes.

Keywords: Hexachlorocyclohexane; Biodegradation; lin genes; IS6100 ; Pseudomonas aeruginosa

The oxygen transfer rate influences the molecular mass of the alginate produced by Azotobacter vinelandii by A. Díaz-Barrera; C. Peña; E. Galindo (pp. 903-910).

The influence of oxygen transfer rate (OTR) on the molecular mass of alginate was studied. In batch cultures without dissolved oxygen tension (DOT) control and at different agitation rates, the DOT was nearly zero and the OTR was constant during biomass growth, hence the cultures were oxygen-limited. The OTR reached different maximum levels (OTR_max) and enabled to establish various relative respiration rates. Overall, the findings showed that OTR influences alginate molecular mass. The mean molecular mass (MMM) of the alginate increased as OTR_max decreased. The molecular mass obtained at 3.0 mmol l⁻¹ h⁻¹ was 7.0 times higher (1,560 kDa) than at 9.0 mmol l⁻¹ h⁻¹ (220 kDa). An increase in molecular mass can be a bacterial response to adverse nutritional conditions such as oxygen limitation.

Keywords: Oxygen transfer rate; Alginate; Molecular mass; Oxygen limitation

Novel aspects of symbiotic (polyvinyl alcohol) biodegradation by Tereza Vaclavkova; Jan Ruzicka; Marketa Julinova; Robert Vicha; Marek Koutny (pp. 911-917).

A new polyvinyl alcohol (PVA)-degrading bacterium was isolated from activated sludge sampled during a waste water treatment process and identified as Sphingomonas sp. Its PVA oxidase activity and alcohol dehydrogenase activity for various low-molecular-weight secondary alcohols were detected. Both activities were associated with cells of the degrader, and they were not extracellular. Under optimal conditions, the isolate was able to degrade 500 mg of PVA per litre in 2 weeks. The strain required pyrroloquinoline quinone (PQQ) and another growth factor, the later could be supplied by a co-isolated Rhodococcus erythropolis strain. The findings stressed the complex nature of environmental PVA degradation and proved that other factors different from PQQ could be important in symbiotic biodegradation of PVA with some sphingomonads.

Keywords: Biodegradation; Polyvinyl alcohol; Sphingomonas ; Rhodococcus ; Symbiosis; Pyrroloquinoline quinone

Change in turnover capacity of crude recombinant dye-decolorizing peroxidase (rDyP) in batch and fed-batch decolorization of Remazol Brilliant Blue R by M. Shakeri; M. Shoda (pp. 919-926).

Decolorization of the representative anthraquinone dye, Remazol Brilliant Blue R (RBBR) was assessed to determine the practical potential of crude recombinant dye-decolorizing peroxidase generated by Aspergillus oryzae (rDyP) in term of turnover capacity of rDyP. The turnover capacity, defined as the milligram of RBBR decolorized per unit of rDyP inactivated over the catalytic life time of rDyP, was quantified under condition by H₂O₂ -mediated rDyP inactivation. In batch culture, equimolar batch addition of H₂O₂ and RBBR yielded complete decolorization of RBBR by rDyP, with a turnover capacity of 4.75. In stepwise fed-batch addition of H₂O₂, the turnover capacity increased to 5.76, and by increasing dye concentration, it reached 14.3. When H₂O₂ was added in continuous fed-batch to minimize rDyP inactivation and 1.6 mM dye was added in stepwise fed-batch mode, the turnover capacity increased to 20.4. At this turnover capacity, 1 l of crude rDyP solution containing 5,000 U could decolorize up to 102 g RBBR in 650 min.

Keywords: Recombinant dye-decolorizing peroxidase (rDyP); Turnover capacity; Remazol Brilliant Blue R; Inactivation by H₂O₂

Microbial source tracking in a small southern California urban watershed indicates wild animals and growth as the source of fecal bacteria by Sunny C. Jiang; Weiping Chu; Betty H. Olson; Jian-Wen He; Samuel Choi; Jenny Zhang; Joanne Y. Le; Phillip B. Gedalanga (pp. 927-934).

Three independent microbial source tracking (MST) methods were applied to a small urban subwatershed in Orange County, California. Fifty-seven water samples collected over summer 2002 were analyzed for human adenovirus and enterovirus. Enterococci and E. coli were isolated for antibiotic resistance analysis (ARA) and for PCR identification of human- and animal-specific toxin genes, respectively. All water samples were PCR negative for human enteroviruses and E. coli human-specific toxin gene. E. coli toxin markers revealed the presence of toxin genes specific to bird, rabbit, and cow. Enterococci ARA results supported this conclusion and indicated that fecal bacteria from bird and wild animal feces as well as soil were the predominant source found in the watershed. An E. coli, isolated from the watershed and inoculated back into the heat-sterilized storm drain water, increased 4 log units within 6 days. Collectively, these results suggest that bird and wild animal feces, soil amendments, and/or fecal coliform growth in the storm drain are the major contributors to the fecal bacterial pollution in downstream areas. However, human adenoviruses were detected on two occasions. Fecal bacterial concentrations were not elevated on these two occasions, suggesting that the elevated levels of fecal indicator bacteria in this small watershed could be unrelated to the source of human adenovirus.

Keywords: Microbial source tracking (MST); Urban watershed; Human virus; E. coli biomarkers; Adenovirus; Enterovirus; Antibiotic resistance analysis (ARA)

Persistence of host-specific Bacteroides–Prevotella 16S rRNA genetic markers in environmental waters: effects of temperature and salinity by Satoshi Okabe; Yoko Shimazu (pp. 935-944).

Host-specific Bacteroides–Prevotella 16S rRNA genetic markers are promising alternative indicators for identifying the sources of fecal pollution because of their high abundance in the feces of warm-blooded animals and high host specificity. However, little is known about the persistence of these genetic markers in environments after being released into environmental waters. The persistence of feces-derived four different host-specific Bacteroides–Prevotella 16S rRNA genetic makers (total, human-, cow-, and pig-specific) in environmental waters was therefore investigated at different incubation temperatures (4, 10, 20, and 30°C) and salinities (0, 10, 20, and 30 ppt) and then compared with the survival of conventional fecal-indicator organisms. The host-specific genetic markers were monitored by using real-time polymerase chain reaction (PCR) assays with specific primer sets. Each host-specific genetic marker showed similar responses in non-filtered river water and seawater: They persisted longer at lower temperatures and higher salinities. In addition, these markers did not increase in all conditions tested. Decay rates for indicator organisms were lower than those for host-specific genetic markers at temperature above 10°C. Furthermore, we investigated whether the PCR-detectable 16S rRNA genetic markers reflect the presence of live target cells or dead target cells in environmental waters. The result revealed that the detection of the Bacteroides–Prevotella 16S rRNA genetic markers in environmental waters mainly reflected the presence of ‘viable but non-culturable’ Bacteroides–Prevotella cells. These findings indicate that seasonal and geographical variations in persistence of these host-specific Bacteroides–Prevotella 16S rRNA genetic markers must be considered when we use them as alternative fecal indicators in environmental waters.

Keywords: Microbial source tracking; Bacteroides–Prevotella 16S rRNA genetic markers; Persistence; Real-time PCR; Temperature and salinity effects

Gene inactivation mediated by Agrobacterium tumefaciens in the filamentous fungi Metarhizium anisopliae by Charley Christian Staats; Angela Junges; Mariana Fitarelli; Marcia Cristina Furlaneto; Marilene Henning Vainstein; Augusto Schrank (pp. 945-950).

The list of fungal species with known complete genome and/or expressed sequence tag collections is extending rapidly during the last couple of years. Postgenomic gene function assignment is an obvious follow-up and depends on methodologies to test gene function in vivo. One of such methods is the generation of null mutants via homologous recombination at the wild–type loci by using inactivation cassettes. In this paper, the ability of Agrobacterium tumefaciens to genetically transform filamentous fungi was exploited to drive homologous recombination at the trp1 locus of the enthomopathogenic fungus Metarhizium anisopliae. The trp1 disruptants exhibited a clearly distinguishable phenotype from wild-type cells and were recovered with high efficiency of homologous recombination (22%). The complementation of such mutants with the wild-type gene generates only transformants with homologous integration.

Keywords: Metarhizium; Agrotransformation; Gene inactivation; Entomopathogen

Effects of dilution on dissolved oxygen depletion and microbial populations in the biochemical oxygen demand determination by Kyo Seong Seo; Ho Nam Chang; Joong Kon Park; Kwang-Ho Choo (pp. 951-956).

The biochemical oxygen demand (BOD) value is still a key parameter that can determine the level of organics, particularly the content of biodegradable organics in water. In this work, the effects of sample dilution, which should be done inevitably to get appropriate dissolved oxygen (DO) depletion, on the measurement of 5-day BOD (BOD₅), was investigated with and without seeding using natural and synthetic water. The dilution effects were also evaluated for water samples taken in different seasons such as summer and winter because water temperature can cause a change in the types of microbial species, thus leading to different oxygen depletion profiles during BOD testing. The predation phenomenon between microbial cells was found to be dependent on the inorganic nutrients and carbon sources, showing a change in cell populations according to cell size after 5-day incubation. The dilution of water samples for BOD determination was linked to changes in the environment for microbial growth such as nutrition. The predation phenomenon between microbial cells was more important with less dilution. BOD₅ increased with the specific amount of inorganic nutrient per microbial mass when the natural water was diluted. When seeding was done for synthetic water samples, the seed volume also affected BOD due to the rate of organic uptake by microbes. BOD₅ increased with the specific bacterial population per organic source supplied at the beginning of BOD measurement. For more accurate BOD measurements, specific guidelines on dilution should be established.

Keywords: Biochemical oxygen demand; Dilution; Microbial population; Nutrient; Predation

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Applied Microbiology and Biotechnology (v.76, #4)