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Applied Microbiology and Biotechnology (v.87, #4)

Biotechnological doxorubicin production: pathway and regulation engineering of strains for enhanced production by Narayan Prasad Niraula; Seon-Hye Kim; Jae Kyung Sohng; Eung-Soo Kim (pp. 1187-1194).

Doxorubicin (DXR) is an anthracycline-type polyketide, typically produced by Streptomyces peucetius ATCC 27952. Like the biosynthesis of other secondary metabolites in Streptomyces species, DXR biosynthesis is tightly regulated, and a very low level of DXR production is maintained in the wild-type strain. Despite that DXR is one of the most broadly used and clinically important anticancer drugs, a traditional strain improvement strategy has long been practiced via recursive random mutagenesis, with little understanding of the molecular genetic basis underlying such enhanced DXR production. Since DXR titer enhancement is imperative in the fermentation industry, attaining a comprehensive understanding and its application of the specific regulatory systems that govern secondary metabolite production is an important aspect of metabolic engineering that can efficiently improve fermentation titers. In this mini-review, various efforts to improve the titers of DXR have been summarized based on biosynthetic and regulatory studies including transcriptional and product analyses.

Keywords: Biosynthesis; Doxorubicin; Feedback regulation; Pathway engineering; Product inhibition; Regulatory engineering; Strain improvement

Engineering cellulolytic ability into bioprocessing organisms by Daniel C. la Grange; Riaan den Haan; Willem H. van Zyl (pp. 1195-1208).

Lignocellulosic biomass is an abundant renewable feedstock for sustainable production of commodities such as biofuels. The main technological barrier that prevents widespread utilization of this resource for production of commodity products is the lack of low-cost technologies to overcome the recalcitrance of lignocellulose. Organisms that hydrolyse the cellulose and hemicelluloses in biomass and produce a valuable product such as ethanol at a high rate and titre would significantly reduce the costs of current biomass conversion technologies. This would allow steps that are currently accomplished in different reactors, often by different organisms, to be combined in a consolidated bioprocess (CBP). The development of such organisms has focused on engineering naturally cellulolytic microorganisms to improve product-related properties or engineering non-cellulolytic organisms with high product yields to become cellulolytic. The latter is the focus of this review. While there is still no ideal organism to use in one-step biomass conversion, several candidates have been identified. These candidates are in various stages of development for establishment of a cellulolytic system or improvement of product-forming attributes. This review assesses the current state of the art for enabling non-cellulolytic organisms to grow on cellulosic substrates.

Keywords: Cellulases; Recombinant microorganisms; Lignocellulosic biomass; Consolidated bioprocessing

Production of chondroitin sulfate and chondroitin by Chiara Schiraldi; Donatella Cimini; Mario De Rosa (pp. 1209-1220).

The production of microbial polysaccharides has recently gained much interest because of their potential biotechnological applications. Several pathogenic bacteria are known to produce capsular polysaccharides, which provide a protection barrier towards harsh environmental conditions, and towards host defences in case of invasive infections. These capsules are often composed of glycosaminoglycan-like polymers. Glycosaminoglycans are essential structural components of the mammalian extracellular matrix and they have several applications in the medical, veterinary, pharmaceutical and cosmetic field because of their peculiar properties. Most of the commercially available glycosaminoglycans have so far been extracted from animal sources, and therefore the structural similarity of microbial capsular polysaccharides to these biomolecules makes these bacteria ideal candidates as non-animal sources of glycosaminoglycan-derived products. One example is hyaluronic acid which was formerly extracted from hen crests, but is nowadays produced via Streptococci fermentations. On the other hand, no large scale biotechnological production processes for heparin and chondrotin sulfate have been developed. The larger demand of these biopolymers compared to hyaluronic acid (tons vs kilograms), due to the higher titre in the final product (grams vs milligrams/dose), and the scarce scientific effort have hampered the successful development of fermentative processes. In this paper we present an overview of the diverse applications and production methods of chondroitin reported so far in literature with a specific focus on novel microbial biotechnological approaches.

Keywords: Chondroitin sulfate; Biotechnological production; Fed-batch process; Biotransformation; Glycosyltransferase; Escherichia coli K4; Pasteurella multocida, ecc.

Proteins with antifungal properties and other medicinal applications from plants and mushrooms by Jack H. Wong; T. B. Ng; Randy C. F. Cheung; X. J. Ye; H. X. Wang; S. K. Lam; P. Lin; Y. S. Chan; Evandro F. Fang; Patrick H. K. Ngai; L. X. Xia; X. Y. Ye; Y. Jiang; F. Liu (pp. 1221-1235).

Living organisms produce a myriad of molecules to protect themselves from fungal pathogens. This review focuses on antifungal proteins from plants and mushrooms, many of which are components of the human diet or have medicinal value. Plant antifungal proteins can be classified into different groups comprising chitinases and chitinase-like proteins, chitin-binding proteins, cyclophilin-like proteins, defensins and defensin-like proteins, deoxyribonucleases, embryo-abundant protein-like proteins, glucanases, lectins, lipid transfer proteins, peroxidases, protease inhibitors, ribonucleases, ribosome-inactivating proteins, storage 2S albumins, and thaumatin-like proteins. Some of the aforementioned antifungal proteins also exhibit mitogenic activity towards spleen cells, nitric oxide inducing activity toward macrophages, antiproliferative activity toward tumor cells, antibacterial activity, and inhibitory activity toward HIV-1 reverse transcriptase. In contrast to the large diversity of plant antifungal proteins, only a small number of mushroom antifungal proteins have been reported. Mushroom antifungal proteins are distinct from their plant counterparts in N-terminal sequence. Nevertheless, some of the mushroom antifungal proteins have been shown to inhibit HIV-1 reverse transcriptase activity and tumor cell proliferation.

Keywords: Antifungal; Medicinal; Plants; Mushrooms

Chemical diversity of biologically active metabolites in the sclerotia of Inonotus obliquus and submerged culture strategies for up-regulating their production by Weifa Zheng; Kangjie Miao; Yubing Liu; Yanxia Zhao; Meimei Zhang; Shenyuan Pan; Yucheng Dai (pp. 1237-1254).

Inonotus obliquus (Fr.) Pilat is a white rot fungus belonging to the family Hymenochaetaceae in the Basidiomycota. In nature, this fungus rarely forms a fruiting body but usually an irregular shape of sclerotial conk called ‘Chaga’. Characteristically, I. obliquus produces massive melanins released to the surface of Chaga. As early as in the sixteenth century, Chaga was used as an effective folk medicine in Russia and Northern Europe to treat several human malicious tumors and other diseases in the absence of any unacceptable toxic side effects. Chemical investigations show that I. obliquus produces a diverse range of secondary metabolites including phenolic compounds, melanins, and lanostane-type triterpenoids. Among these are the active components for antioxidant, antitumoral, and antiviral activities and for improving human immunity against infection of pathogenic microbes. Geographically, however, this fungus is restricted to very cold habitats and grows very slowly, suggesting that Chaga is not a reliable source of these bioactive compounds. Attempts for culturing this fungus axenically all resulted in a reduced production of bioactive metabolites. This review examines the current progress in the discovery of chemical diversity of Chaga and their biological activities and the strategies to modulate the expression of desired pathways to diversify and up-regulate the production of bioactive metabolites by the fungus grown in submerged cultures for possible drug discovery.

Keywords: Inonotus obliquus ; Bioactive metabolites; Submerged cultures; Biosynthetic pathways; Signal transduction; S-Nitrosylation; NMR-based metabonomic analysis

Expression and export: recombinant protein production systems for Aspergillus by André Fleißner; Petra Dersch (pp. 1255-1270).

Several Aspergillus species, in particular Aspergillus niger and Aspergillus oryzae, are widely used as protein production hosts in various biotechnological applications. In order to improve the expression and secretion of recombinant proteins in these filamentous fungi, several novel genetic engineering strategies have been developed in recent years. This review describes state-of-the-art genetic manipulation technologies used for strain improvement, as well as recent advances in designing the most appropriate engineering strategy for a particular protein production process. Furthermore, current developments in identifying bottlenecks in the protein production and secretion pathways are described and novel approaches to overcome these limitations are introduced. An appropriate combination of expression vectors and optimized host strains will provide cell factories customized for each production process and expand the great potential of Aspergilli as biotechnology workhorses to more complex multi-step industrial applications.

Keywords: Aspergillus niger ; Gene expression; Inducible promoters; Cloning vectors; Protein production; Secretion systems

Metallomics: lessons for metalliferous soil remediation by Götz Haferburg; Erika Kothe (pp. 1271-1280).

The term metallomics has been established for the investigation of transcriptome, proteome, and metabolome changes induced by metals. The mechanisms allowing the organisms to cope with metals in the environment, metal resistance factors, will in turn change biogeochemical cycles of metals in soil, coupling the metal pool with the root system of plants. This makes microorganisms key players in introducing metals into food webs, as well as for bioremediation strategies. Research on physiological and metabolic responses of microorganisms on metal stress in soil is thus essential for the selection of optimized consortia applicable in bioremediation strategies such as bioaugmentation or microbially enhanced phytoextraction. The results of metallomics studies will help to develop applications including identification of biomarkers for ecotoxicological studies, bioleaching, in situ soil regeneration, and microbially assisted phytoremediation of contaminated land. This review will therefore focus on the molecular understanding of metal resistance in bacteria and fungi, as can be derived from metallomics studies.

Keywords: Metallomics; Proteomics; Heavy metal resistance; Bioremediation; Phytoextraction; Biostabilization; Bacteria; Fungi

Biotechnological production and applications of N-acetyl-d-neuraminic acid: current state and perspectives by Fei Tao; Yinan Zhang; Cuiqing Ma; Ping Xu (pp. 1281-1289).

N-Acetyl-d-neuraminic acid (Neu5Ac) and its derivates are a very important group of biomolecules because these sugars occupy the terminal positions in numerous macromolecules, such as the glycans of glycoproteins, and are involved in many biological and pathological phenomena. The synthesis and applications of Neu5Ac are attracting much interest due to the potential applications of this compound in the pharmaceutical industry, such as in the synthesis of the anti-flu drug zanamivir. In this review article, we discuss existing knowledge on the biotechnological production and applications of Neu5Ac and also propose some guidelines for future studies.

Keywords: N-Acetyl-d-neuraminic acid; Sialic acid; Biocatalysis; Application

Developments and perspectives of photobioreactors for biofuel production by Michael Morweiser; Olaf Kruse; Ben Hankamer; Clemens Posten (pp. 1291-1301).

The production of biofuels from microalgae requires efficient photobioreactors in order to meet the tight constraints of energy efficiency and economic profitability. Current cultivation systems are designed for high-value products rather than for mass production of cheap energy carriers. Future bioreactors will imply innovative solutions in terms of energy efficiency, light and gas transfer or attainable biomass concentration to lower the energy demand and cut down production costs. A new generation of highly developed reactor designs demonstrates the enormous potential of photobioreactors. However, a net energy production with microalgae remains challenging. Therefore, it is essential to review all aspects and production steps for optimization potential. This includes a custom process design according to production organism, desired product and production site. Moreover, the potential of microalgae to synthesize valuable products additionally to the energetic use can be integrated into a production concept as well as waste streams for carbon supply or temperature control.

Keywords: Biofuels; Light transfer; Mass transfer; Microalgae; Photobioreactor; Renewable energy

Trends and challenges in the microbial production of lignocellulosic bioalcohol fuels by Christian Weber; Alexander Farwick; Feline Benisch; Dawid Brat; Heiko Dietz; Thorsten Subtil; Eckhard Boles (pp. 1303-1315).

Bioalcohols produced by microorganisms from renewable materials are promising substitutes for traditional fuels derived from fossil sources. For several years already ethanol is produced in large amounts from feedstocks such as cereals or sugar cane and used as a blend for gasoline or even as a pure biofuel. However, alcohols with longer carbon chains like butanol have even more suitable properties and would better fit with the current fuel distribution infrastructure. Moreover, ethical concerns contradict the use of food and feed products as a biofuel source. Lignocellulosic biomass, especially when considered as a waste material offers an attractive alternative. However, the recalcitrance of these materials and the inability of microorganisms to efficiently ferment lignocellulosic hydrolysates still prevent the production of bioalcohols from these plentiful sources. Obviously, no known organism exist which combines all the properties necessary to be a sustainable bioalcohol producer. Therefore, breeding technologies, genetic engineering and the search for undiscovered species are promising means to provide a microorganism exhibiting high alcohol productivities and yields, converting all lignocellulosic sugars or are even able to use carbon dioxide or monoxide, and thereby being highly resistant to inhibitors and fermentation products, and easy to cultivate in huge bioreactors. In this review, we compare the properties of various microorganisms, bacteria and yeasts, as well as current research efforts to develop a reliable lignocellulosic bioalcohol producing organism.

Keywords: Biofuel; Lignocellulose; Microorganisms; Fermentation; Metabolic engineering; Xylose; Arabinose; Isobutanol; Bioethanol

Effects of acetic acid, ethanol, and SO₂ on the removal of volatile acidity from acidic wines by two Saccharomyces cerevisiae commercial strains by Alice Vilela-Moura; Dorit Schuller; Arlete Mendes-Faia; Manuela Côrte-Real (pp. 1317-1326).

Herein, we report the influence of different combinations of initial concentration of acetic acid and ethanol on the removal of acetic acid from acidic wines by two commercial Saccharomyces cerevisiae strains S26 and S29. Both strains reduced the volatile acidity of an acidic wine (1.0 g l⁻¹ acetic acid and 11% (v/v) ethanol) by 78% and 48%, respectively. Acetic acid removal by strains S26 and S29 was associated with a decrease in ethanol concentration of 0.7 and 1.2% (v/v), respectively. Strain S26 revealed better removal efficiency due to its higher tolerance to stress factors imposed by acidic wines. Sulfur dioxide (SO₂) in the concentration range 95–170 mg l⁻¹ inhibits the ability of both strains to reduce the volatile acidity of the acidic wine used under our experimental conditions. Therefore, deacidification should be carried out either in wines stabilized by filtration or in wines with SO₂ concentrations up to 70 mg l⁻¹. Deacidification of wines with the better performing strain S26 was associated with changes in the concentration of volatile compounds. The most pronounced increase was observed for isoamyl acetate (banana) and ethyl hexanoate (apple, pineapple), with an 18- and 25-fold increment, respectively, to values above the detection threshold. The acetaldehyde concentration of the deacidified wine was 2.3 times higher, and may have a detrimental effect on the wine aroma. Moreover, deacidification led to increased fatty acids concentration, but still within the range of values described for spontaneous fermentations, and with apparently no negative impact on the organoleptical properties.

Keywords: Volatile acidity removal; Acidic wines; S. cerevisiae ; Acetic acid; Ethanol; SO₂

Production of geranylgeraniol on overexpression of a prenyl diphosphate synthase fusion gene in Saccharomyces cerevisiae by Chikara Ohto; Masayoshi Muramatsu; Shusei Obata; Eiji Sakuradani; Sakayu Shimizu (pp. 1327-1334).

An acyclic diterpene alcohol, (E,E,E)-geranylgeraniol (GGOH), is one of the important compounds used as perfume and pharmacological agents. A deficiency of squalene (SQ) synthase activity allows yeasts to accumulate an acyclic sesquiterpene alcohol, (E,E)-farnesol, in their cells. Since sterols are essential for the growth of yeasts, a deficiency of SQ synthase activity makes the addition of supplemental sterols to the culture media necessary. To develop a GGOH production method not requiring any supplemental sterols, we overexpressed HMG1 encoding hydroxymethylglutaryl-CoA reductase and the genes of two prenyl diphosphate synthases, ERG20 and BTS1, in Saccharomyces cerevisiae. A prototrophic diploid coexpressing HMG1 and the ERG20-BTS1 fusion accumulated GGOH with neither disruption of the SQ synthase gene nor the addition of any supplemental sterols. The GGOH content on the diploid cultivation in a 5-l jar fermenter reached 138.8 mg/l under optimal conditions.

Keywords: Geranylgeraniol; Mevalonate pathway; Hydroxymethylglutaryl-CoA reductase; Prenyl diphosphate synthase; Yeast recombinant

Novel biosynthesis of (R)-ethyl-3-hydroxyglutarate with (R)-enantioselective hydrolysis of racemic ethyl 4-cyano-3-hydroxybutyate by Rhodococcus erythropolis by Hua-Ping Dong; Zhi-Qiang Liu; Yu-Guo Zheng; Yin-Chu Shen (pp. 1335-1345).

(R)-ethyl-3-hydroxyglutarate with highly optical purity (≥99%) can be used as a novel precursor for synthesis of chiral side chain of rosuvastatin. In this study, a novel synthesis route of (R)-ethyl-3-hydroxyglutarate by whole microorganism cells from racemic ethyl 4-cyano-3-hydroxybutyate was created. A strain ZJB-0910 capable of transforming racemic β-hydroxy aliphatic nitrile was isolated by employing a screening method based on a colorimetric reaction of Co²⁺ ion with ammonia, and identified as Rhodococcus erythropolis based on its morphology, physiological tests, Biolog, and the 16S rDNA sequence. After cultivation in a sterilized medium with composition of 20 g glucose, 5 g yeast extract, 0.5 g KH₂PO₄, 0.5 g K₂HPO₄, 0.2 g MgSO₄·7H₂O per liter at 30°C and 150 rpm for 48 h, the whole cells of R. erythropolis ZJB-0910 were prepared as a catalyst in (R)-enantioselective hydrolysis of racemic ethyl 4-cyano-3-hydroxybutyate for synthesis of (R)-ethyl-3-hydroxyglutarate, without bearing hydrolase activity for the ester bond of ethyl 4-cyano-3-hydroxybutyate. Under the optimized biotransformation conditions of pH 7.5, 30°C, and 20 mM substrate concentration, (R)-ethyl-3-hydroxyglutarate with 46.2% yield (ee > 99%) was afforded, and its chemical structure was determined by ESI-MS, NMR, and IR. The apparent Michaelis constant K _m and maximum rate V _max for this biocatalytic reaction were 0.01 M and 85.6 μmol min⁻¹ g⁻¹, respectively.

Keywords: Rosuvastatin; (R)-ethyl-3-hydroxyglutarate; Enantioselective hydrolysis; Colorimetric screening method; Nitrilase; Rhodococcus erythropolis

Directed microbial biosynthesis of deuterated biosurfactants and potential future application to other bioactive molecules by Thomas J. Smyth; Amedea Perfumo; Roger Marchant; Ibrahim M. Banat; Minglei Chen; Robert K. Thomas; Jeffrey Penfold; Paul S. Stevenson; Neil J. Parry (pp. 1347-1354).

Deuterated rhamnolipids were produced using strain AD7 of Pseudomonas aeruginosa, which was progressively adapted to increasing levels of deuterium in D₂O and carbon substrates. Fourteen different deuterated rhamnolipid structures, including structural isomers, were produced which is similar to normal protonated structures. There were two main products monorhamnolipid Rha-C₁₀-C₁₀ and dirhamnolipid Rha₂-C₁₀-C₁₀. The levels of deuteration varied from 16% with 25% D₂O + h-glycerol to 90% with 100% D₂O + d-glycerol. When d-tetradecane was used with H₂O, virtually all the deuterium appeared in the lipid chains while using h-tetradecane + D₂O led to the majority of deuterium in the sugars. The adaptation to growth in deuterium appeared to be metabolic since no genetic changes could be found in the key rhamnolipid biosynthetic genes, the rhamnosyl transferases RhlB and RhlC. Deuterated sophorolipids were similarly produced using Candida bombicola and Candida apicola although in this case, no adaptation process was necessary. Up to 40 different sophorolipids were produced by these yeasts. However, unlike the rhamnolipids, use of D₂O did not lead to any deuteration of the lipid chains, but direct incorporation into the lipid was achieved using d-isostearic acid. The results from these experiments show the feasibility of producing deuterated bioactive compounds from microorganisms coupled with the possibility of manipulating the pattern of labelling through judicious use of different deuterated substrates.

Keywords: Deuterium; Deuterated; Biosurfactants; Bioactives; Rhamnolipids; Sophorolipids

Construction and expression of immunogenic hybrid enterotoxigenic Escherichia coli CFA/I and CS2 colonization fimbriae for use in vaccines by Joshua Tobias; Ann-Mari Svennerholm; Jan Holmgren; Michael Lebens (pp. 1355-1365).

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrheal morbidity in developing countries, especially in children and also of traveler's diarrhea. Colonization factors (CFs) of ETEC, like CFA/I and CS2 which are genetically and structurally related, play a substantial role in pathogenicity, and since intestinal–mucosal immune responses against CFs appear to be protective, much effort has focused on the development of a CF-based ETEC vaccine. We have constructed hybrid operons in which the major CS2 subunit-encoding cotA gene was inserted into the CFA/I operon, either replacing (hybrid I) or being added to the major CFA/I subunit-encoding cfaB gene (hybrid II). Using specific monoclonal antibodies against the major subunits of CFA/I and CS2, high levels of surface expression of both fimbrial subunits were shown in E. coli carrying the hybrid II operon. Oral immunization of mice with formalin-killed bacteria expressing hybrid II fimbriae induced strong CFA/I- and CS2-specific serum IgG + IgM and fecal IgA antibody responses, which were higher than those achieved by similar immunization with the reference strains. Bacteria expressing hybrid fimbriae are potential candidate strains in an oral-killed CF-ETEC vaccine, and the approach represents an attractive and novel means of producing a broad-spectrum ETEC vaccine.

Keywords: ETEC; CFA/I; CS2; Hybrid fimbriae; Vaccine

Site-directed mutagenesis of disulfide bridges in Aspergillus niger NRRL 3135 phytase (PhyA), their expression in Pichia pastoris and catalytic characterization by Edward J. Mullaney; Heather Locovare; Kandan Sethumadhavan; Stephanie Boone; Xin Gen Lei; Abul H. J. Ullah (pp. 1367-1372).

Earlier studies have established the importance of five disulfide bridges (DBs) in Aspergillus niger phytase. In this study, the relative importance of each of the individual disulfide bridge is determined by its removal by site-directed mutagenesis of specific cysteines in the cloned A. niger phyA gene. Individually, these mutant phytases were expressed in a Pichia expression system and their product purified and characterized. The removal of disulfide bridge 2 yielded a mutant phytase with a complete loss of catalytic activity. The other disulfide mutants displayed a broad array of altered catalytic properties including a lower optimum temperature from 58°C to 53°C for bridge number 1, 37°C for bridge number 3 and 4, and 42°C for bridge number 5. The pH versus activity profile was also modified in the DB mutants. The pH profile of the wild-type phytase was modified by the DB mutations. In bridge number 1, 3, and 4, the second peak at pH 2.5 was abolished, and in bridge number 5, the peak at pH 5.0 was abolished completely leaving only the pH 2.5. While the K _m was not affected drastically, the turnover number was lowered significantly in bridge number 3, 4, and 5.

Keywords: Phytase; Histidine acid phosphatase; Disulfide bridge; Aspergillus niger

A novel salt-tolerant endo-β-1,4-glucanase Cel5A in Vibrio sp. G21 isolated from mangrove soil by Zhaoming Gao; Lingwei Ruan; Xiulan Chen; Yuzhong Zhang; Xun Xu (pp. 1373-1382).

Although cellulases have been isolated from various microorganisms, no functional cellulase gene has been reported in the Vibrio genus until now. In this report, a novel endo-β-1,4-glucanase gene, cel5A, 1,362 bp in length, was cloned from a newly isolated bacterium, Vibrio sp. G21. The deduced protein of cel5A contains a catalytic domain of glycosyl hydrolase family 5 (GH5), followed by a cellulose binding domain (CBM2). The GH5 domain shows the highest sequence similarity (69%) to the bifunctional beta 1,4-endoglucanase/cellobiohydrolase from Teredinibacter turnerae T7902. The mature Cel5A enzyme was overexpressed in Escherichia coli and purified to homogeneity. The optimal pH and temperature of the recombinant enzyme were determined to be 6.5–7.5 and 50°C, respectively. Cel5A was stable over a wide range of pH and retained more than 90% of total activity even after treatment in pH 5.5–10.5 for 1 h, indicating high alkali resistance. Moreover, the enzyme was activated after pretreatment with mild alkali, a novel characteristic that has not been previously reported in other cellulases. Cel5A also showed a high level of salt tolerance. Its activity rose to 1.6-fold in 0.5 M NaCl and remained elevated even in 4 M NaCl. Further experimentation demonstrated that the thermostability of Cel5A was improved in 0.4 M NaCl. In addition, Cel5A showed specific activity towards β-1,4-linkage of amorphous region of lignocellulose, and the main final hydrolysis product of carboxymethylcellulose sodium and cellooligosaccharides was cellobiose. As an alkali-activated and salt-tolerant enzyme, Cel5A is an ideal candidate for further research and industrial applications.

Keywords: Cellulase; Endoglucanase; Vibrio ; Mangrove; Salt tolerant; Alkali

Molecular detection and diversity of xylanase genes in alpine tundra soil by Guozeng Wang; Yaru Wang; Peilong Yang; Huiying Luo; Huoqing Huang; Pengjun Shi; Kun Meng; Bin Yao (pp. 1383-1393).

Xylan is a major polysaccharide in plant cell walls, and its degradation is mainly conducted by microbial xylanases in nature. To explore the xylanase diversity in the environment, two sets of degenerate primers were designed based on the microbial xylanase sequences in Pfam database of glycosyl hydrolase (GH) family 10 and 11 and were used to amplify objective gene fragments directly from the alpine tundra soil DNA of the Tianshan Mountains, China. Ninety-six distinct GH 10 and 31 GH 11 xylanase gene fragments were retrieved, and most of them have low identities with known sequences in GenBank. Based on phylogenetic analysis, all of the GH 10 xylanase sequences fell into six clusters and were related to xylanases from Actinobacteria, Proteobacteria, Verrucomicrobia, Bacteroidetes, Firmicutes, and Acidobacteria. Three clusters of GH 11 xylanase sequences were established, and two of them were related with enzymes from fungi. These results indicated the diversity of xylanase genes in this cold environment. Four xylanolytic strains were isolated from the soil, and GH 10 xylanase gene fragments were cloned using the same primers. A full-length gene was obtained and expressed in Escherichia coli, and the recombinant enzyme showed some cold-related characteristics. Our study provides an efficient molecular approach to study xylanase in complex environments and casts an insight into the diversity and distribution of xylanases in a cold environment, which is very meaningful to understand their roles in xylan degradation in nature.

Keywords: Xylanase; Glycosyl hydrolase; Alpine tundra soil; Gene diversity; Degenerate PCR

Identification and characterization of an avian β-defensin orthologue, avian β-defensin 9, from quails by Ruiqin Wang; Deying Ma; Lijuan Lin; Caiyuan Zhou; Zongxi Han; Yuhao Shao; Wenyan Liao; Shengwang Liu (pp. 1395-1405).

In this study, a newly identified avian β-defensin (AvBD) orthologue was isolated from Chinese painted quail (Coturnix chinensis) lung and bone marrow tissues. The complete nucleotide sequence of the gene contained a 204-bp open-reading frame encoding 67 amino acids. Homology, characterization, and comparison of the gene with AvBD from other avian species confirmed that it was quail AvBD9. To analyze and compare the expression pattern of AvBD9 in tissues from young and adult quails, layer hens, and broilers, reverse transcription polymerase chain reaction was performed using mRNA isolated from 21 different tissues. The AvBD9 expression pattern distribution was differed among quails of different ages, layer hens, and broilers. It was widely expressed in all the tissues except the trachea, liver, and kidney and was highly expressed in the lung and heart of young quails. Similarly, it was widely expressed in all the tissues of adult quails except for the liver, kidney, spleen, thymus, and Harderian gland. In layer hens, AvBD9 was widely expressed in all the tissues except the trachea, glandular stomach, and cecum. Similarly, it was widely expressed in all the tissues of broilers except for the trachea, glandular stomach, rectum, cecum, bone marrow, and cecal tonsil. Recombinant AvBD9 (rAvBD9) was produced and purified by expressing the gene in Escherichia coli. Additionally, peptide according to quail AvBD9 sequence was synthesized, named sAvBD9. As expected, both rAvBD9 and sAvBD9 exhibited strong bactericidal properties against 11 strains of bacteria, including Gram-positive and Gram-negative forms.

Keywords: Avian β-defensin 9; Quail; Expression; Recombinant peptide; Synthesized peptide; Antimicrobial activity

Cloning, characterization, and engineering of fungal L-arabinitol dehydrogenases by Byoungjin Kim; Ryan P. Sullivan; Huimin Zhao (pp. 1407-1414).

L-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of L-arabinitol to L-xylulose with concomitant NAD⁺ reduction in fungal L-arabinose catabolism. It is an important enzyme in the development of recombinant organisms that convert L-arabinose to fuels and chemicals. Here, we report the cloning, characterization, and engineering of four fungal LADs from Penicillium chrysogenum, Pichia guilliermondii, Aspergillus niger, and Trichoderma longibrachiatum, respectively. The LAD from P. guilliermondii was inactive, while the other three LADs were NAD⁺-dependent and showed high catalytic activities, with P. chrysogenum LAD being the most active. T. longibrachiatum LAD was the most thermally stable and showed the maximum activity in the temperature range of 55–65°C with the other LADs showed the maximum activity in the temperature range of 40–50°C. These LADs were active from pH 7 to 11 with an optimal pH of 9.4. Site-directed mutagenesis was used to alter the cofactor specificity of these LADs. In a T. longibrachiatum LAD mutant, the cofactor preference toward NADP⁺ was increased by 2.5 × 10⁴-fold, whereas the cofactor preference toward NADP⁺ of the P. chrysogenum and A. niger LAD mutants was also drastically improved, albeit at the expense of significantly reduced catalytic efficiencies. The wild-type LADs and their mutants with altered cofactor specificity could be used to investigate the functionality of the fungal L-arabinose pathways in the development of recombinant organisms for efficient microbial L-arabinose utilization.

Keywords: Arabinose fermentation; Xylitol production; Alcohol dehydrogenase; Cofactor specificity; Ethanol production

Characterization of two aldo–keto reductases from Gluconobacter oxydans 621H capable of regio- and stereoselective α-ketocarbonyl reduction by Paul Schweiger; Harald Gross; Uwe Deppenmeier (pp. 1415-1426).

Two cytosolic NADPH-dependent carbonyl reductases from Gluconobacter oxydans 621H, Gox0644 and Gox1615, were heterologously produced in Escherichia coli. The recombinant proteins were purified to homogeneity and characterized. Gox0644 and Gox1615 were dimers with native molecular masses of 66.1 and 74.5 kDa, respectively. The enzymes displayed broad substrate specificities and reduced α-ketocarbonyls at the keto moiety most proximal to the terminus of the alkyl chain to produce alpha-hydroxy carbonyls, as demonstrated by NMR. With respect to stereoselectivity, protein Gox0644 specifically reduced 2,3-pentanedione to 2R-hydroxy-pentane-3-one, whereas Gox1615 produced 2S-hydroxy-pentane-3-one. Both enzymes also reduced 1-phenyl-1,2-propanedione to 2-hydroxy-1-phenylpropane-1-one, which is a key intermediate in the production of numerous pharmaceuticals, such as antifungal azoles and antidepressants. Gox0644 displayed highest activities with 2,3-diones, α-ketoaldehydes, α-keto esters, and 2,5-diketogluconate. Gox1615 was less active with these substrates, but displayed a broader substrate spectrum reducing a variety of α-diketones and aldehydes.

Keywords: Acetic acid bacteria; Oxidoreductase; Biotransformation; Stereospecific reduction; Chiral

Chain transfer reaction catalyzed by various polyhydroxyalkanoate synthases with poly(ethylene glycol) as an exogenous chain transfer agent by Satoshi Tomizawa; Yuta Saito; Manami Hyakutake; Yoshiyuki Nakamura; Hideki Abe; Takeharu Tsuge (pp. 1427-1435).

Polyhydroxyalkanoate (PHA) synthases catalyze chain transfer (CT) reaction after polymerization reaction of PHA by transferring PHA chain from PHA synthase to a CT agent, resulting in covalent bonding of CT agent to PHA chain at the carboxyl end. Previous studies have shown that poly(ethylene glycol) (PEG) is an effective exogenous CT agent. This study aimed to compare the effects of PEG on CT reaction during poly[(R)-3-hydroxybutyrate] [P(3HB)] synthesis by using six PHA synthases in Escherichia coli JM109. The synthesized P(3HB) polymers were characterized in terms of molecular weight and end-group structure. Supplementation of PEG to the culture medium reduced P(3HB) molecular weights by up to 96% due to PEG-induced CT reaction. The P(3HB) polymers were subjected to ¹H NMR analysis to confirm the formation of a covalent bond between PEG and P(3HB) chain at the carboxyl end. This study revealed the reactivity of PHA synthases to PEG with respect to CT reaction in E. coli.

Keywords: Polyhydoxyalkanoate; Poly(ethylene glycol); Molecular weight; Escherichia coli ; Chain transfer reaction

Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii by Orquídea Ribeiro; Marilyn Wiebe; Marja Ilmén; Lucília Domingues; Merja Penttilä (pp. 1437-1446).

To explore the potential of Ashbya gossypii as a host for the expression of recombinant proteins and to assess whether protein secretion would be more similar to the closely related Saccharomyces cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were successfully expressed in A. gossypii from plasmids containing the two micron sequences from S. cerevisiae, under the S. cerevisiae PGK1 promoter. The native signal sequences of EGI and CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. gossypii. Although CBHI activity was not detected using 4-methylumbelliferyl-β-d-lactoside as substrate, the protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable, the specific activity being comparable to that produced by a similar EGI producing S. cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared with the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae.

Keywords: Ashbya gossypii ; Recombinant protein production; Trichoderma reesei endoglucanase I; Trichoderma reesei cellobiohydrolase I; Cellulases heterologous expression

Retrotransposon expression in ethanol-stressed Saccharomyces cerevisiae by Dragana Stanley; Sarah Fraser; Grant A. Stanley; Paul J. Chambers (pp. 1447-1454).

There are five retrotransposon families in Saccharomyces cerevisiae, three (Ty1, Ty2, and Ty3) of which are known to be transcriptionally active. Early investigations reported yeast retrotransposons to be stress-induced; however, microarray-based studies do not report retrotransposition-related Gene Ontology (GO) categories in the ethanol stress response of S. cerevisiae. In this study, microarray technology was used to investigate the ethanol stress response of S. cerevisiae W303-1A, and the highest stress-induced GO categories, based on z-score, were found to be retrotransposition-related, namely, Retrotransposition Nucleocapsid and Transposition, RNA-Mediated. Further investigation, involving reanalysis of previously published results on the stress response of S. cerevisiae, identified the absence of annotation for retrotransposon genes and associated GO categories and their omission during the printing of spotted arrays as two reasons why these categories in previous gene expression studies on the ethanol stress response of yeast were not reported.

Keywords: Saccharomyces ; Ethanol; Stress; Retrotransposon; Gene expression

Genomic structure and promoter analysis of the dsz operon for dibenzothiophene biodesulfurization from Gordonia alkanivorans RIPI90A by Mahmoud Shavandi; Majid Sadeghizadeh; Khosro Khajeh; Ghasemali Mohebali; Alireza Zomorodipour (pp. 1455-1461).

The bacterium Gordonia alkanivorans RIPI90A has been previously reported as dibenzothiophene-desulfurizing strain. The present study provides a complete investigation of the dsz operon including dsz promoter analysis from desulfurization competent strain belonging to the genus Gordonia. PCR was used to amplify the dszABC genes and adaptor ligation-based PCR-walking strategy used to isolate the dsz promoter. Unlike the dsz operon of Rhodococcus erythropolis, the operon of RIPI90A was located on chromosome. Despite the remarkably high homology between dsz genes of G. alkanivorans RIPI90A and R. erythropolis IGST8, promoter sequences of the strains were not very similar. The dsz promoter of G. alkanivorans RIPI90A shows only 52.5% homology to that of R. erythropolis IGTS8 and Gordonia nitida. Deletion analysis of the dsz promoter from RIPI90A using luciferase as a reporter gene revealed that the dsz promoter was located in regions from −156 to −50.

Keywords: Gordonia alkanivorans ; Biodesulfurization; dsz operon; PCR walking; Promoter analysis; Reporter gene

Expanding the ku70 toolbox for filamentous fungi: establishment of complementation vectors and recipient strains for advanced gene analyses by Neuza D. S. P. Carvalho; Mark Arentshorst; Min Jin Kwon; Vera Meyer; Arthur F. J. Ram (pp. 1463-1473).

Mutants with a defective non-homologous-end-joining (NHEJ) pathway have boosted functional genomics in filamentous fungi as they are very efficient recipient strains for gene-targeting approaches, achieving homologous recombination frequencies up to 100%. For example, deletion of the ku70 homologous gene kusA in Aspergillus niger resulted in a recipient strain in which deletions of essential or non-essential genes can efficiently be obtained. To verify that the mutant phenotype observed is the result of a gene deletion, a complementation approach has to be performed. Here, an intact copy of the gene is transformed back to the mutant, where it should integrate ectopically into the genome. However, ectopic complementation is difficult in NHEJ-deficient strains, and the gene will preferably integrate via homologous recombination at its endogenous locus. To circumvent that problem, we have constructed autonomously replicating vectors useful for many filamentous fungi which contain either the pyrG allele or a hygromycin resistance gene as selectable markers. Under selective conditions, the plasmids are maintained, allowing complementation analyses; once the selective pressure is removed, the plasmid becomes lost and the mutant phenotype prevails. Another disadvantage of NHEJ-defective strains is their increased sensitivity towards DNA damaging conditions such as radiation. Thus, mutant analyses in these genetic backgrounds are limited and can even be obscured by pleiotropic effects. The use of sexual crossings for the restoration of the NHEJ pathway is, however, impossible in imperfect filamentous fungi such as A. niger. We have therefore established a transiently disrupted kusA strain as recipient strain for gene-targeting approaches.

Keywords: Aspergillus niger ; ku70 ; AMA1; Heterokaryon rescue; Transformation

Functional analysis of genes for benzoate metabolism in the albicidin biosynthetic region of Xanthomonas albilineans by Saeed M. Hashimi; Robert G. Birch (pp. 1475-1485).

Albicidins are potent DNA-gyrase-inhibiting antibiotics and phytotoxins synthesised by Xanthomonas albilineans. Functions have been deduced for some clustered biosynthetic genes, including a PKS-NRPS megasynthase, methyltransferases and regulatory genes, and resistance genes including a transporter and a gyrase-binding protein. More puzzling is the presence in this cluster of apparent aromatic metabolism genes. Here, we describe functional analysis of several such genes and propose a model for their role. An apparent benzoate CoA ligase (xabE) proved essential for albicidin production and pathogenicity. A neighbouring operon includes genes for p-aminobenzoate (PABA) metabolism. A PABA synthase fusion (pabAB) restored prototrophy in pabA and pabB mutants of Escherichia coli, proving functionality. Inactivation of pabAB increased susceptibility to sulphanilamide but did not block albicidin production. X. albilineans contains a remote pabB gene which evidently supplies enough PABA for albicidin biosynthesis in culture. Additional capacity from pabAB may be advantageous in more demanding environments such as infected plants. Downstream from pabAB are a known resistance gene (albG) and ubiC which encodes a p-hydroxybenzoate (PHBA) synthase. PHBA protects X. albilineans from inhibition by PABA. Therefore, coordinated expression may protect X. albilineans against toxicity of both the PABA intermediate and the albicidin product, under conditions that induce high-level antibiotic biosynthesis.

Keywords: Benzoate CoA ligase; PABA synthase; PabAB fusion; PHBA synthase; Albicidin; Benzoate metabolism; Xanthomonas albilineans

Fermentation characteristics of Dekkera bruxellensis strains by Johanna Blomqvist; Thomas Eberhard; Johan Schnürer; Volkmar Passoth (pp. 1487-1497).

The influence of pH, temperature and carbon source (glucose and maltose) on growth rate and ethanol yield of Dekkera bruxellensis was investigated using a full-factorial design. Growth rate and ethanol yield were lower on maltose than on glucose. In controlled oxygen-limited batch cultivations, the ethanol yield of the different combinations varied from 0.42 to 0.45 g (g glucose)⁻¹ and growth rates varied from 0.037 to 0.050 h⁻¹. The effect of temperature on growth rate and ethanol yield was negligible. It was not possible to model neither growth rate nor ethanol yield from the full-factorial design, as only marginal differences were observed in the conditions tested. When comparing three D. bruxellensis strains and two industrial isolates of Saccharomyces cerevisiae, S. cerevisiae grew five times faster, but the ethanol yields were 0–13% lower. The glycerol yields of S. cerevisiae strains were up to six-fold higher compared to D. bruxellensis, and the biomass yields reached only 72–84% of D. bruxellensis. Our results demonstrate that D. bruxellensis is robust to large changes in pH and temperature and may have a more energy-efficient metabolism under oxygen limitation than S. cerevisiae.

Keywords: Dekkera bruxellensis ; Saccharomyces cerevisiae ; Ethanol yield; Growth rate; Glycerol yield; Full-factorial design

Effect of methanogenic substrates on anaerobic oxidation of methane and sulfate reduction by an anaerobic methanotrophic enrichment by Roel J. W. Meulepas; Christian G. Jagersma; Ahmad F. Khadem; Alfons J. M. Stams; Piet N. L. Lens (pp. 1499-1506).

Anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) is assumed to be a syntrophic process, in which methanotrophic archaea produce an interspecies electron carrier (IEC), which is subsequently utilized by sulfate-reducing bacteria. In this paper, six methanogenic substrates are tested as candidate-IECs by assessing their effect on AOM and SR by an anaerobic methanotrophic enrichment. The presence of acetate, formate or hydrogen enhanced SR, but did not inhibit AOM, nor did these substrates trigger methanogenesis. Carbon monoxide also enhanced SR but slightly inhibited AOM. Methanol did not enhance SR nor did it inhibit AOM, and methanethiol inhibited both SR and AOM completely. Subsequently, it was calculated at which candidate-IEC concentrations no more Gibbs free energy can be conserved from their production from methane at the applied conditions. These concentrations were at least 1,000 times lower can the final candidate-IEC concentration in the bulk liquid. Therefore, the tested candidate-IECs could not have been produced from methane during the incubations. Hence, acetate, formate, methanol, carbon monoxide, and hydrogen can be excluded as sole IEC in AOM coupled to SR. Methanethiol did inhibit AOM and can therefore not be excluded as IEC by this study.

Keywords: Anaerobic oxidation of methane; Interspecies electron carrier; Methanogenic substrates

Lipidome profiling of Saccharomyces cerevisiae reveals pitching rate-dependent fermentative performance by Hong-Chi Tian; Jian Zhou; Bin Qiao; Ying Liu; Jin-Mei Xia; Ying-Jin Yuan (pp. 1507-1516).

A high cell density strategy has been used in bioethanol production to shorten the fermentation period. To reveal the molecular basis of fermentative behavior in high cell density, the profiling of the phospholipids and sterols of Saccharomyces cerevisiae during fermentation at five different pitching rates (1, 5, 10, 20, and 40 g/L) was investigated. Using LC/ESI/MSⁿ technology, 148 phospholipid species were detected, of which 91 species were quantified, and using the gas chromatography–time-of-flight mass spectrometry procedure, a total of 11 sterols were quantified. Phospholipid samples from different pitching rates were discriminated into three groups using principal component analysis (1, 5 g/L, and the others). The main changes in the lipid profile of yeast cells with higher pitching rates were as follows: (a) the relative contents of phosphatidylglycerol and phosphatidylserine were higher while phosphatidylinositol was lower compared with lower pitching rates, (b) the saturated and the relatively shorter fatty acyl chains of phospholipids decreased, and (c) the content of ergosterol was higher. These findings suggested a regulation of the property of the membrane at the situation of high cell density and a possible approach of self-protection of the yeast cells against the high density stresses.

Keywords: Phospholipids; Sterols; Cell density; Saccharomyces cerevisiae ; Fermentation

Combination of the 2A/furin technology with an animal component free cell line development platform process by Thomas Jostock; Zorica Dragic; Jianmin Fang; Karin Jooss; Burkhard Wilms; Hans-Peter Knopf (pp. 1517-1524).

The recently described 2A/furin technology combines both chains of the antibody in a single open reading frame. Upon translation and secretion, the peptide is processed by the cell to generate native fully functional IgG antibodies. Here, we describe the results of an evaluation study of this technology for an industrial CHO cell line development process. The 2A/furin expression cassette setup was combined with a Novartis vector system. A transfection, selection, and cloning procedure in chemically defined media was established at Novartis and applied for a monoclonal test antibody. The productivity of 2A/furin-vector-derived clones in non-optimized generic shake flask fed-batch models was in a comparable range with clones derived from the reference control vector. Higher clonal production stability was seen for the majority of clones generated with the 2A/furin technology compared to the clones generated with the reference control vector. Product quality was analyzed by SDS-PAGE and no significant difference was detected between the two systems. Thus, it was shown that the 2A/furin technology can be successfully combined with a Novartis CHO expression system and platform. Due to the single ORF setup, the 2A/furin technology may therefore offer a suitable approach to reduce vector size and complexity.

Keywords: Recombinant antibody production; CHO; Cell culture; 2A/furin

I-SceI endonuclease: a new tool for DNA repair studies and genetic manipulations in streptomycetes by Theresa Siegl; Lutz Petzke; Elisabeth Welle; Andriy Luzhetskyy (pp. 1525-1532).

Actinomycetes are Gram-positive bacteria with a complex life cycle. They produce many pharmaceutically relevant secondary metabolites, including antibiotics and anticancer drugs. However, there is a limited number of biotechnological applications available as opposed to genetic model organisms like Bacillus subtilis or Escherichia coli. We report here a system for the functional expression of a synthetic gene encoding the I-SceI homing endonuclease in several streptomycetes. Using the synthetic sce(a) gene, we were able to create controlled genomic DNA double-strand breaks. A mutagenesis system, based on the homing endonuclease I-SceI, has been developed to construct targeted, non-polar, unmarked gene mutations in Streptomyces sp. Tü6071. In addition, we have shown that homologous recombination is a major pathway in streptomycetes to repair an I-SceI-generated DNA double-strand break. This novel I-SceI-based tool will be useful in fundamental studies on the repair mechanism of DNA double-strand breaks and for a variety of biotechnological applications.

Keywords: I-SceI; Actinomycetes; Mutagenesis; Counter-selection; Homologous recombination

A mini-Mu transposon-based method for multiple DNA fragment integration into bacterial genomes by Xiao-Xing Wei; Zhen-Yu Shi; Zheng-Jun Li; Lei Cai; Qiong Wu; Guo-Qiang Chen (pp. 1533-1541).

A method for construction of bacterial strains with multiple DNA inserted into their chromosomes has been developed based on the mini-Mu transposon and FLP/FRT recombination. Exogenous DNA can be integrated by Mu transposition with an FRT cassette containing selection marker and conditional replicative origin (R6Kγori). Subsequently, with the introduction of a helper plasmid bearing gene of FLP recombinase, drug-resistant selection marker is excised from the chromosome. Cells cured of the helper plasmid can undergo the next cycle of transposition and excision of selection marker. Each cycle can add further foreign gene(s) to the chromosome. As an example, resistance genes of chloramphenicol, tetracycline, and gentamicin were successively integrated into the chromosome of Escherichia coli BW25113 by three cycles of insertion and excision as described above. This method proved to be simple and time-saving, which could be applicable to a variety of microorganisms.

Keywords: DNA integration; Mu transposon; Bacterial genome; FLP/FRT recombination; Transposition

Mineralization of PCBs by the genetically modified strain Cupriavidus necator JMS34 and its application for bioremediation of PCBs in soil by Juan Matías Saavedra; Francisca Acevedo; Myriam González; Michael Seeger (pp. 1543-1554).

Polychlorobiphenyls (PCBs) are classified as “high-priority pollutants.” Diverse microorganisms are able to degrade PCBs. However, bacterial degradation of PCBs is generally incomplete, leading to the accumulation of chlorobenzoates (CBAs) as dead-end metabolites. To obtain a microorganism able to mineralize PCB congeners, the bph locus of Burkholderia xenovorans LB400, which encodes one of the most effective PCB degradation pathways, was incorporated into the genome of the CBA-degrading bacterium Cupriavidus necator JMP134-X3. The bph genes were transferred into strain JMP134-X3, using the mini-Tn5 transposon system and biparental mating. The genetically modified derivative, C. necator strain JMS34, had only one chromosomal insertion of bph locus, which was stable under nonselective conditions. This modified bacterium was able to grow on biphenyl, 3-CBA and 4-CBA, and degraded 3,5-CBA in the presence of m-toluate. The strain JMS34 mineralized 3-CB, 4-CB, 2,4′-CB, and 3,5-CB, without accumulation of CBAs. Bioaugmentation of PCB-polluted soils with C. necator strain JMS34 and with the native B. xenovorans LB400 was monitored. It is noteworthy that strain JMS34 degraded, in 1 week, 99% of 3-CB and 4-CB and approximately 80% of 2,4′-CB in nonsterile soil, as well as in sterile soil. Additionally, the bacterial count of strain JMS34 increased by almost two orders of magnitude in PCB-polluted nonsterile soil. In contrast, the presence of native microflora reduced the degradation of these PCBs by strain LB400 from 73% (sterile soil) to approximately 50% (nonsterile soil). This study contributes to the development of improved biocatalysts for remediation of PCB-contaminated environments.

Keywords: PCBs; Chlorobenzoate; Cupriavidus necator ; Mineralization; Genetically modified microorganism; Bioremediation

Performance and microbial community composition dynamics of aerobic granular sludge from sequencing batch bubble column reactors operated at 20 °C, 30 °C, and 35 °C by Sirous Ebrahimi; Sébastien Gabus; Emmanuelle Rohrbach-Brandt; Maryam Hosseini; Pierre Rossi; Julien Maillard; Christof Holliger (pp. 1555-1568).

Two bubble column sequencing batch reactors fed with an artificial wastewater were operated at 20 °C, 30 °C, and 35 °C. In a first stage, stable granules were obtained at 20 °C, whereas fluffy structures were observed at 30 °C. Molecular analysis revealed high abundance of the operational taxonomic unit 208 (OTU 208) affiliating with filamentous bacteria Leptothrix spp. at 30 °C, an OTU much less abundant at 20 °C. The granular sludge obtained at 20 °C was used for the second stage during which one reactor was maintained at 20 °C and the second operated at 30 °C and 35 °C after prior gradual increase of temperature. Aerobic granular sludge with similar physical properties developed in both reactors but it had different nutrient elimination performances and microbial communities. At 20 °C, acetate was consumed during anaerobic feeding, and biological phosphorous removal was observed when Rhodocyclaceae-affiliating OTU 214 was present. At 30 °C and 35 °C, acetate was mainly consumed during aeration and phosphorous removal was insignificant. OTU 214 was almost absent but the Gammaproteobacteria-affiliating OTU 239 was more abundant than at 20 °C. Aerobic granular sludge at all temperatures contained abundantly the OTUs 224 and 289 affiliating with Sphingomonadaceae indicating that this bacterial family played an important role in maintaining stable granular structures.

Keywords: Wastewater treatment; Nutrient removal; Biological dephosphatation; Aerobic granular sludge

Mineralization and transformation of monofluorophenols by Pseudonocardia benzenivorans by Eun-Ju Kim; Jong-Rok Jeon; Young-Mo Kim; Kumarasamy Murugesan; Yoon-Seok Chang (pp. 1569-1577).

The aerobic metabolism of monofluorophenols (mono-FPs) by the actinomycete, Pseudonocardia benzenivorans, was studied. This strain was able to grow on 4-fluorophenol (4-FP) and readily transform 2- and 3-fluorophenol to the corresponding metabolites. The detailed mechanism of mono-FPs degradation by P. benzenivorans was elucidated from enzymatic assays and the identification of reaction intermediates by high-performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry. Two types of fluorocatechols (i.e., 3- and 4-fluorocatechol) were identified as the key transformation products. During 4-FP degradation, only 4-fluorocatechol was detected, and a stoichiometric level of fluoride was released. Both fluorocatechols were observed together in cultures containing 3-fluorophenol (3-FP), while only 3-fluorocatechol was found to accumulate in 2-fluorophenol (2-FP)-containing cultures. Whole-cell extracts of P. benzenivorans expressed catechol 1,2-dioxygenase activity, indicating that the transformation of the three tested mono-FPs proceeded via ortho-cleavage pathway. The results presented in this paper provide comprehensive information regarding the metabolism of mono-FPs by a single bacterium.

Keywords: Biodegradation; Monofluorophenols; 4-Fluorophenol; Pseudonocardia benzenivorans

Efficient degradation of rice straw in the reactors packed by carbon fiber textiles by Kengo Sasaki; Masahiko Morita; Shin-ichi Hirano; Daisuke Sasaki; Naoya Ohmura; Yasuo Igarashi (pp. 1579-1586).

We have reported for the first time that agricultural and cellulosic waste, i.e., rice straw was directly applied to methanogenic bioreactors containing carbon fiber textiles (CFT) as supporting material. Addition of CFT to the methanogenic bioreactors enhanced the conversion of dichromate chemical oxygen demand of the substrate to methane (41%) to a greater extent than bioreactors without CFT (9%). In addition, removal of rice straw as a suspended solid was increased from 31% (in bioreactors without CFT) to 57% (in those with CFT). Methanogenic 16S rRNA gene analysis showed that the abundance of acetoclastic methanogen, genus Methanosarcina, was about 11 times higher in bioreactors with CFT (suspended fraction plus retained fraction to CFT) than in bioreactors without CFT (suspended fraction), resulting in lower concentration of acetate in bioreactors with CFT (0.4 mM) than in those without CFT (29.7 mM). On the other hand, the abundance of hydrogenotrophic methanogen, genus Methanobacterium, in bioreactors with CFT was similar to those without CFT. Bacterial communities in bioreactors with CFT were different from those in bioreactors without CFT. Our results indicated that specific microbial community and cooperative relationships between microorganisms in reactors containing CFT facilitated efficient decomposition of rice straw and its conversion to methane.

Keywords: Rice straw; Methane fermentation; Supporting material; Methanogenic archaea

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Applied Microbiology and Biotechnology (v.87, #4)