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Applied Microbiology and Biotechnology (v.76, #2)

Biocatalytic ketone reduction—a powerful tool for the production of chiral alcohols—part I: processes with isolated enzymes by Katja Goldberg; Kirsten Schroer; Stephan Lütz; Andreas Liese (pp. 237-248).

Enzymes are able to perform reactions under mild conditions, e.g., pH and temperature, with remarkable chemo-, regio-, and stereoselectivity. Because of this feature, the number of biocatalysts used in organic synthesis has rapidly increased during the last decades, especially for the production of chiral compounds. The present review highlights biotechnological processes for the production of chiral alcohols by reducing prochiral ketones. These reactions can be catalyzed by either isolated enzymes or whole cells that exhibit ketone-reducing activity. The use of isolated enzymes is often preferred because of a higher volumetric productivity and the absence of side reactions. Both types of catalysts have also deficiencies limiting their use in synthesis of chiral alcohols. Because reductase-catalyzed reactions are dependent on cofactors, one major task in process development is to provide an effective method for regeneration of the consumed cofactors. In this paper, strategies for cofactor regeneration in biocatalytic ketone reduction are reviewed. Furthermore, different processes carried out on laboratory and industrial scales using isolated enzymes are presented. Attention is turned to process parameters, e.g., conversion, yield, enantiomeric excess, and process strategies, e.g., the application of biphasic systems or methods of in situ (co)product recovery. The biocatalytic production of chiral alcohols utilizing whole cells is presented in part II of this review (Goldberg et al., Appl Microbiol Biotechnol, 2007).

Keywords: Ketone reduction; Cofactor regeneration; Chiral alcohol

Biocatalytic ketone reduction—a powerful tool for the production of chiral alcohols—part II: whole-cell reductions by Katja Goldberg; Kirsten Schroer; Stephan Lütz; Andreas Liese (pp. 249-255).

Enzymes are able to perform reactions under mild conditions, e.g., pH and temperature, with remarkable chemo-, regio-, and stereoselectivity. Due to this feature the number of biocatalysts used in organic synthesis has rapidly increased during the last decades, especially for the production of chiral compounds. The present review highlights biotechnological processes for the production of chiral alcohols by reducing prochiral ketones with whole cells. Microbial transformations feature different characteristics in comparison to isolated enzymes. Enzymes that are used in whole-cell biotransformations are often more stable due to the presence of their natural environment inside the cell. Because reductase-catalyzed reactions are dependent on cofactors, one major task in process development is to provide an effective method for regeneration of the consumed cofactors. Many whole-cell biocatalysts offer their internal cofactor regeneration that can be used by adding cosubstrates, glucose or, in the case of cyanobacteria, simply light. In this paper, various processes carried out on laboratory and industrial scales are presented. Thereby, attention is turned to process parameters, e.g., conversion, yield, enantiomeric excess, and process strategies, e.g., the application of biphasic systems. The biocatalytic production of chiral alcohols utilizing isolated enzymes is presented in part I of this review (Goldberg et al., Appl Microbiol Biotechnol, 2007).

Keywords: Ketone reduction; Whole cell biotransformation; Chiral alcohol

Single molecule techniques for the study of membrane proteins by Ana J. García-Sáez; Petra Schwille (pp. 257-266).

Single molecule techniques promise novel information about the properties and behavior of individual particles, thus enabling access to molecular heterogeneities in biological systems. Their recent developments to accommodate membrane studies have significantly deepened the understanding of membrane proteins. In this short review, we will describe the basics of the three most common single-molecule techniques used on membrane proteins: fluorescence correlation spectroscopy, single particle tracking, and atomic force microscopy. We will discuss the most relevant findings made during the recent years and their contribution to the membrane protein field.

Keywords: Membrane protein; Single molecule technique; Atomic force microscopy; Fluorescence correlation spectroscopy; Single particle tracking; Single molecule microscopy

TNT biotransformation: when chemistry confronts mineralization by Barth F. Smets; Hong Yin; Abraham Esteve-Nuñez (pp. 267-277).

Our understanding of the genetics and biochemistry of microbial 2,4,6-trinitrotoluene (TNT) biotransformation has advanced significantly during the past 10 years, and biotreatment technologies have developed. In this review, we summarize this new knowledge. A number of enzyme classes involved in TNT biotransformation include the type I nitroreductases, the old yellow enzyme family, a respiration-associated nitroreductase, and possibly ring hydroxylating dioxygenases. Several strains harbor dual pathways: nitroreduction (reduction of the nitro group in TNT to a hydroxylamino and/or amino group) and denitration (reduction of the aromatic ring of TNT to Meisenheimer complexes with nitrite release). TNT can serve as a nitrogen source for some strains, and the postulated mechanism involves ammonia release from hydroxylamino intermediates. Field biotreatment technologies indicate that both stimulation of microbial nitroreduction and phytoremediation result in significant and permanent immobilization of TNT via its metabolites. While the possibility for TNT mineralization was rekindled with the discovery of TNT denitration and oxygenolytic and respiration-associated pathways, further characterization of responsible enzymes and their reaction mechanisms are required.

Keywords: TNT; Biotransformation; Nitroreductase; Denitration; Old yellow enzyme; Phytoremediation

Availability and applications of ATP-binding cassette (ABC) transporter blockers by Alicia Ponte-Sucre (pp. 279-286).

ATP-binding cassette (ABC) transporters encompass membrane transport proteins that couple the energy derived from ATP hydrolysis to the translocation of solutes across biological membranes. The functions of these proteins include ancient and conserved mechanisms related to nutrition and pathogenesis in bacteria, spore formation in fungi, and signal transduction, protein secretion and antigen presentation in eukaryotes. Furthermore, one of the major causes of drug resistance and chemotherapeutic failure in both cancer and anti-infective therapies is the active movement of compounds across membranes carried out by ABC transporters. Thus, the clinical relevance of ABC transporters is enormous, and the membrane transporters related to chemoresistance are among the best-studied members of the ABC transporter superfamily. As ABC transporter blockers can be used in combination with current drugs to increase their efficacy, the (possible) impact of efflux pump inhibitors is of great clinical interest. The present review summarizes the progress made in recent years in the identification, design, availability, and applicability of ABC transporter blockers in experimental scenarios oriented towards improving the treatment of infectious diseases caused by microorganisms including parasites.

Keywords: ABC transporters; ABC transporter blockers; Microorganisms; Drug resistance

Bioremediation and monitoring of aromatic-polluted habitats by Vincenza Andreoni; Liliana Gianfreda (pp. 287-308).

Bioremediation may restore contaminated soils through the broad biodegradative capabilities evolved by microorganisms towards undesirable organic compounds. Understanding bioremediation and its effectiveness is rapidly advancing, bringing available molecular approaches for examining the presence and expression of the key genes involved in microbial processes. These methods are continuously improving and require further development and validation of primer- and probe-based analyses and expansion of databases for alternative microbial markers. Phylogenetic marker approaches provide tools to determine which organisms are present or generally active in a community; functional gene markers provide only information concerning the distribution or transcript levels (deoxyribonucleic acid [DNA]- or messenger ribonucleic acid [mRNA]-based approaches) of specific gene populations across environmental gradients. Stable isotope probing methods offer great potential to identify microorganisms that metabolize and assimilate specific substrates in environmental samples, incorporating usually a rare isotope (i.e., ¹³C) into their DNA and RNA. DNA and RNA in situ characterization allows the determination of the species actually involved in the processes being measured. DNA microarrays may analyze the expression of thousands of genes in a soil simultaneously. A global analysis of which genes are being expressed under various conditions in contaminated soils will reveal the metabolic status of microorganisms and indicate environmental modifications accelerating bioremediation.

Keywords: PAHs; BTEX; Bioremediation; Molecular approaches; Monitoring; Soil quality

Potential and capabilities of hydroxynitrile lyases as biocatalysts in the chemical industry by Thomas Purkarthofer; Wolfgang Skranc; Christian Schuster; Herfried Griengl (pp. 309-320).

The application of hydroxynitrile lyases (HNLs) as catalysts for the stereoselective condensation of HCN with carbonyl compounds has been reported as early as 1908. This enzymatic C–C bond coupling reaction furnishes enantiopure cyanohydrins which serve as versatile bifunctional building blocks for chemical synthesis. Screening of natural sources led to the discovery of both (R)- and (S)-selective HNLs, and several distinctly different classes of these enzymes with substantial differences concerning sequence, structure, and mechanism have been found. Especially during the last two centuries, HNLs have been developed into valuable biocatalysts, which can be produced in recombinant form by overexpression in microbial hosts, resulting in the implementation of industrial processes utilizing these enzymes. Recently, protein engineering in combination with in silico methods gave rise to the development of a tailor-made HNL for large-scale manufacturing of a specific target cyanohydrin.

Production of uridine 5′-monophosphate by Corynebacterium ammoniagenes ATCC 6872 using a statistically improved biocatalytic process by Xing Wang; Xiuwen Wang; Mengxin Yin; Zijun Xiao; Cuiqing Ma; Zhixin Lin; Peng George Wang; Ping Xu (pp. 321-328).

Attempts were made with success to develop a two-step biocatalytic process for uridine 5′-monophosphate (UMP) production from orotic acid by Corynebacterium ammoniagenes ATCC 6872: the strain was first cultivated in a high salt mineral medium, and then cells were harvested and used as the catalyst in the UMP production reaction. Effects of cultivation and reaction conditions on UMP production were investigated. The cells exhibited the highest biocatalytic ability when cultivated in a medium containing corn steep liquor at pH 7.0 for 15 h in the exponential phase of growth. To optimize the reaction, both “one-factor-at-a-time” method and statistical method were performed. By “one-factor-at-a-time” optimization, orotic acid, glucose, phosphate ion (equimolar KH₂PO₄ and K₂HPO₄), MgCl₂, Triton X-100 were shown to be the optimum components for the biocatalytic reaction. Phosphate ion and C. ammoniagenes cell were furthermore demonstrated as the most important main effects on UMP production by Plackett–Burman design, indicating that 5-phosphoribosyl-1-pyrophosphate (PRPP) synthesis was the rate-limiting step for pyrimidine nucleotides production. Optimization by a central composition design (CCD) was then performed, and up to 32 mM (10.4 g l⁻¹) UMP was accumulated in 24 h from 38.5 mM (6 g l⁻¹) orotic acid. The yield was threefold higher than the original UMP yield before optimization.

Keywords: Corynebacterium ammoniagenes ; Uridine 5′-monophosphate; Biocatalytic production; Statistical optimization

Enhanced production of ɛ-caprolactone by overexpression of NADPH-regenerating glucose 6-phosphate dehydrogenase in recombinant Escherichia coli harboring cyclohexanone monooxygenase gene by Won-Heong Lee; Jin-Byung Park; Kyungmoon Park; Myoung-Dong Kim; Jin-Ho Seo (pp. 329-338).

Whole-cell conversion of cyclohexanone to ɛ-caprolactone was attempted by recombinant Escherichia coli BL21(DE3) expressing cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871. High concentrations of cyclohexanone and ɛ-caprolactone reduced CHMO-mediated bioconversion of cyclohexanone to ɛ-caprolactone in the resting recombinant E. coli cells. Metabolically active cells were employed by adopting a fed-batch culture to improve the production of ɛ-caprolactone from cyclohexanone. A glucose-limited fed-batch Baeyer–Villiger oxidation where a cyclohexanone level was maintained less than 6 g/l resulted in a maximum ɛ-caprolactone concentration of 11.0 g/l. The maximum ɛ-caprolactone concentration was improved further to 15.3 g/l by coexpression of glucose-6-phosphate dehydrogenase, an NADPH-generating enzyme encoded by the zwf gene which corresponded to a 39% enhancement in ɛ-caprolactone concentration compared with the control experiment performed under the same conditions.

Keywords: Cyclohexanone monooxygenase; Escherichia coli ; Glucose 6-phosphate dehydrogenase; Fed-batch; Substrate/product inhibition; NADPH

H₂ enrichment from synthesis gas by Desulfotomaculum carboxydivorans for potential applications in synthesis gas purification and biodesulfurization by Jan Sipma; M. Begoña Osuna; Sofiya N. Parshina; Gatze Lettinga; Alfons J. M. Stams; Piet N. L. Lens (pp. 339-347).

Desulfotomaculum carboxydivorans, recently isolated from a full-scale anaerobic wastewater treatment facility, is a sulfate reducer capable of hydrogenogenic growth on carbon monoxide (CO). In the presence of sulfate, the hydrogen formed is used for sulfate reduction. The organism grows rapidly at 200 kPa CO, pH 7.0, and 55°C, with a generation time of 100 min, producing nearly equimolar amounts of H₂ and CO₂ from CO and H₂O. The high specific CO conversion rates, exceeding 0.8 mol CO (g protein)⁻¹ h⁻¹, makes this bacterium an interesting candidate for a biological alternative of the currently employed chemical catalytic water–gas shift reaction to purify synthesis gas (contains mainly H₂, CO, and CO₂). Furthermore, as D. carboxydivorans is capable of hydrogenotrophic sulfate reduction at partial CO pressures exceeding 100 kPa, it is also a good candidate for biodesulfurization processes using synthesis gas as electron donor at elevated temperatures, e.g., in biological flue gas desulfurization. Although high maximal specific sulfate reduction rates (32 mmol (g protein)⁻¹ h⁻¹) can be obtained, its sulfide tolerance is rather low and pH dependent, i.e., maximally 9 and 5 mM sulfide at pH 7.2 and pH 6.5, respectively.

Keywords: Desulfotomaculum carboxydivorans ; Carbon monoxide; Hydrogenogen; Thermophilic; Sulfate reduction; Biohydrogen production; Biological flue gas desulfurization

Characterization of alcohol dehydrogenase 1 and 3 from Neurospora crassa FGSC2489 by Yong-Cheol Park; Ka-Yiu San; George N. Bennett (pp. 349-356).

Alcohol dehydrogenase (ADH) is a key enzyme in the production and utilization of alcohols. Some also catalyze the formation of carboxylate esters from alcohols and aldehydes. The ADH1 and ADH3 genes of Neurospora crassa FGSC2489 were cloned and expressed in recombinant Escherichia coli to investigate their alcohol dehydrogenation and carboxylate ester formation abilities. Homology analysis and sequence alignment of amino acid sequence indicated that ADH1 and ADH3 of N. crassa contained a zinc-binding consensus sequence and a NAD⁺-binding motif and showed 54–75% identity with fungi ADHs. N. crassa ADH1 was expressed in E. coli to give a specific activity of 289 ± 9 mU/mg using ethanol and NAD⁺ as substrate and cofactor, respectively. Corresponding experiments on the expression and activity of ADH3 gave 4 mU/mg of specific activity. N. crassa ADH1 preferred primary alcohols containing C3–C8 carbons to secondary alcohols such as 2-propanol and 2-butanol. N. crassa ADH1 possessed 5.3 mU/mg of specific carboxylate ester-forming activity accumulating 0.4 mM of ethyl acetate in 18 h. Substrate specificity of various linear alcohols and aldehydes indicated that short chain-length alcohols and aldehydes were good substrates for carboxylate ester production. N. crassa ADH1 was a primary alcohol dehydrogenase using cofactor NAD⁺ preferably and possessed carboxylate ester-forming activity with short chain alcohols and aldehydes.

Keywords: Ester; Alcohol dehydrogenase; Hemiacetal; Fungi; Primary alcohol

Purification and characterization of aldehyde dehydrogenase with a broad substrate specificity originated from 2-phenylethanol-assimilating Brevibacterium sp. KU1309 by Jun-ichiro Hirano; Kenji Miyamoto; Hiromichi Ohta (pp. 357-363).

Phenylacetaldehyde dehydrogenase (PADH) was purified and characterized from Brevibacterium sp. KU1309, which can grow on the medium containing 2-phenylethanol as the sole carbon source. This enzyme was a homotetrameric protein with a subunit of 61 kDa. The enzyme catalyzed the oxidation of aryl (benzaldehyde, phenylacetaldehyde, 3-phenylpropionaldehyde) and aliphatic (hexanal, octanal, decanal) aldehydes to the corresponding carboxylic acids using NAD⁺ as the electron acceptor. The PADH activity was enhanced by several divalent cationic ions such as Mg²⁺, Ca²⁺, and Mn²⁺. On the other hand, it was inhibited by SH reagents (Hg²⁺, p-chloromercuribenzoate, iodoacetamide, and N-ethylmaleinimide). The substrate specificity of the enzyme is compared with those of various aldehyde dehydrogenases.

Cloning and characterization of three epoxide hydrolases from a marine bacterium, Erythrobacter litoralis HTCC2594 by Jung-Hee Woo; Young-Ok Hwang; Sung Gyun Kang; Hyun Sook Lee; Jang-Cheon Cho; Sang-Jin Kim (pp. 365-375).

Previously, we reported that ten strains belonging to Erythrobacter showed epoxide hydrolase (EHase) activities toward various epoxide substrates. Three genes encoding putative EHases were identified by analyzing open reading frames of Erythrobacter litoralis HTCC2594. Despite low similarities to reported EHases, the phylogenetic analysis of the three genes showed that eeh1 was similar to microsomal EHase, while eeh2 and eeh3 could be grouped with soluble EHases. The three EHase genes were cloned, and the recombinant proteins (rEEH1, rEEH2, and rEEH3) were purified. The functionality of purified proteins was proved by hydrolytic activities toward styrene oxide. EEH1 preferentially hydrolyzed (R)-styrene oxide, whereas EEH3 preferred to hydrolyze (S)-styrene oxide, representing enantioselective hydrolysis of styrene oxide. On the other hand, EEH2 could hydrolyze (R)- and (S)-styrene oxide at an equal rate. The optimal pH and temperature for the EHases occurred largely at neutral pHs and 40–55 °C. The substrate selectivity of rEEH1, rEEH2, and rEEH3 toward various epoxide substrates were also investigated. This is the first representation that a strict marine microorganism possessed three EHases with different enantioselectivity toward styrene oxide.

Keywords: Epoxide hydrolase; Marine bacterium; Erythrobacter ; Overexpression; Genomics

Production in Trichoderma reesei of three xylanases from Chaetomium thermophilum: a recombinant thermoxylanase for biobleaching of kraft pulp by Arja Mäntylä; Marja Paloheimo; Satu Hakola; Emilia Lindberg; Sanna Leskinen; Jarno Kallio; Jari Vehmaanperä; Raija Lantto; Pirkko Suominen (pp. 377-386).

Three endoxylanase genes were cloned from the thermophilic fungus Chaetomium thermophilum CBS 730.95. All genes contained the typical consensus sequence of family 11 glycoside hydrolases. Genomic copies of Ct xyn11A, Ct xyn11B, and Ct xyn11C were expressed in the filamentous fungus T. reesei under the control of the strong T. reesei cel7A (cellobiohydrolase 1, cbh1) promoter. The molecular masses of the Ct Xyn11A, Ct Xyn11B, and Ct Xyn11C proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were 27, 23, and 22 kDa, respectively. Ct Xyn11A was produced almost as efficiently as the homologous xylanase II from a corresponding single-copy transformant strain. Ct Xyn11B production level was approximately half of that of Ct Xyn11A. The amount of Ct Xyn11C was remarkably lower. Ct Xyn11A had the highest temperature optimum and stability of the recombinant xylanases and the highest activity at acid-neutral pH (pH 5–7). It was the most suitable for industrial bleaching of kraft pulp at high temperature.

Acid phosphatase production by recombinant Arxula adeninivorans by Neha Minocha; Parvinder Kaur; T. Satyanarayana; G. Kunze (pp. 387-393).

Acid phosphatase production by recombinant Arxula adeninivorans was carried out in submerged fermentation. Using the Plackett–Burman design, three fermentation variables (pH, sucrose concentration, and peptone concentration) were identified to significantly affect acid phosphatase and biomass production, and these were optimized using response surface methodology of central composite design. The highest enzyme yields were attained in the medium with 3.9% sucrose and 1.6% peptone at pH 3.8. Because of optimization, 3.86- and 4.19-fold enhancement in enzyme production was achieved in shake flasks (17,054 U g⁻¹ DYB) and laboratory fermenter (18,465 U g⁻¹ DYB), respectively.

Keywords: Arxula adeninivorans ; APHO1 ; Secretory acid phosphatase; Phytase; Yeast

Alkaliphilic Bacillus sp. strain KSM-LD1 contains a record number of subtilisin-like serine proteases genes by Yasushi Takimura; Kazuhiro Saito; Mitsuyoshi Okuda; Yasushi Kageyama; Katsuhisa Saeki; Katsuya Ozaki; Susumu Ito; Tohru Kobayashi (pp. 395-405).

The presence of 11 genes encoding subtilisin-like serine proteases was demonstrated by cloning from the genome of alkaliphilic Bacillus sp. strain KSM-LD1. This strain exoproduces the oxidatively stable alkaline protease LD-1 (Saeki et al. Curr Microbiol, 47:337–340, 2003). Among the 11 genes, six genes encoding alkaline proteases (SA, SB, SC, SD, SE, and LD-1) were expressed in Bacillus hosts. However, the other five genes for subtilisin-like proteases (SF, SG, SH, SI, and SJ) were expressed in neither Bacillus hosts nor Escherichia coli. The deduced amino acid sequences of SA, SB, SC, SF, SG, SH, SI, and SJ showed similarity to those of other subtilisin-like proteases from Bacillus strains with only 38 to 86% identity. The deduced amino acid sequence of SD was completely identical to that of an oxidatively stable alkaline protease from Bacillus sp. strain SD521, and that of SE was almost identical to that of a high-molecular mass subtilisin from Bacillus sp. strain D-6 with 99.7% identity. There are four to nine subtilisin-like serine protease genes in the reported genomes of Bacillus strains. At least 11 genes for the enzymes present in the genome of Bacillus sp. strain KSM-LD1, and this is the greatest number identified to date.

Keywords: Serine protease; Subtilisin; Genome; Alkaliphile; Bacillus

Carbon-oxygen bond formation by fungal laccases: cross-coupling of 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide with the solvents water, methanol, and other alcohols by Katrin Manda; Dirk Gördes; Annett Mikolasch; Elke Hammer; Enrico Schmidt; Kerstin Thurow; Frieder Schauer (pp. 407-416).

Laccase-catalyzed reactions lead to oxidation of the substrate via a cation radical, which has been described to undergo proton addition to form a quinonoid derivative or nucleophilic attack by itself producing homomolecular dimers. In this study, for the substrate 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide, we show that, besides the quinonoid form of substrate, all products formed are nonhomomolecular ones. Indeed, without addition of a reaction partner, heteromolecular products are formed from the quinonoid form of the laccase-substrate and the solvents water or methanol present in the incubation assay. Consequently, in laccase catalyzed syntheses performed in aqueous solutions or in the presence of methanol or other alcohols, undesirable heteromolecular coupling reactions between the laccase substrate and solvents must be taken into account. Additionally, it could be shown at the example of methanol and other alcohols that C-O-bound cross-coupling of dihydroxylated aromatic substances with the hydroxyl group of aliphatic alcohols can be catalyzed by fungal laccases.

Keywords: Laccase; Homomolecular product; Cross-coupling; Alcohols; C-O-bound

Functional analysis of very long-chain fatty acid elongase gene, HpELO2, in the methylotrophic yeast Hansenula polymorpha by Phatthanon Prasitchoke; Yoshinobu Kaneko; Minetaka Sugiyama; Takeshi Bamba; Eiichiro Fukusaki; Akio Kobayashi; Satoshi Harashima (pp. 417-427).

We describe the cloning and functional characterization of the fatty acid elongase gene HpELO2, a homologue of the HpELO1 gene required for the production of C24:0 in the yeast Hansenula polymorpha. The open reading frame (ORF) of HpELO2 consists of 1,035 bp, encoding 344 amino acids, sharing about 65% identity with that of Saccharomyces cerevisiae Elo2. Expression of HpELO2 rescued the lethality of the S. cerevisiae elo2Δ elo3Δ double disruptant. An accumulation of C18:0 and a significant increase and decrease in the levels of C24:0 and C26:0, respectively, were observed in the Hpelo2Δ disruptant. These results supported an idea that HpELO2 encodes a fatty acid elongase involved in the elongation of C18:0 to very long-chain fatty acids. The Hpelo1Δ Hpelo2Δ double disruption was nonviable, suggesting that HpELO1 and HpELO2 are the only two genes necessary for the biosynthesis in H. polymorpha. Interestingly, transcription of HpELO2 and HpELO1 were found to be transiently up-regulated by exogenous long-chain fatty acids; however, this up-regulation was not observed with HpELO1 and HpELO2 genes driven by the constitutively expressed promoter of the HpACT gene, suggesting that exogenous fatty acids specifically trigger the transcriptional induction of HpELO1 and HpELO2 through their promoter regions.

Keywords: Methylotrophic yeast; Hansenula polymorpha ; Elongase for very long-chain fatty acids; Transient transcriptional induction

Blue–white selection of regulatory genes that affect the expression of dehalogenase IVa of Burkholderia cepacia MBA4 by Yun-wing Faan; Manda Yu; Jimmy S. H. Tsang (pp. 429-437).

We have developed a method for rapid screening of genes that affected the expression of dehalogenase IVa of Burkholderia cepacia MBA4. The promoter region of the dehalogenase gene was used to drive the expression of a beta-galactosidase gene. A plasmid containing this reporter was first electroporated into MBA4, and a Tn5 containing suicidal plasmid was introduced subsequently. The use of electroporation was necessary because Escherichia coli mediated transconjugation was ineffective in plasmid-carrying MBA4. The number of integrants generated was directly proportional to the amount of plasmid DNA used. Integrants with an elevated beta-galactosidase activity were isolated. Mutants with a disruption in a putative iron-transporter gene and in a putative response regulator receiver gene were identified. The basal dehalogenase transcript levels of these mutants were higher than the wild type. These mutants also grow faster than the wild type in chloroacetate-containing medium. This methodology of isolating regulatory mutants is theoretically feasible and convenient for any kinds of bacteria.

Keywords: Burkholderia ; Electro-transformation; Electroporation; Regulatory gene screening; Transposon-mutagenesis

Bifunctional enzyme fusion of 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase by Izumi Orita; Naoki Sakamoto; Nobuo Kato; Hiroya Yurimoto; Yasuyoshi Sakai (pp. 439-445).

The formaldehyde-fixing enzymes, 3-Hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), are the key enzymes catalyzing sequential reactions in the ribulose monophosphate (RuMP) pathway. In this study, we generated two fused gene constructs of the hps and phi genes (i.e., hps–phi and phi–hps) from a methylotrophic bacterium Mycobacterium gastri MB19. The gene product of hps–phi exhibited both HPS and PHI activities at room temperature and catalyzed the sequential reactions more efficiently than a simple mixture of the individual enzymes. The gene product of phi–hps failed to display any enzyme activity. Escherichia coli strains harboring the hps–phi gene consumed formaldehyde more efficiently and exhibited better growth in a formaldehyde-containing medium than the host strain. Our results demonstrate that the engineered fusion gene has the possibility to be used to establish a formaldehyde-resistance detoxification system in various organisms.

Keywords: 3-Hexulose-6-phosphate synthase; 6-phospho-3-hexuloisomerase; Ribulose monophosphate pathway; Mycobacterium gastri ; Fusion enzyme

Comparative transcriptome analysis of Desulfovibrio vulgaris grown in planktonic culture and mature biofilm on a steel surface by Weiwen Zhang; David E. Culley; Lei Nie; Johannes C. M. Scholten (pp. 447-457).

Biofilm build-up of sulphate-reducing bacteria (SRB) on metal surfaces may lead to severe corrosion of iron. To understand the processes at molecular level, in this study, a whole-genome oligonucleotide microarray was used to examine differential expression patterns between planktonic populations and mature biofilm of Desulfovibrio vulgaris on a steel surface. Statistical analysis revealed that 472 genes were differentially expressed (1.5-fold or more with a q value less than 0.025) by comparing the biofilm cells with the planktonic cells. Among the differentially expressed genes were several that corresponded to genes identified in many aerobic bacterial biofilms (i.e., Pseudomonas species and Escherichia coli) such as genes encoding flagellin, a flagellar motor switch protein, chemotaxis proteins involved in cell motility, as well as genes involved in exopolysaccharide biosynthesis. In addition, the biofilm-bound cells of D. vulgaris exhibited decreased transcription of genes involved in protein synthesis, energy metabolism and sulfate reduction, as well as genes involved in general stress responses. These findings were all consistent with early suggestion that the average physiology of the biofilm cells were similar to cells reduced in growth. Most notably, up-regulation of large number of outer membrane proteins was observed in the D. vulgaris biofilm. Although their function is still unknown, the higher expression of these genes in the biofilm could implicate important roles in the formation and maintenance of multi-cellular consortium on a steel surface. The study provided insights into the metabolic networks associated with the formation and maintenance of a D. vulgaris biofilm on a steel surface.

Keywords: Transcriptome analysis; Biofilm; Corrosion; Desulfovibrio vulgaris

Study of the antifungal activity of Acinetobacter baumannii LCH001 in vitro and identification of its antifungal components by C. H. Liu; X. Chen; T. T. Liu; B. Lian; Yucheng Gu; V. Caer; Y. R. Xue; B. T. Wang (pp. 459-466).

An Acinetobacter strain, given the code name LCH001 and having the potential to be an endophytic antagonist, has been isolated from healthy stems of the plant Cinnamomum camphora (L.) Presl, guided by an in vitro screening technique. The bacterium inhibited the growth of several phytopathogenic fungi such as Cryphonectria parasitica, Glomerella glycines, Phytophthora capsici, Fusarium graminearum, Botrytis cinerea, and Rhizoctonia solani. Biochemical, physiological, and 16S rDNA sequence analysis proved that it is Acinetobacter baumannii. When the filtrate from the fermentation broth of strain LCH001 was tested in vitro and in vivo, it showed strong growth inhibition against several phytopathogens including P. capsici, F. graminearum, and R. solani, indicating that suppression of the growth of the fungi was due to the presence of antifungal compounds in the culture broth. Moreover, the antifungal activity of the culture filtrate was significantly correlated with the cell growth of strain LCH001. The active metabolites in the filtrate were relatively thermally stable, but were sensitive to acidic conditions. Three antifungal compounds were isolated from the culture broth by absorption onto macropore resin, ethanol extraction, chromatography on silica gel or LH-20 columns, and crystallization. The structures of the bioactive compounds were identified by spectroscopic methods as isomers of iturin A, namely, iturin A2, iturin A3, and iturin A6. The characterization of an unusual endophytic bacterial strain LCH001 and its bioactive components may provide an alternative resource for the biocontrol of plant diseases.

Keywords: Acinetobacter baumannii LCH001; Antifungal activity; Iturin A

Characterization of technetium(vII) reduction by cell suspensions of thermophilic bacteria and archaea by Nikolay A. Chernyh; Sergei N. Gavrilov; Vladimir V. Sorokin; Konstantin E. German; Claire Sergeant; Monique Simonoff; Frank Robb; Alexander I. Slobodkin (pp. 467-472).

Washed cell suspensions of the anaerobic hyperthermophilic archaea Thermococcus pacificus and Thermoproteus uzoniensis and the anaerobic thermophilic gram-positive bacteria Thermoterrabacterium ferrireducens and Tepidibacter thalassicus reduced technetium [⁹⁹Tc(VII)], supplied as soluble pertechnetate with molecular hydrogen as an electron donor, forming highly insoluble Tc(IV)-containing grayish-black precipitate. Apart from molecular hydrogen, T. ferrireducens reduced Tc(VII) with lactate, glycerol, and yeast extract as electron donors, and T. thalassicus reduced it with peptone. Scanning electron microscopy and X-ray microanalysis of cell suspensions of T. ferrireducens showed the presence of Tc-containing particles attached to the surfaces of non-lysed cells. This is the first report on the reduction in Tc(VII) by thermophilic microorganisms of the domain Bacteria and by archaea of the phylum Euryarchaeota.

Keywords: Technetium reduction; Tc(VII) reduction; Thermophilic microorganisms; Radionuclide immobilization

Biotechnological intensification of biogas production by Zoltán Bagi; Norbert Ács; Balázs Bálint; Lenke Horváth; Krisztina Dobó; Katalin R. Perei; Gábor Rákhely; Kornél L. Kovács (pp. 473-482).

The importance of syntrophic relationships among microorganisms participating in biogas formation has been emphasized, and the regulatory role of in situ hydrogen production has been recognized. It was assumed that the availability of hydrogen may be a limiting factor for hydrogenotrophic methanogens. This hypothesis was tested under laboratory and field conditions by adding a mesophilic (Enterobacter cloacae) or thermophilic hydrogen-producing (Caldicellulosyruptor saccharolyticus) strain to natural biogas-producing consortia. The substrates were waste water sludge, dried plant biomass from Jerusalem artichoke, and pig manure. In all cases, a significant intensification of biogas production was observed. The composition of the generated biogas did not noticeably change. In addition to being a good hydrogen producer, C. saccharolyticus has cellulolytic activity; hence, it is particularly suitable when cellulose-containing biomass is fermented. The process was tested in a 5-m³ thermophilic biogas digester using pig manure slurry as a substrate. Biogas formation increased at least 160–170% upon addition of the hydrogen-producing bacteria as compared to the biogas production of the spontaneously formed microbial consortium. Using the hydrogenase-minus control strain provided evidence that the observed enhancement was due to interspecies hydrogen transfer. The on-going presence of C. saccharolyticus was demonstrated after several months of semicontinuous operation.

Keywords: Biogas; Hydrogen; Interspecies electron transfer; Intensification; Syntrophy; Thermophilic

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Applied Microbiology and Biotechnology (v.76, #2)

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Applied Microbiology and Biotechnology (v.76, #2)