Expanding the genetic code of Saccharomyces cerevisiae with methionine analogues
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Abstract We replaced the single N‐terminal methionine in heterologously expressed human Cu/Zn superoxide dismutase with the non‐canonical methionine analogues homopropargylglycine and norleucine in the yeast Saccharomyces cerevisiae . Our non‐canonical amino acid incorporation protocol involves a two‐step procedure. In the first step, the methionine auxotrophic yeast cells are accumulated in synthetic medium containing methionine while the target protein production is shut off. After a short methionine depletion phase, the cells are transferred to inducing medium that contains the methionine analogue instead of methionine and target protein expression is switched on. The initially low level incorporation of ∼12% could be elevated to 40% by increasing the non‐canonical amino acid concentration in the medium by 10‐fold. With this approach we were able to produce up to 5 mg substituted protein per litre of yeast culture. Copyright © 2008 John Wiley & Sons, Ltd.Keywords:
Auxotrophy
Norleucine
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Cells of the yeast Saccharomyces cerevisiae are transformable by DNA under non‐artificial conditions
Transformants of bakers' yeast (Saccharomyces cerevisiae) can be generated when non-growing cells metabolize sugars (without additional nutrients) in the presence of plasmid DNA. These results suggest that there is a mechanism by which DNA can naturally be taken up by the yeast cell. Natural transformation does not take place in common complete or minimal yeast culture media such as YPD and YNB. The starvation conditions used in our experiments thus seem to be an important prerequisite for such transformation events. Copyright © 2000 John Wiley & Sons, Ltd.
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ABSTRACT Estimating availability of methionine is relevant to feed formulation since diets can be supplemented with crystalline methionine to meet the minimum requirements of rapidly growing birds. Bacterial assays have been developed to measure the bioavailable levels of several essential amino acids in feeds, including methionine. The E. coli methionine auxotroph strain used in this study exhibited a linear extent of growth response to increasing concentrations of methionine added to the minimal test media, in the range of 0 to 4 μg/mL. In addition the growth rates of the E. coli auxotroph were significantly (P < 0.01) different when the methionine concentrations were varied (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 μg/mL) in minimal media. To assay feeds, feed grade methionine was added to poultry feed mixtures and samples were diluted with M9 media. Using this assay for estimating crystalline methionine added to feed, the extent of growth of the methionine auxotroph was correlated with the levels of crystalline methionine supplemented in the feed (R 2 = 0.9873). For all supplementation levels methionine recovery percentages ranged from 71 to 80% indicating that the bacterial assay response to crystalline methionine was relatively constant in the presence of the feed matrix. The overall results indicate that the rapid detection of crystalline methionine added to feeds is possible using this E. coli methionine auxotroph growth‐based assay.
Auxotrophy
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Norleucine
Ethionine
Methionine sulfoxide
Alanine
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One of the key feature of prions is the ability to be stable in two alternative conformations. Besides the intensively studied mammalian prions, there are also prion proteins present in the yeast Saccharomyces cerevisiae. Research in this field has lead to opposite hypotheses that explain the sense of presence of [PSI+] prion in yeast cells. Some authors postulate e of role of the prions in the evolution of S. cerevisiae, whereas other investigators point out the negative influence of these particles upon the yeast physiology. In recent years, yeast prions are used for anti-prion drug screening, because of common features with mammalian prions. This work presents the most intensively studied fields of the research carried out on [PSI+] prion in yeast.
Prion Proteins
Fungal prion
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We have isolated cDNAs encoding human myristoyl-CoA:protein N-myristoyltransferase (NMT, EC 2.3.1.97) by complementing the nmtl-181 mutation of Saccharomyces cerevisiae, which causes temperature-sensitive myristic acid auxotrophy. Human NMT is derived from a single-copy gene, contains 416 amino acids, is 44% identical to S. cerevisiae NMT (yeast NMT), and can complement the lethal phenotype of an nmtl null mutation. Human and yeast NMTs have overlapping yet distinct protein substrate specificities as judged by a coexpression system that reconstitutes protein N-myristoylation in Escherichia coli. Both enzymes contain a glycine five residues from the C terminus. Gly----Asp or Lys mutagenesis in these orthologous NMTs produces marked reductions in their activities in E. coli as well as temperature-sensitive myristic acid auxotrophy in S. cerevisiae. These results indicate highly conserved structure-function relationships in vivo and underscore the usefulness of these functional assays for identifying factors that regulate protein N-myristoylation in mammalian systems.
Auxotrophy
Myristic acid
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Methionine auxotrophic mutant for the bioassay of methionine and vitamine was selected from Escherichia coli by U.V. irradiation, and the physiological characteristics of the mutant were investigated. The mutant was grown in proportion to the concentration of methionine in minimal medium up to 100 ng/ml methionine. However, any amino acids and any intermediates related in methionine biosynthesis were not effective on the mutant cell growth. The mutant in the minimal medium containing 40 ng/ml methionine and 1g/ml folic acid, was grown in proportion to the range from 0 to 400 pg/ml vitamin
Auxotrophy
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Ethyl methanesulfonate
Methyl methanesulfonate
Strain (injury)
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Methionine sulfoxide
Ethionine
Methionine synthase
Norleucine
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Control of methionine biosynthesis in Escherichia coli K12 was reinvestigated by using methionine-analogue-resistant mutants. Norleucine (NL) and a-methylmethionine (MM) were found to inhibit methionine biosynthesis directly whereas ethionine (Et) competitively inhibited methionine utilization. Adenosylation of Et to generate S-adenosylethionine (AdoEt) by cell-free enzyme from E. coli K12 was demonstrated. Tolerance of increasing concentrations of NL by E. coli K12 mutants is expressed serially as phenotypes NLR, NLREtR, NLRMMR and finally NLREtRMMR. All spontaneous NLR mutants had a metK mutation, whereas NTG-induced mutants had mutations in both the metK and metJ genes. The kinetics of methionine adenosylation by the E. coli K12 cell-free enzyme were found to be similar to those reported for the yeast enzyme, showing the typical lag phase at low methionine concentration and disappearance of this phase when AdoMet was included in the incubation mixture. NL extended the lag phase, and lowered the rate of subsequent methionine adenosylation, but did not affect the shortening of the lag phase of adenosylation by AdoMet.
Ethionine
Norleucine
Methionine synthase
Methionine Adenosyltransferase
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