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Applied Microbiology and Biotechnology (v.95, #6)

Biosynthesis and metabolic pathways of pivalic acid by Tomáš Řezanka; Irena Kolouchová; Alena Čejková; Karel Sigler (pp. 1371-1376).

Occurrence, biosynthesis, and biodegradation of pivalic acid and other compounds, having a quaternary carbon atom by different bacteria, are described. We have summarized the relevant data that have so far been published, presenting them in a graphical form, i.e., as biodegradation pathways including B₁₂-dependent isomerization and desaturation that lead to the degradation of pivalic acid and similar compounds to products with other than quaternary carbon atoms, i.e., compounds whose catabolism is well known.

Keywords: Pivalic acid; Isooctane; Biosynthesis; Biodegradation

Engineering yeasts for raw starch conversion by W. H. van Zyl; M. Bloom; M. J. Viktor (pp. 1377-1388).

Next to cellulose, starch is the most abundant hexose polymer in plants, an import food and feed source and a preferred substrate for the production of many industrial products. Efficient starch hydrolysis requires the activities of both α-1,4 and α-1,6-debranching hydrolases, such as endo-amylases, exo-amylases, debranching enzymes, and transferases. Although amylases are widely distributed in nature, only about 10 % of amylolytic enzymes are able to hydrolyse raw or unmodified starch, with a combination of α-amylases and glucoamylases as minimum requirement for the complete hydrolysis of raw starch. The cost-effective conversion of raw starch for the production of biofuels and other important by-products requires the expression of starch-hydrolysing enzymes in a fermenting yeast strain to achieve liquefaction, hydrolysis, and fermentation (Consolidated Bioprocessing, CBP) by a single organism. The status of engineering amylolytic activities into Saccharomyces cerevisiae as fermentative host is highlighted and progress as well as challenges towards a true CBP organism for raw starch is discussed. Conversion of raw starch by yeast secreting or displaying α-amylases and glucoamylases on their surface has been demonstrated, although not at high starch loading or conversion rates that will be economically viable on industrial scale. Once efficient conversion of raw starch can be demonstrated at commercial level, engineering of yeast to utilize alternative substrates and produce alternative chemicals as part of a sustainable biorefinery can be pursued to ensure the rightful place of starch converting yeasts in the envisaged bio-economy of the future.

Keywords: Amylases; Recombinant yeast; Raw starch conversion; Consolidated bioprocessing

The chromatin code of fungal secondary metabolite gene clusters by Agnieszka Gacek; Joseph Strauss (pp. 1389-1404).

Secondary metabolite biosynthesis genes in fungi are usually physically linked and organized in large gene clusters. The physical linkage of genes involved in the same biosynthetic pathway minimizes the amount of regulatory steps necessary to regulate the biosynthetic machinery and thereby contributes to physiological economization. Regulation by chromatin accessibility is a proficient molecular mechanism to synchronize transcriptional activity of large genomic regions. Chromatin regulation largely depends on DNA and histone modifications and the histone code hypothesis proposes that a certain combination of modifications, such as acetylation, methylation or phosphorylation, is needed to perform a specific task. A number of reports from several laboratories recently demonstrated that fungal secondary metabolite (SM) biosynthesis clusters are controlled by chromatin-based mechanisms and histone acetyltransferases, deacetylases, methyltransferases, and proteins involved in heterochromatin formation were found to be involved. This led to the proposal that establishment of repressive chromatin domains over fungal SM clusters under primary metabolic conditions is a conserved mechanism that prevents SM production during the active growth phase. Consequently, transcriptional activation of SM clusters requires reprogramming of the chromatin landscape and replacement of repressive histone marks by activating marks. This review summarizes recent advances in our understanding of chromatin-based SM cluster regulation and highlights some of the open questions that remain to be answered before we can draw a more comprehensive picture.

Keywords: Secondary metabolism; Chromatin; Aspergillus ; Histone modifications

Current state and perspectives on erythropoietin production by Jae Seong Lee; Tae Kwang Ha; Seung Joo Lee; Gyun Min Lee (pp. 1405-1416).

Erythropoietin is a major regulator of erythropoiesis which maintains the body’s red blood cell mass and tissue oxygenation at an optimum level. Recombinant human erythropoietin (rhEPO), which is a widely used therapeutic agent for the treatment of anemia and which represents one of the largest biopharmaceuticals markets, is produced from recombinant Chinese hamster ovary cells. rhEPO is a glycoprotein with complex glycan structure, which is responsible for its therapeutic efficacy, including the in vivo activity and half-life. In order to obtain an optimal and consistent glycoform profile of rhEPO and concurrently maintain a high production yield, various approaches in drug development and cell culture technology have been attempted. Recent advances in rhEPO production are classified into three types: the development of improved rhEPO molecules by protein engineering; improvement of production host cells by genetic engineering; and culture condition optimization by fine control of the production mode/system, process parameters, and culture media. In this review, we focus on rhEPO production strategies as they have progressed thus far. Furthermore, the current status of the market and outlook on rhEPO and its derivatives are discussed.

Keywords: Erythropoietin; CHO cells; Glycosylation; EPO market

Biotechnological production of arbutins (α- and β-arbutins), skin-lightening agents, and their derivatives by Dong-Ho Seo; Jong-Hyun Jung; Jae-Eun Lee; Eun-Jung Jeon; Wooki Kim; Cheon-Seok Park (pp. 1417-1425).

Arbutins (α- and β-arbutins) are glycosylated hydroquinones that are commercially used in the cosmetic industry. These compounds have an inhibitory function against tyrosinase, a critical enzyme for generating pigments, which leads to the prevention of melanin formation, resulting in a whitening effect on the skin. Although β-arbutin is found in various plants including bearberry, wheat, and pear, α-arbutin and other arbutin derivatives are synthesized by chemical and enzymatic methods. This article presents a mini-review of recent studies on the production of α-arbutin and other α- and β-arbutin derivatives via enzymatic bioconversion methods. In addition, the structures of α- and β-arbutin derivatives and their biological activities are discussed. The catalytic characteristics of various enzymes used in the biosynthesis of arbutin derivatives are also reviewed.

Keywords: Amylosucrase; α-Arbutin; β-Arbutin; Hydroquinone; Skin-lightening agent

Application of microorganisms towards synthesis of chiral terpenoid derivatives by Renata Kuriata-Adamusiak; Daniel Strub; Stanisław Lochyński (pp. 1427-1436).

Biotransformations are a standard tool of green chemistry and thus are following the rules of sustainable development. In this article, we describe the most common types of reactions conducted by microorganisms applied towards synthesis of chiral terpenoid derivatives. Potential applications of obtained products in various areas of industry and agriculture are shown. We also describe biological activity of presented compounds. Stereoselective hydroxylation, epoxidation, Baeyer–Villiger oxidation, stereo- and enantioselective reduction of ketones, and various kinetic resolutions carried out by bacteria and fungi have been reviewed. Mechanistic considerations regarding chemical and enzymatic reactions are presented. We also briefly describe modern approaches towards enhancing desired enzymatic activity in order to apply modified biocatalysts as an efficient tool and green alternative to chemical catalysts used in industry.

Keywords: Biotransformations; Terpenoids; Microorganisms; Stereochemistry

Prophylactic and therapeutic efficacy of the epitope vaccine CTB-UA against Helicobacter pylori infection in a BALB/c mice model by Le Guo; Kunmei Liu; Guangxian Xu; Xiaokang Li; Jiajie Tu; Feng Tang; Yingying Xing; Tao Xi (pp. 1437-1444).

Epitope vaccine based on the enzyme urease of Helicobacter pylori is a promising option for prophylactic and therapeutic vaccination against H. pylori infection. In our previous study, the epitope vaccine CTB-UA, which was composed of the mucosal adjuvant cholera toxin B subunit (CTB) and an epitope (UreA_183–203) from the H. pylori urease A subunit (UreA) was constructed. This particular vaccine was shown to have good immunogenicity and immunoreactivity and could induce specific neutralizing antibodies, which exhibited effectively inhibitory effects on the enzymatic activity of H. pylori urease. In this study, the prophylactic and therapeutic efficacy of the epitope vaccine CTB-UA was evaluated in a BALB/c mice model. The experimental results indicated that oral prophylactic or therapeutic immunization with CTB-UA significantly decreased H. pylori colonization compared with oral immunization with PBS. The results also revealed that the protection was correlated with antigen-specific IgG, IgA, and mucosal secretory IgA antibody responses. CTB-UA may be a promising vaccine candidate for the control of H. pylori infection.

Keywords: Epitope vaccine; Helicobacter pylori ; Cholera toxin B subunit; Urease A subunit; Neutralizing antibody

Yeast cells as microcapsules. Analytical tools and process variables in the encapsulation of hydrophobes in S. cerevisiae by Federica Ciamponi; Craig Duckham; Nicola Tirelli (pp. 1445-1456).

Yeast cells can be used as biocompatible and biodegradable containers for the microencapsulation of a variety of actives. Despite the wide application of this process, e.g. in the food industry, mechanism and controlling factors are yet poorly known. In this study we have studied kinetics and mechanistic aspects of the spontaneous internalization of terpenes (as model hydrophobic compounds) in Saccharomyces cerevisiae, quantifying their encapsulation through HPLC analysis and fluorescent staining of lipidic bodies with Nile Red, while in parallel monitoring cell viability. Our results showed that this encapsulation process is essentially a phenomenon of passive diffusion with negligible relevance of active transport. Further, our evidence shows that the major determinant of the encapsulation kinetics is the solubility of the hydrophobe in the cell wall, which is inversely related to partition coefficient (log P).

Keywords: Encapsulation; Yeast; Cell wall; Flavours; Diffusion

Novel whole-cell biocatalysts with recombinant hydroxysteroid dehydrogenases for the asymmetric reduction of dehydrocholic acid by Michael Braun; Boqiao Sun; Bernd Anselment; Dirk Weuster-Botz (pp. 1457-1468).

Ursodeoxycholic acid is an important pharmaceutical so far chemically synthesized from cholic acid. Various biocatalytic alternatives have already been discussed with hydroxysteroid dehydrogenases (HSDH) playing a crucial role. Several whole-cell biocatalysts based on a 7α-HSDH-knockout strain of Escherichia coli overexpressing a recently identified 7β-HSDH from Collinsella aerofaciens and a NAD(P)-bispecific formate dehydrogenase mutant from Mycobacterium vaccae for internal cofactor regeneration were designed and characterized. A strong pH dependence of the whole-cell bioreduction of dehydrocholic acid to 3,12-diketo-ursodeoxycholic acid was observed with the selected recombinant E. coli strain. In the optimal, slightly acidic pH range dehydrocholic acid is partly undissolved and forms a suspension in the aqueous solution. The batch process was optimized making use of a second-order polynomial to estimate conversion as function of initial pH, initial dehydrocholic acid concentration, and initial formate concentration. Complete conversion of 72 mM dehydrocholic acid was thus made possible at pH 6.4 in a whole-cell batch process within a process time of 1 h without cofactor addition. Finally, a NADH-dependent 3α-HSDH from Comamonas testosteroni was expressed additionally in the E. coli production strain overexpressing the 7β-HSDH and the NAD(P)-bispecific formate dehydrogenase mutant. It was shown that this novel whole-cell biocatalyst was able to convert 50 mM dehydrocholic acid directly to 12-keto-ursodeoxycholic acid with the formation of only small amounts of intermediate products. This approach may be an efficient process alternative which avoids the costly chemical epimerization at C-7 in the production of ursodeoxycholic acid.

Keywords: Biocatalysis; Cofactor regeneration; Hydroxysteroid dehydrogenase; Ursodeoxycholic acid; Response surface methodology

Directed evolution and structural prediction of cellobiohydrolase II from the thermophilic fungus Chaetomium thermophilum by Xiu-Juan Wang; Yan-Jie Peng; Li-Qing Zhang; An-Na Li; Duo-Chuan Li (pp. 1469-1478).

Cellulases can be engineered with enhanced properties for broad use in scientific and industrial applications. In this study, the wild-type cbh2 gene of the thermophilic fungus Chaetomium thermophilum encoding cellobiohydrolase II (CBHII) was mutagenized through in vitro directed evolution. The resulting Pichia pastoris yeast library was screened, and two transformants were selected for enhanced CBHII activities that were not attributed to increased gene copy numbers. The optimum fermentation times of the two mutant transformants were shortened to 4–5 days after methanol induction compared to 6 days for the wild-type. The optimum reaction temperature (60 °C) and pH level (5 or 6) of the mutant CBHII proteins, designated CBHIIX16 and CBHIIX305, were higher than those of wild-type CBHII (50 °C and pH 4). Kept at 80 °C for 1 h, CBHIIX16 and CBHIIX305 retained >50% of their activities, while the wild-type CBHII lost all activity. Sequence analysis of CBHIIX16 and CBHIIX305 revealed that they contained five and six mutated amino acids, respectively. Structural modeling confirmed the presence of carbohydrate binding type-1 and catalytic domains, where the hydrogen bond numbers between the 227th and 203rd amino acids were increased, which perhaps contributed to the elevated enzyme stability. Therefore, the two CBHII mutants selected for increased enzymatic activities also demonstrated elevated optimum reaction temperature and pH levels and enhanced thermal stability. These properties may be beneficial in practical applications for CBHII.

Keywords: Cellobiohydrolase II; Chaetomium thermophilum ; Directed evolution; Structure prediction

Expanding the set of rhodococcal Baeyer–Villiger monooxygenases by high-throughput cloning, expression and substrate screening by A. Riebel; H. M. Dudek; G. de Gonzalo; P. Stepniak; L. Rychlewski; M. W. Fraaije (pp. 1479-1489).

To expand the available set of Baeyer–Villiger monooxygenases (BVMOs), we have created expression constructs for producing 22 Type I BVMOs that are present in the genome of Rhodococcus jostii RHA1. Each BVMO has been probed with a large panel of potential substrates. Except for testing their substrate acceptance, also the enantioselectivity of some selected BVMOs was studied. The results provide insight into the biocatalytic potential of this collection of BVMOs and expand the biocatalytic repertoire known for BVMOs. This study also sheds light on the catalytic capacity of this large set of BVMOs that is present in this specific actinomycete. Furthermore, a comparative sequence analysis revealed a new BVMO-typifying sequence motif. This motif represents a useful tool for effective future genome mining efforts.

Keywords: Biocatalysis; Baeyer–Villiger monooxygenase; Sulfoxidation; Enantioselectivity

Efficient biocatalyst for large-scale synthesis of cephalosporins, obtained by combining immobilization and site-directed mutagenesis of penicillin acylase by Davide A. Cecchini; Roberto Pavesi; Sara Sanna; Simona Daly; Roberto Xaiz; Massimo Pregnolato; Marco Terreni (pp. 1491-1500).

We describe the rational design of a new efficient biocatalyst and the development of a sustainable green process for the synthesis of cephalosporins bearing a NH₂ group on the acyl side chain. The new biocatalyst was developed starting from the WT penicillin acylase (PA) from Escherichia coli by combining enzyme mutagenesis, in position α146 and β24 (βF24A/αF146Y), and immobilization on an appropriate modified industrial support, glyoxyl Eupergit C250L. The obtained derivative was used in the kinetically controlled synthesis of cephalexin, cefprozil and cefaclor and compared to the WT-PA and an already described mutant, PA-βF24A, with improved properties. The new biocatalyst posses a very high ratio between the rates of the synthesis and two undesired hydrolyses (acylating ester and the amidic product). In particular, a very low amidase activity was observed with PA-βF24A/αF146Y and, consequently, the hydrolysis of the produced antibiotic was avoided during the process. Taking advantage of this property, higher conversions in the synthesis of cephalexin (99% versus 76%), cefaclor (99% versus 65%) and cefprozil (99% versus 60%) were obtained compared to the WT enzyme. Furthermore, the new mutant also show a higher synthetic activity compared to PA-βF24A immobilized on the same support, allowing the maximum yields to be achieved in very short reaction times. The production of cephalexin with the immobilized βF24A/αF146Y acylase has been developed on a pre-industrial scale (30 l). After 20 cycles, the average yield was 93%. The biocatalyst showed good stability properties and no significant decrease in performance.

Keywords: Biocatalyst; Penicillin acylase; Enzyme mutagenesis; Enzyme immobilization; Cephalosporins

Characterization of porcine circovirus type 2 (PCV2) capsid particle assembly and its application to virus-like particle vaccine development by Pei-Ching Wu; Wei-Li Lin; Chi-Ming Wu; Jiun-Ni Chi; Maw-Sheng Chien; Chienjin Huang (pp. 1501-1507).

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. The sole structural capsid protein of PCV2, Cap, consists of major antigenic domains, but little is known about the assembly of capsid particles. The purpose of this study is to produce a large amount of Cap protein using Escherichia coli expression system for further studying the essential sequences contributing to formation of particles. By using codon optimization of rare arginine codons near the 5′-end of the cap gene for E. coli, a full-length Cap without any fusion tag recombinant protein (Cap1-233) was expressed and proceeded to form virus-like particles (VLPs) in normal Cap appearance that resembled the authentic PCV2 capsid. The N-terminal deletion mutant (Cap51-233) deleted the nuclear localization signal (NLS) domain, while the internal deletion mutant (CapΔ51-103) deleted a likely dimerization domain that failed to form VLPs. The unique Cys108 substitution mutant (CapC/S) exhibited most irregular aggregates, and only few VLPs were formed. These results suggest that the N-terminal region within the residues 1 to 103 possessing the NLS and dimerization domains are essential for self-assembly of stable Cap VLPs, and the unique Cys108 plays an important role in the integrity of VLPs. The immunogenicity of PCV2 VLPs was further evaluated by immunization of pigs followed by challenge infection. The Cap1-233-immunized pigs demonstrated specific antibody immune responses and are prevented from PCV2 challenge, thus implying its potential use for a VLP-based PCV2 vaccine.

Keywords: Porcine circovirus type 2 (PCV2); Cap protein; Capsid assembly; Virus-like particles (VLPs); Codon optimization

Generation of an actagardine A variant library through saturation mutagenesis by Steven Boakes; Tania Ayala; Mark Herman; Antony N. Appleyard; Michael J. Dawson; Jesús Cortés (pp. 1509-1517).

The lantibiotic actagardine A is nineteen amino acids in length and comprises three intertwined C-terminal methyllanthionine-bridged rings and an N-terminal lanthionine-bridged ring. Produced by the actinomycete Actinoplanes garbadinensis ATCC 31049, actagardine A demonstrates antibacterial activity against important Gram-positive pathogens. This activity combined with its ribosomal synthesis makes it an attractive target for the generation of lantibiotic variants with improved biological activity. A variant generation system designed to allow the specific substitution of amino acids at targeted sites throughout the actagardine A peptide has been used to generate a comprehensive library by site-directed mutagenesis. With the exception of residues involved in bridge formation, each amino acid in the actagardine A peptide as well as the alanine (ala(0)) at position −1 relative to the mature peptide, has been systematically substituted with all remaining 19 amino acids. A total of 228 mutants have been engineered with 44 produced in good yield. The mutant V15F in particular demonstrates improved activity against a range of notable Gram-positive pathogens including Clostridium difficile, when evaluated alongside actagardine A. The scope of variants generated provides an insight into the flexibility of the actagardine A processing machinery and will undoubtedly assist in future mutational studies.

Keywords: Antibacterial; Lantibiotic; Actagardine A; Peptide engineering; Actinoplanes garbadinensis

Rhamnolipid production: effect of oxidative stress on virulence factors and proteome of Pseudomonas aeruginosa PA1 by Graziela Jardim Pacheco; Rodrigo Siqueira Reis; Ana Carolina Loureiro Brito Fernandes; Surza Lucia Gonçalves da Rocha; Marcos Dias Pereira; Jonas Perales; Denise Maria Guimarães Freire (pp. 1519-1529).

Under specific environmental conditions, Pseudomonas aeruginosa produces a biodegradable surfactant rhamnolipid. Evidences suggest that this biosurfactant is involved in protecting cells against oxidative stress; however, the effects of oxidative stress on its production and other virulence factors are still unclear. Here we show that rhamnolipid production is dependent on the aeration surface when P. aeruginosa is cultured in shaken flasks, as well as in production of elastases and alkaline proteases. The production of alginate, lipase, and pyocyanin was not detected in our shaken-flask experiments. P. aeruginosa was treated with hydrogen peroxide to trigger its oxidative stress response, and the proteome profile was analyzed. We identified 14 proteins that were expressed differently between samples that were treated and not treated with peroxide; these proteins are potentially involved in the rhamnolipid production/secretion pathway and oxidative stress.

Keywords: Pseudomonas aeruginosa ; Rhamnolipid; Oxidative stress; Virulence factors; Proteomics

High-temperature sorbose fermentation with thermotolerant Gluconobacter frateurii CHM43 and its mutant strain adapted to higher temperature by Hiromi Hattori; Toshiharu Yakushi; Minenosuke Matsutani; Duantip Moonmangmee; Hirohide Toyama; Osao Adachi; Kazunobu Matsushita (pp. 1531-1540).

We succeeded in obtaining a strain adapted to higher temperature from a thermotolerant strain, Gluconobacter frateurii CHM43, for sorbose fermentation. The adapted strain showed higher growth and l-sorbose production than original CHM43 strain at higher temperature around 38.5–40 °C. It was also shown to be useful even with the fermentation without temperature control. To understand the sorbose fermentation ability of the adapted strain at higher temperature, d-sorbitol-oxidizing respiratory chain was compared with the CHM43 strain and the adapted strain. We found that the activity of pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase (GLDH), which is a primary dehydrogenase of the respiratory chain and responsible for l-sorbose production, was decreased when the temperature increased, but the decreased activity of GLDH was recovered by the addition of PQQ. Since the adapted strain was found to produce more PQQ than the CHM43 strain, it was suggested that the adapted strain keeps GLDH as holoenzyme with the increased PQQ production, and thus produces more l-sorbose and grows better under higher temperature.

Keywords: Quinoprotein glycerol dehydrogenase; PQQ; Sorbose fermentation; Thermotolerance; Adaptation; Gluconobacter

Adaptation dynamics of Clostridium butyricum in high 1,3-propanediol content media by Afroditi Chatzifragkou; George Aggelis; Chryssavgi Gardeli; Maria Galiotou-Panayotou; Michael Komaitis; Seraphim Papanikolaou (pp. 1541-1552).

Aim of the present study was to evaluate the effect of exogenous additions of 1,3-propanediol (1,3-PDO) on microbial growth and metabolites production of Clostridium butyricum VPI 1718 strain, during crude glycerol fermentation. Preliminary batch cultures in anaerobic Duran bottles revealed that early addition of 1,3-PDO caused growth cessation in rather low quantities (15 g/L), while 1,3-PDO additions during the middle exponential growth phase up to 70 g/L resulted in an almost linear decrease of the specific growth rate (μ), accompanied by reduced glycerol assimilation, with substrate consumption being used mainly for energy of maintenance requirements. During batch trials in a 3-L bioreactor, the strain proved able to withstand more than 70 g/L of both biologically produced and externally added 1,3-PDO, whereas glycerol assimilation and metabolite production were carried on at a lower rate. Adaptation of the strain in high 1,3-PDO concentration environments was validated during its continuous cultivation with pulses of 1,3-PDO in concentrations of 31 and 46 g/L, where no washout phenomena were noticed. As far as C. butyricum cellular lipids were concerned, during batch bioreactor cultivations, 1,3-PDO addition was found to favor the biosynthesis of unsaturated fatty acids. Also, fatty acid composition was studied during continuous cultures, in which additions of 1,3-PDO were performed at steady states. Lipids were globally more saturated compared to batch cultures, while by monitoring of the transitory phases, it was noticed that the gradual diol washout had an evident impact in the alteration of the fatty acid composition, by rendering them more unsaturated.

Keywords: Clostridium butyricum ; 1,3-Propanediol; Crude glycerol; Cellular lipids

Transcriptional and preliminary functional analysis of the six genes located in divergence of phoR/phoP in Streptomyces lividans by Emmanuelle Darbon; Cécile Martel; Aleksandra Nowacka; Sylvine Pegot; Patrice L. Moreau; Marie-Joëlle Virolle (pp. 1553-1566).

Streptomyces lividans senses and adjusts to a situation of Pi limitation via the expression of genes of the pho regulon controlled by the two-component system PhoR/PhoP. Interestingly, an in silico analysis of the proteins encoded by the six genes located in divergence of phoR/phoP revealed that the latter bear features often found in metalloproteins involved in the sensing/resistance to oxidative stress. We determined whether genes of this region were belonging to the pho regulon and whether the encoded proteins do play a role in the resistance to oxidative stress. For this purpose, a transcriptional analysis of these genes was carried out on the carbon and nitrogen rich medium R2YE and on a minimal medium (MM). On R2YE, the expression of the genes phoU to sco4225 was much higher than on MM and constant throughout growth. On this medium, the expression of phoU was totally PhoP-dependent whereas the expression of sco4227 and sco4226 was partially PhoP-dependent, taking place from the phoU promoter region. In contrast, on MM, the expression of sco4227 and sco4226 was PhoP-independent whereas that of phoU remained PhoP-dependent and showed, as phoR/phoP, a peak of expression at 48 h. sco4225, sco4224, and sco4223 were transcribed from their own promoter independently of PhoP in both media. The mutants of five out of six genes of the region (Δsco4226 mutant could not be obtained) grew poorly in the presence of exogenous oxidants, suggesting a role of the encoded proteins in the resistance to oxidative stress, especially on the rich medium R2YE.

Keywords: Streptomyces ; Pho regulon; Pi limitation; Oxidative stress

Identification of nicotine biotransformation intermediates by Agrobacterium tumefaciens strain S33 suggests a novel nicotine degradation pathway by Shuning Wang; Haiyan Huang; Kebo Xie; Ping Xu (pp. 1567-1578).

Nicotine, a major alkaloid in tobacco plants and the main toxic chemical in tobacco wastes, can be transformed by bacteria into hydroxylated-pyridine intermediates, which are important precursors for the chemical synthesis of valuable drugs and insecticides. Such biotransformation could be a useful approach to utilize tobacco and its wastes. In this study, we explored nicotine degradation by a recently isolated Agrobacterium tumefaciens S33 by identifying the intermediates during its growth on nicotine and during transformation of nicotine with its resting cells. Five hydroxylated-pyridine intermediates were detected through multiple approaches, including GC-HR-MS, HPLC, and ESI-Q-TOF MS analyses. Surprisingly, these identified intermediates suggest that strain S33 employs a novel pathway that is different from the two characterized pathways described in Arthrobacter and Pseudomonas. Based on these findings, we propose that strain S33 is able to transform nicotine to 6-hydroxy-pseudooxynicotine first via the pyridine pathway through 6-hydroxy-L-nicotine and 6-hydroxy-N-methylmyosmine, and then, it turns to the pyrrolidine pathway with the formation of 6-hydroxy-3-succinoylpyridine and 2,5-dihydroxypyridine. The activities of the key enzymes, nicotine dehydrogenase, 6-hydroxy-L-nicotine oxidase, and 6-hydroxy-3-succinoylpyridine hydroxylase, were demonstrated in the cell extract of strain S33 and by partially enriched enzymes. Moreover, the cell extract could transform 6-hydroxy-pseudooxynicotine into 6-hydroxy-3-succinoylpyridine by coupling with 6-hydroxy-L-nicotine oxidation reaction by 6-hydroxy-L-nicotine oxidase. These results indicated that strain S33 can transform nicotine into renewable hydroxylated-pyridine intermediates by the special pathway, in which at least three intermediates, 6-hydroxy-L-nicotine, 6-hydroxy-3-succinoylpyridine, and 2,5-dihydroxypyridine, have potential to be further chemically modified into useful compounds.

Keywords: Nicotine; Biotransformation; Biodegradation; Agrobacterium tumefaciens ; Intermediate; Pathway; Functionalized pyridine

A universal primer multiplex PCR method for typing of toxinogenic Pseudomonas aeruginosa by Hui Shi; Quoclinh Trinh; Wentao Xu; Baiqiang Zhai; Yunbo Luo; Kunlun Huang (pp. 1579-1587).

Pseudomonas aeruginosa is a well-known opportunistic pathogen that can cause acute nosocomial necrotizing pneumonia and genetic disorder cystic fibrosis of lung patients. Pathogenic interactions between P. aeruginosa and hosts are often guided by the secreted virulence determinants that interact with specific host targets. Exotoxin A, pyocyanin, elastase, and type III secretion system are the most significant virulence determinants and cause great concern. However, P. aeruginosa in various environments has high genotypic diversity, leading to deficiency of exotoxin genes for some P. aeruginosa strains. In current study, a universal primer–multiplex PCR method (UP-MPCR) was employed for the detection of five significant enterotoxin genes (toxA, phzM, lasB, ExoU, and ExoS) and one internal control gene ecfX in P. aeruginosa. Owing to the application of universal primer (UP), different targeted products have identical amplified efficiency and the sensitivity of multiplex PCR is improved. In addition, the complexity of multiplex PCR system is reduced and the compatibility of primers in a reaction is greatly increased. This UP-MPCR method can detect the presence of five P. aeruginosa enterotoxin genes in a single assay more rapidly and sensitively than conventional methods. In 214 drinking water and environmental isolates, the ExoU, ExoS, phzM, toxA, and lasB genes were detected in 20 (9 %), 180 (84 %), 179 (84 %), 196 (92 %), and 171 (80 %) isolates, respectively.

Keywords: P. aeruginosa ; UP-MPCR; Exotoxin genes; Drinking water; Environment

Plant exudates promote PCB degradation by a rhodococcal rhizobacteria by Jean-Patrick Toussaint; Thi Thanh My Pham; Diane Barriault; Michel Sylvestre (pp. 1589-1603).

Rhodococcus erythropolis U23A is a polychlorinated biphenyl (PCB)-degrading bacterium isolated from the rhizosphere of plants grown on a PCB-contaminated soil. Strain U23A bphA exhibited 99% identity with bphA1 of Rhodococcus globerulus P6. We grew Arabidopsis thaliana in a hydroponic axenic system, collected, and concentrated the plant secondary metabolite-containing root exudates. Strain U23A exhibited a chemotactic response toward these root exudates. In a root colonizing assay, the number of cells of strain U23A associated to the plant roots (5.7 × 10⁵ CFU g⁻¹) was greater than the number remaining in the surrounding sand (4.5 × 10⁴ CFU g⁻¹). Furthermore, the exudates could support the growth of strain U23A. In a resting cell suspension assay, cells grown in a minimal medium containing Arabidopsis root exudates as sole growth substrate were able to metabolize 2,3,4′- and 2,3′,4-trichlorobiphenyl. However, no significant degradation of any of congeners was observed for control cells grown on Luria–Bertani medium. Although strain U23A was unable to grow on any of the flavonoids identified in root exudates, biphenyl-induced cells metabolized flavanone, one of the major root exudate components. In addition, when used as co-substrate with sodium acetate, flavanone was as efficient as biphenyl to induce the biphenyl catabolic pathway of strain U23A. Together, these data provide supporting evidence that some rhodococci can live in soil in close association with plant roots and that root exudates can support their growth and trigger their PCB-degrading ability. This suggests that, like the flagellated Gram-negative bacteria, non-flagellated rhodococci may also play a key role in the degradation of persistent pollutants.

Keywords: Rhodococcus erythropolis ; Polychlorinated biphenyl (PCB); Rhizoremediation; Root exudates; Plant secondary metabolites (PSMs); Flavonoids; Flavanone

Surface display of monkey metallothionein α tandem repeats and EGFP fusion protein on Pseudomonas putida X4 for biosorption and detection of cadmium by Xiaochuan He; Wenli Chen; Qiaoyun Huang (pp. 1605-1613).

Monkey metallothionein α domain tandem repeats (4mMTα), which exhibit high cadmium affinity, have been displayed for the first time on the surface of a bacterium using ice nucleation protein N-domain (inaXN) protein from the Xanthomonas campestris pv (ACCC—10049) as an anchoring motif. The shuttle vector pIME, which codes for INAXN–4mMTα–EGFP fusion, was constructed and used to target 4mMTα and EGFP on the surface of Pseudomonas putida X4 (CCTCC—209319). The surface location of the INAXN–4mMTα–EGFP fusion was further verified by western blot analysis and immunofluorescence microscopy. The growth of X4 showed resistance to cadmium presence. The presence of surface-exposed 4mMTα on the engineered strains was four times higher than that of the wild-type X4. The Cd²⁺ accumulation by X4/pIME was not only four times greater than that of the original host bacterial cells but was also remarkably unaffected by the presence of Cu²⁺ and Zn²⁺. Moreover, the surface-engineered strains could effectively bind Cd²⁺ under a wide range of pH levels, from 4 to 7. P. putida X4/pIME with surface-expressed 4mMTα–EGFP had twice the cadmium binding capacity as well as 1.4 times the fluorescence as the cytoplasmic 4mMTa–EGFP. These results suggest that P. putida X4 expressing 4mMTα–EGFP with the INAXN anchor motif on the surface would be a useful tool for the remediation and biodetection of environmental cadmium contaminants.

Keywords: Cadmium; Fluorescence; Surface display; Ice nucleation protein; Metallothionein; Pseudomonas putida

Changes in functional diversity of soil microbial community with addition of antibiotics sulfamethoxazole and chlortetracycline by Feng Liu; Jinshui Wu; Guang-Guo Ying; Zhuanxi Luo; Hong Feng (pp. 1615-1623).

Potential effects of antibiotics on agricultural soil microflora have recently become increasing concerns with antibiotic-contaminated biosolid now being used in agricultural land. However, changes of soil microbial community function caused by the antibiotic-associated disturbance are less addressed. This paper investigated the changes in microbial functional diversity by spiking sulfamethoxazole (SMX) and chlortetracycline (CTC) in a loam paddy soil and then incubating for 21 days. The dose–effect and time-dependent changes of antibiotic-associated disturbance on soil microbial community were analyzed with the soils sampled at 7 and 21 days using Biolog EcoPlate. At day 7 following treatment, SMX decreased functional diversity of soil microbial community, and the treatment of 100 mg SMX kg⁻¹ dry soil had a significant inhibition of average well color development (AWCD) and Shannon index as compared to the control (p < 0.05). The SMX changed to improve soil microbial community function at day 21. CTC had less effect on soil microbial community function during the whole incubation period. Antibiotic dissipation and adsorption in soil, which may decrease their microbial bioavailability, led to the temporary change of antibiotic effect on functional diversity of soil microbial community. Principal component analysis clearly revealed the difference of antibiotic dose–effects on the soil microbial community function. The findings demonstrated that soil microbial community showed more sensitivity to SMX than CTC, and soil microbial community function could recover or improve due to antibiotic dissipation in soil.

Keywords: Antibiotic-associated disturbance; Soil microbial community; Functional diversity; Biolog EcoPlate; Dissipation

Selection of a whole-cell biocatalyst for methyl parathion biodegradation by Jijian Yang; Ruihua Liu; Hong Jiang; Yao Yang; Chuanling Qiao (pp. 1625-1632).

Whole-cell biocatalyst has the potential to become a cost-effective alternative to conventional enzyme methods for solving ecological and energy issues. However, cytosolic-expressing biocatalyst systems are critically disadvantaged due to the low permeability of the cell membrane. To overcome substrate transport barrier, periplasmic secretion and surface display biocatalysts were developed by expressing signal peptides or anchor proteins in Escherichia coli. In this work, six carriers were compared in regard to whole-cell activity of methyl parathion hydrolase (MPH). Our results indicate that the surface display systems yielded one to three times whole-cell activity than the periplasmic secretion systems. Although periplasmic secretion systems showed generally more stable than surface display systems, surface display appeared more suitable for whole-cell biocatalyst. It should note that the applicability of the DsbA/PhoA/AIDA-I leader to MPH expression is shown here for the first time. In addition, the result provided a useful reference for other whole-cell biocatalyst selection.

Keywords: Methyl parathion hydrolase; Periplasmic secretion; Surface display; Biocatalyst; Biodegradation

Performances of two biotrickling filters in treating H₂S-containing waste gases and analysis of corresponding bacterial communities by pyrosequencing by Jianjun Li; Guangyun Ye; Duanfang Sun; Guoping Sun; Xiaowei Zeng; Jian Xu; Shizhong Liang (pp. 1633-1641).

Two identical biotrickling filters named BTFa and BTFb were run in parallel to examine their performances in removing hydrogen sulfide. BTFa was filled with ceramic granules, and BTFb was filled with volcanic rocks. The results showed that BTFb was more robust than BTFa under acidic conditions. At empty bed residence times (EBRTs) of 20 and 15 s, the removal efficiency of BTFa was close to 100%. At EBRTs of 10 and 5 s, the removal efficiency of BTFa slightly decreased. The removal efficiencies of BTFa decreased by different degrees at the end of each stage, dropping to 94%, 81%, 60%, and 71%, respectively. However, the H₂S removal efficiency in BTFb consistently reached 99% throughout the experiment. Pyrosequencing analyses indicated that members of Thiomonas dominated in both BTFs, but the relative abundance of Acidithiobacillus was higher in BTFb than in BTFa.

Keywords: Hydrogen sulfide; Biotrickling filter; Bacterial community; Pyrosequencing

Molecular cloning, purification, and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2 by Su In Lee; Sung Hee Choi; Eun Yeol Lee; Hee Sook Kim (pp. 1643-1653).

A gene for a polyMG-specific alginate lyase possessing a novel structure was identified and cloned from Stenotrophomas maltophilia KJ-2 by using PCR with homologous nucleotide sequences-based primers. The recombinant alginate lyase consisting of 475 amino acids was purified on Ni-Sepharose column and exhibited the highest activity at pH 8 and 40 °C. Interestingly, the recombinant alginate lyase was expected to have a similar catalytic active site of chondroitin B lyase but did not show chondroitin lyase activity. In the test of substrate specificity, the recombinant alginate lyase preferentially degraded the glycosidic bond of polyMG-block than polyM-block and polyG-block. The chemical structures of the degraded alginate oligosaccharides were elucidated to have mannuronate (M) at the reducing end on the basis of NMR analysis, supporting that KJ-2 polyMG-specific alginate lyase preferably degraded the glycosidic bond in M–G linkage than that in G–M linkage. The KJ-2 polyMG-specific alginate lyase can be used in combination with other alginate lyases for a synergistic saccharification of alginate.

Keywords: Alginate; Alginate lyase; PolyMG-specific alginate lyase; Saccharification; Stenotrophomas maltophilia KJ-2

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Applied Microbiology and Biotechnology (v.95, #6)