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Applied Microbiology and Biotechnology (v.69, #3)
Thiosugars: new perspectives regarding availability and potential biochemical and medicinal applications by Zbigniew J. Witczak; James M. Culhane (pp. 237-244).
Thiosugars, containing a sulfur atom as heteroatom or a disaccharide linked via a sulfur bridge, possess unique physicochemical properties such as water solubility, which differs from conventional functionalized monosaccharides. The differences in biological activities between thiosugars and their oxygen analogs depend on geometric, conformational, and flexibility differences. They depend also on their electronic differences, the sulfide function being less electronegative and more polarizable than the ethereal moiety. Many functionalized thiosugars occur naturally and are potential targets for the development of carbohydrate-based therapeutics. Among the few new examples of the potential new targets are salacinol and kotalanol, tagetitoxin, thiolactomycin and analogues, mycothiol and analogues, and S-nitrosothiols. These new developments and representative examples of functionalized thiosugar prototypes as potential new targets are presented in this mini review.
Recombinant microbial systems for the production of human collagen and gelatin by Julio Báez; David Olsen; James W. Polarek (pp. 245-252).
The use of genetically engineered microorganisms is a cost-effective, scalable technology for the production of recombinant human collagen (rhC) and recombinant gelatin (rG). This review will discuss the use of yeast (Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha) and of bacteria (Escherichia coli, Bacillus brevis) genetically engineered for the production of rhC and rG. P. pastoris is the preferred production system for rhC and rG. Recombinant strains of P. pastoris accumulate properly hydroxylated triple helical rhC intracellularly at levels up to 1.5 g/l. Coexpression of recombinant collagen with recombinant prolyl hydroxylase results in the synthesis of hydroxylated collagen with thermal stability similar to native collagens. The purified hydroxylated rhC forms fibrils that are structurally similar to fibrils assembled from native collagen. These qualities make rhC attractive for use in many medical applications. P. pastoris can also be engineered to secrete high levels (3 to 14 g/l ) of collagen fragments with defined length, composition, and physiochemical properties that serve as substitutes for animal-derived gelatins. The replacement of animal-derived collagen and gelatin with rhC and rG will result in products with improved safety, traceability, reproducibility, and quality. In addition, the rhC and rG can be engineered to improve the performance of products containing these biomaterials.
High yield of long-chain polyunsaturated fatty acids by labyrinthulids on soybean lecithin-dispersed agar medium by Yasuyuki Kumon; Toshihiro Yokochi; Toro Nakahara (pp. 253-258).
The aim of the present research was to provide an effective long-chain polyunsaturated fatty acid (LCPUFA) production by labyrinthulids using soybean lecithin (SBL). Use of SBL-dispersed agar medium resulted in higher LCPUFA production than soybean oil. Among the components of SBL, phosphatidylinositol (PI) and triacylglycerol (TG) were revealed to be essential factors for high growth of labyrinthulids. PI was effective for surface growth, and TG was effective for three-dimensional growth. The presence of some elements like carotenoids in SBL and the smaller droplet size of dispersed SBL were also attributed to be factors for the higher LCPUFA productivity on SBL medium. LCPUFA productivity and the volume of the oval form of the cells increased with increasing SBL concentration up to 40 g/l. Under optimum conditions, LCPUFA production of the L25 strain, isolated from Ogasawara Island in Japan, reached 2.91 g/l after 21 days.
Polyglutamic acid (PGA) production by Bacillus sp. SAB-26: application of Plackett–Burman experimental design to evaluate culture requirements by Nadia A. Soliman; Mahmoud M. Berekaa; Yasser R. Abdel-Fattah (pp. 259-267).
A locally isolated thermostable Bacillus strain producing polyglutamic acid (PGA) was characterized and identified based on 16S rRNA sequencing. Phylogenetic analysis revealed its closeness to Bacillus licheniformis. To evaluate the effect of different culture conditions on the production of PGA, Plackett–Burman factorial design was carried out. Fifteen variables were examined for their significance on PGA production. Among those variables, K2HPO4, KH2PO4, (NH4)2SO4 and casein hydrolysate were found to be the most significant variables that encourage PGA production. A correlation between cellular growth, PGA and the produced traces of polysaccharides was illustrated. An inverse relationship practice between cell dry weights and the produced PGA was demonstrated. On the other hand, a direct proportional relation was shown between polysaccharides on one side and cell dry weight and produced PGA on the other. The preoptimized medium, based on statistical analysis, showed a production of 33.5 g/l PGA, which is more than three times the basal medium.
Cell adaptation to solvent, substrate and product: a successful strategy to overcome product inhibition in a bioconversion system by Carla C. C. R. de Carvalho; Alessandro Poretti; M. Manuela R. da Fonseca (pp. 268-275).
Carvone has previously been found to highly inhibit its own production at concentrations above 50 mM during conversion of a diastereomeric mixture of (−)-carveol by whole cells of Rhodococcus erythropolis. Adaptation of the cells to the presence of increasing concentrations of carveol and carvone in n-dodecane prior to biotransformation proved successful in overcoming carvone inhibition. By adapting R. erythropolis cells for 197 h, an 8.3-fold increase in carvone production rate compared to non-adapted cells was achieved in an air-driven column reactor. After an incubation period of 268 h, a final carvone concentration of 1.03 M could be attained, together with high productivity [0.19 mg carvone h−1 (ml organic phase)−1] and high yield (0.96 g carvone g carveol−1).
Contribution of manganese peroxidase and laccase to dye decoloration by Trametes versicolor by Paul-Philippe Champagne; Juliana Akit Ramsay (pp. 276-285).
During dye decoloration by Trametes versicolor ATCC 20869 in modified Kirk’s medium, manganese peroxidase (MnP) and laccase were produced, but not lignin peroxidase, cellobiose dehydrogenase or manganese-independent peroxidase. Purified MnP decolorized azo dyes [amaranth, reactive black 5 (RB5) and Cibacron brilliant yellow] in Mn2+-dependent reactions but did not decolorize an anthraquinone dye [Remazol brilliant blue R (RBBR)]. However, the purified laccase decolorized RBBR five to ten times faster than the azo dyes and the addition of a redox mediator, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), did not alter decoloration rates. Amaranth and RB5 were decolorized the most rapidly by MnP since they have a hydroxyl group in an ortho position and a sulfonate group in the meta position relative to the azo bond. During a typical batch decoloration with the fungal culture, the ratio of laccase:MnP was 10:1 to 20:1 (based on enzyme activity) and increased to greater than 30:1 after decoloration was complete. Since MnP decolorized amaranth about 30 times more rapidly than laccase per unit of enzyme activity, MnP should have contributed more to decoloration than laccase in batch cultures.
Poly(3-hydroxyalkanoate) polymerase synthesis and in vitro activity in recombinant Escherichia coli and Pseudomonas putida by Qun Ren; Guy de Roo; Jan B. van Beilen; Manfred Zinn; Birgit Kessler; Bernard Witholt (pp. 286-292).
We tested the synthesis and in vitro activity of the poly(3-hydroxyalkanoate) (PHA) polymerase 1 from Pseudomonas putida GPo1 in both P. putida GPp104 and Escherichia coli JMU193. The polymerase encoding gene phaC1 was expressed using the inducible PalkB promoter. It was found that the production of polymerase could be modulated over a wide range of protein levels by varying inducer concentrations. The optimal inducer dicyclopropylketone concentrations for PHA production were at 0.03% (v/v) for P. putida and 0.005% (v/v) for E. coli. Under these concentrations the maximal polymerase level synthesized in the E. coli host (6% of total protein) was about three- to fourfold less than that in P. putida (20%), whereas the maximal level of PHA synthesized in the E. coli host (8% of total cell dry weight) was about fourfold less than that in P. putida (30%). In P. putida, the highest specific activity of polymerase was found in the mid-exponential growth phase with a maximum of 40 U/g polymerase, whereas in E. coli, the maximal specific polymerase activity was found in the early stationary growth phase (2 U/g polymerase). Our results suggest that optimal functioning of the PHA polymerase requires factors or a molecular environment that is available in P. putida but not in E. coli.
Identification and characterization of two polyhydroxyalkanoate biosynthesis loci in Pseudomonas sp. strain 3Y2 by Soazig C. Delamarre; Hyun-Joo Chang; Carl A. Batt (pp. 293-303).
A Pseudomonas strain, 3Y2, that produced polyhydroxyalkanoate (PHA) polymers consisting of 3-hydroxybutyric acid (3HB) and medium-chain-length 3-hydroxyalkanoate (mcl-HA) units, with up to 30% 3HB, was isolated. Two PHA biosynthesis loci (pha Ps-1 and pha Ps-2) from 3Y2 were cloned by polymerase chain reaction amplification techniques. The pha Ps-2 locus was similar to the PHA biosynthesis loci of other PHA-producing Pseudomonas strains, with five tandem open reading frames (ORFs) located in the order ORF1 Ps-2-phaC1 Ps-2-phaZ Ps-2-phaC2 Ps-2-phaD Ps-2. The pha Ps-1 locus that contains phaC1 Ps-1-phaZ Ps-1 appears to have arisen by a duplication event that placed it downstream of a gene (ORF1 Ps-1), encoding a putative glucose-methanol-choline flavoprotein oxidoreductase. The PHA synthases 1 encoded by phaC1 Ps-1 and phaC1 Ps-2 were investigated by heterologous expression in Wautersia eutropha PHB−4. Both synthases displayed similar substrate specificities for incorporating 3HB and mcl-HA units into PHA. The ability of PhaC1 Ps-1 to confer PHA synthesis, however, appeared reduced compared to that of PhaC1 Ps-2, since cells harboring PhaC1 Ps-1 accumulated 2.5 to 4.6 times less PHA than cells expressing PhaC1 Ps-2. Primary sequence analysis revealed that PhaC1 Ps-1 had markedly diverged from the other PHA synthases with a relatively high substitution rate (14.9 vs 2% within PhaC1 Ps-2). The mutations affected a highly conserved C-terminal region and the surroundings of the essential active site cysteine (Cys296) with a loss of hydrophobicity. This led us to predict that if phaC1 Ps-1 produces a protein product in the native strain, it is likely that PhaC1 Ps-1 may be destined for elimination by the accumulation of inactivating mutations, although its specialization to accommodate different substrates cannot be eliminated.
The potential of Pseudozyma yeastlike epiphytes for the production of heterologous recombinant proteins by T. J. Avis; Y. L. Cheng; Y. Y. Zhao; S. Bolduc; B. Neveu; R. Anguenot; C. Labbé; F. Belzile; R. R. Bélanger (pp. 304-311).
Although Basidiomycetes represent the most evolved class of fungi, they have been neglected with regard to recombinant gene expression. In this work, basidiomycetous yeasts belonging to Pseudozyma spp. were studied with respect to their amenability to heterologous protein production. Single plasmid or cotransformation experiments routinely afforded 100 to 200 independent transformants for the two tested species of Pseudozyma. Green fluorescent protein (GFP) was expressed in the correctly folded conformation, as demonstrated by fluorescence microscopy, and hen egg white lysozyme (HEWL) was expressed in its active form, as revealed by its lytic activity on Micrococcus lysodeikticus cells. Protease analysis established that Pseudozyma spp. contained equivalent or less extracellular protease activity than yeasts and far less protease activity than ascomycetous filamentous fungi in similar culture conditions. This proteolytic activity was inhibited by over 97% with a combination of PMSF and Pepstatin A. N-glycosylation patterns of native Pseudozyma flocculosa secreted proteins were comprised of one or a few short glycan chains that possess a classic eukaryotic structure typical of higher fungi and animal cells. This is the first report of a Basidiomycete that possesses multiple intrinsic characteristics necessary for use as a heterologous gene expression system.
The sequence upstream of the −10 consensus sequence modulates the strength and induction time of stationary-phase promoters in Escherichia coli by Gerhard Miksch; Frank Bettenworth; Karl Friehs; Erwin Flaschel (pp. 312-320).
We constructed a library of synthetic stationary-phase promoters for Escherichia coli. For designing the promoters, the known −10 consensus sequence, as well as the extended −10 region, and an A/T-rich region downstream of the −10 region were kept constant, whereas sequences from −37 to −14 were partially or completely randomised. For detection and selection of stationary-phase promoters, green fluorescent protein (GFP) with enhanced fluorescence was used. To establish the library, 33 promoters were selected, which differ in strength from 670 to more than 13,000 specific fluorescence units, indicating that the strength of promoters can be modulated by the sequence upstream of the −10 region. DNA sequencing revealed a preferential insertion of nucleotides depending on the position. By expressing the promoters in an rpoS-deficient strain, a special group of stationary-phase promoters was identified, which were expressed exclusively or preferentially by RNA polymerase holoenzyme Eσs. The DNA sequence of these promoters differed significantly in the region from −25 to −16. Furthermore, it was shown that the DNA curvature of the promoter region had no effect on promoter strength. The broad range of promoter activities make these promoters very suitable for fine-tuning of gene expression and for cost-effective large-scale applications in industrial bioprocesses.
TNT and nitroaromatic compounds are chemoattractants for Burkholderia cepacia R34 and Burkholderia sp. strain DNT by Thammajun Leungsakul; Brendan G. Keenan; Barth F. Smets; Thomas K. Wood (pp. 321-325).
Nitroaromatic compounds are toxic and potential carcinogens. In this study, a drop assay was used to detect chemotaxis toward nitroaromatic compounds for wild-type Burkholderia cepacia R34, wild-type Burkholderia sp. strain DNT, and a 2,4-dinitrotoluene (2,4-DNT) dioxygenase mutant strain (S5). The three strains are chemotactic toward 2,4,6-trinitrotoluene (TNT), 2,3-DNT, 2,4-DNT, 2,5-DNT, 2-nitrotoluene (NT), 4NT, and 4-methyl-5-nitrocatechol (4M5NC), but not toward 2,6-DNT. Of these, only 2,4-DNT is a carbon and energy source for B. cepacia R34 and Burkholderia sp. strain DNT, and 4M5NC is an intermediate in the 2,4-DNT degradation pathway. It was determined that the 2,4-DNT dioxygenase genes are not required for the chemotaxis for these nitroaromatic compounds because the DNT DDO mutant S5 has a chemotactic response toward 2,4-DNT although 2,4-DNT is not metabolized by S5; hence, 2,4-DNT itself is the chemoattractant. This is the first report of chemotaxis toward TNT, 2,3-DNT, 2,4-DNT, 2,5-DNT, 2NT, 4NT, and 4M5NC.
Reductive transformation of TNT by Escherichia coli resting cells: kinetic analysis by Hong Yin; Thomas K. Wood; Barth F. Smets (pp. 326-334).
Microbial 2,4,6-trinitrotoluene (TNT) biotransformation via sequential nitro-reduction appears a ubiquitous process, but the kinetics of these transformations have been poorly understood or described. TNT transformation by Escherichia coli was monitored and a kinetic model for reductive TNT depletion was developed and experimentally calibrated in this report. Using resting cells of aerobically pregrown E. coli, TNT was quickly reduced to hydroxylaminodinitrotoluenes. The standard Michaelis–Menten model was modified to include three additional parameters: product toxicity (T c), substrate inhibition (K i), and intracellular reducing power (RH) limitation. Experimentally measured product toxicity (5.2 μmol TNT/mg cellular protein) closely matched the best-fit model value (2.84 μmol TNT/mg cellular protein). Parameter identifiability and reliability (k m, K s, T c, and K i) was evaluated and confirmed through sensitivity analyses and via Monte Carlo simulations. The resulting kinetic model adequately described TNT reduction kinetics by E. coli resting cells in the absence or presence of reducing power limitation.
Transient accumulation of γ-butyrolactone during degradation of bis(4-chloro-n-butyl) ether by diethylether-grown Rhodococcus sp. strain DTB by M. Moreno-Horn; L.-A. Garbe; R. Tressl; H. Görisch (pp. 335-340).
Rhodococcus sp. strain DTB (DSM 44534) grows aerobically on diethylether as sole source of carbon and energy. Dense cell suspension experiments showed that the induced ether-cleaving enzyme system attacks a broad range of ethers like tetrahydrofuran, phenetole and chlorinated alkylethers including Cα-substituted alkylethers. Identification of metabolites revealed that degradation of the ethers started by an initial attack of the ether bond. Diethylether-grown cells degraded bis(4-chloro-n-butyl) ether via an initial ether scission followed by the transient accumulation of γ-butyrolactone as intermediate at nearly stoichiometric concentrations.
Effect of medium osmolarity on the bioproduction of glycerol and ethanol by Hansenula anomala growing on glucose and ammonium by H. Djelal; A. Amrane; F. Lahrer; G. Martin (pp. 341-349).
The osmotolerant yeast Hansenula anomala survives in media at low water activity resulting from increasing NaCl concentrations in the culture medium by producing compatible solutes. High salinity resulted in the use of a large part of the assimilated carbon substrate (glucose) for cell maintenance (28%), required for intracellular synthesis compounds and for osmotic cell regulation. The maintenance coefficient for non-growth-associated glucose consumption was found to be 0.38 mmol glucose g biomass−1 h−1. For decreasing water activity, there is a competition between the pathways leading to glycerol and ethanol production, until an experimental ethanol/total glycerol ratio reached a value 3.4 for 2 mol l−1 NaCl (close to the theoretical value of 4)—illustrating the osmodependent channelling of carbon towards polyols production. This competition leads to a cessation of ethanol production during the stationary state before that of glycerol. Since osmotic adjustment occurred mainly during growth, glycerol production during stationary state can be clearly related to another mechanism other than osmotic: it was excreted by a fermentative mechanism to ensure energy for cell maintenance.
Investigation on the mechanism of H2S removal by biological activated carbon in a horizontal biotrickling filter by Huiqi Duan; Rong Yan; Lawrence Choon Chiaw Koe (pp. 350-357).
The use of supporting media for the immobilization of microorganisms is widely known to provide a surface for microbial growth and a shelter that protects the microorganisms from inhibitory compounds. In our previous studies, activated carbon (AC) alone used as a support medium for H2S biological removal was proved prompt and efficient in a bench-scale biofilter and biotrickling filter. In this study, the mechanisms of H2S elimination using microbial immobilized activated carbon, i.e., biological activated carbon (BAC), are investigated. A series of BAC as supporting medium were taken from the inlet to outlet of a bench-scale horizontal biotrickling filter to examine the different effects of physical/chemical adsorption and microbial degradation on the overall removal of H2S. The surface properties of BAC together with virgin and exhausted carbon (after H2S breakthrough test, non-microbial immobilization) were characterized using the sorption of nitrogen (Braunner–Emmett–Teller test), scanning electron microscopy (SEM), surface pH, thermal, carbon–hydrogen–nitrogen–sulfur (CHNS) elemental and Fourier transform infrared (FTIR) analyses. Tests of porosity and surface area provide detailed information about the pore structure of BAC along the bed facilitating the understanding of potential pore blockages due to biofilm coating. A correlation between the available surface area and pore volume with the extent of microbial immobilization and H2S uptake is evidenced. SEM photographs show the direct carbon structure and biofilm coated on carbon surface. FTIR spectra, differential thermogravimetric curves and CHNS results indicate less diversity of H2S oxidation products on BAC than those previously observed on exhausted carbon from H2S adsorption only. The predominant oxidation product on BAC is sulfuric acid, and biofilm is believed to enhance the oxidation of H2S on carbon surface. The combination of biodegradation and physical adsorption of using BAC in removal of H2S could lead to a long-term (i.e., years) good performance of biotrickling filters and biofilters based on BAC compared to carbon adsorption only.
Phylogenetical approach to isolation of white-rot fungi capable of degrading polychlorinated dibenzo-p-dioxin by Ichiro Kamei; Hiroto Suhara; Ryuichiro Kondo (pp. 358-366).
A degradation experiment on PCDDs and phylogenetical analyses were carried out on newly isolated 2,7-dichlorodibenzo-p-dioxin (2,7-diCDD)-degrading white-rot fungi, strains BMC3014, BMC9152, and BMC9160. When these fungi were incubated with tri- or tetraCDDs, the substrates were degraded efficiently, and hydroxylated metabolites were detected. On the other hand, 1,3,6,8-tetrachlorodibenzo-p-dioxin was not decreased, and no metabolites were detected. Phylogenetic analysis of internal transcribed spacers (ITSs) containing rRNA gene sequence (ITS-rDNA) clarified that these strains belonged to the genus Phlebia and were closely related to the fungi Phlebia lindtneri, strains MZ-227 and MG-60, which had both been isolated as 2,7-diCDD-degrading fungi in our previous study. Based on this phylogenetical relationship, other Phlebia genera species were used for a degradation experiment on 2,7-diCDD and 1,3,6,8-tetraCDD. Phlebia acerina and Phlebia brevispora degraded 2,7-diCDD about 40 and 80%, respectively, over 14 days of incubation. It became clear that P. brevispora can degrade 1,3,6,8-tetraCDD and transform it to monohydroxy-tetraCDD, monomethoxy-tetraCDD, dimethoxy-tetraCDD, dimethoxy-triCDD, and 3,5-dichlorocatechol in the treatment cultures. In this paper, we could clearly prove for the first time by identifying the metabolites that white-rot fungus P. brevispora could degrade the recalcitrant dioxin, 1,3,6,8-tetraCDD.