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Applied Microbiology and Biotechnology (v.63, #6)
Biotechnology and molecular biology of the α-glucosidase inhibitor acarbose by U. F. Wehmeier; W. Piepersberg (pp. 613-625).
The α-glucosidase inhibitor acarbose, O-{4,6-dideoxy-4[1s-(1,4,6/5)-4,5,6-trihydroxy-3-hydroxymethyl-2-cyclohexen-1-yl]-amino-α-d-glucopyranosyl}-(1→4)-O-α-d-glucopyranosyl-(1→4)-d-glucopyranose, is produced in large-scale fermentation by the use of strains derived from Actinoplanes sp. SE50. It has been used since 1990 in many countries in the therapy of diabetes type II, in order to enable patients to better control blood sugar contents while living with starch-containing diets. Thus, it is one of the latest successful products of bacterial secondary metabolism to be introduced into the pharmaceutical world market. Cultures of Actinoplanes sp. also produce various other acarbose-like components, of which component C is hard to separate during downstream processing, which is one of the most modern work-up processes developed to date. The physiology, genetics and enzymology of acarbose biosynthesis and metabolism in the producer have been studied to some extent, leading to the proposal of a new pathway and metabolic cycle, the “carbophore”. These data could give clues for further biotechnological developments, such as the suppression of side-products, enzymological or biocombinatorial production of new metabolites and the engineering of production rates via genetic regulation in future.
Avermectin: biochemical and molecular basis of its biosynthesis and regulation by Y. J. Yoon; E.-S. Kim; Y.-S. Hwang; C.-Y. Choi (pp. 626-634).
Avermectin and its analogues, produced by Streptomyces avermitilis, are major commercial antiparasitic agents in the field of animal health, agriculture, and human infections. They are 16-membered pentacyclic lactone compounds derived from polyketide and linked to a disaccharide of the methylated deoxysugar l-oleandrose. Labeling studies, analyses of the biosynthetically blocked mutants, and the identification of the avermectin gene cluster allows characterization of most of the biosynthetic pathway. Recent completion of S. avermitilis genome sequencing is also expected to help in revealing the precise biosynthetic sequence and the complicated regulatory mechanism for avermectin biosynthesis, which has been long-awaited to be elucidated. The well characterized avermectin biosynthetic pathway and availability of S. avermitilis genome information in combination with the recent development of combinatorial biosynthesis should allow us to redesign more potent avermectin analogues and to engineer S. avermitilis as a more efficient host for the production of important commercial analogues.
Production of lipid compounds in the yeast Saccharomyces cerevisiae by M. Veen; C. Lang (pp. 635-646).
This review describes progress using the yeast Saccharomyces cerevisiae as a model organism for the fast and efficient analysis of genes and enzyme activities involved in the lipid biosynthetic pathways of several donor organisms. Furthermore, we assess the impact of baker′s yeast on the production of novel, high-value lipid compounds. Yeast can be genetically modified to produce selected substances in relatively high amounts. A major advantage in choosing yeast as an object for metabolic engineering is the fact that the lipid pathways in this organism have been described in detail and are well characterized. We focus on the de novo production of three major families of lipid products. These are: (1) sterols, providing some previously known and some novel applications as examples of the lipid pathway enhancement that occurs naturally in yeast, (2) the reconstitution of the biosynthetic pathway of steroid hormones and (3) the biosynthesis of polyunsaturated fatty acids, leading to the biosynthesis of different omega-3 and omega-6 fatty acids which do not occur naturally in yeast. We utilize the current knowledge and point out perspectives and problems for future biotechnological applications in the field of lipid compounds.
Functional classification of the microbial feruloyl esterases by V. F. Crepin; C. B. Faulds; I. F. Connerton (pp. 647-652).
Feruloyl esterases have potential uses over a broad range of applications in the agri-food industries. In recent years, the number of microbial feruloyl esterase activities reported has increased and, in parallel, even more related protein sequences may be discerned in the growing genome databases. Based on substrate utilisation data and supported by primary sequence identity, four sub-classes have been characterised and termed type-A, B, C and D. The proposed sub-classification scheme is discussed in terms of the evolutionary relationships existing between carbohydrate esterases.
Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping by T. C. Ezeji; N. Qureshi; H. P. Blaschek (pp. 653-658).
Acetone butanol ethanol (ABE) was produced in an integrated fed-batch fermentation-gas stripping product-recovery system using Clostridium beijerinckii BA101, with H2 and CO2 as the carrier gases. This technique was applied in order to eliminate the substrate and product inhibition that normally restricts ABE production and sugar utilization to less than 20 g l−1 and 60 g l−1, respectively. In the integrated fed-batch fermentation and product recovery system, solvent productivities were improved to 400% of the control batch fermentation productivities. In a control batch reactor, the culture used 45.4 g glucose l−1 and produced 17.6 g total solvents l−1 (yield 0.39 g g−1, productivity 0.29 g l−1 h−1). Using the integrated fermentation-gas stripping product-recovery system with CO2 and H2 as carrier gases, we carried out fed-batch fermentation experiments and measured various characteristics of the fermentation, including ABE production, selectivity, yield and productivity. The fed-batch reactor was operated for 201 h. At the end of the fermentation, an unusually high concentration of total acids (8.5 g l−1) was observed. A total of 500 g glucose was used to produce 232.8 g solvents (77.7 g acetone, 151.7 g butanol, 3.4 g ethanol) in 1 l culture broth. The average solvent yield and productivity were 0.47 g g−1 and 1.16 g l−1 h−1, respectively.
Production of nisin with continuous adsorption to Amberlite XAD-4 resin using Lactococcus lactis N8 and L. lactis LAC48 by M. Tolonen; P. E. J. Saris; M. Siika-aho (pp. 659-665).
The production of nisin, biomass and lactic acid in pH-controlled and uncontrolled batch fermentation and batch fermentation (pH 5.5) with continuous removal of nisin was examined in the parent strain Lactococcus lactis N8 and LAC48. Strain LAC48 in batch fermentor (pH not controlled) gave a maximum nisin concentration of 2.5×106 IU g dcw−1. The nisin concentration remained high (2.0×106 IU g dcw−1) after the logarithmic growth phase (10–22 h), whereas nisin production of strain N8 decreased after the logarithmic growth phase. The maximum nisin production of strain LAC48 was not directly related to the biomass formation and not associated with growth. In order to study end product inhibition in nisin production, a system was built for adsorption of nisin during fermentation. The adsorbent Amberlite XAD-4 was found to have an effective binding capacity for nisin. Cells of LAC48 and N8 compensated for the removal of nisin, indicating that nisin production also occurs in the stationary phase.
Synthesis of FimH receptor-active manno-oligosaccharides by reverse hydrolysis using α-mannosidases from Penicillium citrinum, Aspergillus phoenicis and almond by V. Maitin; V. Athanasopoulos; R. A. Rastall (pp. 666-671).
Recombinant Penicillium citrinum α-1,2-mannosidase, expressed in Aspergillus oryzae, was employed to carry out regioselective synthesis of α-d-mannopyranosyl-(1→2)-d-mannose. Yields (w/w) of 16.68% disaccharide, 3.07% trisaccharide and 0.48% tetrasaccharide were obtained, with α1→2 linkages present at 98.5% of the total linkages formed. Non-specific α-mannosidase from almond was highly efficient in reverse hydrolysis and oligosaccharide yields of 45–50% were achieved. The products of the almond mannosidase were a mixture of disaccharides (30.75%, w/w), trisaccharides (12.26%, w/w) and tetrasaccharides (1.89%, w/w) with 1→2, 1→3 and 1→6 isomers. α-1,2-linkage specific mannosidase from P. citrinum and α-1,6-linkage-specific mannosidase from Aspergillus phoenicis were used in combination to hydrolyse the respective linkages from the mixture of isomers, resulting in α-d-mannopyranosyl-(1→3)-d-mannose in 86.4% purity. The synthesised oligosaccharides can potentially inhibit the adhesion of pathogens by acting as "decoys" of receptors of type-1 fimbriae carried by enterobacteria.
Enrichment of microbial community generating electricity using a fuel-cell-type electrochemical cell by B. H. Kim; H. S. Park; H. J. Kim; G. T. Kim; I. S. Chang; J. Lee; N. T. Phung (pp. 672-681).
A fuel cell was used to enrich a microbial consortium generating electricity, using organic wastewater as the fuel. Within 30 days of enrichment the maximum current of 0.2 mA was generated with a resistance of 1 kΩ. Current generation was coupled to a fall in chemical oxygen demand from over 1,700 mg l−1 down to 50 mg l−1. Denaturing gradient gel electrophoresis showed a different microbial population in the enriched electrode from that in the sludge used as the inoculum. Electron microscopic observation showed a biofilm on the electrode surface and microbial clumps. Nanobacteria-like particles were present on the biofilm surface. Metabolic inhibitors and electron acceptors inhibited the current generation. 16S ribosomal RNA gene analysis showed a diverse bacterial population in the enrichment culture. These findings demonstrate that an electricity-generating microbial consortium can be enriched using a fuel cell and that the electrochemical activity is a form of anaerobic electron transfer.
Purification and partial characterization of the Pyrococcus horikoshii methylmalonyl-CoA epimerase by T. A. Bobik; M. E. Rasche (pp. 682-685).
Methylmalonyl-CoA epimerase (MCE) from the hyperthermophilic archaeon, Pyrococcus horikoshii, was expressed at high levels in Escherichia coli, purified, and partially characterized. The P. horikoshii MCE enzyme was a homodimer with an apparent molecular mass of 31,700 Da. The K m of the enzyme for methylmalonyl-CoA was 79 μM and the k cat was 240 s−1. The P. horikoshii enzyme was extremely heat-stable and withstood boiling for 60 min without detectable loss in activity.
Purification and characterization of a type B feruloyl esterase (StFAE-A) from the thermophilic fungus Sporotrichum thermophile by E. Topakas; H. Stamatis; P. Biely; P. Christakopoulos (pp. 686-690).
A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity. The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0±1.5 kDa, with a mass of 33±1 kDa on SDS-PAGE. The pI of the enzyme was estimated by cation-exchange chromatofocusing to be at pH 3.1. The enzyme activity was optimal at pH 6.0 and 55–60 °C. The purified esterase was stable at the pH range 5.0–7.0. The enzyme retained 70% of activity after 7 h at 50 °C and lost 50% of its activity after 45 min at 55 °C and after 12 min at 60 °C. Determination of k cat/K m revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively. No activity on methyl sinapinate was detected. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and it hydrolyzed 4-nitrophenyl 5-O-trans-feruloyl-α-l-arabinofuranoside (NPh-5-Fe-Araf) 2-fold more efficiently than NPh-2-Fe-Araf. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from S. thermophile (a maximum of 34% total ferulic acid released after 1 h incubation). StFAE-A by itself could release FA, but at a level almost 47-fold lower than that obtained in the presence of xylanase. The potential of StFAE-A for the synthesis of various phenolic acid esters was tested using a ternary water-organic mixture consisting of n-hexane, 1-butanol and water as a reaction system.
Production of l-asparaginase in Enterobacter aerogenes expressing Vitreoscilla hemoglobin for efficient oxygen uptake by H. Geckil; S. Gencer (pp. 691-697).
This study is the first utilizing Vitreoscilla hemoglobin in a heterologous bacterium, Enterobacter aerogenes, to determine the effect of such a highly efficient oxygen-uptake system on the production of l-asparaginase, an enzyme that has attracted considerable attention due to its anti-tumor activity. Here, we show that the Vitreoscilla hemoglobin expressing strain has from 10-fold to more than two orders of magnitude lower l-asparaginase activity than the wild type or the control without the Vitreoscilla hemoglobin gene under different aeration conditions. Aeration and agitation were also determining factors for enzyme production. The enzyme activity was reduced considerably under both full aerobic and anaerobic conditions, while the highest enzyme activity was determined in cultures under low aeration and low agitation. Also, the effect of different concentrations of glucose on enzyme production showed catabolic repression. Glucose at 1% caused almost total inhibition of enzyme activity, while at 0.1% it showed a slightly stimulatory effect on enzyme production, compared with glucose-free medium.
Production of isoamyl acetate in ackA-pta and/or ldh mutants of Escherichia coli with overexpression of yeast ATF2 by R. V. Vadali; C. E. Horton; F. B. Rudolph; G. N. Bennett; K.-Y. San (pp. 698-704).
The gene coding for alcohol acetyltransferase (ATF2), which catalyzes the esterification of isoamyl alcohol and acetyl coenzyme A (acetyl-CoA), was cloned from Saccharomyces cerevisiae and expressed in Escherichia coli. This genetically engineered strain of E. coli produced the ester isoamyl acetate when isoamyl alcohol was added externally to the cell culture medium. Various competing pathways at the acetyl-CoA node were inactivated to increase the intracellular acetyl-CoA pool and divert more carbon flux to the ester synthesis pathway. Several strains with deletions in the ackA-pta and/or ldh pathways and bearing the ATF2 on a high-copy-number plasmid were constructed and studied. Compared to the wild-type, ackA-pta and nuo mutants produced higher amounts of ester and an ackA-pta-ldh-nuo mutant lower amounts. Isoamyl acetate production correlated well with intracellular coenzyme A (CoA) and acetyl-CoA levels. The ackA-pta-nuo mutant had the highest intracellular CoA/acetyl-CoA level and hence produced the highest amount of ester (1.75 mM) during the growth phase under oxic conditions and during the production phase under anoxic conditions.
DNA analysis of the genes encoding acidocin LF221 A and acidocin LF221 B, two bacteriocins produced by Lactobacillus gasseri LF221 by A. Č. Majhenič; K. Venema; G. E. Allison; B. B. Matijašić; I. Rogelj; T. R. Klaenhammer (pp. 705-714).
Lactobacillus gasseri LF221, an isolate from the feces of a child, produces two bacteriocins. Standard procedures for molecular techniques were used to locate, clone and sequence the fragments of LF221 chromosomal DNA carrying the acidocin LF221 A and B structural genes, respectively. Sequencing analysis revealed the gene of acidocin LF221 A to be an open reading frame encoding a protein composed of 69 amino acids, including a 16-amino-acid N-terminal extension. The acidocin LF221 B gene was found to encode a 65-amino-acid bacteriocin precursor with a 17-amino-acid N-terminal leader peptide. DNA homology searches showed similarities of acidocin LF221 A to brochocin B, lactococcin N and thermophilin B, whereas acidocin LF221 B exhibited some homology to lactacin F and was virtually identical to gassericin X. The peptides encoded by orfA1 and orfB3 showed characteristics of class II bacteriocins and are suspected to be the complementary peptides of acidocin A and B, respectively. orfA3 and orfB5 are proposed to encode putative immunity proteins for the acidocins. Acidocin LF221 A and acidocin LF221 B are predicted to be members of the two-component class II bacteriocins, where acidocin LF221 A appears to be a novel bacteriocin. L. gasseri LF221 is being developed as a potential probiotic strain and a food/feed preservative. Detailed characterization of its acidocins is an important piece of background information useful in applying the strain into human or animal consumption. The genetic information on both acidocins also enables tracking of the LF221 strain in mixed populations and complex environments.
Use of the ammonia-oxidizing bacterial-specific phylogenetic probe Nso1225 as a primer for fingerprint analysis of ammonia-oxidizer communities by L. Calvó; X. Vila; C. A. Abella; L. Jesús Garcia-Gil (pp. 715-721).
Autotrophic ammonia-oxidizing bacteria (AOB) are an essential component of nitrifying wastewater treatment systems. The molecular tools used in group-specific studies are mostly based on the sequence of the 16S rRNA gene, but they have not proved to be fully specific. In this study, the sequence of the FISH probe Nso1225R was used as a reverse primer in order to analyze the AOB composition of several environmental samples by denaturing gradient gel electrophoresis (DGGE). For this purpose, samples from several environments, including aerated reactors, water treatment wetlands, and pilot plants, both aerobic and anaerobic, were analyzed. PCR fragments displayed a DGGE pattern consisting of bands melting between 30 and 40% denaturant, and a series of unresolved bands above 45%, mostly corresponding to AOB and β-non-AOB, respectively. This second set of bands corresponded to environments subjected to severe oxygen restrictions. AOB sequences showed similarity percentages higher than 92% with those of known β-AOB. Nso1225R, therefore, proved to be a good molecular phylogenetic marker for AOB samples from well-aerated systems, showing a higher specificity than the group-specific primers used previously.
Molecular identification of pathogenic and nonpathogenic strains of Vibrio harveyi using PCR and RAPD by G. Hernández; J. Olmos (pp. 722-727).
Fifteen environmental samples of Vibrio spp. isolated from healthy and diseased shrimps were tested for pathogenicity to juvenile shrimps. Two isolates, strains Z2 and Z3, were observed to be pathogenic, causing 100% mortality of the target host compared to the control strain Vibrio harveyi ATCC 14126. Environmental and type strains were subjected to molecular characterization by restriction fragment length polymorphism (RFLP) and PCR using primers targeted to different virulence, transcriptional regulator, or quorum sensing genes from V. harveyi. Primers designed for luxN were specific and identified all the environmental strains as V. harveyi. The random amplified polymorphic DNA (RAPD) method was used to differentiate between pathogenic and nonpathogenic strains of V. harveyi. These methodologies allowed us to detect and distinguish strains virulent and avirulent to juvenile shrimp.
Secretory production of Aspergillus oryzae xylanase XynF1, xynF1 cDNA product, in the basidiomycete Coprinus cinereus by M. Kikuchi; N. Kitamoto; K. Shishido (pp. 728-733).
The signal peptide of Aspergillus oryzae endo-(1,4)-β-xylanase XynF1 contains a C-terminal serine-arginine that directs efficient secretion of the enzyme into the culture medium. In the basidiomycete Coprinus cinereus, however, there is little secretion of XynF1 into the culture medium. Modification of the C-terminal sequence of the signal peptide to lysine-arginine resulted in efficient secretion of C. cinereus XynF1, suggesting the presence of a KEX2-like protease in this fungus.
Response to different environmental stress conditions of industrial and laboratory Saccharomyces cerevisiae strains by A. Garay-Arroyo; A. A. Covarrubias; I. Clark; I. Niño; G. Gosset; A. Martinez (pp. 734-741).
Two sets of Saccharomyces cerevisiae strains were compared for their physiological responses to different stress conditions. One group is composed of three strains adapted to controlled laboratory conditions (CEN.PK, LR88 and RS58), whereas the other consisted of five industrial strains (IND1101, SuperStart, LO24, LO41 and Azteca). Most industrial strains showed higher tolerance to heat shock and to an oxidative environment than laboratory strains. Excluding CEN.PK, a similar behavior was observed regarding ethanol production in high sugar concentrations (180 g/l glucose). Addition of acetate (10 g/l) or furfural (2 g/l), in concentrations similar to those found in sugar cane bagasse hydrolysates, decreased cell mass formation and growth rate in almost all strains. CEN.PK and SuperStart showed the highest sensitivity when grown in furfural-containing medium. Acetic acid treatment severely affected cell mass formation and reduced growth rate in all strains; CEN.PK and LO24 were the most resistant. The specific ethanol production rate was not affected by furfural addition. However, specific ethanol production rates decreased in response to acetic acid in four industrial strains, and increased in all laboratory strains and in LO24. No significant correlation was found between the stress tolerance of the strains tested and the transcript accumulation of genes selected by their involvement in the response to each of the stressful environments applied.
The roles of oxygen and alginate-lyase in determining the molecular weight of alginate produced by Azotobacter vinelandii by M. A. Trujillo-Roldán; S. Moreno; G. Espín; E. Galindo (pp. 742-747).
An Azotobacter vinelandii mutant lacking alginate-lyase (SML2) and the wild type (ATCC 9046) were used to discriminate between the roles of the polymerase complex and alginate-lyase in the synthesis of alginate in cultures conducted under controlled dissolved oxygen tension (DOT). To avoid the presence of pre-synthesized alginates, all cultures were inoculated with washed cells. For cultures carried out at 3% DOT using the mutant, a well defined family of alginates of high mean molecular weight (MMW) were obtained (985 kDa). Under 1% and 5% DOT, the mutant produced unique families of alginates with lower MMW (150 and 388 kDa). A similar behavior was observed using the wild type: a production of well defined families of alginates of high MMW at 3% DOT (1,250 kDa) and lower MMW at 1% and 5% DOT (370 and 350 kDa). At the end of the ATCC 9046 fermentations, alginate was depolymerized by the action of lyases. Overall, the evidence indicated that polymerization of alginate is carried out by producing families of polysaccharide in a narrow MMW range, and that it is highly dependent on DOT. The role of alginate-lyase (present in the wild type) is restricted to a post-polymerization step.
The effect of CreA in glucose and xylose catabolism in Aspergillus nidulans by W. Prathumpai; M. McIntyre; J. Nielsen (pp. 748-753).
The catabolism of glucose and xylose was studied in a wild type and creA deleted (carbon catabolite de-repressed) strain of Aspergillus nidulans. Both strains were cultivated in bioreactors with either glucose or xylose as the sole carbon source, or in the presence of both sugars. In the cultivations on single carbon sources, it was demonstrated that xylose acted as a carbon catabolite repressor (xylose cultivations), while the enzymes in the xylose utilisation pathway were also subject to repression in the presence of glucose (glucose cultivations). In the wild type strain growing on the sugar mixture, glucose repression of xylose utilisation was observed; with xylose utilisation occurring only after glucose was depleted. This phenomenon was not seen in the creA deleted strain, where glucose and xylose were catabolised simultaneously. Measurement of key metabolites and the activities of key enzymes in the xylose utilisation pathway revealed that xylose metabolism was occurring in the creA deleted strain, even at high glucose concentrations. Conversely, in the wild type strain, activities of the key enzymes for xylose metabolism increased only when the effects of glucose repression had been relieved. Xylose was both a repressor and an inducer of xylanases at the same time. The creA mutation seemed to have pleiotropic effects on carbohydratases and carbon catabolism.
Sensitivity of chronically HIV-1 infected HeLa cells to electrical stimulation by E. Kumagai; M. Tominaga; S. Harada (pp. 754-758).
Use of combination anti-retroviral drug regimens including protease inhibitors dramatically decreased morbidity and mortality rates in HIV-1 infected individuals. However, such combination therapies appear to have many side effects, in addition to the emergence of resistant HIV-1 strains. Therefore, in this study we sought to elucidate novel therapeutic principles against HIV-1 infection. We examined the effects of electrical stimulation on both chronically HIV-1LAI infected HeLa cells (P6 HeLa/HIV-1LAI) and uninfected cells (P6 HeLa). Cells were cultured on an optically transparent electrode and application of potential at 1.0 V vs Ag/AgCl was performed over time periods ranging from 10 min to 60 min. Both P6 HeLa/HIV-1LAI and P6 HeLa cells were progressively damaged as the duration of electrical stimulation increased. However, P6 HeLa/HIV-1LAl cells were much more influenced by electrical stimulation than P6 HeLa cells. The difference in damage rate was most obvious at 30 min of electrical stimulation, with damaged cells accounting for about 87% and 4% of P6 HeLa/HIV-1LAI and P6 HeLa cells, respectively. After the application of potential for 20 min, the proliferation of P6 HeLa/HIV-1LAI cells was markedly inhibited, while the P6 HeLa cells proliferated to an extent similar to that of uninfected cells without application of potential. These results indicate that sensitivity to electrical stimulation is much higher in chronically HIV-1 infected cells than in uninfected cells. This could be considered as a useful new approach against HIV-1 infection.