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Applied Microbiology and Biotechnology (v.65, #4)
Recombinant expression systems in the pharmaceutical industry by F. R. Schmidt (pp. 363-372).
In terms of downstream processing efficiency, secretory expression systems offer potential advantages for the production of recombinant proteins, compared with inclusion body forming cytosolic systems. However, for high-volume therapeutics like insulin, the product yields of the majority of the potentially available secretory systems is not yet fully competitive. Current strategies to improve productivity and secretion efficiency comprise: (1) enhancement of gene expression rates, (2) optimization of secretion signal sequences, (3) coexpression of chaperones and foldases, (4) creation of protease deficient mutants to avoid premature product degradation and (5) subsequent breeding and mutagenesis. For the production of non-glycosylated proteins and proteins, which are natively glycosylated but are also pharmacologically active without glycosylation, prokaryotes, which usually lack metabolic pathways for glycosylation, are theoretically the most suitable organisms and offer two alternatives: either Escherichia coli strains are conditioned to be efficient secreters or efficient native secreters like Bacillus species are accordingly developed. To fully exploit the secretory capacity of fungal species, a deeper understanding of their posttranslational modification physiology will be necessary to steer the degree and pattern of glycosylation, which influences both folding and secretion efficiency. Insect and mammalian cells display posttranslational modification patterns very similar or identical to humans, but in view of the entailed expenditures, their employment can only be justified if their modification machinery is required to ensure a desired pharmacological activity.
Synthesis of flavor and fragrance esters using Candida antarctica lipase by Araceli Larios; Hugo S. García; Rosa María Oliart; Gerardo Valerio-Alfaro (pp. 373-376).
Candida antarctica lipase fraction B (CAL-B) showed substrate specificity in the synthesis of esters in hexane involving reactions of short-chain acids having linear (acetic and butyric acids) and branched chain (isovaleric acid) structures, an unsaturated (tiglic acid) fatty acid, and phenylacetic acid with n-butanol and geraniol. The variation in the conversion to the esters was ca. 10%. Similar results were observed in a study of the alcohol specificity of the enzyme for esterification of acetic and butyric acids with four alcohols: n-butyl, isopentyl, 2-phenylethyl, and geraniol. Enantioselectivity of CAL-B in hexane with a range of chiral α-substituted or β-substituted carboxylic acids and n-butyl alcohol was analyzed. The results show that CAL-B can be employed as a robust biocatalyst in esterification reactions due to the high conversions obtained in the synthesis of short-chain flavor esters in an organic solvent, although this enzyme exhibited modest enantioselectivity with chiral short-chain carboxylic acids.
The generation of high biomass from chlororespiring bacteria using a continuous fed-batch bioreactor by Qiang He; Robert A. Sanford (pp. 377-382).
A continuous fed-batch reactor system was developed to rapidly obtain dense chlororespiring cultures of Anaeromyxobacter dehalogenans strain 2CP-C. A syringe pump continuously delivered concentrated 2,6-dichlorophenol (50–150 mM) to an anaerobic reactor vessel at a rate that sustained linear growth but prevented the substrate toxicity of chlorophenol. Dechlorination was not significantly inhibited by end product phenol up to 8 mM. A cell density of 76.8 mg protein l−1 was obtained in 24 h. Specific growth rates averaged 0.033 h−1at 50% substrate limitation, which was in agreement with the maximum specific growth rate of 0.068 h−1. This reactor system provides an efficient, cost-effective, and convenient method to rapidly obtain dense dechlorinating biomass and is promising to accelerate investigations of enzymes involved in chlororespiration.
An optimization study of solid-state fermentation: xanthophylls extraction from marigold flowers by Navarrete-Bolaños José Luis; Jiménez-Islas Hugo; Botello-Alvarez Enrique; Rico-Martínez Ramiro; Paredes-López Octavio (pp. 383-390).
Marigold flowers are the main natural source of xanthophylls, and marigold saponified extract is used as an additive in several food and pharmaceutical industries. In this work, the use of a solid-state fermentation (ensilage) process for increasing the yield of xanthophylls extracted from fermented marigold flowers was examined. The process consisted of a mixed culture of three microorganisms (Flavobacterium IIb, Acinetobacter anitratus, and Rhizopus nigricans), part of the normal microbiota associated with the marigold flower. These microorganisms had been previously isolated, and were identified as relevant for the ensilage process due to their capacity to produce cellulolytic enzymes. Based on experimental design strategies, optimum operation values were determined for aeration, moisture, agitation, and marigold-to-inoculum ratio in the proposed solid-state fermentation equipment, leading to a xanthophylls yield of 17.8-g/kg dry weight. The optimum achieved represents a 65% increase with respect to the control. HPLC analysis indicated conservation of extracted oleoresin. Based on the experimental results, interactions were identified that could be associated with the heat and mass-transfer reactions taking place within the bioreactor. The insight gained allows conditions that limit growth and metabolic activity to be avoided.
Mutational analysis of the hydantoin hydrolysis pathway in Pseudomonas putida RU-KM3S by G. F. Matcher; S. G. Burton; R. A. Dorrington (pp. 391-400).
The biocatalytic conversion of 5-mono-substituted hydantoins to the corresponding d-amino acids or l-amino acids involves first the hydrolysis of hydantoin to a N-carbamoylamino acid by an hydantoinase or dihydropyrimidinase, followed by the conversion of the N-carbamoylamino acid to the amino acid by N-carbamylamino acid amidohydrolase (N-carbamoylase). Pseudomonas putida strain RU-KM3S, with high levels of hydantoin-hydrolysing activity, has been shown to exhibit non-stereoselective hydantoinase and l-selective N-carbamoylase activity. This study focused on identifying the hydantoinase and N-carbamoylase-encoding genes in this strain, using transposon mutagenesis and selection for altered growth phenotypes on minimal medium with hydantoin as a nitrogen source. Insertional inactivation of two genes, dhp and bup, encoding a dihydropyrimidinase and β-ureidopropionase, respectively, resulted in loss of hydantoinase and N-carbamoylase activity, indicating that these gene products were responsible for hydantoin hydrolysis in this strain. dhp and bup are linked to an open reading frame encoding a putative transport protein, which probably shares a promoter with bup. Two mutant strains were isolated with increased levels of dihydropyrimidinase but not β-ureidopropionase activity. Transposon mutants in which key elements of the nitrogen regulatory pathway were inactivated were unable to utilize hydantoin or uracil as a nitrogen source. However, these mutations had no effect on either the dihydropyrimidinase or β-ureidopropionase activity. Disruption of the gene encoding dihydrolipoamide succinyltransferase resulted in a significant reduction in the activity of both enzymes, suggesting a role for carbon catabolite repression in the regulation of hydantoin hydrolysis in P. putida RU-KM3S cells.
Streptomyces lividans and Brevibacterium lactofermentum as heterologous hosts for the production of X22 xylanase from Aspergillus nidulans by M. Díaz; S. A. I. Adham; D. Ramón; J. A. Gil; R. I. Santamaría (pp. 401-406).
The Aspergillus nidulans gene xlnA coding for the fungal xylanase X22 has been cloned and expressed in two heterologous bacterial hosts: Streptomyces lividans and Brevibacterium lactofermentum. Streptomyces strains yielded 10 units/ml of xylanase when the protein was produced with its own signal peptide, and 19 units/ml when its signal peptide was replaced by the one for xylanase Xys1 from Streptomyces halstedii. B. lactofermentum was also able to produce xylanase X22, affording 6 units/ml upon using either the Aspergillus xlnA signal peptide or Streptomyces xysA. These production values are higher than those previously reported for the heterologous expression of the A. nidulans xlnA gene in Saccharomyces cerevisiae (1 unit/ml). Moreover, the X22 enzyme produced by Streptomyces lividans showed oenological properties, indicating that this Streptomyces recombinant strain is a good candidate for the production of this enzyme at the industrial scale.
Production of autoproteolytically subunit-assembled 7-β-(4-carboxybutanamido)cephalosporanic acid (GL-7ACA) acylase from Pseudomonas sp. C427 using a chitin-binding domain by Koji Nagao; Michio Yamashita; Mitsuyoshi Ueda (pp. 407-413).
7-β-(4-Carboxybutanamido)cephalosporanic acid (GL-7ACA) acylase from Pseudomonas sp. C427 is known as a proteolytically processed bacterial enzyme. GL-7ACA acylase from Pseudomonas sp. C427 (C427) consists of α- and β-subunits that are processed from a precursor peptide by removing the spacer peptide. A chitin-binding domain (CBD) of chitinase A1 derived from Bacillus circulans was genetically fused into four different positions of the C427-encoding gene. In the four enzymes thereby produced, Nα427, SP427, Cα427, and Cβ427, it was fused, respectively, to the N-terminal region of the α-subunit; the C-terminal region of the α-subunit; the three-amino-acid upper region of the C-terminal of the α-subunit; and to the C-terminal region of the β-subunit. All of the fusion enzymes, expressed in Eschericha coli, were successfully processed into active forms and had GL-7ACA acylase activity. The affinity-binding activity to crystalline chitin was affected by the fusing position of CBD. Nα427, Cα427, and Cβ427 remained fused to the CBD after their processing steps and could bind to chitin, but in the case of SP427 the fused CBD was cleaved away during the processing steps and binding activity was no longer observed. These results indicate that CBD is functional in such autoproteolytically subunit-assembled acylases.
Inactivation of pycA, encoding pyruvate carboxylase activity, increases poly-β-hydroxybutyrate accumulation in Azotobacter vinelandii on solid medium by D. Segura; G. Espín (pp. 414-418).
Strain AJ1678, an Azotobacter vinelandii mutant overproducing the storage polymer poly-β-hydroxybutyrate (PHB) in solid but not liquid complex medium with sucrose, was isolated after mini-Tn5 mutagenesis of strain UW136. Cloning and nucleotide sequencing of the affected locus led to identification of pycA, encoding a protein with high identity to the biotin carboxylase subunit of pyruvate carboxylase enzyme (PYC). A gene (pycB) whose product is similar to the biotin-carrying subunit of PYC is present immediately downstream from pycA. An assay of pyruvate carboxylase activity and an avidin-blot analysis confirmed that pycA and pycB encode the two subunits of this enzyme. In many organisms, PYC catalyzes ATP-dependent carboxylation of pyruvate to generate oxaloacetate and is responsible for replenishing oxaloacetate for continued operation of the tricarboxylic acid cycle. We propose that the pycA mutation causes a slow-down in the TCA cycle activity due to a low oxaloacetate concentration, resulting in a higher availability of acetyl-CoA for the synthesis of poly-β-hydroxybutyrate.
Establishment of an overall transformation system for an oil-producing filamentous fungus, Mortierella alpina 1S-4 by Seiki Takeno; Eiji Sakuradani; Shoichi Murata; Misa Inohara-Ochiai; Hiroshi Kawashima; Toshihiko Ashikari; Sakayu Shimizu (pp. 419-425).
Oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. To determine its physiological properties and to clarify the biosynthetic pathways for polyunsaturated fatty acids, a transformation system for this fungus was established using a derivative of it, i.e., a ura5 − mutant lacking orotate phosphoribosyl transferase (OPRTase, EC.2.4.2.10) activity. Transformation with a vector containing the homologous ura5 gene as a marker was successfully performed using microprojectile bombardment, other methods frequently used for transformation, such as the protoplasting, lithium acetate, or electroporation methods, not giving satisfactory results. As a result, two types of transformants were obtained: a few stable transformants overexpressing the ura5 gene, and many unstable transformants showing OPRTase activity comparable to that of the wild-type strain. The results of quantitative PCR indicated that the stable transformants could retain the ura5 genes originating from the transformation vector regardless of the culture conditions. On the other hand, unstable transformants easily lost the marker gene under uracil-containing conditions, as expected. In this paper, we report that an overall transformation system for this fungus was successfully established, and propose how to select useful transformants as experimental and industrial strains.
Effect of different levels of NADH availability on metabolite distribution in Escherichia coli fermentation in minimal and complex media by S. J. Berríos-Rivera; A. M. Sánchez; G. N. Bennett; K.-Y. San (pp. 426-432).
A range of intracellular NADH availability was achieved by combining external and genetic strategies. The effect of these manipulations on the distribution of metabolites in Escherichia coli was assessed in minimal and complex medium under anoxic conditions. Our in vivo system to increase intracellular NADH availability expressed a heterologous NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii in E. coli. The heterologous FDH pathway converted 1 mol formate into 1 mol NADH and carbon dioxide, in contrast to the native FDH where cofactor involvement was not present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 mol to 4 mol NADH/mol glucose consumed. In the current study, we found that yields of greater than 4 mol NADH were achieved when carbon sources more reduced than glucose were combined with our in vivo NADH regeneration system. This paper demonstrates experimentally that different levels of NADH availability can be achieved by combining the strategies of feeding the cells with carbon sources which have different oxidation states and regenerating NADH through the heterologous FDH pathway. The general trend of the data is substantially similar for minimal and complex media. The NADH availability obtained positively correlates with the proportion of reduced by-products in the final culture. The maximum theoretical yield for ethanol is obtained from glucose and sorbitol in strains overexpressing the heterologous FDH pathway.
Isolation and partial characterization of bacteriocins from Pediococcus species by M. Jamuna; K. Jeevaratnam (pp. 433-439).
Lactic acid bacteria have received increased attention as a potential food preservative due to their strong antagonistic activity against many food-spoilage and pathogenic organisms. Three Pediococcus species, P. acidilactici NCIM 2292, P. pentosaceous. NCIM 2296 and P. cervisiae NCIM 2171, were evaluated for bacteriocin production. Inhibitory substance were produced during the late growth phase and maximum production occurred at 37 °C after 36–48 h of incubation. Bacteriocins partially purified from these species by cold-acetone precipitation at 0 °C and cell adsorption desorption techniques have a broad inhibitory spectrum against microorganisms, including gram-negative bacteria such as Escherichia coli and Pseudomonas. Proteolytic enzymes inactivated these peptides, but amylase and lipase did not show any effect. The bacteriocins were stable over a wide pH range (3–8) and apparently most active at pH 4.0–5.0. They were heat-stable (1 h at ~80 °C and autoclaving) at pH 5.0. No loss in activity was observed when stored under refrigeration (4–8 °C). Tris-Tricine SDS-PAGE revealed the molecular masses of these peptides to be between 3.5 and 5.0 kDa.
Relation between bacterial strain resistance to solvents and biodesulfurization activity in organic medium by Murielle Bouchez-Naïtali; Samir Abbad-Andaloussi; Michel Warzywoda; Frédéric Monot (pp. 440-445).
Microorganisms used in biodesulfurization of petroleum products have to withstand high concentrations of hydrocarbons. The capacities of seven desulfurizing strains of Rhodococcus to be active in the presence of solvents were evaluated. Octanol and toluene (log P=2.9) were selected as toxic solvents. The effect of the solvents was determined by measuring either inhibition of growth or the decrease in respiratory activity of the cells. Differences among strains in their resistance to solvent responses were observed, but these variations were dependent on the test used. Resistance to solvents was then compared to the capacity of the different strains to retain biodesulfurization activity in the presence of hexadecane. Inhibition of desulfurization by high concentrations of hexadecane was found to be well correlated to the sensitivity of the strains to respiration inhibition by toluene, but not to growth inhibition. This result also showed that the respirometric test was a rapid and reliable test to select solvent-resistant strains for use as resting cells in biocatalysis processes, such as biodesulfurization, in organic media.
Parameters and mechanistic studies on the oxidative ring cleavage of synthetic heterocyclic naphthoquinones by Streptomyces strains by Céline Fosse; Laurence Le Texier; Sébastien Roy; Marcel Delaforge; Sébastien Grégoire; Michel Neuwels; Robert Azerad (pp. 446-456).
Screening of fungal and bacterial strains allowed selection of two Streptomyces strains (S. platensis and S. cinnamonensis) that oxidatively cleave, in moderate to high yields (up to 65% in 24 h), the quinonic ring of a thiazole fused 1,4-naphthoquinone compound, INO5042, used as a model compound for a series of homologous substituted heterocyclic naphthoquinones. The respective products of these whole-cell biotransformations were identified as isomeric phenol-carboxylic acids resulting from a C–C bond cleavage at a position vicinal to each one of the carbonyl groups. The culture and incubation conditions have been optimised and the mechanism of this biotransformation investigated using oxygen isotope incorporation. The results of 18O2 incorporation indicate a dioxygenase reaction, the mechanism of which is discussed in relation with that of hydroquinone-epoxidases, a family of oxygenating enzymes involved in the biosynthesis of polyketide antibiotics in Streptomyces.
Death of Escherichia coli during rapid and severe dehydration is related to lipid phase transition by L. Beney; Y. Mille; P. Gervais (pp. 457-464).
This study reports the effects of exposure to increasing osmotic pressure on the viability and membrane structure of Escherichia coli. Changes in membrane structure after osmotic stress were investigated by electron transmission microscopy, measurement of the anisotropy of the membrane fluorescent probe DPH (1,6-diphenyl-1,3,5-hexatriene) inserted in E. coli, and Fourier infrared spectroscopy (FTIR). The results show that, above a critical osmotic pressure of 35 MPa, the viability of the bacterium is drastically reduced (2 log decrease in survivors). Electron micrographs revealed a severe contraction of the cytoplasm and the formation of membrane vesicles at 40 MPa. Changes in DPH anisotropy showed that osmotic dehydration to 40 MPa promoted a decrease in the membrane fluidity of integral cells of E. coli. FTIR measurements showed that at 10–40 MPa a transition from lamellar liquid crystal to lamellar gel among the phospholipids extracted from E. coli occurred. Bacterial death resulting from dehydration can be attributed to the conjunction between membrane deformation, caused by the volumetric contraction, and structural changes of the membrane lipids. The influence of the latter on the formation of membrane vesicles and on membrane permeabilization at lethal osmotic pressure is discussed, since vesiculation is hypothetically responsible for cell death.
Stability and activity of an Enterobacter aerogenes-specific bacteriophage under simulated gastro-intestinal conditions by K. Verthé; S. Possemiers; N. Boon; M. Vaneechoutte; W. Verstraete (pp. 465-472).
A bacteriophage, designated UZ1 and showing lytic activity against a clinically important strain (BE1) of Enterobacter aerogenes was isolated from hospital sewage. The stability and lytic activity against this strain under simulated gastro-intestinal conditions was evaluated. After addition of bacteriophage UZ1 to a liquid feed at gastric pH 2, the phage was immediately inactivated and could not be recovered. However, by use of an antacid to neutralize stomach acidity, no significant changes in phage titer were observed after 2 h incubation at 37 °C. After supplementing pancreatic juice and further incubation for 4 h, the phage titer remained stable. The persistence of UZ1 in a mixed microbial ecosystem that was representative for the large intestine was monitored using an in vitro simulation of the human intestinal microbial ecosystem. A pulse administration of bacteriophage UZ1 at a concentration of 105 plaque-forming units (PFU)/ml to reactor 3 (which simulates the ascending colon) showed that, in the absence of the host, bacteriophage UZ1 persisted for 13 days in the simulated colon, while the theoretical washout was calculated at 16 days. To assess its lytic activity in an intestinal microbial ecosystem, a green fluorescent protein (gfp)-labeled E. aerogenes BE1 strain was constructed and gfp-specific primers were designed in order to quantify the host strain using real-time PCR. It was observed that bacteriophage UZ1 was able to replicate and showed lytic activity against E. aerogenes BE1/gfp in an intestinal microbial ecosystem. Indeed, after 17 h a 2 log unit reduction of E. aerogenes BE1/gfp was measured as compared with the assay without bacteriophage UZ1, while the phage titer increased by 2 log units at an initial multiplicity of infection of 0.07 PFU/colony-forming unit. This is the first report of an in vitro model to study bacteriophage activity in the complex intestinal microbial community.
An isolate of Aspergillus flavus used to reduce aflatoxin contamination in cottonseed has a defective polyketide synthase gene by Kenneth C. Ehrlich; Peter J. Cotty (pp. 473-478).
Contamination of certain foods and feeds with the highly toxic and carcinogenic family of Aspergillus mycotoxins, the aflatoxins, can place a severe economic burden on farmers. As one strategy to reduce aflatoxin contamination, the non-aflatoxin-producing A. flavus isolate AF36 is currently being applied to agricultural fields to competitively exclude aflatoxin-producing Aspergillus species. We now show that the polyketide synthase gene (pksA) required for aflatoxin biosynthesis in AF36, and in other members of the same vegetative compatibility group, possesses a nucleotide polymorphism near the beginning of the coding sequence. This nucleotide change introduces a premature stop codon into the coding sequence, thereby preventing enzyme production and aflatoxin accumulation.
Degradation of acrylic copolymers by white-rot fungi by Carsten Mai; Wiebke Schormann; Andrzej Majcherczyk; Alois Hüttermann (pp. 479-487).
Various water-soluble homopolymers and copolymers of acrylamide (AAm) and acrylic acid (AA) which contained phenolic sites, such as guaiacol, lignin sulfonate (LS) and 3,4-dihydroxybenzoic acid (3,4-DHBA), were tested with regard to their degradability by white-rot fungi. Compared with Phanerochaete chrysosporium, Pleurotus ostreatus caused a significantly higher decrease in the average molecular weight (M̄w) of most of the copolymers and the homopolymer under the applied culture conditions. However, the M̄w of poly(guaiacol/AAm) increased significantly during incubation with Pl ostreatus. P. chrysosporium was able to reduce only the M̄w of the poly(LS/AA) to a significant degree and not that of the other polymers. The mineralization rate of AAm and AA copolymers and terpolymers of AAm, AA and phenolics (LS, 3,4-DHBA, guiacol), which were tested with P. ostreatus and Trametes versicolor, turned out to be low (0.8–3.2%). While the rates of mineralization were similar among all polymers, the decrease in radioactivity from the culture media was higher with the terpolymers bearing phenolic sites. UV spectra of the culture media revealed that the phenolic sites in the terpolymers were significantly degraded by both fungi. Obviously, the degradation of phenolics within the polymer chain caused a higher decrease in M̄w but did not significantly increase the mineralization rate.
Microbial population dynamics during fed-batch operation of commercially available garbage composters by T. Narihiro; T. Abe; Y. Yamanaka; A. Hiraishi (pp. 488-495).
Microbial populations in terms of quantity, quality, and activity were monitored during 2 months of start-up operation of commercially available composters for fed-batch treatment of household biowaste. All the reactors, operated at a waste-loading rate of 0.7 kg day−1 (wet wt), showed a mass reduction efficiency of 88–93%. The core temperature in the reactors fluctuated between 31°C and 58°C due to self-heating. The pH declined during the early stage of operation and steadied at pH 7.4–9.3 during the fully acclimated stage. The moisture content was 48–63% early in the process and 30–40% at the steady state. Both direct total counts and plate counts of bacteria increased via two phases (designated phases I, II) and reached an order of magnitude of 1011 cells g−1 (dry wt) at the steady state. Microbial community changes during the start-up period were studied by culture-independent quinone profiling and denatured gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA. In all the reactors, ubiquinones predominated during phase I, whereas partially saturated menaquinones became predominant during phase II. This suggested that there was a drastic population shift from ubiquinone-containing Proteobacteria to Actinobacteria during the start-up period. The DGGE analysis of the bacterial community in one of the reactors also demonstrated a drastic population shift during phase I and the predominance of members of the phyla Proteobacteria and Bacteroidetes during the overall period. But this molecular analysis failed to detect actinobacterial clones from the reactor at any stage.