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Applied Microbiology and Biotechnology (v.68, #2)

Mutational biosynthesis—a tool for the generation of structural diversity in the biosynthesis of antibiotics by S. Weist; R. D. Süssmuth (pp. 141-150).

Natural products represent an important source of drugs in a number of therapeutic fields, e.g. antiinfectives and cancer therapy. Natural products are considered as biologically validated lead structures, and evolution of compounds with novel or enhanced biological properties is expected from the generation of structural diversity in natural product libraries. However, natural products are often structurally complex, thus precluding reasonable synthetic access for further structure-activity relationship studies. As a consequence, natural product research involves semisynthetic or biotechnological approaches. Among the latter are mutasynthesis (also known as mutational biosynthesis) and precursor-directed biosynthesis, which are based on the cellular uptake and incorporation into complex antibiotics of relatively simple biosynthetic building blocks. This appealing idea, which has been applied almost exclusively to bacteria and fungi as producing organisms, elegantly circumvents labourious total chemical synthesis approaches and exploits the biosynthetic machinery of the microorganism. The recent revitalization of mutasynthesis is based on advancements in both chemical syntheses and molecular biology, which have provided a broader available substrate range combined with the generation of directed biosynthesis mutants. As an important tool in supporting combinatorial biosynthesis, mutasynthesis will further impact the future development of novel secondary metabolite structures.

Amphotericin B by A. Lemke; A. F. Kiderlen; O. Kayser (pp. 151-162).

Invasive fungal infections are a major cause of morbidity and mortality in immunodeficient individuals (such as AIDS patients) and in transplant recipients or tumor patients undergoing immunosuppressive chemotherapy. Amphotericin B is one of the oldest, yet most efficient antimycotic agents. However, its usefulness is limited due to dose-dependent side-effects, notably nephrotoxicity. In order to improve its safety margin, new pharmaceutical formulations of amphotericin B have been designed especially to reduce its detrimental effects on the kidneys. Since the 1980s, a wide variety of new amphotericin B formulations have been brought forward for clinical testing, many of which were approved and reached market value in the 1990s. This review describes and discusses the molecular genetics, pharmacological, toxicological, and clinical aspects of amphotericin B itself and many of its innovative formulations.

Curdlan and other bacterial (1→3)-β-d-glucans by M. McIntosh; B. A. Stone; V. A. Stanisich (pp. 163-173).

Three structural classes of (1→3)-β-d-glucans are encountered in some important soil-dwelling, plant-associated or human pathogenic bacteria. Linear (1→3)-β-glucans and side-chain-branched (1→3,1→2)-β-glucans are major constituents of capsular materials, with roles in bacterial aggregation, virulence and carbohydrate storage. Cyclic (1→3,1→6)-β-glucans are predominantly periplasmic, serving in osmotic adaptation. Curdlan, the linear (1→3)-β-glucan from Agrobacterium, has unique rheological and thermal gelling properties, with applications in the food industry and other sectors. This review includes information on the structure, properties and molecular genetics of the bacterial (1→3)-β-glucans, together with an overview of the physiology and biotechnology of curdlan production and applications of this biopolymer and its derivatives.

Bioconversion of lutein using a microbial mixture—maximizing the production of tobacco aroma compounds by manipulation of culture medium by Eduardo Rodríguez-Bustamante; Gabriela Maldonado-Robledo; Marco Antonio Ortiz; Carlos Díaz-Ávalos; Sergio Sanchez (pp. 174-182).

The generation of aroma compounds by carotenoid cleavage in the 9–10 position was studied, due to the importance of these compounds in the flavor and fragrance industry. The bioconversion of the carotenoid lutein to C₁₃ norisoprenoids utilizing a microbial mixture composed of Trichosporon asahii and Paenibacillus amylolyticus was carried out by a fermentation process. Applying an experimental design methodology, the effects of nutritional factors on the production of aroma compounds present in the tobacco profile were studied. After an assessment of the significance of each nutritional factor, the levels of the variables yielding the maximum response were calculated. Glucose, tryptone, and yeast extract exerted a strong negative effect over the objective function, with glucose being the strongest. Lutein possessed a positive effect over the tobacco aroma production, while sodium chloride and trace elements showed no influence over the process. The yield attained after culture medium manipulation was almost ten-fold higher, compared with the base medium; and the aroma mixture was characterized as: 7,8-dihydro-β-ionol (95.2%), 7,8-dihydro-β-ionone (3.7%), and β-ionone (1.1%).

Induction and potentiation of diterpenoid tanshinone accumulation in Salvia miltiorrhiza hairy roots by β-aminobutyric acid by Xiuchun Ge; Jianyong Wu (pp. 183-188).

The non-protein amino acid β-aminobutyric acid (BABA) is a proven inducer of plant defense against pathogens. This work examines its effect on the production of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures, both separately and in combination with a yeast elicitor (YE, the carbohydrate fraction of yeast extract). In the absence of YE, BABA at 0.1, 1 and 2 mM caused a dose-dependent enhancement of tanshinone accumulation, with up to a 4.5-fold increase (from 0.24 to 1.09 mg/g DW) in total content of three major tanshinones (cryptotanshinone, tanshinone I and tanshinone IIA) in the hairy roots. The combination of BABA with YE treatment further enhanced tanshinone production, but only when the BABA treatment was applied to the culture a few days before the YE treatment. Compared with methyl jasmonate, BABA was more effective in enhancing tanshinone production. A 3-day pretreatment with 1 mM BABA followed by YE-treatment, increased the total tanshinone content of roots by 9.4 times to 2.26 mg/g cells, and the volumetric tanshinone yield of culture by 6.3 times (from 3.2 to 20.1 mg/l). The results suggest that BABA can strongly potentiate elicitor-induced secondary metabolism in plant tissue cultures.

Optimization of extracellular production of recombinant asparaginase in Escherichia coli in shake-flask and bioreactor by Amardeep Khushoo; Yogender Pal; K. J. Mukherjee (pp. 189-197).

Various host–vector combinations were tested to maximize the extracellular production of recombinant asparaginase in Escherichia coli. Expression of recombinant asparaginase fused to pelB leader sequence under the inducible T7lac promoter in BLR (DE3) host cells resulted in optimum extracellular production in shake-flasks. Fed-batch studies were carried out using this recombinant strain and an exponential feeding strategy was used to maintain a specific growth rate of 0.3 h⁻¹. To check the effect of the time of induction on expression, cultures were induced with 1 mM isopropyl-β-D-thiogalactopyranoside at varying cell optical densities (OD₆₀₀: 33, 60, 90, 135). Although the specific product formation rates declined with increasing OD of induction, a maximum volumetric activity of 8.7×10⁵ units l⁻¹, corresponding to ∼5.24 g l⁻¹ of recombinant asparaginase, was obtained when induction was done at an OD₆₀₀ of 90. The recombinant protein was purified directly from the culture medium, using a rapid two-step purification strategy, which resulted in a recovery of ∼70% and a specific activity of ∼80% of that of the native enzyme.

Chlorogenate hydrolase-catalyzed synthesis of hydroxycinnamic acid ester derivatives by transesterification, substitution of bromine, and condensation reactions by N. Kishimoto; Y. Kakino; K. Iwai; T. Fujita (pp. 198-202).

A chlorogenate hydrolase (EC 3.1.1.42) synthesized 2-phenylethyl caffeate (2-CAPE) from 5-chlorogenic acid (5-CQA) and 2-phenylethyl alcohol (2-PA) (by transesterification), from 5-CQA and 2-phenylethyl bromide (2-PBr) (by substitution of bromine), and from caffeic acid (CA) and 2-PA or 2-PBr (by condensation) as well as hydrolysis of 5-CQA. Some reaction conditions including pH, temperature, substrate and solvent concentrates, and reaction time were optimized for the production of 2-CAPE. A maximal molar yield of 50% was achieved by transesterification, 4.7% by substitution of bromine, and 13% by condensation. Among the parameters studied for optimization, the pH of the buffer solution and concentration of 2-PA or 2-PBr affected the production of 2-CAPE. The optimum pH for the hydrolysis reaction was within the neutral range (pH 6.5), whereas the residual three reactions were only catalyzed within the acidic range (pH 3.0–4.0). The optimum concentrations of 2-PA and 2-PBr for three reactions were 5–70 vol% and no 2-CAPE was produced in the 2-PA or 2-PBr solutions containing powdered enzyme. The enzyme may bind to the caffeoyl moiety of 5-CQA or CA to form an enzyme–substrate complex. It then catalyzes four different reactions corresponding to the reaction conditions.

A novel lipase/acyltransferase from the yeast Candida albicans: expression and characterisation of the recombinant enzyme by Jean Louis Roustan; Agustin Rascon Chu; Guy Moulin; Frédéric Bigey (pp. 203-212).

A gene encoding an extracellular lipase (CaLIP4) from Candida albicans was successfully expressed in Saccharomyces cerevisiae after mutagenesis of its unusual CUG serine codon into a universal one. The ability of this lipase, which shares 60% sequence homology with the lipase/acyltransferase from Candida parapsilosis, to synthesise esters was investigated. CaLIP4 behaved as a true lipase, displaying activity towards insoluble triglycerides and having no activity in the presence of short-chain fatty acid (FA) esters and phosphatidylcholine. Methyl, ethyl and propyl esters were efficiently used. The lipase exhibited highest selectivity for unsaturated FA. With saturated FAs, C14–C16 acyl chains were preferred. In a biphasic aqueous/lipid system, CaLIP4 displayed a high alcoholysis activity with a range of alcohols (e.g. methanol, ethanol, propanol and isopropanol) as acyl acceptor. During the course of the alcoholysis reaction, new esters are produced at concentrations above the thermodynamic equilibrium of the esterification reaction, indicating that ester synthesis does not proceed by esterification but mainly by direct acyltransfer. Ester synthesis is under kinetic control due to the high rate of alcoholysis. Unwanted hydrolysis is limited by competition between the acyl acceptor (alcohol) and water for the acyltransfer reaction, favouring the alcohol.

Cloning and expression of a trehalose synthase from Pseudomonas stutzeri CJ38 in Escherichia coli for the production of trehalose by Jin-Ho Lee; Kwang-Ho Lee; Chang-Gyeom Kim; Se-Young Lee; Geun-Joong Kim; Young-Hoon Park; Sung-Oh Chung (pp. 213-219).

A novel strain was isolated, Pseudomonas stutzeri CJ38, that enabled direct transformation of maltose to trehalose. In comparison with others reported to date, CJ38 provided a novel trehalose synthase (TSase) without any byproduct, including glucose. Activity analysis, using either maltose or trehalose as a substrate, showed a reversible reaction. There was also no detectable activity of related enzymes with liquid starch and maltooligosaccharides as substrates. Using a malPQ-negative host and MacConkey medium, the TSase gene was cloned in Escherichia coli from CJ38. The resulting sequence contained an open reading frame consisted of 689 amino acids with a calculated molecular mass of 76 kDa. A search for related sequences in various gene and protein data banks revealed a novel family of enzymes that was predicted putatively as a glycosidase or TSase family, with no biochemical evidence. The recombinant enzyme exhibited a high activity toward the substrate maltose, about 50-fold higher than the parent strain and resulted in a high conversion yield (72%) at a relatively high substrate concentration (20%). These results provided the possibility that the strain was effectively used as a potential biocatalyst for the production of trehalose from maltose in a one-step reaction.

Expression of SARS-coronavirus nucleocapsid protein in Escherichia coli and Lactococcus lactis for serodiagnosis and mucosal vaccination by Huadong Pei; Jingfang Liu; Yun Cheng; Chaomin Sun; Chen Wang; Yueping Lu; Jie Ding; Jian Zhou; Hua Xiang (pp. 220-227).

The nucleocapsid (N) protein of the severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) is an important antigen for the early diagnosis of SARS and the development of vaccines. It was expressed in Escherichia coli as a fusion with human glutathione S-transferase (hGST) and was confirmed by Western blotting analysis. This recombinant N protein (hGST-N) was purified and used to measure the SARS-CoV N-specific antibody in the sera of eight SARS patients by enzyme-linked immunosorbent assay. Specific antibody response to this purified recombinant N protein was 100% positive in the SARS patients’ sera, while none of the control sera from 30 healthy people gave a positive reaction in the same assay. The SARS-CoV N protein was also expressed in Lactococcus lactis in the cytoplasm or secreted into the medium. The N-producing strain MG1363/pSECN and the purified hGST-N protein were respectively administered to mice, either orally or intranasally. Results indicated that orally delivered MG1363/pSECN induced significant N-specific IgG in the sera. In conclusion, our work provides a novel strategy to produce the SARS-CoV N protein for serodiagnosis and for L. lactis-based mucosal vaccines.

Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum by Jörg Mampel; Hartwig Schröder; Stefan Haefner; Uwe Sauer (pp. 228-236).

Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite d,l-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of l-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.

Comparison of heterotrophic and photoautotrophic induction on astaxanthin production by Haematococcus pluvialis by C. D. Kang; J. S. Lee; T. H. Park; S. J. Sim (pp. 237-241).

During light induction for astaxanthin formation in Haematococcus pluvialis, we substituted photoautotrophic induction for heterotrophic induction using acetate, both to prevent contamination by heterotrophs due to addition of organic carbon and to enhance carbon assimilation in the induced cells. Strong photoautotrophic induction was performed by N-deprivation of photoautotrophically grown Haematococcus cells followed by supplementation with bicarbonate (HCO₃⁻) or CO₂. Bicarbonate-induced cells contained more astaxanthin than acetate-induced cells, and even further enhancement of astaxanthin accumulation was achieved by continuous CO₂ supply. The maximum astaxanthin content (77.2 mg g⁻¹ biomass, 3.4-fold higher than with heterotrophic induction) was obtained under conditions of 5% CO₂, yielding astaxanthin concentration and productivity of 175.7 mg l⁻¹ and 6.25 mg l⁻¹ day⁻¹, respectively. The results indicate that photoautotrophic induction is more effective than heterotrophic induction for astaxanthin synthesis in H. pluvialis.

SspA, an outer membrane protein, is highly induced under salt-stressed conditions and is essential for growth under salt-stressed aerobic conditions in Rhodobacter sphaeroides f. sp. denitrificans by M. Tsuzuki; XY. Xu; K. Sato; M. Abo; M. Arioka; H. Nakajima; K. Kitamoto; A. Okubo (pp. 242-250).

We have previously shown that an outer membrane protein, SspA, is prominently induced by salt stress in a photosynthetic bacterium, Rhodobacter sphaeroides f. sp. denitrificans IL106 (R. sphaeroides). In this study, we investigated the physiological role of SspA under various stress conditions. Using recombinant SspA expressed in Escherichia coli as an antigen, the polyclonal antiserum of SspA was prepared. Western blot analysis demonstrated that SspA was highly induced by salt stress under both anaerobic and aerobic conditions. SspA was also induced, but to a lesser extent, by osmotic and acid stress. It is reduced under heat and cold compared to non-stressed conditions. While sspA-disrupted R. sphaeroides grew normally under anaerobic conditions in either the presence or absence of stress, it displayed significantly retarded growth under aerobic conditions in the dark, especially when osmotic or salt stress were imposed. In addition, the sspA disruptant, but not the wild type, formed cell aggregates when grown under both anaerobic and aerobic conditions, and this phenotype was significantly enhanced under salt-stressed aerobic conditions. Together, our findings suggest that SspA is critical under salt-stressed, aerobic growth conditions.

Oligomeric compounds formed from 2,5-xylidine (2,5-dimethylaniline) are potent enhancers of laccase production in Trametes versicolor ATCC 32745 by Albert Kollmann; François-Didier Boyer; Paul-Henri Ducrot; Lucien Kerhoas; Claude Jolivalt; Isabelle Touton; Jacques Einhorn; Christian Mougin (pp. 251-258).

Numerous chemicals, including the xenobiotic 2,5-xylidine, are known to induce laccase production in fungi. The present study was conducted to determine whether the metabolites formed from 2,5-xylidine by fungi could enhance laccase activity. We used purified laccases to transform the chemical and then we separated the metabolites, identified their chemical structure and assayed their effect on enzyme activity in liquid cultures of Trametes. versicolor. We identified 13 oligomers formed from 2,5-xylidine. (4E)-4-(2,5-dimethylphenylimino)-2,5-dimethylcyclohexa-2,5-dienone at 1.25×10⁻⁵ M was an efficient inducer, resulting in a nine-fold increase of laccase activity after 3 days of culture. Easily synthesized in one step (67% yield), this compound could be used in fungal bioreactors to obtain a great amount of laccases for biochemical or biotechnological purposes, with a low amount of inducer.

Exopolysaccharide biosynthesis by Lactobacillus helveticus ATCC 15807 by M. I. Torino; F. Mozzi; G. Font de Valdez (pp. 259-265).

Exopolysaccharide (EPS) production and the activities of the enzymes involved in sugar nucleotide biosynthesis in Lactobacillus helveticus ATCC 15807 under controlled pH conditions were investigated. Batch fermentations using lactose as energy source showed higher EPS synthesis by L. helveticus ATCC 15807 at pH 4.5 with respect to pH 6.2, the enzyme α-phosphoglucomutase (α-PGM) being correlated with both total and specific EPS production. When glucose was used as carbon source instead of lactose, the lower EPS synthesis obtained was linked to a decrease in α-PGM and galactose 1-phosphate-uridyltransferase (GalT) activities, the reduction of the latter being more pronounced. Higher EPS production by L. helveticus ATCC 15807 at the acidic constant pH of 4.5 requires that both α-PGM and GalT activities are high. These enzymes are needed to synthesize UDP-glucose and UDP-galactose for supplying the corresponding monomers for EPS biosynthesis. Although differences are observed in EPS production by this strain regarding the energy source (lactose or glucose), the monomeric composition of the polymers produced is independent of the carbohydrate used. The obtained results contribute to a better understanding of the physiological factors that affect EPS biosynthesis by lactobacilli, which could help in the correct handling of the fermentation parameters within the fermented dairy industry.

Fine measurement of ergosterol requirements for growth of Saccharomyces cerevisiae during alcoholic fermentation by Christelle Deytieux; Ludivine Mussard; Marie-José Biron; Jean-Michel Salmon (pp. 266-271).

Yeasts can incorporate a wide variety of exogenous sterols under strict anaerobiosis. Yeasts normally require oxygen for growth when exogenous sterols are limiting, as this favours the synthesis of lipids (sterols and unsaturated fatty acids). Although much is known about the oxygen requirements of yeasts during anaerobic growth, little is known about their exact sterol requirements in such conditions. We developed a method to determine the amount of ergosterol required for the growth of several yeast strains. We found that pre-cultured yeast strains all contained similar amounts of stored sterols, but exhibited different ergosterol assimilation efficiencies in enological conditions [as measured by the ergosterol concentration required to sustain half the number of generations attributed to ergosterol assimilation (P₅₀)]. P₅₀ was correlated with the intensity of sterol synthesis. Active dry yeasts (ADYs) contained less stored sterols than their pre-cultured counterparts and displayed very different ergosterol assimilation efficiencies. We showed that five different batches of the same industrial Saccharomyces cerevisiae ADY exhibited significantly different ergosterol requirements for growth. These differences were mainly attributed to differences in initial sterol reserves. The method described here can therefore be used to quantify indirectly the sterol synthesis abilities of yeast strains and to estimate the size of sterol reserves.

Corrosion risk associated with microbial souring control using nitrate or nitrite by Casey Hubert; Mehdi Nemati; Gary Jenneman; Gerrit Voordouw (pp. 272-282).

Souring, the production of hydrogen sulfide by sulfate-reducing bacteria (SRB) in oil reservoirs, can be controlled through nitrate or nitrite addition. To assess the effects of this containment approach on corrosion, metal coupons were installed in up-flow packed-bed bioreactors fed with medium containing 8 mM sulfate and 25 mM lactate. Following inoculation with produced water to establish biogenic H₂S production, some bioreactors were treated with 17.5 mM nitrate or up to 20 mM nitrite, eliminating souring. Corrosion rates were highest near the outlet of untreated bioreactors (up to 0.4 mm year⁻¹). Nitrate (17.5 mM) eliminated sulfide but gave pitting corrosion near the inlet of the bioreactor, whereas a high nitrite dose (20 mM) completely eliminated microbial activity and associated corrosion. More gradual, step-wise addition of nitrite up to 20 mM resulted in the retention of microbial activity and localized pitting corrosion, especially near the bioreactor inlet. We conclude that: (1) SRB control by nitrate or nitrite reduction shifts the corrosion risk from the bioreactor outlet to the inlet (i.e. from production to injection wells) and (2) souring treatment by continuous addition of a high inhibitory nitrite dose is preferable from a corrosion-prevention point of view.

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Applied Microbiology and Biotechnology (v.68, #2)