Applied Microbiology and Biotechnology (v.97, #15)

Metagenomic technology and genome mining: emerging areas for exploring novel nitrilases by Jin-Song Gong; Zhen-Ming Lu; Heng Li; Zhe-Min Zhou; Jin-Song Shi; Zheng-Hong Xu (6603-6611).
Nitrilase is one of the most important members in the nitrilase superfamily and it is widely used for bioproduction of commodity chemicals and pharmaceutical intermediates as well as bioremediation of nitrile-contaminated wastes. However, its application was hindered by several limitations. Searching for new nitrilases and improving their application performances are the driving force for researchers. Genetic data resources in various databases are quite rich in post-genomic era. Besides, more than 99 % of microbes in the environment are unculturable. Metagenomic technology and genome mining are thus becoming burgeoning areas and provide unprecedented opportunities for searching more useful novel nitrilases due to the abundance of already existing but unexplored gene resources, namely uncharacterized genome information in the database and unculturable microbes in the natural environment. These techniques seem to be innovative and highly efficient. This study reviews the current status and future directions of metagenomics and genome mining in nitrilase exploration. Moreover, it discussed their utilization in coping with the challenges for nitrilase application. In the next several years, with the rapid development of nitrile biocatalysis, these two techniques would be bound to attract increasing attentions and even become a dominant trend for finding more novel nitrilases. Also, this review would provide guidance for exploitation of other commercially important enzymes.
Keywords: Biocatalysis; Biocatalyst discovery; Genome mining; Metagenomics; Nitrilase

Ernst Chain: a great man of science by Nelson Kardos; Arnold L. Demain (6613-6622).
This paper is a tribute to the scientific accomplishments of Ernst Chain and the influence he exerted over the fields of industrial microbiology and biotechnology. Chain is the father of the modern antibiotic era and all the benefits that these therapeutic agents have brought, i.e., longer life spans, greater levels of public health, widespread modern surgery, and control of debilitating infectious diseases, including tuberculosis, gonorrhea, syphilis, etc. Penicillin was the first antibiotic to become commercially available, and its use ushered in the age of antibiotics. The discovery of penicillin’s bactericidal action had been made by Alexander Fleming in London in 1928. After publishing his observations in 1929, no further progress was made until the work was picked up in 1939 by scientists at Oxford University. The group was headed by Howard Florey, and Chain was the group’s lead scientist. Chain was born and educated in Germany, and he fled in 1933 as a Jewish refugee from Nazism to England. Other important members of the Oxford research team were Norman Heatley and Edward Abraham. The team was able to produce and isolate penicillin under conditions of scarce resources and many technical challenges. Sufficient material was collected and tested on mice to successfully demonstrate penicillin’s bactericidal action on pathogens, while being nontoxic to mammals. Chain directed the microbiological methods for producing penicillin and the chemical engineering methods to extract the material. This technology was transferred to US government facilities in 1941 for commercial production of penicillin, becoming an important element in the Allied war effort. In 1945, the Nobel Prize for medicine was shared by Fleming, Florey, and Chain in recognition of their work in developing penicillin as a therapeutic agent. After World War II, Chain tried to persuade the British government to fund a new national antibiotic industry with both research and production facilities. As resources were scarce in postwar Britain, the British government declined the project. Chain then took a post in 1948 at Rome’s Instituto Superiore di Sanitá, establishing a new biochemistry department with a pilot plant. During that period, his department developed important new antibiotics (including the first semisynthetic antibiotics) as well as improved technological processes to produce a wide variety of important microbial metabolites that are still in wide use today. Chain was also responsible for helping several countries to start up a modern penicillin industry following World War II, including the Soviet Union and the People’s Republic of China. In 1964, Chain returned to England to establish a new biochemistry department and industrial scale fermentation pilot plant at Imperial College in London. Imperial College became the preeminent biochemical department in Europe. Chain was also a pioneer in changing the relationship between government, private universities, and private industry for collaboration and funding to support medical research. Ernst Chain has left a lasting impact as a great scientist and internationalist.
Keywords: Ernst Chain; Howard Florey; Alexander Fleming; Penicillin; Antibiotics; Nobel Prize; Oxford University; Imperial College; Instituto Superiore di Sanitá

Properties, metabolisms, and applications of l-proline analogues by Thi Mai Hoa Bach; Hiroshi Takagi (6623-6634).
Due to the unique role of l-proline in the folding and structure of protein, a variety of synthetic proline analogues have been developed. l-Proline analogues have been proven to be valuable reagents for studying cellular metabolism and the regulation of macromolecule synthesis in both prokaryotic and eukaryotic cells. In addition to these fundamental researches, they are useful compounds for industrial use. For instance, microorganisms that overproduce l-proline have been obtained by isolating mutants resistant to l-proline analogues. They are also promising candidates for tuning the biological, pharmaceutical, or physicochemical properties of naturally occurring or de novo designed peptides. Among l-proline analogues, l-azetidine-2-carboxylic acid (l-AZC) is a toxic non-proteinogenic amino acid originally found in lily of the valley plants and trans-4-hydroxy-l-proline (4-l-THOP) is the most abundant component of mammalian collagen. Many hydroxyprolines (HOPs), such as 4-l-THOP and cis-4-hydroxy-l-proline (4-l-CHOP), are useful chiral building blocks for the organic synthesis of pharmaceuticals. In addition, l-AZC and 4-l-CHOP, which are potent inhibitors of cell growth, have been tested for their antitumor activity in tissue culture and in vivo. In this review, we describe the recent discoveries regarding the physiological properties and microbial production and metabolism of l-proline analogues, particularly l-AZC and HOPs. Their applications in fundamental research and industrial use are also discussed.
Keywords: l-Proline analogues; l-Azetidine-2-carboxylic acid; Hydroxyprolines; cis-4-Hydroxy-l-proline; cis-4-Hydroxy-d-proline; trans-4-Hydroxy-l-proline; trans-4-Hydroxy-d-proline; N-acetyl cis-4-hydroxy-l-proline; l-Proline cis-4-hydroxylase; N-acetyltransferase Mpr1

Encapsulation in a natural, preformed, multi-component and complex capsule: yeast cells by Bao Ngoc Pham-Hoang; Cynthia Romero-Guido; Hanh Phan-Thi; Yves Waché (6635-6645).
From the first observation about 40 years ago that yeast cells were interesting protective structures that could be used in several industrial applications, processes have been developed enabling technologists to incorporate several compounds possessing different physico-chemical (hydrophobic/hydrophilic) properties. Technologists screened yeast diversity to choose strains possessing the best potential and modified their physiological state to increase the uptake capability and the envelope plasticity, for instance by increasing the amount of lipids. Physico-chemical treatments were also used to improve the uptake and decrease the yeast natural material impact on the final products. For example, yeast cells could be “emptied” of their plasmic material. Yeast cells can also be coated with an additional polymeric material to increase resistance to heat treatment or decrease material liberation.These capsules can be used for several applications including carbonless paper, perfuming tissues and drug targeting, but the main industrial application deals currently with flavour encapsulation, although encapsulation in yeast is also interesting for the global food industry trend for health products.This paper proposes to review the use of yeast as an encapsulation structure focusing particularly on the properties of the yeast capsule and their impact on loading, protection, targeting and release.
Keywords: Encapsulation; Yeast; Flavour; Antioxidant; Pigments; Lipids

Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.
Keywords: Fibrinolytic activity; Esterolytic assays; Fluorimetric assays; Fibrin plate method; Euglobulin clot lysis time; Thrombolysis

Heavy metal pollution is a matter of concern in industrialised countries. Contrary to organic pollutants, heavy metals are not metabolically degraded. This fact has two main consequences: its bioremediation requires another strategy and heavy metals can be indefinitely recycled. Yeast cells of Saccharomyces cerevisiae are produced at high amounts as a by-product of brewing industry constituting a cheap raw material. In the present work, the possibility of valorising this type of biomass in the bioremediation of real industrial effluents containing heavy metals is reviewed. Given the auto-aggregation capacity (flocculation) of brewing yeast cells, a fast and off-cost yeast separation is achieved after the treatment of metal-laden effluent, which reduces the costs associated with the process. This is a critical issue when we are looking for an effective, eco-friendly, and low-cost technology. The possibility of the bioremediation of industrial effluents linked with the selective recovery of metals, in a strategy of simultaneous minimisation of environmental hazard of industrial wastes with financial benefits from reselling or recycling the metals, is discussed.
Keywords: Biosorption; Heavy metals bioremediation; Industrial effluents; Metals recycling; Saccharomyces cerevisiae ; Yeast

Knocking out analysis of tryptophan permeases in Escherichia coli for improving L-tryptophan production by Pengfei Gu; Fan Yang; Fangfang Li; Quanfeng Liang; Qingsheng Qi (6677-6683).
Three permeases, Mtr, TnaB, and AroP, are involved in the uptake of L-tryptophan in Escherichia coli. These permeases possess individual function for cell transportation and metabolism, and affect extracellular L-tryptophan accumulation. In this study, by knocking out three tryptophan permeases separately and simultaneously in L-tryptophan-producing strain E. coli GPT1002, we analyzed the effect of permease knock out on L-tryptophan uptake, cell growth, and L-tryptophan production. We found that TnaB is the main transporter that is responsible for the uptake of L-tryptophan. Inactivation of tnaB improved the L-tryptophan production significantly, and inactivation of aroP has an additive effect on tnaB mutant. Quantitative real-time PCR analysis confirmed that knocking out permeases affects gene transcription and cell metabolism in many metabolic pathways. The tryptophan permease-deficient GPT1017 mutant exhibited the highest L-tryptophan production at 2.79 g l−1, which is 51.6 % higher than that produced by the control strain. In 5-l bioreactor fermentation, the L-tryptophan production in GPT1017 reached 16.3 g l−1.
Keywords: E. coli ; L-Tryptophan; TnaB; AroP; Mtr

(R)-[3,5-bis(trifluoromethyl)phenyl] ethanol is a crucial intermediate for the synthesis of Aprepitant. An efficient biocatalytic process for (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol was developed via the asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone, catalyzed by whole cells of newly isolated Trichoderma asperellum ZJPH0810 using ethanol and glycerol as dual cosubstrate for cofactor recycling. A fungal strain ZJPH0810, showing asymmetric biocatalytic activity of 3,5-bis(trifluoromethyl) acetophenone to its corresponding (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol, was isolated from a soil sample. Based on its morphological and physiological characteristics and internal transcribed spacer sequence, this isolate was identified as T. asperellum ZJPH0810, which afforded an NADH-dependent (R)-stereospecific carbonyl reductase and was a promising biocatalyst for the synthesis of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol. Some key reaction parameters involved in the bioreduction catalyzed by T. asperellum ZJPH0810 were subsequently optimized. The effectiveness of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol production was significantly enhanced by employing a novel dual cosubstrate-coupled system for cofactor recycling. The established efficient bioreduction system contained 50 mM of 3,5-bis(trifluoromethyl) acetophenone and 60 g l−1 of resting cells, employing ethanol (6.0 %, v/v) and glycerol (0.5 %, v/v) as dual cosubstrate. The bioreduction was performed in distilled water medium, at 30 °C and 200 rpm. Under the above conditions, a best yield of 93.4 % was obtained, which is nearly a 3.5-fold increase in contrast to no addition of cosubstrate. The ee value of the product reached above 98 %. This biocatalytic process shows great potential in the production of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol, a valuable chiral building block in the pharmaceutical industry.
Keywords: Trichoderma asperellum ; Bioreduction; Dual-cosubstrate (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol

Reactions upstream of glycerate-1,3-bisphosphate drive Corynebacterium glutamicum d-lactate productivity under oxygen deprivation by Yota Tsuge; Shougo Yamamoto; Masako Suda; Masayuki Inui; Hideaki Yukawa (6693-6703).
We previously demonstrated the simplicity of oxygen-deprived Corynebacterium glutamicum to produce d-lactate, a primary building block of next-generation biodegradable plastics, at very high optical purity by introducing heterologous D-ldhA gene from Lactobacillus delbrueckii. Here, we independently evaluated the effects of overexpressing each of genes encoding the ten glycolytic enzymes on d-lactate production in C. glutamicum. We consequently show that while the reactions catalyzed by glucokinase (GLK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase had positive effects on d-lactate productivity by increasing 98, 39, 15, 13, and 10 %, respectively, in 10 h reactions in minimal salts medium, the reaction catalyzed by pyruvate kinase had large negative effect by decreasing 70 %. The other glycolytic enzymes did not affect d-lactate productivity when each of encoding genes was overexpressed. It is noteworthy that all reactions associated with positive effects are located upstream of glycerate-1,3-bisphosphate in the glycolytic pathway. The d-lactate yield also increased by especially overexpressing TPI encoding gene up to 94.5 %. Interestingly, overexpression of PFK encoding gene reduced the yield of succinate, one of the main by-products of d-lactate production, by 52 %, whereas overexpression of GAPDH encoding gene increased succinate yield by 26 %. Overexpression of GLK encoding gene markedly increased the yield of dihydroxyacetone and glycerol by 10- and 5.8-fold in exchange with decreasing the d-lactate yield. The effect of overexpressing glycolytic genes was also evaluated in 80 h long-term reactions. The variety of effects of overexpressing each of genes encoding the ten glycolytic enzymes on d-lactate production is discussed.
Keywords: Corynebacterium glutamicum ; Oxygen deprivation; d-Lactate production; Metabolic engineering; Glycolytic enzyme

A novel strategy for enhancing extracellular secretion of recombinant proteins in Escherichia coli by Lingqia Su; Chenhua Xu; Ronald W. Woodard; Jian Chen; Jing Wu (6705-6713).
Secretion of cytoplasmic expressed proteins into culture medium has significant commercial advantages in large-scale production of proteins. Our previous study demonstrated that the membrane permeability of Escherichia coli could be significantly improved when Thermobifida fusca cutinase, without a signal peptide, was expressed in cytoplasm. This study investigated the extracellular production of other recombinant proteins, including both secretory and cytosolic proteins, with co-expression of cutinase. When the secretory enzymes, xylanase and α-amylase, were co-expressed with cutinase, the culture period was shortened by half, and the productivity was 7.9 and 2.0-fold to that of their individual control without co-expression, respectively. When the normally cytosolic proteins, xylose isomerase and trehalose synthase, were co-expressed with cutinase, more than half of the target proteins were “secreted” into the culture medium. Moreover, by using β-galactosidase to detect membrane leakage, the improved secretion of the above model proteins was confirmed not to be due to cell lysis. The study provides a novel strategy for enhancing extracellular secretion of recombinant proteins in E. coli.
Keywords: T. fusca cutinase; Co-expression; Extracellular secretion; Secretory protein; Cytosolic protein

Enhanced 2,3-butanediol production in fed-batch cultures of free and immobilized Bacillus licheniformis DSM 8785 by Ileana-Maria Jurchescu; Jan Hamann; Xiye Zhou; Tim Ortmann; Anja Kuenz; Ulf Prüße; Siegmund Lang (6715-6723).
2,3-Butanediol (2,3-BD) is a valuable bulk chemical with particular use in industry. 2,3-BD has a potential as solvent and fuel additive, as carrier for pharmaceuticals, or as feedstock for the production of synthetic rubber. Until now, the highest 2,3-BD concentrations were obtained with risk group 2 microorganisms (e.g., Klebsiella oxytoca). In this study, the nonpathogenic bacterium Bacillus licheniformis DSM 8785 was used for 2,3-BD production from glucose. In batch experiments, a maximum 2,3-BD concentration of 72.6 g/L was reached from 180 g/L glucose after 86 h. The yield was 0.42 g/g glucose and the productivity was 0.86 g/(L h). During fed-batch cultivation, 2,3-BD production could be increased up to 144.7 g/L, with a productivity of 1.14 g/(L h). Additionally, repeated batch/fed-batch experiments were conducted using immobilized B. licheniformis in the form of LentiKats®. Results showed a high activity and stability of the immobilizates even after multiple medium replacements, as well as 2,3-BD concentrations, yields, and productivities similar to those obtained with free cells. To our knowledge, these results show the highest 2,3-BD concentration reported so far using a risk group 1 microorganism in general and B. licheniformis in particular. Furthermore, productivity lies in the same range with data reported from risk group 2 strains, which makes B. licheniformis DSM 8785 a suitable candidate for large-scale fermentation processes.
Keywords: 2,3-Butanediol; Glucose; Bacillus licheniformis ; Fed-batch; Immobilized cells; LentiKats®

Solid-state fermentation of industrial solid wastes from the fruits of milk thistle Silybum marianum for feed quality improvement by Fang Li; Feng Li; Ting Zhao; Guanghua Mao; Ye Zou; Daheng Zheng; Mohammed Takase; Weiwei Feng; Xiangyang Wu; Liuqing Yang (6725-6737).
The industrial solid wastes generated during the production of silymarin from the fruits of milk thistle Silybum marianum was used as the substrate. Preparation and evaluation of the feeds produced by solid-state fermentation (SSF) of the industrial solid wastes was carried out. The protein content of the fermented feed (FF) from a combination of Aspergillus niger and Candida tropicalis was the highest among the examined strains. The optimal process parameters for protein enrichment with SSF using A. niger and C. tropicalis included incubation temperature of 30.8 °C, fermentation time of 87.0 h, and initial moisture content of 59.7 %. Under these conditions, the value additions of FF occurred. The fiber of FF was decreased by 25.07 %, while the digestibility of protein, protein content, and the ratio of total essential amino acids to total amino acids were increased by 79.85, 16.22, and 8.21 %, respectively. The analysis indicated that FF contained 1.44 mg/kg flavonoids and 0.5 mg/kg silybin, which significantly increased by 2.42 and 1.63 times, respectively than those in unfermented substrates. FF recorded reduced molecular weight of proteins from 20.1 to 44.3 kDa to below 14.3 kDa. The results of feeding trial of FF replacement with soybean meal in broilers diets for 8 weeks showed that FF significantly improved carcass characteristics including abdominal fat rate, serum biochemical parameters including aspartate transaminase, blood urea nitrogen and high density lipoprotein cholesterol, and immune responses of broilers. A potential feed quality improvement was achieved through mixed strains SSF of industrial solid wastes of S. marianum fruits.
Keywords: Milk thistle Silybum marianum fruit; Industrial solid wastes; Mixed strains fermentation; Protein enrichment; Feed for broilers; Feed quality improvement

Enhancement of succinate production by metabolically engineered Escherichia coli with co-expression of nicotinic acid phosphoribosyltransferase and pyruvate carboxylase by Jiangfeng Ma; Dongmei Gou; Liya Liang; Rongming Liu; Xu Chen; Changqing Zhang; Jiuhua Zhang; Kequan Chen; Min Jiang (6739-6747).
Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. The total concentration of NAD(H) was 9.8-fold higher in BA016 than in BA002, and the NADH/NAD+ ratio decreased from 0.60 to 0.04. Under anaerobic conditions, BA016 consumed 17.50 g l−1 glucose and produced 14.08 g l−1 succinate with a small quantity of pyruvate. Furthermore, when the reducing agent dithiothreitol or reduced carbon source sorbitol was added, the cell growth and carbon source consumption rate of BA016 was reasonably enhanced and succinate productivity increased.
Keywords: Succinate; NADH/NAD+ ; Anaerobic fermentation; NAPRTase; Pyruvate carboxylase

Biochemical characterization of a thermostable β-1,3-xylanase from the hyperthermophilic eubacterium, Thermotoga neapolitana strain DSM 4359 by Fumiyoshi Okazaki; Nanami Nakashima; Chiaki Ogino; Yutaka Tamaru; Akihiko Kondo (6749-6757).
The biochemical properties of a putative β-1,3-xylanase from the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 were determined from a recombinant protein (TnXyn26A) expressed in Escherichia coli. This enzyme showed specific hydrolytic activity against β-1,3-xylan and released β-1,3-xylobiose and β-1,3-xylotriose as main products. It displayed maximum activity at 85 °C during a 10-min incubation, and its activity half-life was 23.9 h at 85 °C. Enzyme activity was stable in the pH range 3–10, with pH 6.5 being optimal. Enzyme activity was significantly inhibited by the presence of N-bromosuccinimide (NBS). The insoluble β-1,3-xylan K m value was 10.35 mg/ml and the k cat value was 588.24 s−1. The observed high thermostability and catalytic efficiency of TnXyn26A is both industrially desirable and also aids an understanding of the chemistry of its hydrolytic reaction.
Keywords: β-1,3-Xylanase; Thermostable enzyme; Thermotoga neapolitana ; Hyperthermophilic bacterium; β-1,3-Xylan

Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus by André R. L. Damásio; Cleiton Márcio Pinto Braga; Lívia B. Brenelli; Ana Paula Citadini; Fernanda Mandelli; Junio Cota; Rodrigo Ferreira de Almeida; Victor Hugo Salvador; Douglas Antonio Alvaredo Paixao; Fernando Segato; Adriana Zerlotti Mercadante; Mario de Oliveira Neto; Wanderley Dantas do Santos; Fabio M. Squina (6759-6767).
The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production.
Keywords: Aspergillus clavatus ; Feruloyl esterase; Sugarcane bagasse; Wheat arabinoxylan; Phenolic compounds; Antioxidant capacity

Biosynthesis of nucleoside analogues via thermostable nucleoside phosphorylase by Shaozhou Zhu; Dawei Song; Cuiyu Gong; Pingwah Tang; Xingzhou Li; Jianjun Wang; Guojun Zheng (6769-6778).
Biocatalyzed synthesis of nucleoside analogues was carried out using two thermostable nucleoside phosphorylases from the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix K1. The synthesis of the 2,6-diaminopurine nucleoside and 5-methyluridine was used as a reaction model to test the process. Both the purine nucleoside phosphorylase (apPNP) and uridine phosphorylase (apUP) were functionally expressed in Escherichia coli. The recombinant enzymes were characterized after purification, and both enzymes showed high thermostability and broad substrate specificity. Both enzymes retained 100 % of their activity after 60 min at high temperature, and the optimum temperature for the enzymes was 90–100 °C. The nucleoside phosphorylases obtained from A. pernix are valuable industrial biocatalysts for high-temperature reactions that produce nucleoside drugs in high yields.
Keywords: Nucleoside analogues; Purine nucleoside phosphorylase; Uridine phosphorylase; Biosynthesis; Thermostable

The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli: a comparison with the traditional gene fusion technology by Sofia J. Costa; André Almeida; António Castro; Lucília Domingues; Hüseyin Besir (6779-6791).
The Escherichia coli host system is an advantageous choice for simple and inexpensive recombinant protein production but it still presents bottlenecks at expressing soluble proteins from other organisms. Several efforts have been taken to overcome E. coli limitations, including the use of fusion partners that improve protein expression and solubility. New fusion technologies are emerging to complement the traditional solutions. This work evaluates two novel fusion partners, the Fh8 tag (8 kDa) and the H tag (1 kDa), as solubility enhancing tags in E. coli and their comparison to commonly used fusion partners. A broad range comparison was conducted in a small-scale screening and subsequently scaled-up. Six difficult-to-express target proteins (RVS167, SPO14, YPK1, YPK2, Frutalin and CP12) were fused to eight fusion tags (His, Trx, GST, MBP, NusA, SUMO, H and Fh8). The resulting protein expression and solubility levels were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after protein purification and after tag removal. The Fh8 partner improved protein expression and solubility as the well-known Trx, NusA or MBP fusion partners. The H partner did not function as a solubility tag. Cleaved proteins from Fh8 fusions were soluble and obtained in similar or higher amounts than proteins from the cleavage of other partners as Trx, NusA or MBP. The Fh8 fusion tag therefore acts as an effective solubility enhancer, and its low molecular weight potentially gives it an advantage over larger solubility tags by offering a more reliable assessment of the target protein solubility when expressed as a fusion protein.
Keywords: Escherichia coli ; Fusion protein; Fh8 fusion tag; Traditionally used fusion tags; Protein solubility; Tag removal

Surface charge engineering of a Bacillus gibsonii subtilisin protease by Felix Jakob; Ronny Martinez; John Mandawe; Hendrik Hellmuth; Petra Siegert; Karl-Heinz Maurer; Ulrich Schwaneberg (6793-6802).
In proteins, a posttranslational deamidation process converts asparagine (Asn) and glutamine (Gln) residues into negatively charged aspartic (Asp) and glutamic acid (Glu), respectively. This process changes the protein net charge affecting enzyme activity, pH optimum, and stability. Understanding the principles which affect these enzyme properties would be valuable for protein engineering in general. In this work, three criteria for selecting amino acid substitutions of the deamidation type in the Bacillus gibsonii alkaline protease (BgAP) are proposed and systematically studied in their influence on pH-dependent activity and thermal resistance. Out of 113 possible surface amino acids, 18 (11 Asn and 7 Gln) residues of BgAP were selected and evaluated based on three proposed criteria: (1) The Asn or Gln residues should not be conserved, (2) should be surface exposed, and (3) neighbored by glycine. “Deamidation” in five (N97, N253, Q37, Q200, and Q256) out of eight (N97, N154, N250, N253, Q37, Q107, Q200, and Q256) amino acids meeting all criteria resulted in increased proteolytic activity. In addition, pH activity profiles of the variants N253D and Q256E and the combined variant N253DQ256E were dramatically shifted towards higher activity at lower pH (range of 8.5–10). Variant N253DQ256E showed twice the specific activity of wild-type BgAP and its thermal resistance increased by 2.4 °C at pH 8.5. These property changes suggest that mimicking surface deamidation by substituting Gln by Glu and/or Asn by Asp might be a simple and fast protein reengineering approach for modulating enzyme properties such as activity, pH optimum, and thermal resistance.
Keywords: Deamidation; Deamination; pH optimum; Protease; Surface charge engineering

Authentic human basic fibroblast growth factor produced by secretion in Bacillus subtilis by Keith W. Y. Kwong; K. L. Ng; C. C. Lam; Yule Y. Wang; W. K. R. Wong (6803-6811).
Bacillus subtilis is generally accepted as an inborn host candidate employed for secretory production of heterologous proteins. However, this ideal host system has never been employed for commercial production of medically useful proteins. In this communication, we report for the first time the employment of an engineered B. subtilis system, in conjunction with a facile cell-wall destabilization protocol, to successfully obtain an alluring yield of 40 mg l−1 of secreted human basic fibroblast growth factor (hbFGF) in the culture supernatant. The product was not only shown to exhibit potent bioactivity but also revealed to possess a protein sequence identical to that of mature native hbFGF (Mat-hbFGF). Our findings may pave way for the development of a cost-effective process for producing Mat-hbFGF, which is currently sold at an unusually expensive price of over US $1 million g−1, for medical and skin care applications.
Keywords: Human basic fibroblast growth factor; hbFGF; FGF-2; Bacillus subtilis ; Escherichia coli ; Extracellular expression; Secretion; Authentic; Peptidoglycan destabilization

Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system by Hiroaki Mon; JaeMan Lee; Mai Fukushima; Yudai Nagata; Mie Fujii; Jian Xu; Oumi Nishi; Kazuhiro Iiyama; Takahiro Kusakabe (6813-6822).
Mutation and polymorphism detection by nucleases has become a more important tool in clinical and biological researches. There are several kinds of single-stranded nucleases for detecting mismatched DNAs. One of them, CEL II, was isolated from Apium graveolens and cleaves DNA with high specificity at sites of mismatch. High-throughput mutation scanning requires large quantity of CEL II endonuclease. Here, we demonstrate high-level expression of CEL II using silkworm-baculovirus system. The recombinant CEL II secreted in silkworm hemolymph was glycosylated and susceptible to N-glycosidase F. Additionally, larger metal ions such as Ca2+ and Sr2+ were able to replace Mg2+ and enhanced mismatch cleavage activity of CEL II. These results indicate that the silkworm-baculovirus platform is a good alternative system to obtain the functional CEL II.
Keywords: Mismatch endonuclease; Silkworm; Cel II; TALEN; Baculovirus

Deletion of an architectural unit, leucyl aminopeptidase (SCO2179), in Streptomyces coelicolor increases actinorhodin production and sporulation by Eunjung Song; Thangamani Rajesh; Bo-Rahm Lee; Eun-jung Kim; Jong-Min Jeon; Sung-Hee Park; Hyung-Yeon Park; Kwon-Young Choi; Yun-Gon Kim; Yung-Hun Yang; Byung-Gee Kim (6823-6833).
Several reports state that three architectural units, including integration host factor, leucyl aminopeptidase (PepA), and purine regulator, are involved in transcriptional process with RNA polymerase in Escherichia coli. Similarly, Streptomyces species possess the same structural units. We previously identified a protein, Streptomyces integration host factor (sIHF), involved in antibiotic production and sporulation. Subsequently, the function of PepA (SCO2179) was examined in detail. PepA is highly conserved among various Streptomyces spp., but it has not yet been characterized in Streptomyces coelicolor. While it is annotated as a putative leucyl aminopeptidase because it contains a peptidase M17 superfamily domain, this protein did not exhibit leucyl aminopeptidase activity. SCO2179 deletion mutant showed increased actinorhodin production and sporulation, as well as more distinct physiological differences, particularly when cultured on N-acetylglucosamine (GlcNAc) minimal media. The results of two-dimensional gel analysis and reverse transcription PCR showed that the SCO2179 deletion increased protein and mRNA levels of ftsZ, ssgA, and actinorhodin (ACT)-related genes such as actII-ORF4, resulting in increased actinorhodin production and spore formation in minimal media containing GlcNAc.
Keywords: Streptomyces ; Leucyl aminopeptidase; Antibiotic production; N-acetylglucosamine; Spore formation

In the fission yeast Schizosaccharomyces pombe, the gld1 + gene encoding glycerol dehydrogenase is repressed by glucose and induced by ethanol and 1-propanol. The promoter region of gld1 + was cloned into a multicopy vector designated as pEG1 for evaluation as an ethanol-inducible expression vector using EGFP as a model heterologous protein. Expression of EGFP was repressed in the presence of high glucose and induced in the presence of ethanol, low-glucose, and 1-propanol in the absence of glucose. Addition of ethanol to cells harboring pEG1–EGFP was found to be the most effective means for inducing EGFP production. Protein yields were found to increase in proportion to ethanol concentration. As a further test of effectiveness, secreted recombinant human growth hormone was produced using the pEG1 expression vector in medium containing glycerol and ethanol. The pEG1 gene expression system is an effective tool for the production of heterologous proteins under glucose-limiting conditions, including medium containing glycerol as a carbon source.
Keywords: Schizosaccharomyces pombe ; Fission yeast; Glycerol; Ethanol; Gene expression system; gld1 +

A newly designed site-specific recombination system is presented which allows multiple targeted markerless deletions. The most frequently used tool for removing selection markers or to introduce genes by recombination-mediated cassette exchange is the Cre/loxP system. Many mutant loxP sites have been created for this purpose. However, this study presents a chimeric mutant loxP site denoted mroxP-site. The mroxP site consists of one Cre (loxP/2) and one MrpA (mrpS/2) binding site separated by a palindromic 6-bp spacer sequence. Two mroxP-sites can be recombined by Cre recombinase in head-to-tail as well as in head-to-head orientation. In the head-to-head orientation and the loxP half-sites inside, Cre removes the loxP half-sites during site-specific recombination, creating a new site, mrmrP. The new site is essentially a mrpS site with a palindromic spacer and cannot be used by Cre for recombination anymore. It does, however, present a substrate for the recombinase MrpA. This new system has been successfully applied introducing multiple targeted gene deletions into the Escherichia coli genome. Similar to Cre/loxP and FLP/FRT, this system may be adapted for genetic engineering of other pro- and eukaryotes.
Keywords: Site-specific recombination; Cre; Multimer resolution protein MrpA; Targeted deletion

The epothilones, compounds with anticancer mechanisms similar to that of paclitaxel (Taxol), are produced by strains of the myxobacterium Sorangium cellulosum, and the gene cluster responsible for epothilone biosynthesis is organised as a large operon. In this work, we showed that the 440-bp promoter regions of the operons from eight S. cellulosum strains have 94.27 % DNA sequence identity and 50 % variability in promoter activity in Escherichia coli. A primer extension analysis revealed two transcriptional start sites (TSSs) at 246 (TSS1) and 193 bp (TSS2) upstream of the translation start site (TLS), respectively. Promoter truncation determined that the basal promoter from the So0157-2 strain is located within a 264-bp region containing weak promoter activity; whereas in the 38-bp region upstream, the 264-bp promoter was required for the strong promoter activity, which was dramatically increased by 11-fold in average. There was a conserved stem–loop structure between TSS2 and the TLS, which was identified in E. coli as a negative regulatory element. In addition, the upstream non-conserved 357-bp non-coding region contributes to the promoter activity, increasing it by 1.5-fold. In conclusion, the expression of the epothilone operon non-coding region in E. coli is regulated by a double promoter (with −35 and −10 regions and two distinct TSSs), a stem–loop structure, and a distal non-coding region.
Keywords: Sorangium cellulosum ; Epothilones; Biosynthetic gene cluster; Promoter

Overexpression of stress-related genes enhances cell viability and velum formation in Sherry wine yeasts by Jesús Fierro-Risco; Ana María Rincón; Tahía Benítez; Antonio C. Codón (6867-6881).
Flor formation and flor endurance have been related to ability by Saccharomyces cerevisiae flor yeasts to resist hostile conditions such as oxidative stress and the presence of acetaldehyde and ethanol. Ethanol and acetaldehyde toxicity give rise to formation of reactive oxygen species (ROS) and loss of cell viability. Superoxide dismutases Sod1p and Sod2p and other proteins such as Hsp12p are involved in oxidative stress tolerance. In this study, genes SOD1, SOD2, and HSP12 were overexpressed in flor yeast strains FJF206, FJF414 and B16. In the SOD1 and SOD2 transformant strains superoxide dismutases encoded by genes SOD1 and SOD2 increased their specific activity considerably as a direct result of overexpression of genes SOD1 and SOD2, indirectly, catalase, glutathione reductase, and glutathione peroxidase activities increased too. The HSP12 transformant strains showed higher levels of glutathione peroxidase and reductase activities. These transformant strains showed an increase in intracellular glutathione content, a reduction in peroxidized lipid concentration, and higher resistance to oxidative stress conditions. As a result, flor formation by these strains took place more rapidly than by their parental strains, velum being thicker and with higher percentages of viable cells. In addition, a slight decrease in ethanol and glycerol concentrations, and an increase in acetaldehyde were detected in wines matured under velum formed by transformant strains, as compared to their parental strains. In the industry, velum formed by transformant strains with increased viability may result in acceleration of both metabolism and wine aging, thus reducing time needed for wine maturation.
Keywords: Flor industrial yeast; Flor velum formation and stability; Cell viability; Stress-related genes; SOD1 ; SOD2 ; HSP12

NADPH-dependent reactions play important roles in production of industrially valuable compounds. In this study, we used phosphofructokinase (PFK)-deficient strains to direct fructose-6-phosphate to be oxidized through the pentose phosphate pathway (PPP) to increase NADPH generation. pfkA or pfkB single deletion and double-deletion strains were tested for their ability to produce lycopene. Since lycopene biosynthesis requires many NADPH, levels of lycopene were compared in a set of isogenic strains, with the pfkA single deletion strain showing the highest lycopene yield. Using another NADPH-requiring process, a one-step reduction reaction of 2-chloroacrylate to 2-chloropropionic acid by 2-haloacrylate reductase, the pfkA pfkB double-deletion strain showed the highest yield of 2-chloropropionic acid product. The combined effect of glucose-6-phosphate dehydrogenase overexpression or lactate dehydrogenase deletion with PFK deficiency on NADPH bioavailability was also studied. The results indicated that the flux distribution of fructose-6-phosphate between glycolysis and the pentose phosphate pathway determines the amount of NAPDH available for reductive biosynthesis.
Keywords: PFK; NADPH bioavailability; pfkA ; pfkB ; G6PDH; E. coli

The immobilization of quinone compounds is regarded as a promising strategy to accelerate anaerobic decolorization of xenobiotic compounds azo dyes in the presence of quinone-reducing microorganisms. However, little is known about the basic response of these microorganisms to immobilized quinones in the presence of azo dyes. In the present study, whole-genome DNA microarrays were used to investigate a quinone-reducing bacterium Escherichia coli K-12 transcription response to immobilized anthraquinone-2-sulfonate (AQSim) reduction and azo dye acid red 18 (AR 18) decolorization. Transcriptome analysis showed that AQSim was more accessible for the cells of E. coli K-12 than AR 18. Despite there being some differences between AQSim and soluble AQS mediated decolorization of AR 18, AQSim reduction and AR 18 decolorization, more similarity could be observed in the four processes. Among over 60 % shared genes, several groups of genes exhibited high expression levels, including those genes encoding terminal reductases, menaquinone biosynthesis, formate dehydrogenases and outer membrane proteins. Especially, nrfABCD, frdBCD and dsmABC encoding terminal reductases were significantly upregulated. Further gene deletion experiments demonstrated that the above three groups of genes were involved in AQSim-mediated AR 18 decolorization. In addition, significant upregulation of stress response genes was observed, which indicated the adaptation of E. coli K-12 to AQSim and AR 18 exposures.
Keywords: Escherichia coli K-12; Azo dye; Immobilized anthraquinone-2-sulfonate; Response; Microarray; Transcription

High cell density cultivation of a novel Aurantiochytrium sp. strain TC 20 in a fed-batch system using glycerol to produce feedstock for biodiesel and omega-3 oils by Kim Jye Lee Chang; Geoff Dumsday; Peter D. Nichols; Graeme A. Dunstan; Susan I. Blackburn; Anthony Koutoulis (6907-6918).
A recently isolated Australian Aurantiochytrium sp. strain TC 20 was investigated using small-scale (2 L) bioreactors for the potential of co-producing biodiesel and high-value omega-3 long-chain polyunsaturated fatty acids. Higher initial glucose concentration (100 g/L compared to 40 g/L) did not result in markedly different biomass (48 g/L) or fatty acid (12–14 g/L) yields by 69 h. This comparison suggests factors other than carbon source were limiting biomass production. The effect of both glucose and glycerol as carbon sources for Aurantiochytrium sp. strain TC 20 was evaluated in a fed-batch process. Both glucose and glycerol resulted in similar biomass yields (57 and 56 g/L, respectively) by 69 h. The agro-industrial waste from biodiesel production—glycerol—is a suitable carbon source for Aurantiochytrium sp. strain TC 20. Approximately half the fatty acids from Aurantiochytrium sp. strain TC 20 are suitable for development of sustainable, low emission sources of transportation fuels and bioproducts. To further improve biomass and oil production, fortification of the feed with additional nutrients (nitrogen sources, trace metals and vitamins) improved the biomass yield from 56 g/L (34 % total fatty acids) to 71 g/L (52 % total fatty acids, cell dry weight) at 69 h; these yields are to our knowledge around 70 % of the biomass yields achieved, however, in less than half of the time by other researchers using glycerol and markedly greater than achieved using other industrial wastes. The fast growth and suitable fatty acid profile of this newly isolated Aurantiochytrium sp. strain TC 20 highlights the potential of co-producing the drop-in biodiesel and high value omega-3 oils.
Keywords: Labyrinthulomycetes; Thraustochytrid; Biodiesel; Glycerol; Docosahexaenoic acid

Undesirable butter-tasting vicinal diketones are produced as by-products of valine and isoleucine biosynthesis during wort fermentation. One promising method of decreasing diacetyl production is through control of wort valine content since valine is involved in feedback inhibition of enzymes controlling the formation of diacetyl precursors. Here, the influence of valine supplementation, wort amino acid profile and free amino nitrogen content on diacetyl formation during wort fermentation with the lager yeast Saccharomyces pastorianus was investigated. Valine supplementation (100 to 300 mg L−1) resulted in decreased maximum diacetyl concentrations (up to 37 % lower) and diacetyl concentrations at the end of fermentation (up to 33 % lower) in all trials. Composition of the amino acid spectrum of the wort also had an impact on diacetyl and 2,3-pentanedione production during fermentation. No direct correlation between the wort amino acid concentrations and diacetyl production was found, but rather a negative correlation between the uptake rate of valine (and also other branched-chain amino acids) and diacetyl production. Fermentation performance and yeast growth were unaffected by supplementations. Amino acid addition had a minor effect on higher alcohol and ester composition, suggesting that high levels of supplementation could affect the flavour profile of the beer. Modifying amino acid profile of wort, especially with respect to valine and the other branched-chain amino acids, may be an effective way of decreasing the amount of diacetyl formed during fermentation.
Keywords: Diacetyl; Valine; Amino acid; Beer; Lager; Fermentation

Dynamics of lactic acid bacteria populations in Rioja wines by PCR-DGGE, comparison with culture-dependent methods by Lucía González-Arenzana; Rosa López; Pilar Santamaría; Isabel López-Alfaro (6931-6941).
Lactic acid bacteria populations of red wine samples from industrial fermentations, including two different vinification methods were studied. For this investigation, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) analysis was employed to supplement previous results that were obtained by culture-dependent methods. PCR-DGGE was aimed to study two targeted genes, 16S ribosomal DNA (rDNA) and rpoB, and the results were useful to evaluate the microbial populations in wine samples. Moreover, an improvement of a detection limit determined so far for DGGE analysis was obtained with the method described in this study, what made possible to identify lactic acid bacteria populations below 101 colony-forming unit/mL. The species Oenococcus oeni was the most frequently detected bacterium, but identifications close to species Oenococcus kitaharae and Lactococcus lactis that are not often found in wine were firstly identified in samples of this research. PCR-DGGE allowed to detect 9 out of 11 lactic acid bacteria species identified in this study (nine by PCR-16S rDNA/DGGE and four by PCR-rpoB/DGGE), while five species were detected using the modified de Man, Rogosa and Sharpe agar. Therefore, the two methods were demonstrated to be complementary. This finding suggests that analysis of the lactic acid bacteria population structure in wine should be carried out using both culture-dependent and culture-independent techniques with more than one primer pair.
Keywords: Lactic acid bacteria; PCR-DGGE; 16S rDNA; rpoB gene; Wine

Hollow fiber culture accelerates differentiation of Caco-2 cells by Xudong Deng; Guoliang Zhang; Chong Shen; Jian Yin; Qin Meng (6943-6955).
Caco-2 cells usually require 21 days of culture for developing sufficient differentiation in traditional two-dimensional Transwell culture, deviating far away from the quick differentiation of enterocytes in vivo. The recently proposed three-dimensional cultures of Caco-2 cells, though imitating the villi/crypt-like microstructure of intestinal epithelium, showed no effect on accelerating the differentiation of Caco-2 cells. In this study, a novel culture of Caco-2 cells on hollow fiber bioreactor was applied to morphologically mimic the human small intestine lumen for accelerating the expression of intestine functions. The porous hollow fibers of polyethersulfone (PES), a suitable membrane material for Caco-2 cell culture, successfully promoted cells to form confluent monolayer on the inner surface. The differentiated functions of Caco-2 cells, represented by alkaline phosphatase, γ-glutamyltransferase, and P-glycoprotein activity, were greatly higher in a 10-day hollow fiber culture than in a 21-day Transwell culture. Moreover, the Caco-2 cells on PES hollow fibers expressed higher F-actin and zonula occludens-1 protein than those on Transwell culture, indicative of an increased mechanical stress in Caco-2 cells on PES hollow fibers. The accelerated differentiation of Caco-2 cells on PES hollow fibers was unassociated with membrane chemical composition and surface roughness, but could be stimulated by hollow fiber configuration, since PES flat membranes with either rough or smooth surface failed to enhance the differentiation of Caco-2. Therefore, the accelerated expression of Caco-2 cell function on hollow fiber culture might show great values in simulation of the tissue microenvironment in vivo and guide the construction of intestinal tissue engineering apparatus.
Keywords: Polyethersulfone; Hollow fiber membrane; Caco-2 cell; Acceleration of cell differentiation

Evidence on antimicrobial properties and mode of action of a chitosan obtained from crustacean exoskeletons on Pseudomonas syringae pv. tomato DC3000 by A. Y. Mansilla; L. Albertengo; M. S. Rodríguez; A. Debbaudt; A. Zúñiga; C. A. Casalongué (6957-6966).
Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) causes bacterial speck of tomato, a widely spread disease that causes significant economical losses worldwide. It is representative of many bacterial plant diseases for which effective controls are still needed. Despite the antimicrobial properties of chitosan has been previously described in phytopathogenic fungi, its action on bacteria is still poorly explored. In this work, we report that the chitosan isolated from shrimp exoskeletons (70 kDa and 78 % deacetylation degree) exerts cell damage on Pto DC3000. Chitosan inhibited Pto DC3000 bacterial growth depending on its concentration, medium-pH, and presence of metal ion (Mg+2). Biochemical and cellular changes resulting in cell aggregation and impaired bacterial growth were also viewed. In vivo studies using fluorescent probes showed cell aggregation, increase in membrane permeability, and cell death, suggesting the chitosan antibacterial activity is due to its interaction as a polycation with Pto DC3000 membranes. Transmission electron microscopic analysis revealed that chitosan also caused morphological changes and damage in bacterial surfaces. Also, the disease incidence in tomato inoculated with Pto DC3000 was significantly reduced in chitosan pretreated seedlings, revealing a promising action of chitosan as nontoxic biopesticide in tomato plants. Indeed, a wider comprehensive knowledge of the mechanism of action of chitosan in phytopathogenic bacterial cells will increase the chances of its successful application to the control of spread disease in plants.
Keywords: Chitosan; Membrane permeability; Pseudomonas syringae pv. tomato DC3000; Solanum lycopersicum

The complete genome of Acinetobacter oleivorans DR1 contains AqsR and AqsI genes, which are LuxR and LuxI homolog, respectively. In a previous study, we demonstrated that quorum sensing (QS) signals play an important role in biofilm formation and hexadecane biodegradation. However, the regulation of genes controlled by the QS system in DR1 remains unexplored. We constructed an aqsR mutant and performed RNA sequencing analysis to understand the QS system. A total of 353 genes were differentially expressed during the stationary phase of wild-type cells compared to that of the aqsR mutant. AqsR appears to be an exceptionally important regulator because knockout of aqsR affected global gene expression. Genes involved in posttranslational modification, chaperones, cell wall structure, secondary metabolites biosynthesis, and stress defense were highly upregulated only in the wild type. Among upregulated genes, both the AOLE_03905 (putative surface adhesion protein) and the AOLE_11355 (L-asparaginase) genes have putative LuxR binding sites at their promoter regions. Soluble AqsR proteins were successfully purified in Escherichia coli harboring both aqsR and aqsI. Comparison of QS signals in an AqsI–AqsR co-overexpression strain with N-acyl homoserine lactone standards showed that the cognate N-acyl homoserine lactone binding to AqsR might be 3OH C12HSL. Our electrophoretic mobility shift assays with purified AqsR revealed direct binding of AqsR to those promoter regions. Our data showed that AqsR functions as an important regulator and is associated with several phenotypes, such as hexadecane utilization, biofilm formation, and sensitivity to cumene hydroperoxide.
Keywords: Bacteria; Quorum sensing; Transcriptional regulation; Soil; N-acyl homoserine lactone; Oxidative stress

Production of hydrogen from domestic wastewater in a pilot-scale microbial electrolysis cell by E. S. Heidrich; J. Dolfing; K. Scott; S. R. Edwards; C. Jones; T. P. Curtis (6979-6989).
Addressing the need to recover energy from the treatment of domestic wastewater, a 120-L microbial electrolysis cell was operated on site in Northern England, using raw domestic wastewater to produce virtually pure hydrogen gas (100 ± 6.4 %) for a period of over 3 months. The volumetric loading rate was 0.14 kg of chemical oxygen demand (COD) per cubic metre per day, just below the typical loading rates for activated sludge of 0.2–2 kg COD m−3 day−1, at an energetic cost of 2.3 kJ/g COD, which is below the values for activated sludge 2.5–7.2 kJ/g COD. The reactor produced an equivalent of 0.015 L H2 L−1 day−1, and recovered around 70 % of the electrical energy input with a coulombic efficiency of 55 %. Although the reactor did not reach the breakeven point of 100 % electrical energy recovery and COD removal was limited, improved hydrogen capture and reactor design could increase the performance levels substantially. Importantly, for the first time, a ‘proof of concept’ has been made, showing that this technology is capable of energy capture as hydrogen gas from low strength domestic wastewaters at ambient temperatures.
Keywords: Microbial electrolysis cell; Hydrogen; Wastewater; Energy

An investigation of bacterial diversity in compost was performed using molecular chronometer in order to reveal its phylogeny. Thirty-three bacterial isolates isolated from compost were analyzed by 16S rRNA gene sequencing which revealed phylogenetic lineage of class Bacilli, γ, β-Proteobacteria, and Actinobacteria. Among these lineages, isolates belonging to class Bacilli consisted of species from genera Staphylococcus, Bacillus, Terribacillus, and Lysinibacillus. From phylum Actinobacteria: Microbacterium barkeri and Kocuria sp. were identified. Other bacterial groups had phylogenetic linkage with genera Comamonas and Acidovorax (class β-Proteobacteria); Serratia, Klebsiella, and Enterobacter (class γ-Proteobacteria). Similar isolates were analyzed through ARDRA. Amplified product of 16S rRNA gene from each isolates was subjected to cleavage by enzymes HpaII, HinfI, and MspI in separate reaction tubes. HpaII generated 2–6 bands ranging from 90–688 bp, HinfI generated 2–5 bands of 71–1,038 bp, and MspI 2–7 bands of 69–793 bp. The restriction patterns from HpaII, HinfI, and MspI were normalized separately and combined by means of pattern recognition software “Diversity Database.” HpaII had highest discrimination index (0.72) than HinfI (0.68) and MspI (0.65), and the combination of all three showed discrimination index (0.69). Numerical analysis of ARDRA patterns demonstrated sufficient phylogenetic information for characterizing bacterial diversity. Phylogenetic relationship obtained among isolates through ARDRA was compared with 16S rRNA gene sequence and ARDRA results showed sufficiently similar 16S rRNA gene sequence analysis, but not an overlapping. It has been observed that ARDRA technique facilitates the identification of bacteria in less than 36 h as compared to traditional 16S rRNA gene sequencing.
Keywords: Bacterial diversity; Phylogeny; Ribosomal DNA; Amplified rDNA restriction analysis

The membraneless bioelectrochemical reactor stimulates hydrogen fermentation by inhibiting methanogenic archaea by Kengo Sasaki; Masahiko Morita; Daisuke Sasaki; Naoya Ohmura; Yasuo Igarashi (7005-7013).
The membraneless bioelectrochemical reactor (Ml-BER) is useful for dark hydrogen fermentation. The effect of the electrochemical reaction on microorganisms in the Ml-BER was investigated using glucose as the substrate and compared with organisms in a membraneless non-bioelectrochemical reactor (Ml-NBER) and bioelectrochemical reactor (BER) with a proton exchange membrane. The potentials on the working electrode of the Ml-BER and BER with membrane were regulated to −0.9 V (versus Ag/AgCl) to avoid water electrolysis with a carbon electrode. The Ml-BER showed suppressed methane production (19.8 ± 9.1 mg-C·L−1·day−1) and increased hydrogen production (12.6 ± 3.1 mg-H·L−1·day−1) at pHout 6.2 ± 0.1, and the major intermediate was butyrate (24.9 ± 2.4 mM), suggesting efficient hydrogen fermentation. In contrast, the Ml-NBER showed high methane production (239.3 ± 17.9 mg-C·L−1·day−1) and low hydrogen production (0.2 ± 0.0 mg-H·L−1·day−1) at pHout 6.3 ± 0.1. In the cathodic chamber of the BER with membrane, methane production was high (276.3 ± 20.4 mg-C·L−1·day−1) (pHout, 7.2 ± 0.1). In the anodic chamber of the BER with membrane (anode-BER), gas production was low because of high lactate production (43.6 ± 1.7 mM) at pHout 5.0 ± 0.1. Methanogenic archaea were not detected in the Ml-BER and anode-BER. However, Methanosarcina sp. and Methanobacterium sp. were found in Ml-NBER. Prokaryotic copy numbers in the Ml-BER and Ml-NBER were similar, as were the bacterial community structures. Thus, the electrochemical reaction in the Ml-BER affected hydrogenotrophic and acetoclastic methanogens, but not the bacterial community.
Keywords: Hydrogen; Fermentation; Bioelectrochemical system; Microbial community; Methanogen

Anaerobic ammonium-oxidizing (anammox) bacteria, aerobic ammonia-oxidizing archaea (AOA), and ammonia-oxidizing bacteria (AOB) are three groups of ammonium/ammonia-oxidizing prokaryotes (AOPs) that are involved in the nitrogen cycle. This research compared the AOP communities in a constructed freshwater wetland with a natural coastal marine wetland in the subtropical Hong Kong. Both vegetated/rhizosphere and nonvegetated sediments were investigated to identify the effects of different macrophytes on the AOP communities. The polymerase chain reaction (PCR)-amplified gene fragments of 16S rRNA and archaeal and bacterial amoA (encoding the ammonia monooxygenase alpha subunit) were applied as molecular biomarkers to analyze the AOPs’ phylogeny and diversity. Quantitative PCR was used to determine the abundances of AOPs in the sediments. The results showed that the relatively more heterogeneous freshwater wetland contained a broader range of phylotypes, higher diversity, more complex community structures, and more unevenly distributed abundances of AOPs than the coastal wetland. The effects of vegetation on the community structures of AOPs were plant-specific. The exotic Typha angustifolia affected the community structures of all AOPs and enhanced their abundances in the rhizosphere region. Both Phragmites australis and Cyperus malaccensis showed some effects on the community structures of AOB, but minimal effects on those of anammox bacteria or AOA. Kandelia obovata had almost no detectable effect on all AOPs due to their smaller size. This study suggested that the freshwater and coastal marine wetlands may have different contributions to the inorganic N removal due to the variations in AOP communities and plant types.
Keywords: Anammox bacteria; Ammonia-oxidizing archaea; Ammonia-oxidizing bacteria; amoA ; Mangrove; Diversity; Wetland; Subtropical

GeoChip-based analysis of the functional gene diversity and metabolic potential of soil microbial communities of mangroves by Shijie Bai; Jiangwei Li; Zhili He; Joy D. Van Nostrand; Yun Tian; Guanghui Lin; Jizhong Zhou; Tianling Zheng (7035-7048).
Mangroves are unique and highly productive ecosystems and harbor very special microbial communities. Although the phylogenetic diversity of sediment microbial communities of mangrove habitats has been examined extensively, little is known regarding their functional gene diversity and metabolic potential. In this study, a high-throughput functional gene array (GeoChip 4.0) was used to analyze the functional diversity, composition, structure, and metabolic potential of microbial communities in mangrove habitats from mangrove national nature reserves in China. GeoChip data indicated that these microbial communities were functionally diverse as measured by the number of genes detected, unique genes, and various diversity indices. Almost all key functional gene categories targeted by GeoChip 4.0 were detected in the mangrove microbial communities, including carbon (C) fixation, C degradation, methane generation, nitrogen (N) fixation, nitrification, denitrification, ammonification, N reduction, sulfur (S) metabolism, metal resistance, antibiotic resistance, and organic contaminant degradation. Detrended correspondence analysis (DCA) of all detected genes showed that Spartina alterniflora (HH), an invasive species, did not harbor significantly different microbial communities from Aegiceras corniculatum (THY), a native species, but did differ from other species, Kenaelia candel (QQ), Aricennia marina (BGR), and mangrove-free mud flat (GT). Canonical correspondence analysis (CCA) results indicated the microbial community structure was largely shaped by surrounding environmental variables, such as total nitrogen (TN), total carbon (TC), pH, C/N ratio, and especially salinity. This study presents a comprehensive survey of functional gene diversity of soil microbial communities from different mangrove habitats/species and provides new insights into our understanding of the functional potential of microbial communities in mangrove ecosystems.
Keywords: Mangroves; GeoChip; Functional gene; Microbial communities; Invasive species; Native species

Nutrient resupplementation arrests bio-oil accumulation in Phaeodactylum tricornutum by J. Valenzuela; R. P. Carlson; R. Gerlach; K. Cooksey; B. M. Peyton; B. Bothner; M. W. Fields (7049-7059).
Phaeodactylum tricornutum is a marine diatom in the class Bacillariophyceae and is important ecologically and industrially with regards to ocean primary production and lipid accumulation for biofuel production, respectively. Triacylglyceride (TAG) accumulation has been reported in P. tricornutum under different nutrient stresses, and our results show that lipid accumulation can occur with nitrate or phosphate depletion. However, greater lipid accumulation was observed when both nutrients were depleted as observed using a Nile Red assay and fatty acid methyl ester (FAME) profiles. Nitrate depletion had a greater effect on lipid accumulation than phosphate depletion. Lipid accumulation in P. tricornutum was arrested upon resupplementation with the depleted nutrient. Cells depleted of nitrogen showed a distinct shift from a lipid accumulation mode to cellular growth post-resupplementation with nitrate, as observed through increased cell numbers and consumption of accumulated lipid. Phosphate depletion caused lipid accumulation that was arrested upon phosphate resupplementation. The cessation of lipid accumulation was followed by lipid consumption without an increase in cell numbers. Cells depleted in both nitrate and phosphate displayed cell growth upon the addition of both nitrate and phosphate and had the largest observed lipid consumption upon resupplementation. These results indicate that phosphate resupplementation can shut down lipid accumulation but does not cause cells to shift into cellular growth, unlike nitrate resupplementation. These data suggest that nutrient resupplementation will arrest lipid accumulation and that switching between cellular growth and lipid accumulation can be regulated upon the availability of nitrogen and phosphorus.
Keywords: Algae; Diatom; Lipid; Biofuel; Nitrate; Phosphate

Although successful production of fatty alcohols in metabolically engineered Escherichia coli with heterologous expression of fatty acyl-CoA reductase has been reported, low biosynthetic efficiency is still a hurdle to be overcome. In this study, we examined the characteristics of two fatty acyl-CoA reductases encoded by Maqu_2220 and Maqu_2507 genes from Marinobacter aquaeolei VT8 on fatty alcohol production in E. coli. Fatty alcohols with diversified carbon chain length were obtained by co-expressing Maqu_2220 with different carbon chain length-specific acyl-ACP thioesterases. Both fatty acyl-CoA reductases displayed broad substrate specificities for C12–C18 fatty acyl chains in vivo. The optimized mutant strain of E. coli carrying the modified tesA gene and fadD gene from E. coli and Maqu_2220 gene from Marinobacter aquaeolei VT8 produced fatty alcohols at a remarkable level of 1.725 g/L under the fermentation condition.
Keywords: Escherichia coli ; Fatty alcohol; Fatty acyl-CoA reductase; Marinobacter aquaeolei VT8