Cytosolic NADPH balancing in Penicillium chrysogenum cultivated on mixtures of glucose and ethanol

The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary 13C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied. This indicates that the cytosolic NADPH supply is independent of the amount of assimilated ethanol. The cofactor assignment in the model of van Gulik et al. (Biotechnol Bioeng 68(6):602–618, 2000) was supported using the published genome annotation of P. chrysogenum. Metabolic flux analysis showed that NADPH requirements in the cytosol remain nearly the same in these experiments due to constant biomass growth. Based on the cytosolic NADPH balance, it is known that the cytosolic aldehyde dehydrogenase in P. chrysogenum is NAD +  dependent. Metabolic modeling shows that changing the NAD + -aldehyde dehydrogenase to NADP + -aldehyde dehydrogenase can increase the penicillin yield on substrate

NONSENSE MUTATION IN THE REGULATORY GENE <i>ETH</i>2 INVOLVED IN METHIONINE BIOSYNTHESIS IN <i>SACCHAROMYCES CEREVISIAE</i>

ABSTRACT Ethionine-resistant mutants, mapping at the locus eth2—the product of which is involved in pleiotropic regulation of methionine biosynthesis—have been isolated in a strain carrying five ochre nonsense mutations. Selection for nonsense suppressors in such a strain led to characterization of several allele-specific but gene non-specific suppressors which are active on the recessive heteroallele eth2–2 (resulting in partial recovery of sensitivity toward ethionine) as well as on the five other suppressible alleles. Two of these suppressors are unlinked to the eth2 gene and either dominant or semi-dominant. It is concluded that the mutation eth2–2 resulted in a nonsense codon. Enzyme studies indicate that this mutation results in a complete absence of an active product of gene eth2, in contrast with the effect of a former mutation eth2–1 which was interpreted as leading to a modified product of this gene (Cherest, Surdin-Kerjan and de Robichon-Szulmajster 1971). This conclusion is based on the absence of repressibility of methionine group I enzymes and the observation that in a heteroallelic diploid, eth2–1 expression is not masked by eth2–2. The nonsense suppressors studied lead to at least partial recovery of repressibility of methionine group I enzymes. All these results support the idea that the product of gene ETH2 is an aporepressor protein.</jats:p

Existence of two levels of repression in the biosynthesis of methionine in Saccharomyces cerevisiae: effect of lomofungin on enzyme synthesis

Derepression of a methionine biosynthetic enzyme (homocysteine synthase) has been studied after repression either by exogenous methionine or by exogenous S-adenosylmethionine (SAM). Lomofungin, which inhibits the synthesis of ribosomal precursor and messenger ribonucleic acid but not of protein in Saccharomyces cerevisiae, has been used in this system. It has been shown that the addition of this antibiotic prevents the derepression of homocysteine synthase after repression by exogenous methionine but not after repression by exogenous SAM. These experiments with lomofungin and the kinetics of repression after addition of methionine or SAM to the growth medium provide evidence that the repression induced by exogenous methionine acts at the transcriptional level whereas the repression induced by exogenous SAM acts at the translational level.</jats:p

Genetic and Regulatory Aspects of Methionine Biosynthesis in <i>Saccharomyces cerevisiae</i>

Methionine biosynthesis and regulation of four enzymatic steps involved in this pathway were studied in Saccharomyces cerevisiae , in relation to genes concerned with resistance to ethionine ( eth 1 and eth 2 ). Data presented in this paper and others favor a scheme which excludes cystathionine as an obligatory intermediate. Kinetic data are presented for homocysteine synthetase [K m ( O -acetyl- l -homoserine) = 7 × 10 −3 m ; K i ( l -methionine) = 1.9 × 10 −3 m ]. Enzymes catalyzing steps 3, 4, 5, and 9 were repressible by methionine. Enzyme 4 (homoserine- O -transacetylase) and enzyme 9 (homocysteine synthetase) were simultaneously derepressed in strains carrying the mutant allele eth 2 r . Studies on diploid strains confirmed the dominance of the eth 2 s allele over eth 2 r . Regulation of enzyme 3 (homoserine dehydrogenase) and enzyme 5 (adenosine triphosphate sulfurylase) is not modified by the allele eth 2 r . The other gene eth 1 did not appear to participate in regulation of these four steps. Gene enzyme relationship was determined for three of the four steps studied (steps 3, 4, and 9). The structural genes concerned with the steps which are under the control of eth 2 ( met 8 : enzyme 9 and met a : enzyme 4) segregate independently, and are unlinked to eth 2 . These results are compatible with the idea that the gene eth 2 is responsible for the synthesis of a pleiotropic methionine repressor and suggest the existence of at least two different methionine repressors in S. cerevisiae . Implications of these findings in general regulatory mechanisms have been discussed. </jats:p