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Applied Microbiology and Biotechnology (v.55, #3)

Metabolic engineering by J. Nielsen (pp. 263-283).

Metabolic engineering has developed as a very powerful approach to optimising industrial fermentation processes through the introduction of directed genetic changes using recombinant DNA technology. Successful metabolic engineering starts with a careful analysis of cellular function; based on the results of this analysis, an improved strain is designed and subsequently constructed by genetic engineering. In recent years some very powerful tools have been developed, both for analysing cellular function and for introducing directed genetic changes. In this paper, some of these tools are reviewed and many examples of metabolic engineering are presented to illustrate the power of the technology. The examples are categorised according to the approach taken or the aim: (1) heterologous protein production, (2) extension of substrate range, (3) pathways leading to new products, (4) pathways for degradation of xenobiotics, (5) improvement of overall cellular physiology, (6) elimination or reduction of by-product formation, and (7) improvement of yield or productivity.

Solid-state fermentation: a promising microbial technology for secondary metabolite production by T. Robinson; D. Singh; P. Nigam (pp. 284-289).

Solid state (substrate) fermentation (SSF) has been used successfully for the production of enzymes and secondary metabolites. These products are associated with the stationary phase of microbial growth and are produced on an industrial scale for use in agriculture and the treatment of disease. Many of these secondary metabolites are still produced by submerged liquid fermentations (SmF) even though production by this method has been shown to be less efficient than SSF. As large-scale production increases further, so do the costs and energy demands. SSF has been shown to produce a more stable product, requiring less energy, in smaller fermenters, with easier downstream processing measures. In this article we review an important area of biotechnology, since the recent evidence indicates that bacteria and fungi, growing under SSF conditions, are more than capable of supplying the growing global demand for secondary metabolites.

Production of 3-nitrocatechol by oxygenase-containing bacteria: optimization of the nitrobenzene biotransformation by Nocardia S3 by J. Kieboom; H. van den Brink; J. Frankena; J. de Bont (pp. 290-295).

Twenty-one microorganisms were screened for their ability to convert nitroaromatics into 3-nitrocatechol as a result of the action of an oxygenase. Cultures containing toluene dioxygenases and phenol monooxygenases accumulated 3-nitrocatechol during incubation with nitrobenzene and nitrophenol, respectively. Nocardia S3 was selected and studied in more detail. Toluene-pregrown cultures were able to degrade nitrobenzene with a concomitant formation of 3-nitrocatechol. The rates of nitrobenzene utilization decreased throughout the biotransformation period and finally the accumulation ceased. The gradual deterioration of the biotransformation rates was not a consequence of depletion of the NADH pool, but was due to the accumulation of 3-nitrocatechol. The inhibition of nitrobenzene biotransformation by 3-nitrocatechol greatly impacts 3-nitrocatechol production processes .

Involvement of branched-chain amino acid aminotransferases in the production of fusel alcohols during fermentation in yeast by A. Eden; L. Van Nedervelde; M. Drukker; N. Benvenisty; A. Debourg (pp. 296-300).

Organoleptic compounds produced by yeast during the fermentation of wort have a great impact on beer smell and taste. Among them, fusel alcohols are the major abundant volatile compounds. The availability of Saccharomyces cerevisiae mutants in which the genes coding for the two branched-chain amino acid aminotransferases have been deleted offers the possibility of further defining the role of these enzymes in the formation of higher alcohols. Comparing the production profiles of different strains, it is clear that they are not all influenced in the same way by branched-chain amino acid aminotransferase mutations. First of all, as propanol is synthesised from α-ketobutyrate, the first metabolic intermediate in the anabolic pathway of isoleucine, neither the eca39 nor eca40 mutations have any effect on the production of this higher alcohol. On the other hand, it can be concluded that the eca40 mutation has a drastic effect on the production of isobutanol. To a certain extent, the same conclusion can be made for the production of active amyl alcohol and isoamyl alcohol, although the results suggest that another route could lead to the formation of these two higher alcohols.

Improved β-thujaplicin production in Cupressus lusitanica suspension cultures by fungal elicitor and methyl jasmonate by J. Zhao; K. Fujita; J. Yamada; K. Sakai (pp. 301-305).

Production of a novel antimicrobial tropolone, β-thujaplicin, in Cupressus lusitanica suspension cultures was studied by using a variety of chemicals and fungal elicitors. Sodium alginate, chitin, and methyl jasmonate resulted in 2-, 2.5-, and 3-fold higher β-thujaplicin production, respectively, than in the control. Significantly improved β-thujaplicin production (187 mg l^–1) was obtained using a high cell density (180–200 g l^–1) and fungal elicitor treatment [10 mg (g fresh cells)^–1] in a production medium with a high ferrous ion concentration (0.3 mM). This improved volumetric productivity was 3- to 4-fold higher than obtained under standard conditions. A synergistic effect of fungal elicitor and ferrous ion on β-thujaplicin production was also suggested by our study.

Combination of Trichoderma harzianum endochitinase and a membrane-affecting fungicide on control of Alternaria leaf spot in transgenic broccoli plants by A. Mora; E. Earle (pp. 306-310).

Progeny from transgenic broccoli (cv. Green Comet) expressing a Trichoderma harzianum endochitinase gene were used to assess the interaction between endochitinase and the fungicide Bayleton in the control of Alternaria brassicicola. In vitro assays have shown synergistic effects of endochitinase and fungicides on fungal pathogens. Our study examined the in planta effects of endochitinase and Bayleton, individually and in combination. Two month old transgenic and non-transgenic plants were sprayed with ED₅₀ levels of Bayleton and/or inoculated with an A. brassicicola spore suspension. Disease levels in non-sprayed transgenic plants were not statistically different from sprayed transgenic plants nor from sprayed non-transgenic controls. Thus endochitinase-transgenic plants alone provided a significant reduction of disease severity, comparable to the protection by fungicide on non-transgenic plants. Comparison of the expected additive and observed effects revealed no synergism between endochitinase and Bayleton (at ED₅₀ level), and usually less than an additive effect. Some transgenic lines sprayed with fungicide at doses higher than ED₅₀ showed resistance similar to the non-sprayed transgenic lines, again suggesting no synergistic effect. Lack of synergism may be due to incomplete digestion of the cell wall by endochitinase, so that the effect of Bayleton at the cell membrane is not enhanced.

Enhanced degradation of naphthalene by immobilization of Pseudomonas sp. strain NGK1 in polyurethane foam by S. Manohar; C. Kim; T. Karegoudar (pp. 311-316).

A Pseudomonas sp. strain NGK1 (NCIM 5120) capable of degrading naphthalene was immobilized in polyurethane foam. The naphthalene-degrading activity of the freely suspended cells was compared with that of immobilized cells in batches in shaken culture and in a continuous culture system in a packed-bed reactor. Increasing concentrations of naphthalene were better tolerated and more quickly degraded by immobilized cell cultures than by free cells. An initial naphthalene concentration of 25 mM was completely degraded by freely suspended cells (4×10¹⁰ cfu ml^–1) and polyurethane-foam-immobilized cells (0.8–1×10¹² cfu g^–1 foam cubes) after 4 days and 2 days of incubation, respectively. Free cells degraded a maximum of 30 mM naphthalene after 4 days of incubation with 50 mM naphthalene, and no further degradation was observed even after 15 days of incubation, whereas foam-immobilized cells brought about the complete degradation of 50 mM initial naphthalene after 6 days of incubation. Furthermore, with 25 mM naphthalene, the polyurethane-foam-immobilized cells were reused 45 times over a period of 90 days without losing naphthalene-degrading activity. By contrast, with the same amount of naphthalene, alginate-, agar-, and polyacrylamide-entrapped cells could be reused for 18, 12, and 23 times over a period of 44, 28, and 50 days, respectively. During continuous degradation in a packed-bed reactor, foam-immobilized cells degraded 80 mM naphthalene at a rate of 150 ml^–1 h^–1. With the same flow rate and 40 mM naphthalene, this system operated efficiently and continuously for about 120 days, whereas the packed-bed reactor with alginate-, agar-, and polyacrylamide-entrapped cells could be operated only for 45, 40, and 60 days respectively. Thus, more efficient degradation of naphthalene could be achieved by immobilizing cells of Pseudomonas sp. strain NGK1 in polyurethane foam, rather than in the other matrices tested.

Reactivity of Trametes laccases with fatty and resin acids by Stina Karlsson; Bjarne Holmbom; Peter Spetz; Annikka Mustranta; Johanna Buchert (pp. 317-320).

Lipophilic extractives commonly referred to as wood pitch or wood resin can have a negative impact on paper machine runnability and product quality. The lipophilic extractives are composed mainly of fatty acids, resin acids, sterols, steryl esters and triglycerides. In this work, the suitability of laccases for the modification of fatty and resin acids was studied, using two model fractions. In the treatments, resin and fatty acid dispersions were treated with two different laccases, i.e. laccases from Trametes hirsuta and T. villosa. Different chromatographic methods were used to elucidate the effects of laccase treatments on the chemistry of the fatty and resin acids. Both laccases were able to modify the fatty and resin acids to some extent. In the case of fatty acids, a decrease in the amount of linoleic, oleic and pinolenic acids was observed, whereas the modification of resin acids resulted in a reduced amount of conjugated resin acids.

Biotransformations catalyzed by cloned p-cymene monooxygenase from Pseudomonas putida F1 by Toshiyuki Nishio; Ashok Patel; Ying Wang; Peter C. Lau (pp. 321-325).

p-Cymene monooxygenase (CMO) from Pseudomonas putida F1 consists of a hydroxylase (CymA1) and a reductase component (CymA2) which initiate p-cymene (p-isopropyltoluene) catabolism by oxidation of the methyl group to p-isopropylbenzyl alcohol (p-cumic alcohol). To study the possible diverse range of substrates catalyzed by CMO, the cymA1A2 genes were cloned in an Escherichia coli pT7-5 expression system and the cells were used in transformation experiments. The tested substrates include different substituents on the aromatic ring at the 2 (ortho), 3 (meta) or 4 (para) position relative to the methyl moiety. As a result, a distinct preference was observed for substrates containing at least an alkyl or heteroatom substituent at the para-position of toluene. The conversion rate of 4-chlorotoluene or 4-methylthiotoluene to the corresponding benzyl alcohol was found to be as good as the canonical substrate, p-cymene. But 3-chlorotoluene, 4-fluorotoluene and 4-nitrotoluene were relatively poor substrates. CMO is also capable of producing styrene oxide from styrene. However, the oxidation of 4-chlorostyrene to 4-chlorostyrene oxide was by far the fastest among the substrates used in this study. The various biotransformation products were identified by a combined solid phase microextraction/gas chromatographic-mass spectrometric analytical technique.

Proteome and transcriptome based analysis of Bacillus subtilis cells overproducing an insoluble heterologous protein by Britta Jürgen; Renate Hanschke; Matti Sarvas; Michael Hecker; Thomas Schweder (pp. 326-332).

Bacillus subtilis and related Bacillus species are frequently used as hosts for the industrial production of recombinant proteins. In this study the cellular response of B. subtilis to the overproduction of an insoluble heterologous protein was investigated. For this purpose PorA, an outer membrane protein from Neisseria meningitidis, which accumulates after overexpression in the cytoplasm of B. subtilis mainly in the form of inclusion bodies, was used. The molecular response to overexpression of porA has been analysed at the transcriptional level using the DNA macro array technique and at the translational level by two-dimensional polyacrylamide gel electrophoresis. It was found that the expression of the heat shock genes of class I (dnaK, groEL and grpE) and class III (clpP and clpC) are increased under overproducing conditions. Furthermore, the protein levels of the two ribosomal proteins RpsB and RplJ are increased in the PorA overproducing cells. The transcriptome analysis indicated that mRNA levels of genes encoding pyrimidine and purine synthesis enzymes but also from ribosomal protein genes have elevated levels under overproducing conditions. Finally, the association of the protease ClpP and its ATPase subunits ClpC and ClpX with the PorA inclusion bodies was demonstrated by means of the immunogold labelling technique.

HO gene polymorphism in Saccharomyces industrial yeasts and application of novel HO genes to convert homothallism to heterothallism in combination with the mating-type detection cassette by Yukio Tamai; Keiko Tanaka; Yoshinobu Kaneko; Satoshi Harashima (pp. 333-340).

Southern blot analysis of industrial yeasts showed that all top-fermenting yeasts, distiller's yeasts and a proportion of wine yeasts tested in the present study produced a hybridization signal (approximately 7 kb), corresponding to a Saccharomyces cerevisiae-type HO gene (Sc-HO). It also showed that bottom-fermenting yeasts gave rise to 7-kb and 4-kb hybridization signals, corresponding to the Sc-HO gene and the lager yeast HO gene (Lg-HO), respectively. Two wine yeasts produced a 4-kb hybridization signal, corresponding to Lg-HO; and one wine yeast produced 2.5-kb and 1.5-kb hybridization bands, corresponding to a S. uvarum-type HO gene (Uv-HO). Partial nucleotide sequences of HO genes amplified from these wine yeasts perfectly matched those of Lg-HO and Uv-HO, respectively. HO disruption vectors were constructed by inserting a dominant selective marker PGK1p-neo and the mating-type detection cassette MF α 1p-PHO5 within the Lg-HO or Uv-HO gene. From transformants carrying a single-disrupted ho gene, mating-competent progenies were easily obtained through meiosis. Moreover, mating-competent derivatives appearing at very low frequency could be obtained from a double-disrupted ho transformant without meiosis (even from a wine yeast lacking sporulation ability), because the sensitive phosphatase-staining method allowed detection of the Pho⁺ mating-competent derivatives from confluent colonies by the random spore method. Our study describes a rapid and convenient method for isolating mating-competent clones from industrial yeasts.

Citrate, a possible precursor of astaxanthin in Phaffia rhodozyma: influence of varying levels of ammonium, phosphate and citrate in a chemically defined medium. by L. Flores-Cotera; R. Martín; S. Sánchez (pp. 341-347).

The influence of ammonium, phosphate and citrate on astaxanthin production by the yeast Phaffia rhodozyma was investigated. The astaxanthin content in cells and the final astaxanthin concentration increased upon reduction of ammonium from 61 mM to 12.9 mM (from 140 µg/g to 230 µg/g and 1.2 µg/ml to 2.3 µg/ml, respectively). Similarly, both the astaxanthin content and astaxanthin concentration increased by reducing phosphate from 4.8 mM to 0.65 mM (160 µg/g to 215 µg/g and 1.7 µg/ml to 2.4 µg/ml, respectively). Low concentrations of ammonium or phosphate also increased the fatty acid content in cells. By analogy with lipid synthesis in other oleaginous yeasts, an examination of the data for varying nitrogen and phosphate levels suggested that citrate could be the source of carbon for fatty acids and carotenoid synthesis. Supporting this possibility was the fact that supplementation of citrate in the medium at levels of 28 mM or higher notably increased the final pigment concentration and pigment content in cells. Increased carotenoid synthesis at low ammonium or phosphate levels, and stimulation by citrate were both paralleled by decreased protein synthesis. This suggested that restriction of protein synthesis could play an important role in carotenoid synthesis by P. rhodozyma.

Isolation and characterization of two aerobic bacterial strains that completely degrade ethyl tert-butyl ether (ETBE) by M. Kharoune; L. Kharoune; J. Lebeault; A. Pauss (pp. 348-353).

Two bacterial strains, E₁ and E₂, isolated from gasoline-polluted soil completely degraded ethyl tert-butyl ether (ETBE), as the sole source of carbon and energy, at specific rates of about 80 mg g^–1 and 58 mg g^–1 of cell protein day^–1, respectively. On the basis of morphological and phenotypic characteristics, strain E₁ was tentatively identified as Comamonas testosteroni and strain E₂ as belonging to Centre for Disease Control group A-5. The inhibitory effect of metyrapone on the degradative ability of both strains was the first evidence indicating the involvement of a soluble cytochrome P-450 in the cleavage of the ETBE ether bond. This observation was confirmed by spectrophotometric analysis of reduced cell extracts that gave, in the presence of carbon monoxide, a major absorbance peak at about 450 nm. Both strains were also able to degrade, as the sole source of carbon and energy, ETBE's major metabolic intermediates (tert-butyl alcohol and tert-butyl formate) and other gasoline oxygenates (methyl tert-butyl ether and tert-amyl methyl ether). The degradation rates varied considerably, with both strains exhibiting a preferential activity for ETBE's metabolic intermediates.

Screening, overexpression and characterization of an N-acylamino acid racemase from Amycolatopsis orientalis subsp. lurida by S. Verseck; A. Bommarius; M.-R. Kula (pp. 354-361).

Thirty-one different actinomycete strains were used in a genetic screening using PCR and Southern hybridization methods to detect N-acetylamino acid racemases (AAR) in order to obtain enzymes with different properties. Cloning and sequencing of a 2.5 kb EcoRI DNA fragment from Amycolatopsis orientalis subsp. lurida revealed the coding gene of an N-acetylamino acid racemase, which had identities to the aar gene of Amycolatopsis sp. TS-1-60 [Tokuyama and Hatano (1995) Appl Microbiol Biotechnol 42:884–889] of 86% at the level of DNA, and 90% at the level of amino acids. The heterologous overexpression in Escherichia coli resulted in a specific activity of about 0.2 U/mg of this racemase. A two-step purification with heat treatment followed by anion-exchange chromatography led to almost homogeneous enzyme. The optimum pH of the enzyme was 8.0 and it was stable at 50°C for 30 min. The relative molecular mass of the native enzyme and the subunit was calculated to be 300 kDa and 40 kDa by gel filtration and SDS–PAGE, respectively. The isoelectric point (pI) of the AAR was 4.4. It catalyzed the racemization of optically active N-acetylamino acids such as N-acetyl-L- or -D-methionine and N-acetyl-L-phenylalanine. Further characterization of the racemase demonstrated a requirement for divalent metal ions (Co²⁺, Mn²⁺, Mg²⁺) for activity and inhibition by EDTA and p-hydroxymercuribenzoic acid. AAR is sensitive to substrate inhibition at concentrations exceeding 200 mM.

Automatic method for evaluating the activity of sourdough strains based on gas pressure measurements by Marion Wick; Jean-Jacques Vanhoutte; Alain Adhemard; Georges Turini; Jean-Michel Lebeault (pp. 362-368).

A new method is proposed for the evaluation of the activity of sourdough strains, based on gas pressure measurements in closed air-tight reactors. Gas pressure and pH were monitored on-line during the cultivation of commercial yeasts and heterofermentative lactic acid bacteria on a semi-synthetic medium with glucose as the major carbon source. Relative gas pressure evolution was compared both to glucose consumption and to acidification and growth. It became obvious that gas pressure evolution is related to glucose consumption kinetics. For each strain, a correlation was made between maximum gas pressure variation and amount of glucose consumed. The mass balance of CO₂ in both liquid and gas phase demonstrated that around 90% of CO₂ was recovered. Concerning biomass production, a linear relationship was found between log colony-forming units/ml and log pressure for both yeasts and bacteria during the exponential phase; and for yeasts, relative gas pressure evolution also followed optical density variation.

Biodegradation of tert-butyl alcohol and related xenobiotics by a methylotrophic bacterial isolate by P. Piveteau; F. Fayolle; J.-P. Vandecasteele; F. Monot (pp. 369-373).

A new aerobic bacterial strain, CIP I-2052, isolated from an activated sludge sample, was able to use tert-butyl alcohol (TBA), a product of methyl tert-butyl ether (MTBE) and ethyl tert-butyl ether (ETBE) degradation, as its sole carbon and energy source. Cobalt ions stimulated TBA mineralization. The maximum growth and TBA degradation rates were 0.032±0.004 h^–1 and 35.8±8.5 mg TBA·g^–1 (cell dry mass) per h, respectively. The growth yield on TBA was 0.54±0.02 g·g^–1. Strain CIP I-2052 exhibited a particular substrate specificity towards alcohols. It degraded tertiary alcohols, TBA and tert-amyl alcohol (TAA), but neither their primary and secondary alcohol homologues, nor ethanol. However, one-carbon compounds, namely methanol and formate, were degraded by strain CIP I-2052, showing the methylotrophic nature of this isolate. The properties of this new strain suggest that it could be used for bioremediation of contaminated aquifers.

Long-term repeated biodesulfurization by immobilized Rhodococcus erythropolis KA2-5-1 cells by Manabu Naito; Takuo Kawamoto; Kazuhito Fujino; Morio Kobayashi; Kenji Maruhashi; Atsuo Tanaka (pp. 374-378).

In this study, biodesulfurization (BDS) was carried out using immobilized Rhodococcus erythropolis KA2-5-1 in n-tetradecane containing dibenzothiophene (DBT) as a model oil (n-tetradecane/immobilized cell biphasic system). The cells were immobilized by entrapping them with calcium alginate, agar, photo-crosslinkable resin prepolymers (ENT-4000 and ENTP-4000), and urethane prepolymers (PU-3 and PU-6); and it was found that ENT-4000-immobilized cells had the highest DBT desulfurization activity in the model oil system without leakage of cells from the support. Furthermore, ENT-4000-immobilized cells could catalyze BDS repeatedly in this system for more than 900 h with reactivation; and recovery of both the biocatalyst and the desulfurized model oil was easy. This study would give a solution to the problems in BDS, such as the troublesome process of recovering desulfurized oil and the short life of BDS biocatalysts.

Concurrent sorption of Ni²⁺ and Cu²⁺ by Chlorella vulgaris from a binary metal solution by S. Mehta; J. Gaur (pp. 379-382).

Kinetics and capacity of Ni²⁺ and Cu²⁺ sorption by Chlorella vulgaris were studied using single and binary metal solutions at various concentrations of these metal ions. The second-order rate law best described the kinetics of metal sorption from both single and binary metal systems. C. vulgaris preferentially sorbed Cu²⁺ over Ni²⁺ in the binary system. In comparison to the single metal system, the amounts of Ni²⁺ and Cu²⁺ sorbed at equilibrium (q _e) were respectively 73% and 25%, and the initial rate of sorption (h) was ca. 50% in the case of the binary metal system. The test metals inhibited sorption of each other, thereby indicating competition between Ni²⁺ and Cu²⁺for sorption onto non-specific binding sites. The present study showed that C. vulgaris has specific as well as non-specific sites for the binding of Ni²⁺ and Cu²⁺. Participation of these sites for sorption depended on the ratio of Ni²⁺ and Cu²⁺ in solution. The maximum metal sorption capacity of C. vulgaris was 6.75 mmol g^–1 from the binary metal solution at the tested biomass concentration (100 mg dry weight l^–1). Total metal sorption was enhanced with increasing total concentration of both the metals up to 1.6 mM, beyond which a decrease occurred. Two-dimensional contour plots were successfully used for the first time for the evaluation of metal sorption potential.

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Applied Microbiology and Biotechnology (v.55, #3)