Effect of squalene on astaxanthin production by a mutant of Phaffia rhodozyma

The effect of different sesquiterpenes on carotenoid synthesis in Phaffia rhodozyma was studied. Addition of squalene to the culture medium resulted in a decrease in the echinenone and trans-astaxanthin concentrations, whereas -carotene remained unchanged. The role of squalene as an inhibitor of ketocarotenoid synthesis in Ph. rhodozyma is discussed.     

Astaxanthin production by a Phaffia rhodozyma mutant on grape juice

During fermenter cultivation of Phaffia rhodozyma on a grape juice medium, the presence of glucose initially delayed fructose utilization, although fructose was consumed before glucose depletion. Total pigment and astaxanthin production were growth associated and reached maximum values of 15.9 g/ml and 9.8 g/ml, respectively, after depletion of the carbon source. The total cellular pigment and astaxanthin content increased during the stationary growth phase due to a decrease in biomass, reaching final values of 2120 g/g and 1350 g/g, respectively, without the volumetric concentration in the culture changing. The final cell yield was 0.33 g/g sugar utilized. High sugar concentrations in shake-flasks as well as O₂ limitation decreased the astaxanthin content of the cells. Addition of yeast extract to a grape juice minimal medium markedly increased the maximum specific growth rate, total pigment and astaxanthin content of the cells. An excess of ammonia decreased the intracellular astaxanthin content, which reached a maximal value in cultures with no residual glucose or ammonia.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa;     

Photoisomerization of lycopene during carotenogenesis in mutant C-6D of Scenedesmus obliquus

Mutant C-6D of the unicellular green alga Scenedesmus obliquus has lost the ability to form cyclic carotenoids during heterotrophic growth in the dark. In the dark it accumulates acyclic intermediates, i.e. lycopene, neurosporene and ζ-carotene. The lycopene and two neurosporene forms were identified to be cis-isomers. Upon transfer to light, intermediates decrease and a normal set of carotenoids is synthesized. Inhibition of the cyclization reaction by nicotine reveals a lightinduced isomerization of cis-lycopene to trans-lycopene. Since the spectral characteristics of these two isomers differ drastically the isomerization can be followed in vivo by measuring a light-induced absorbance change. This absorbance change has its maximum at 520 nm and shows fast kinetics under high light intensities reaching a saturation level after about 2 min. Fluence-response curves for this absorbance change were performed for different wavelengths of actinic light. From the linear parts of these curves an action spectrum was caculated showing maxima at 670, 630 and 440 nm originating from chlorophyll and a maximum at shorter wavelengths (400–510 nm) which is interpreted to derive from ζ-carotene. A model for the light regulation of carotenogenesis in mutant C-6D is presented and the relation to the so-called 520-change observed in many plants is discussed.     

Purification and characterization of Mycobacterium tuberculosis KatG, KatG(S315T), and Mycobacterium bovis KatG(R463L)

Isoniazid, a first-line antibiotic used for the treatment of tuberculosis, is a prodrug that requires activation by the Mycobacterium tuberculosis enzyme KatG. The KatG(S315T) mutation causes isoniazid resistance while the KatG(R463L) variation is thought to be a polymorphism. Much of the work to date focused on isoniazid activation by KatG has utilized recombinant enzyme overexpressed in Escherichia coli. In this work, native KatG and KatG(S315T) were purified from M. tuberculosis, and KatG(R463L) was purified from Mycobacterium bovis. The native molecular weight, enzymatic activity, optical, resonance Raman, and EPR spectra, K(D) for isoniazid binding, and isoniazid oxidation rates were measured and compared for each native enzyme. Further, the properties of the native enzymes were compared and contrasted with those reported for recombinant KatG, KatG(S315T), and KatG(R463L) in order to assess the ability of the recombinant enzymes to act as good models for the native enzymes.     

挖掘与调控乙酸胁迫响应基因提高重组酿酒酵母合成番茄红素水平

【背景】乙酰辅酶A是酿酒酵母异源合成番茄红素的重要中间产物,胞质中乙酰辅酶A主要来自乙酰辅酶A合成酶催化乙酸合成。【目的】通过外源添加乙酸盐结合调控乙酸胁迫应答基因增加胞内乙酰辅酶A含量,改善细胞生长,促进番茄红素合成。【方法】在合成番茄红素的重组酵母菌中过表达乙酰辅酶A合成酶编码基因(acs2),在发酵过程中添加10g/L乙酸盐,结合转录组学分析挖掘乙酸胁迫响应基因,进行单一和组合调控。【结果】添加乙酸盐后,重组菌Y02中番茄红素含量增加了19.14%,但细胞生长受到抑制,转录组学结果表明adk2、fap7、hem13、elo3、pdc5、set5、pmt5、hst4、clb2和swe1表达水平增加,因此构建了单基因和双基因过表达菌株,其中Y02-set5-hst4菌在添加乙酸盐后细胞生长得到了显著改善,同时胞内乙酰辅酶A浓度提高了78.21%,番茄红素含量和产量达到12.62 mg/g-DCW和108.67 mg/L,与对照菌Y02相比分别提高了42.76%和67.13%。同时该菌中甲羟戊酸途径中关键基因erg12、erg20和hmg1的表达量与对照菌相比分别上调了1.70、1.4...     

Turnover of lipids in Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354

The rates of breakdown and renewal of individual lipids in cultures of Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354 were investigated by means of a pulse labelling technique using palmitate-1-¹⁴C. The results indicated that in growing cultures of both strains phospholipids were broken down, and cardiolipin had a very rapid turnover. In chase experiments, almost 45% and 40% of the radioactivity of this component were lost respectively from M. smegmatis and M. phlei during one generation time of the cell. The other two major components, phosphatidyl ethanolamine and phosphatidylinositol mannosides showed relatively low turnover. The loss of radioactivity from phosphatidylinositol mannosides was greater in M. phlei than in M. smegmatis but the loss of radioactivity from phosphatidyl ethanolamine was higher in M. smegmatis. The pattern of loss of radioactivity from lipids was almost the same in both strains, the difference being only in the extent of loss. The differences in the cellular localization of the phospholipids indicate their different roles within the cell. Results obtained with the glyceride fraction indicated a very rapid turnover of triglycerides in both strains.Abbreviations CL Cardiolipin - PE Phosphatidyl ethanolamine - PIMx phosphatidylinositol mannosides - PIM₂A phosphatidylinositol dimannoside tetra acylated - PIM₂B phosphatidylinositol dimannoside tri acylated - PIM₅ phosphatidylinositol pentamannoside tetra acylated     

Functional insights by comparison of modeled structures of 18kDa small heat shock protein and its mutant in Mycobacterium leprae

In this work we are proposing Homology modeled structures of Mycobacterium leprae 18kDa heat shock protein and its mutant. The more closely related structure of the small heat shock protein (sHSP) belonging to the eukaryotic species from wheat sHSP16.9 and 16.3kDa ACR1 protein from Mycobacterium tuberculosis were used as template structures. Each model contains an N-terminal domain, alpha-crystalline domain and a C-terminal tail. The models showed that a single point mutation from serine to proline at 52^nd position causes structural changes. The structural changes are observed in N-terminal region and alpha-crystalline domains. Serine in 52^nd position is observed in β4 strand and Proline in 52^nd position is observed in loop. The number of residues contributing α helix at N-terminal region varies in both models. In 18S more number of residues is present in α helix when compared to 18P. The loop regions between β3 and β4 strands of both models vary in number of residues present in it. Number of residues contributing β4 strand in both models vary. β6 strand is absent in both models. Major functional peptide region of alpha crystalline domains of both models varies. These differences observed in secondary structures support their distinct functional roles. It also emphasizes that a point mutation can cause structural variation.     

Copper but not iron limitation increases astaxanthin production by Phaffia rhodozyma in a chemically defined medium

The influence of copper (0–32 M) and iron (0–108 M) on growth and astaxanthin production by Phaffia rhodozyma was studied. Copper below 3.2 M increased the astaxanthin content of the cells (from 220 to 287 g g^–1) but at the expense of a slightly decreased growth (from 11.3 to 10.2 mg ml^–1). In contrast, iron below 1 M decreased both the growth and astaxanthin content of the cells. Using copper limitation instead of toxic respiratory inhibitors to improve astaxanthin production has obvious advantages from the product quality, environmental and process operation points of view.     

An orphan gyrB in the Mycobacterium smegmatis genome uncovered by comparative genomics

DNA gyrase is an essential topoisomerase found in all bacteria. It is encoded bygyrB andgyrA genes. These genes are organized differently in different bacteria. Direct comparison ofMycobacterium tuberculosis andMycobacterium smegmatis genomes reveals presence of an additionalgyrB inM. smegmatis flanked by novel genes. Analysis of the amino acid sequence of GyrB from different organisms suggests that the orphan GyrB inM. smegmatis may have an important cellular role.     

VNTR analysis differentiates Mycobacterium ulcerans and IS2404 positive mycobacteria

In recent years, numerous IS2404 positive mycobacteria have been identified, compromising the detection of Mycobacterium ulcerans. In this study, variable number of tandem repeats (VNTR) analysis was successfully applied on cultures and tissue specimens to differentiate all currently known IS2404 positive mycobacteria from M. ulcerans.     

Mathematical Modelling of Mycobacterium tuberculosis VNTR Loci Estimates a Very Slow Mutation Rate for the Repeats

Minisatellites are highly variable tandem repeats used for over 20 years in humans for DNA fingerprinting. In prokaryotes fingerprinting techniques exploiting VNTR (variable number of tandem repeats) polymorphisms have become widely used recently in bacterial typing. However although many investigations into the mechanisms underlying minisatellite variation in humans have been performed, relatively little is known about the processes that mediate bacterial minisatellite polymorphism. An understanding of this is important since it will influence how the results from VNTR experiments are interpreted. The minisatellites of Mycobacterium tuberculosis are well characterized since they are some of the few polymorphic loci in what is otherwise a very homogeneous organism. Using VNTR results from a well-defined and characterized set of M. tuberculosis strains we show that the repeats at a locus are likely to evolve by stepwise contraction or expansion in the number of repeats. A stochastic continuous-time population mathematical model was developed to simulate the evolution of the repeats. This allowed estimation of the tendency of the repeats to increase or decrease and the rate at which they change. The majority of loci tend to lose rather than gain repeats. All of the loci mutate extremely slowly, with an average rate of 2.3 x 10(-8), which is 350 times slower than that of a set of VNTR repeats with similar diversity observed experimentally in Escherichia coli. This suggests that the VNTR profile of a strain of M. tuberculosis will be indicative of its clonal lineage and will be unlikely to vary in epidemiologically-related strains.     

Evidence of Metyrapone Reduction by two Mycobacterium strains shown by 1H NMR

In situ 1H NMR monitoring of metyrapone incubations with resting-cells of two strains of Mycobacterium, Mycobacterium aurum MO1 and Mycobacterium sp. RP1, showed the biotransformation of this compound, and more precisely the carbonyl-reduction of metyrapone into the corresponding alcohol, metyrapol. This reduction produced both enantiomers. The use of inhibitors allowed us to show the multiple enzymatic activities involved in this biotransformation including carbonyl reductase (EC 1.1.1.1.84) from the short-chain dehydrogenase superfamily and aldehyde reductase (EC 1.1.1.2) from the aldo-keto reductase superfamily.     

The production of a new extracellular putative long-chain saturated polyester by smooth variants of Mycobacterium vaccae interferes with Th1-cytokine production

Mycobacterium vaccae is of major pharmaceutical interest as an immunotherapeutic agent. Although M. vaccae 15483 ATCC(T) strain displays smooth and rough colonial morphologies on solid culture media, it is not known in which conditions M. vaccae switches from one colonial morphotype to the other or whether there are biological differences, especially immunological, between them. We have found that the change from a smooth to rough stable variant occurs spontaneously at 30 degrees C. The analysis of the composition of the cell wall in both variants showed that the smooth morphotype presents an extracellular material that has never previously been described and was identified as a long-chain saturated polyester that, interestingly, is not produced by the rough morphotype. Our results also indicate that this compound could be implicated in the spreading ability of smooth colonies. Proliferation, IFN-gamma and IL-12(p40) production by splenocyte cultures was significantly higher in mice immunised with the rough variant compared with those immunised with the smooth one. This latter finding suggests that the different colonial morphology of M. vaccae may affect the immunomodulatory effects induced from M. vaccae preparations.     

Superoxide dismutase from Mycobacterium species,strain Takeo

Superoxide dismutase from Mycobacterium species, strain Takeo, has been purified to homogeneity as judged by disc gel electrophoresis and ultracentrifugation. The enzyme was found to have a molecular weight of approximately 61 500 by sedimentation equilibrium and to contain manganese by atomic absorption and electron spin resonance spectra. The amino acid composition was also determined. The enzyme was considerably stable to the treatment with sodium dodecyl sulfate; unless incubating at 80°C for 2 min, it was not completely dissociated into the subunits. The molecular weight of the subunit was found to be approximately 21 000. Antibodies against the superoxide dismutase were produced by immunization of rabbits with the enzyme, and the -globulin fraction was purified. Superoxide dismutase preparations obtained from various species of mycobacteria and nocardia cross-reacted to different degrees with these antibodies on the Ouchterlony double diffusion plates. Comparative immunological studies indicated that strain Takeo might be most closely related to Myobacterium smegmatis among species of mycobacteria and nocardia tested. The antibodies against superoxide dismutase may be used as a valuable tool for the classification of mycobacteria.     

Sake brewing characteristics of a new type of cerulenin-resistant sake yeast mutant

Several new types of cerulenin-resistant mutants of sake yeast were isolated. These mutants showed respiratory deficiency and could grow on media containing a higher concentration of antibiotics than could the parent. Sakes brewed by the mutants produced less succinate than by both the parent yeast and the mutants with respiratory deficiency induced by ethidium bromide. In addition, the acidity of these mutants was decreased. Since low acidity is favourable in both sake and wine, these mutants might be applicable for both sake and wine brewing.     

Novel DNA glycosylases from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Oxidized bases are removed from DNA of Escherichia coli by enzymes formamidopyrimidine DNA glycosylase (Eco-Fpg) and endonuclease VIII (Eco-Nei) of the same structural family Fpg/Nei. New homologs of these enzymes not characterized earlier have been found in genomes of Actinobacteria. We have cloned and expressed two paralogs (Mtu-Nei2 and Mtu-Fpg2) from 36KAZ and KHA94 isolates of Mycobacterium tuberculosis and studied their ability to participate in DNA repair. Under heterologous expression in E. coli, Mtu-Nei2 decreased the rate of spontaneous mutagenesis in the rpoB gene, whereas Mtu-Fpg2 moderately increased it, possibly due to absence of residues crucially important for catalysis in this protein. Mtu-Nei2 was highly active toward double-stranded DNA substrates containing dihydrouracil residues and apurine-apyrimidine sites and was less efficient in cleavage of substrates containing 8-oxoguanine and uracil residues. These lesions, as well as 8-oxoadenine residues, were also recognized and removed by the enzyme from single-stranded DNA. Fpg and Nei homologs from M. tuberculosis can play an important role in protection of bacteria against genotoxic stress caused by oxidative burst in macrophages.     

Stimulation of the growth and solamargine production by Solanum paludosum multiple shoot cultures using a new culture medium

Organogenic callus cultures of Solanum paludosum were obtained from root, hypocotyle and cotyledon explants of plantlets cultured in sterile conditions. These callus cultures developed multiple shoots which proliferated in Murashige and Skoog basal liquid medium. These multiple shoots produced solamargine, the main steroidal glycoalkaloid present in the unripe fruits.The optimization of the macronutrient composition of the liquid medium was performed by a method derived from the plant composition. This approach results in the establishment of an appropriate medium (SPOM medium) suitable for the improvement of both growth and solamargine production by multiple shoot cultures of S. paludosum.     

Development of fermentation process for PLA-degrading enzyme production by a new thermophilic Actinomadura sp. T16-1

The fermentation process for a poly (L-lactide) (PLA)-degrading enzyme production by a newly isolate of thermophilic PLA-degrading Actinomadura sp. T16-1 was investigated. The strain produced 33.9 U/mL of enzyme activity after cultivation at 50°C under shaking of 150 rpm for 96 h in a medium consisting of (w/v) 0.05% PLA film, 0.2% gelatin, 0.4% (NH₄)₂SO₄, 0.4% K₂HPO₄, 0.2 % KH₂PO₄, and 0.02% MgSO₄ · 7H₂O. The optimal concentration of PLA film and gelatin obtained by response surface methodology (RSM) for the highest production of PLA-degrading enzyme was 0.035% (w/v) and 0.238% (w/v), respectively. Under these conditions, the model predicted 40.4 U/mL of PLA-degrading activity and the verification of the optimization showed 44.6 U/mL of PLA-degrading enzymatic activity in the flasks experiment. The maximum PLA-degrading activity reached 150 U/mL within 72 h cultivation in the 3-L airlift fermenter.     

Oxalic acid production from lipids by a mutant of Aspergillus niger at different pH

Crude rapeseed oil and post-refining fatty acids were used as substrates for oxalic acid production by a mutant of Aspergillus niger. Both the final concentration and the yield of the product were highest at pH 4 to 5. With a medium containing 50 g lipids l^–1, production reached a maximum of 68 g oxalic acid l^–1 after 7 d. A high yield of the product (up to 1.4 g oxalic acid g^–1 lipids consumed) was achieved with oil and fatty acids combined.     

Expression and characterization of Rv2430c, a novel immunodominant antigen of Mycobacterium tuberculosis

About 10% of the coding sequence of Mycobacterium tuberculosis corresponds to hitherto unknown members of the PE and PPE protein families which display significant sequence and length variation at their C-terminal region. It has been suggested that this could possibly represent a rich source of antigenic variation within the pathogen. We describe the purification and biophysical characterization of the recombinant PPE protein coded by hypothetical ORF Rv2430c, a member of the PPE gene family that was earlier shown to induce a strong B cell response. Expression of the recombinant PPE protein in Escherichia coli led to its localization in inclusion bodies and subsequent refolding using dialysis after its extraction from the same resulted in extensive precipitation. Therefore, an on-column refolding strategy was used, after which the protein was found to be in the soluble form. CD spectrum of the recombinant protein displayed predominantly alpha helical content (81%) which matched significantly with in silico and web-based secondary structure predictions. Furthermore, fluorescence emission spectra revealed that aromatic amino acids are buried inside the protein, which are exposed to aqueous environment under 8M urea. These results, for the first time, provide evidence on the structural features of PPE family protein which, viewed with its reported immunodominant characteristics, have implications for other proteins of the PE/PPE family.