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Applied Microbiology and Biotechnology (v.94, #6)

Field applicability of Compound-Specific Isotope Analysis (CSIA) for characterization and quantification of in situ contaminant degradation in aquifers by M. Braeckevelt; A. Fischer; M. Kästner (pp. 1401-1421).

Microbial processes govern the fate of organic contaminants in aquifers to a major extent. Therefore, the evaluation of in situ biodegradation is essential for the implementation of Natural Attenuation (NA) concepts in groundwater management. Laboratory degradation experiments and biogeochemical approaches are often biased and provide only indirect evidence of in situ degradation potential. Compound-Specific Isotope Analysis (CSIA) is at present among the most promising tools for assessment of the in situ contaminant degradation within aquifers. One- and two-dimensional (2D) CSIA provides qualitative and quantitative information on in situ contaminant transformation; it is applicable for proving in situ degradation and characterizing degradation conditions and reaction mechanisms. However, field application of CSIA is challenging due to a number of influencing factors, namely those affecting the observed isotope fractionation during biodegradation (e.g., non-isotope-fractionating rate-limiting steps, limited bioavailability), potential isotope effects caused by processes other than biodegradation (e.g., sorption, volatilization, diffusion), as well as non-isotope-fractionating physical processes such as dispersion and dilution. This mini-review aims at guiding practical users towards the sound interpretation of CSIA field data for the characterization of in situ contaminant degradation. It focuses on the relevance of various constraints and influencing factors in CSIA field applications and provides advice on when and how to account for these constraints. We first evaluate factors that can influence isotope fractionation during biodegradation, as well as potential isotope-fractionating and non-isotope-fractionating physical processes governing observed isotope fractionation in the field. Finally, the potentials of the CSIA approach for site characterization and the proper ways to account for various constraints are illustrated by means of a comprehensive CSIA field study at the benzene, toluene, ethylbenzene, and xylene (BTEX)-contaminated site Zeitz.

Keywords: Groundwater; Biodegradation; Natural Attenuation; Isotope fractionation factor; Rayleigh equation; Aquifer

Microbial steroid transformations: current state and prospects by Marina V. Donova; Olga V. Egorova (pp. 1423-1447).

Studies of steroid modifications catalyzed by microbial whole cells represent a well-established research area in white biotechnology. Still, advances over the last decade in genetic and metabolic engineering, whole-cell biocatalysis in non-conventional media, and process monitoring raised research in this field to a new level. This review summarizes the data on microbial steroid conversion obtained since 2003. The key reactions of structural steroid functionalization by microorganisms are highlighted including sterol side-chain degradation, hydroxylation at various positions of the steroid core, and redox reactions. We also describe methods for enhancement of bioprocess productivity, selectivity of target reactions, and application of microbial transformations for production of valuable pharmaceutical ingredients and precursors. Challenges and prospects of whole-cell biocatalysis applications in steroid industry are discussed.

Keywords: Steroid; Microbial transformation; Bioconversion; Sterol; Side-chain degradation; Hydroxylation; Dehydrogenation; Sterol catabolism; Whole-cell biocatalysis

Development of Monascus fermentation technology for high hypolipidemic effect by Chun-Lin Lee; Tzu-Ming Pan (pp. 1449-1459).

Monascus species has been used as the traditional food fungus in Eastern Asia for several centuries. Monascus-fermented products are gradually developed as the popular functional food for the prevention of cardiovascular disease, but we know that culture condition affects the hypolipidemic effect of Monascus-fermented product. In the past, the cholesterol-lowering agent—monacolin K—is regarded as the most important hypolipidemic agent. Two natural yellow pigments—monascin and ankaflavin—are also proven as novel hypolipidemic agents in recent years. However, the hypolipidemic effect of Monascus-fermented product should contribute from monacolin K, monascin, ankaflavin, and other unknown functional ingredients. In addition to hypolipidemic effect, the safety concern of Monascus-fermented product is involved in the levels of mycotoxin—citrinin. The hypolipidemic effect and the production of these functional metabolites or mycotoxin are influenced by many factors such as the choice of culture substrates, carbon and nitrogen source, pH value, extra nutrients, and so on. Therefore, this review focused on the effect of various culture conditions and nutrients on the functional metabolites production, hypolipidemic effect as well as citrinin concentration, and further organized the fermentation technologies used by previous studies for the promotion of hypolipidemic effect and safety.

Keywords: Monascus; Monacolin K; Monascin; Ankaflavin; Citrinin; Hypolipidemic effect

Recent advances on applications and biotechnological production of d-psicose by Wanmeng Mu; Wenli Zhang; Yinghui Feng; Bo Jiang; Leon Zhou (pp. 1461-1467).

d-Psicose is a hexoketose monosaccharide sweetener, which is a C-3 epimer of d-fructose and is rarely found in nature. It has 70 % relative sweetness but 0.3 % energy of sucrose, and is suggested as an ideal sucrose substitute for food products. It shows important physiological functions, such as blood glucose suppressive effect, reactive oxygen species scavenging activity, and neuroprotective effect. It also improves the gelling behavior and produces good flavor during food process. This article presents a review of recent studies on the properties, physiological functions, and food application of d-psicose. In addition, the biochemical properties of d-tagatose 3-epimerase family enzymes and the d-psicose-producing enzyme are compared, and the biotechnological production of d-psicose from d-fructose is reviewed.

Keywords: d-Psicose; d-Tagatose 3-epimerase family enzyme; Property; Physiological function; Biotechnological production

Real-time monitoring and control of microbial bioprocesses with focus on the specific growth rate: current state and perspectives by Moira Monika Schuler; Ian William Marison (pp. 1469-1482).

Understanding the growth characteristics of microorganisms is an essential step in bioprocessing, not only because product formation may be growth-associated but also because they might influence cell physiology and thereby product quality. The specific growth rate, a key variable of many bioprocesses, cannot be measured directly and relies on the estimation through other measurable variables such as biomass, substrate, or product concentrations. Techniques for real-time estimation of the specific growth rate in microbial fed-batch cultures are discussed in the present paper. The advantages and limitations of different models and various monitoring techniques are discussed, highlighting the importance of the specific growth rate in the development of fast, reliable, and robust processes for the production of high-value products such as recombinant proteins.

Keywords: Growth model; Monod model; Recombinant protein production; Process analytical technology (PAT)

Current knowledge of the Escherichia coli phosphoenolpyruvate–carbohydrate phosphotransferase system: peculiarities of regulation and impact on growth and product formation by Adelfo Escalante; Ania Salinas Cervantes; Guillermo Gosset; Francisco Bolívar (pp. 1483-1494).

In Escherichia coli, the phosphoenolpyruvate–carbohydrate phosphotransferase system (PTS) is responsible for the transport and phosphorylation of sugars, such as glucose. PTS activity has a crucial role in the global signaling system that controls the preferential consumption of glucose over other carbon sources. When the cell is exposed to carbohydrate mixtures, the PTS prevents the expression of catabolic genes and activity of non-PTS sugars transport systems by carbon catabolite repression (CCR). This process defines some metabolic and physiological constraints that must be considered during the development of production strains. In this review, we summarize the importance of the PTS in controlling and influencing both PTS and non-PTS sugar transport processes as well as the mechanisms of transcriptional control involved in the expression of catabolic genes of non-PTS sugars in E. coli. We discuss three main approaches applied efficiently to avoid these constraints resulting in obtaining PTS⁻ glc⁺ mutants useful for production purposes: (1) adaptive selection in chemostat culture system of PTS⁻ mutants, resulting in the selection of strains that recovered the ability to grow in glucose, along with the simultaneous consumption of two carbon sources and reduced acetate production; (2) replacement in PTS⁻ strains of the native GalP promoter by strong promoters or the substitution of this permease by recombinant glucose transport system; and (3) enhancement of Crp (crp+) in mgsA, pgi, and ptsG mutants, resulting in derivative strains that abolished CCR, allowing the simultaneous consumption of mixtures of sugars with low acetate production.

Keywords: Escherichia coli ; PTS; Carbon catabolite repression; PTS⁻ mutants; Metabolite production

Patterns of carbohydrate and fatty acid changes under nitrogen starvation in the microalgae Haematococcus pluvialis and Nannochloropsis sp. by Lee Recht; Aliza Zarka; Sammy Boussiba (pp. 1495-1503).

The aim of this research was to study the impact of nitrogen starvation on the production of two major secondary metabolites, fatty acids and carbohydrates, in two microalgae: Nannochloropsis sp. and Haematococcus pluvialis. The major response to nitrogen starvation in both algae occurred within the first 2 days, accompanied by a sharp reduction in chlorophyll content. However, the pattern of the response differed between the two microalgae. In H. pluvialis, the first response to nitrogen starvation was intensive production of carbohydrates, accumulating to up to 63% of dry weight by day 1; on day 2, the total carbohydrate content decreased and was partially degraded, possibly to support fatty acid synthesis. Under these conditions, H. pluvialis accumulated up to 35% total fatty acids in the biomass. In Nannochloropsis sp., the immediate and major response, which was maintained throughout the entire period of exposure to stress, was production of fatty acids, accumulating up to 50% of dry weight, while carbohydrate content in the biomass remained stable at 18%. In addition, we tested the effect of the lipid-synthesis inhibitor sesamol, known to inhibit malic enzyme, on the balance between total fatty acid and carbohydrate contents in H. pluvialis and Nannochloropsis sp. In both cultures, sesamol inhibited fatty acid accumulation, but the carbohydrate content was reduced as well, albeit to a lesser extent. These findings demonstrate the complexity of the stress–response and the potential link between fatty acid and carbohydrate synthesis.

Keywords: Nitrogen starvation; Nannochloropsis sp.; Haematococcus pluvialis ; Fatty acid; Carbohydrate; Sesamol

In vitro and in vivo comparisons of the effects of the fruiting body and mycelium of Antrodia camphorata against amyloid β-protein-induced neurotoxicity and memory impairment by Li-Chun Wang; Shen-En Wang; Jyh-Jye Wang; Tsung-Yu Tsai; Chun-Hong Lin; Tzu-Ming Pan; Chun-Lin Lee (pp. 1505-1519).

Antrodia camphorata is a particular and precious medicinal mushroom, and its fruiting body was found to provide more efficient protection from oxidative stress and inflammation than its mycelium because of its higher content of triterpenoids, total phenols, and so on. In the previous in vitro studies, the mycelium of A. camphorata is proven to provide strong neuroprotection in neuron cells and suggested to have the potential of protection against neurotoxicity of amyloid β-protein (Aβ) known as the risk factor toward Alzheimer’s disease (AD) development. However, the in vivo study and the comparison study with the fruiting body have not yet been investigated. This study compared the effect of the fruiting body and mycelium of A. camphorata on alleviating the Aβ40-induced neurocytotoxicity in the in vitro Aβ-damaged neuron cell model (PC-12 cell treated with Aβ40) and memory impairment in the in vivo AD animal model induced with a continuous brain infusion of Aβ40. In the results of in vitro and in vivo studies, the fruiting body possessed stronger anti-oxidative and anti-inflammatory abilities for inhibiting neurocytotoxicity in Aβ40-treated PC-12 cells and Aβ40 accumulation in Aβ40-infused brain than mycelium. Moreover, hyperphosphorylated tau (p-tau) protein expression, known as an important AD risk factor, was suppressed by the treatment of fruiting body rather than that of mycelium in the in vitro and in vivo studies. These comparisons supported the reasons why the fruiting body resulted in a more significant improvement effect on working memory ability than mycelium in the AD rats.

Keywords: Alzheimer’s disease; Amyloid; Antrodia camphorata ; Fruiting body; Mycelium

Pathway redesign for deoxyviolacein biosynthesis in Citrobacter freundii and characterization of this pigment by Pei-xia Jiang; Hai-sheng Wang; Su Xiao; Ming-yue Fang; Rui-ping Zhang; Shu-ying He; Kai Lou; Xin-Hui Xing (pp. 1521-1532).

Violacein (Vio) is an important purple pigment with many potential bioactivities. Deoxyviolacein, a structural analog of Vio, is always synthesized in low concentrations with Vio in wild-type bacteria. Due to deoxyviolacein’s low production and difficulties in isolation and purification, little has been learned regarding its function and potential applications. This study was the first effort in developing a stable and efficient biosynthetic system for producing pure deoxyviolacein. A recombinant plasmid with vioabce genes was constructed by splicing using an overlapping extension-polymerase chain reaction, based on the Vio-synthesizing gene cluster of vioabcde, originating from Duganella sp. B2, and was introduced into Citrobacter freundii. With the viod gene disrupted in the Vio synthetic pathway, Vio production was completely abolished and the recombinant C. freundii synthesized only deoxyviolacein. Interestingly, vioe gene expression was strongly stimulated in the viod-deleted recombinant strain, indicating that viod disruptions could potentially induce polar effects upon the downstream vioe gene within this small operon. Deoxyviolacein production by this strain reached 1.9 g/L in shaker flasks. The product exhibited significant acid/alkali and UV resistance as well as significant inhibition of hepatocellular carcinoma cell proliferation at low concentrations of 0.1–1 μM. These physical characteristics and antitumor activities of deoxyviolacein contribute to illuminating its potential applications.

Keywords: Violacein; Deoxyviolacein; Biosynthesis; Heterologous expression; Pathway redesign

Impact of aeration and agitation on metabolic heat and protease secretion of Aspergillus tamarii in a real-time biological reaction calorimeter by Balaji Dhandapani; Surianarayanan Mahadevan; Sathish Sundar Dhilipkumar; Suseela Rajkumar; Asit Baran Mandal (pp. 1533-1542).

The effects of aeration and agitation on metabolic heat, alkaline protease production and morphology for Aspergillus tamarii MTCC5152 are reported in this manuscript. Measurement of metabolic heat has been attempted by the continuous and dynamic heat balance method in a biological real-time reaction calorimeter. At lower agitation intensities, growth-related processes were dominating. As a result the protease activity and the product heat yields were lower than those for 350 and 450 rpm. Although biomass growth was necessary to obtain maximum protease yield, agitation seemed to play a vital role in the protease production process. Energy dissipation per circulation function of the process is also deduced from power input. At optimal conditions, 350 rpm and 1 vvm, the gassed power required was 0.133 W. Pellet morphology and protease production were studied under different aeration and agitation intensities of A. tamarii. Pellet structure was considerably influenced by DO, a higher DO level resulted in denser pellets (1,018.4 kg/m³) leading to higher protease activity. Coupling of hydrodynamics and bio-reaction highlighted the complex relationship between energy dissipation, substrate uptake rate and fungal physiology. This study emphasised the potential of biocalorimetry as a reliable monitoring and robust control tool for aerobic fermentation of A. tamarii, using agricultural by-products.

Keywords: Morphology; EDCF; Metabolic heat; Alkaline protease

Cloning and expression in Pichia pastoris of an Irpex lacteus rhamnogalacturonan hydrolase tolerant to acetylated rhamnogalacturonan by J. Normand; E. Bonnin; P. Delavault (pp. 1543-1552).

In order to produce a recombinant rhamnogalacturonase from the basidiomycete Irpex lacteus using a molecular approach, PCR primers were designed based on a sequence alignment of four known ascomycete rhamnogalacturonases. Using 5′ and 3′ rapid amplification of cDNA ends (RACE) experiments, a 1,437-bp full-length cDNA containing an open reading frame of 1,329 bp was isolated. The corresponding putative protein sequence is of 443 amino acids and contains a secretion signal sequence of 22 amino acids. The theoretical mass of this protein is 44.6 kDa with a theoretical isoelectric point of 6.2. The amino acid sequence shared not only significant identities with ascomycete and basidiomycete putative rhamnogalacturonases but also complete similarity with peptides obtained from a recently purified rhamnogalacturonase from I. lacteus. The recombinant protein was successfully expressed in active form in Pichia pastoris. SDS-PAGE assay demonstrated that the recombinant enzyme was secreted in the culture medium and had a molar mass of 56 kDa. This recombinant rhamnogalacturonan hydrolase exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40°C and 50°C, which correspond to that of the native rhamnogalacturonase from I. lacteus. The study of its specificity through reaction products analysis showed that it was highly tolerant to the presence of acetyl groups on its substrate, even more than the native enzyme.

Keywords: Rhamnogalacturonase; Acetylated rhamnogalacturonans; Irpex lacteus ; Pichia pastoris

An isofenphos-methyl hydrolase (Imh) capable of hydrolyzing the P–O–Z moiety of organophosphorus pesticides containing an aryl or heterocyclic group by Rong Li; Yuan Liu; Jian Zhang; Kai Chen; Shunpeng Li; Jiandong Jiang (pp. 1553-1564).

Organophosphorus pesticide (OP) hydrolases play key roles in the degradation and decontamination of agricultural and household OPs and in the detoxification of chemical warfare agents. In this study, an isofenphos-methyl hydrolase gene (imh) was cloned from the isocarbophos-degrading strain of Arthrobacter sp. scl-2 using the polymerase chain reaction method. Isofenphos-methyl hydrolase (Imh) showed 98% sequence identity with the isofenphos hydrolase from Arthrobacter sp. strain B-5. Imh was highly expressed in Escherichia coli BL21 (DE3), and the His₆-tagged Imh was purified (1.7 mg/ml) with a specific activity of 14.35 U/mg for the substrate isofenphos-methyl. The molecular mass of the denatured Imh is about 44 kDa, and the isoelectric point (pI) value was estimated to be 3.4. The optimal pH and temperature for hydrolysis of isofenphos-methyl were pH 8.0 and 35 °C, respectively. The secondary structure of Imh shows that Imh is a metallo-dependent hydrolase, and it was found that Imh was completely inhibited by the metalloprotease inhibitor 1,10-phenanthroline (0.5 mM), and the catalytic activity was restored by the subsequent addition of Zn²⁺. Interestingly, Imh had a relatively broader substrate specificity and was capable of hydrolyzing 12 of the tested oxon and thion OPs with the P–O–Z moiety instead of the P–S(C)–Z moiety. Furthermore, it was found that the existence of an aryl or heterocyclic group in the leaving group (Z) is also important in determining the substrate specificity. Among all the substrates hydrolyzed by Imh, it was assumed that Imh preferred P–O–Z substrates still with a phosphamide bond (P–N), such as isofenphos-methyl, isofenphos, isocarbophos, and butamifos. The newly characterized Imh has a great potential for use in the decontamination and detoxification of agricultural and household OPs and is a good candidate for the study of the catalytic mechanism and substrate specificity of OP hydrolases.

Keywords: Organophosphate detoxification; Organophosphorus pesticide hydrolase; Isofenphos-methyl hydrolase (imh); Substrate specificity

Expression and characterization of human proinsulin fused to thioredoxin in Escherichia coli by Aldana Trabucchi; Luciano Lucas Guerra; Natalia Inés Faccinetti; Ruben Francisco Iacono; Edgardo Poskus; Silvina Noemí Valdez (pp. 1565-1576).

Native proinsulin (PI) belongs to the class of the difficult-to-express proteins in Escherichia coli. Problems mainly arise due to its high proteolytic decay and troubles to reproduce the native disulphide pattern. In the present study, human PI was produced in E. coli as a fusion thioredoxin protein (Trx-PI). Such chimeric protein was obtained from the intracellular soluble fraction, and it was purified in one step by affinity chromatography on immobilized phenylarsine oxide. Trx-PI was also recovered from inclusion bodies and purified by anion exchange chromatography. The product identity and integrity were verified by mass analysis (22,173.5 Da) and mapping with Staphylococcus aureus V8 protease. Native PI folding was evaluated by biochemical and also by immunochemical analysis using specific sera from PI antibody-positive diabetic patients that recognise conformational discontinue epitopes. Dose–response curves showed identity between standard PI and Trx-PI. Moreover, surface plasmon resonance technique verified the correct conformation of the recombinant protein. The biochemical and immunochemical assays demonstrated the integrity of the chimera and the epitopes involved in the interaction with antibodies. In conclusion, it was possible to obtain with high-yield purified human PI as a fusion protein in E. coli and useful for analytical purposes.

Keywords: Thioredoxin proinsulin; Protein expression; Immunoreactivity; Diabetes mellitus; Autoantibodies

Preferential and rapid degradation of raw rice starch by an α-amylase of glycoside hydrolase subfamily GH13_37 by Yin Lei; Hui Peng; Ying Wang; Yuantao Liu; Fang Han; Yazhong Xiao; Yi Gao (pp. 1577-1584).

The α-amylase (AmyP) from a marine metagenomic library belongs to the recently classified glycoside hydrolase subfamily GH13_37. The degradation abilities of AmyP on a broad range of raw starch granules were examined at 40 °C and pH 7.5. It was found that AmyP is a raw starch-degrading enzyme, exhibiting a unique and remarkable ability to preferentially and very rapidly digest raw rice starch. The specific activity of raw rice starch was reached 118.5 ± 0.6 U mg⁻¹, which was much higher than that of other raw starches. The final hydrolysis degrees were obtained in 4 h for 1 % raw rice starch and 1 h for 8 % concentration, indicating a very rapid speed of hydrolysis. The presence of a starch residue resistant was the main limiting factor for complete hydrolysis, although end product inhibition also existed, especially at high starch concentrations. AmyP randomly attacks unique or susceptible sites on raw rice starch granules, and releases glucose, maltose, and maltotriose as end products. This is the first biochemical characterization of the raw starch-degrading ability of an α-amylase of family GH13_37. The specific ability towards raw rice starch has never been described before, and this makes AmyP a promising candidate for use as a novel enzyme in rice starch processing.

Keywords: α-Amylase; Raw starch digestion; Rice starch; Inhibition

Improvements in ethanol production from xylose by mating recombinant xylose-fermenting Saccharomyces cerevisiae strains by Hiroko Kato; Hiroaki Suyama; Ryosuke Yamada; Tomohisa Hasunuma; Akihiko Kondo (pp. 1585-1592).

To improve the ability of recombinant Saccharomyces cerevisiae strains to utilize the hemicellulose components of lignocellulosic feedstocks, the efficiency of xylose conversion to ethanol needs to be increased. In the present study, xylose-fermenting, haploid, yeast cells of the opposite mating type were hybridized to produce a diploid strain harboring two sets of xylose-assimilating genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase. The hybrid strain MN8140XX showed a 1.3- and 1.9-fold improvement in ethanol production compared to its parent strains MT8-1X405 and NBRC1440X, respectively. The rate of xylose consumption and ethanol production was also improved by the hybridization. This study revealed that the resulting improvements in fermentation ability arose due to chromosome doubling as well as the increase in the copy number of xylose assimilation genes. Moreover, compared to the parent strain, the MN8140XX strain exhibited higher ethanol production under elevated temperatures (38 °C) and acidic conditions (pH 3.8). Thus, the simple hybridization technique facilitated an increase in the xylose fermentation activity.

Keywords: Xylose; Ethanol production; Saccharomyces cerevisiae ; Diploid; Mating

Transcription analysis of hyaluronan biosynthesis genes in Streptococcus zooepidemicus and metabolically engineered Lactococcus lactis by Shashi Bala Prasad; K. B. Ramachandran; Guhan Jayaraman (pp. 1593-1607).

The has operon genes in the hyaluronan (HA) producer, Streptococcus zooepidemicus, encode for some of the critical enzymes in the HA biosynthetic pathway. Heterologous expression of different combinations of multiple has genes has resulted in increasing HA production to varying degrees in different recombinant strains. In this work, a recombinant Lactococcus lactis strain (SJR6) was constructed, with insertion of three has operon genes (hasABD) from S. zooepidemicus. The SJR6 strain was found to be a better HA producer than two previously constructed recombinant L. lactis strains (SJR2 and SJR3), containing hasAB and hasABC genes, respectively, but exhibited lower HA production than the native HA producer S. zooepidemicus. To understand the differences in HA yield between the various strains, transcriptions of the HA biosynthesis genes (has genes and their homologues) were compared at different phases of exponential growth of the L. lactis and S. zooepidemicus cultures. The mRNA levels of all the heterologous has genes were expectedly far higher than their corresponding homologues in the L. lactis strains. The relative mRNA level of the hasB-homologue, viz. ugd (encoding UDP-glucose dehydrogenase), was found to be much lower than that of other homologues, corroborating earlier reports which indicate tight transcriptional regulation of the ugd gene in L. lactis. Interestingly, all the has gene homologues were found to be up-regulated in all the recombinant L. lactis strains, when compared with the corresponding genes in the untransformed strain, L. lactis NZ9000. A transcription analysis of S. zooepidemicus cultures revealed that the has operon was down-regulated in the mid-exponential growth phase in comparison to the early- and late-exponential growth phases. The transcription analyses in this study have provided insights for the design of recombinant strains with higher HA productivity.

Keywords: Hyaluronan; Metabolic engineering; Lactococcus lactis ; Streptococcus zooepidemicus ; Transcription analysis; Real-time PCR

Characterization of lytic Pseudomonas aeruginosa bacteriophages via biological properties and genomic sequences by Natia Karumidze; Julie. A. Thomas; Nino Kvatadze; Marina Goderdzishvili; Kevin W. Hakala; Susan T. Weintraub; Zemphira Alavidze; Stephen C. Hardies (pp. 1609-1617).

Pseudomonas aeruginosa is an important cause of infections, especially in patients with immunodeficiency or diabetes. Antibiotics are effective in preventing morbidity and mortality from Pseudomonas infection, but because of spreading multidrug-resistant bacterial strains, bacteriophages are being explored as an alternative therapy. Two newly purified broad host range Pseudomonas phages, named vB_Pae-Kakheti25 and vB_Pae-TbilisiM32, were characterized as candidates for use in phage therapy. Morphology, host range, growth properties, thermal stability, serology, genomic sequence, and virion composition are reported. When phages are used as bactericides, they are used in mixtures to overcome the development of resistance in the targeted bacterial population. These two phages are representative of diverse siphoviral and podoviral phage families, respectively, and hence have unrelated mechanisms of infection and no cross-antigenicity. Composing bactericidal phage mixtures with members of different phage families may decrease the incidence of developing resistance through a common mechanism.

Keywords: Pseudomonas aeruginosa ; Bacteriophage; Phage therapy; Host range; Phage resistance

The two-step biotransformation of monosodium glutamate to GABA by Lactobacillus brevis growing and resting cells by Ying Zhang; Lei Song; Qiang Gao; Shao Mei Yu; Lei Li; Nian Fa Gao (pp. 1619-1627).

Gama-aminobutyric acid (GABA) is a natural functional amino acid. In the current study, Lactobacillus brevis TCCC13007, a high GABA-producing strain, was isolated from naturally pickled Chinese vegetables. A two-step cellular bioconversion process was established using L. brevis TCCC13007 for the production of GABA. First, L. brevis cells were grown anaerobically in 7% monosodium glutamate (MSG)-containing medium at an initial pH of 6.0 and a controlled pH of 4.6 for 16 to 66 h; approximately 38 g L⁻¹ of GABA was obtained after 66 h of fermentation at a conversion rate of 98.6%. In the second stage of the process, about 7.6 g L⁻¹ of GABA was produced three more times at a conversion rate of 92.2% using the same batch of resting cells in the substrate-containing buffer under optimized conditions. Thus, the total GABA yield reached 61 g L⁻¹. A model system for the biotransformation of MSG to GABA was established using L. brevis TCCC13007 resting cells. The reaction rates were found to follow the classic Michaelis–Menten equation at low substrate concentrations (<80 mM). Kinetic analysis of the biotransformation revealed that L. brevis TCCC13007 resting cells produced GABA similar to that produced by purified glutamate decarboxylase from L. brevis.

Keywords: Gama-aminobutyric acid (GABA); Lactobacillus brevis ; Glutamate; Biotransformation; Kinetic model

Comparison and characterization of α-amylase inducers in Aspergillus nidulans based on nuclear localization of AmyR by Yuriko Murakoshi; Tomohiro Makita; Masashi Kato; Tetsuo Kobayashi (pp. 1629-1635).

AmyR, a fungal transcriptional activator responsible for induction of amylolytic genes in Aspergillus nidulans, localizes to the nucleus in response to the physiological inducer isomaltose. Maltose, kojibiose, and d-glucose were also found to trigger the nuclear localization of GFP-AmyR. Isomaltose- and kojibiose-triggered nuclear localization was not inhibited by the glucosidase inhibitor, castanospermine, while maltose-triggered localization was inhibited. Thus, maltose itself does not appear to be an direct inducer, but its degraded or transglycosylated product does. Non-metabolizable d-glucose analogues were also able to trigger the nuclear localization, implying that these sugars, except maltose, directly function as the inducers of AmyR nuclear entry. The inducing activity of d-glucose was 4 orders-of-magnitude weaker compared with isomaltose. Although d-glucose has the ability to induce α-amylase production, this activity would generally be masked by CreA-dependent carbon catabolite repression. Significant induction of α-amylase by d-glucose was observed in creA-defective A. nidulans.

Keywords: Aspergillus nidulans ; Amylase; Inducer; AmyR; Nuclear localization

Fast and accurate preparation fatty acid methyl esters by microwave-assisted derivatization in the yeast Saccharomyces cerevisiae by Sakda Khoomrung; Pramote Chumnanpuen; Suwanee Jansa-ard; Intawat Nookaew; Jens Nielsen (pp. 1637-1646).

We present a fast and accurate method for preparation of fatty acid methyl esters (FAMEs) using microwave-assisted derivatization of fatty acids present in yeast samples. The esterification of free/bound fatty acids to FAMEs was completed within 5 min, which is 24 times faster than with conventional heating methods. The developed method was validated in two ways: (1) through comparison with a conventional method (hot plate) and (2) through validation with the standard reference material (SRM) 3275-2 omega-3 and omega-6 fatty acids in fish oil (from the Nation Institute of Standards and Technology, USA). There were no significant differences (P > 0.05) in yields of FAMEs with both validations. By performing a simple modification of closed-vessel microwave heating, it was possible to carry out the esterification in Pyrex glass tubes kept inside the closed vessel. Hereby, we are able to increase the number of sample preparations to several hundred samples per day as the time for preparation of reused vessels was eliminated. Pretreated cell disruption steps are not required, since the direct FAME preparation provides equally quantitative results. The new microwave-assisted derivatization method facilitates the preparation of FAMEs directly from yeast cells, but the method is likely to also be applicable for other biological samples.

Keywords: Fatty acid methyl esters; Fatty acid analysis; Microwave-assisted derivatization; Saccharomyces cerevisiae

Biodegradation of melamine and its hydroxy derivatives by a bacterial consortium containing a novel Nocardioides species by Kazuhiro Takagi; Kunihiko Fujii; Ken-ichi Yamazaki; Naoki Harada; Akio Iwasaki (pp. 1647-1656).

Melamine has recently been recognized as a food contaminant with adverse human health effects. Melamine contamination in some crops arises from soil and water pollution from various causes. To remove melamine from the polluted environment, a novel bacterium, Nocardioides sp. strain ATD6, capable of degrading melamine was enriched and isolated from a paddy soil sample. The enrichment culture was performed by the soil–charcoal perfusion method in the presence of triazine-degrading bacteria previously obtained. Strain ATD6 degraded melamine and accumulated cyanuric acid and ammonium, via the intermediates ammeline and ammelide. No gene known to encode for triazine-degrading enzymes was detected in strain ATD6. A mixed culture of strain ATD6 and a simazine-degrading Methyloversatilis sp. strain CDB21 completely degraded melamine, but the degradation rate of cyanuric acid was slow. The degradation of melamine and its catabolites by the mixed culture was greatly enhanced by including Bradyrhizobium japonicum strain CSB1 in the inoculum and adding ethanol to the culture medium. The melamine-degrading consortium consisting of strains ATD6, CDB21, and CSB1 appears to be potentially safer than other known melamine-degrading bacteria for the bioremediation of farmland and other contaminated sites, as no known pathogens were included in the consortium.

Keywords: Melamine; Cyanuric acid; Biodegradation; Nocardioides sp. ; Bacterial consortium

Unravelling the reasons for disproportion in the ratio of AOB and NOB in aerobic granular sludge by Mari K. H. Winkler; João P. Bassin; Robbert Kleerebezem; Dimitry Y. Sorokin; Mark C. M. van Loosdrecht (pp. 1657-1666).

In this study, we analysed the nitrifying microbial community (ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) within three different aerobic granular sludge treatment systems as well as within one flocculent sludge system. Granular samples were taken from one pilot plant run on municipal wastewater as well as from two lab-scale reactors. Fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) showed that Nitrobacter was the dominant NOB in acetate-fed aerobic granules. In the conventional system, both Nitrospira and Nitrobacter were present in similar amounts. Remarkably, the NOB/AOB ratio in aerobic granular sludge was elevated but not in the conventional treatment plant suggesting that the growth of Nitrobacter within aerobic granular sludge, in particular, was partly uncoupled from the lithotrophic nitrite supply from AOB. This was supported by activity measurements which showed an approximately threefold higher nitrite oxidizing capacity than ammonium oxidizing capacity. Based on these findings, two hypotheses were considered: either Nitrobacter grew mixotrophically by acetate-dependent dissimilatory nitrate reduction (ping-pong effect) or a nitrite oxidation/nitrate reduction loop (nitrite loop) occurred in which denitrifiers reduced nitrate to nitrite supplying additional nitrite for the NOB apart from the AOB.

Keywords: Aerobic granular sludge; Mixotrophic; Nitrite loop; AOB; NOB; PHA

Ethanol production from wood hydrolysate using genetically engineered Zymomonas mobilis by Hideshi Yanase; Hitoshi Miyawaki; Mitsugu Sakurai; Akinori Kawakami; Mari Matsumoto; Kenji Haga; Motoki Kojima; Kenji Okamoto (pp. 1667-1678).

An ethanologenic microorganism capable of fermenting all of the sugars released from lignocellulosic biomass through a saccharification process is essential for secondary bioethanol production. We therefore genetically engineered the ethanologenic bacterium Zymomonas mobilis such that it efficiently produced bioethanol from the hydrolysate of wood biomass containing glucose, mannose, and xylose as major sugar components. This was accomplished by introducing genes encoding mannose and xylose catabolic enzymes from Escherichia coli. Integration of E. coli manA into Z. mobilis chromosomal DNA conferred the ability to co-ferment mannose and glucose, producing 91 % of the theoretical yield of ethanol within 36 h. Then, by introducing a recombinant plasmid harboring the genes encoding E. coli xylA, xylB, tal, and tktA, we broadened the range of fermentable sugar substrates for Z. mobilis to include mannose and xylose as well as glucose. The resultant strain was able to ferment a mixture of 20 g/l glucose, 20 g/l mannose, and 20 g/l xylose as major sugar components of wood hydrolysate within 72 h, producing 89.8 % of the theoretical yield. The recombinant Z. mobilis also efficiently fermented actual acid hydrolysate prepared from cellulosic feedstock containing glucose, mannose, and xylose. Moreover, a reactor packed with the strain continuously produced ethanol from acid hydrolysate of wood biomass from coniferous trees for 10 days without accumulation of residual sugars. Ethanol productivity was at 10.27 g/l h at a dilution rate of 0.25 h⁻¹.

Keywords: Bioethanol; Lignocellulose; Hydrolysates; Mannose; Xylose; Ethanol; Metabolic engineering; Zymomonas mobilis

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Applied Microbiology and Biotechnology (v.94, #6)

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Applied Microbiology and Biotechnology (v.94, #6)