No abstract

Saccharomyces cerevisiae contains two structural genes, ACS1 and ACS2, each encoding an active acetylcoenzyme A synthetase. Characterization of enzyme activities in cell-free extracts from strains expressing either of the two genes revealed differences in the catalytic properties of the two enzymes. The K m for acetate of Acs1p was about 30-fold lower than that of Acs2p and Acs1p, but not Acs2p, could use propionate as a substrate. Enzyme activity measurements and mRNA analyses showed that ACS1 and ACS2 were both expressed during carbon-limited growth on glucose, ethanol, and acetate in aerobic chemostat cultures. In anaerobic glucose-limited cultures, only the ACS2 gene was expressed. Based on these facts, the products of the ACS1 and ACS2 genes were identified as the previously described "aerobic" and "non-aerobic" forms of acetylcoenzyme A synthetase, respectively. Batch and glucosepulse experiments revealed that transcription of ACS1 is subject to glucose repression. A mutant strain lacking Acs2p was unable to grow on glucose in batch cultures, but grew readily in aerobic glucose-limited chemostat cultures, in which the low residual glucose concentration alleviated glucose repression. Experiments in which ethanol was pulsed to aerobic ethanol-limited chemostat cultures indicated that, in addition to glucose, ethanol also repressed ACS1 transcription, although to a lesser extent. In contrast, transcription of ACS2 was slightly induced by ethanol and glucose. Absence of ACS2 prevented complete glucose repression of ACS1, indicating that ACS2 (in)directly is involved in the transcriptional regulation of ACS1.When Saccharomyces cerevisiae grows on acetate or ethanol, ATP-dependent activation of acetate to acetyl-coenzyme A is catalyzed by acetyl-coenzyme A synthetase (EC 6.2.1.1). In addition to serving as the fuel for the citric acid cycle, acetylcoenzyme A is an essential building block for the synthesis of lipids and some amino acids. During growth on glucose, direct formation of acetyl-coenzyme A from pyruvate is catalyzed by the mitochondrial pyruvate dehydrogenase complex. Alternatively, conversion of pyruvate into acetyl-coenzyme A can be accomplished by the concerted action of the enzymes of the pyruvate dehydrogenase bypass: pyruvate decarboxylase, acetaldehyde dehydrogenase, and acetyl-coenzyme A synthetase (1, 2). Recent work has indicated that the latter pathway is essential for growth, probably for the provision of cytosolic acetylcoenzyme A required for lipid synthesis (3, 4).In S. cerevisiae, a completely respiratory sugar metabolism is only observed at relatively low growth rates in aerobic, sugarlimited cultures (e.g. chemostat cultures). Upon exposure of such cultures to high sugar concentrations, metabolism becomes respirofermentative and pyruvate metabolism occurs predominantly via pyruvate decarboxylase (5, 6). Under such conditions acetate is formed as a by-product, indicating that the in vivo activity of acetaldehyde dehydrogenase exceeds that of acetyl-coenzyme A synthetase. Acetate produ...

Background: Since the discovery of the antibacterial activity of penicillin by Fleming 80 years ago, improvements of penicillin titer were essentially achieved by classical strain improvement through mutagenesis and screening. The recent sequencing of Penicillium chrysogenum strain Wisconsin1255-54 and the availability of genomics tools such as DNA-microarray offer new perspective.

In the filamentous fungus Penicillium chrysogenum, microbodies are essential for penicillin biosynthesis. To better understand the role of these organelles in antibiotics production, we determined the matrix enzyme contents of P. chrysogenum microbodies. Using a novel in silico approach, we first obtained a catalogue of 200 P. chrysogenum proteins with putative microbody targeting signals (PTSs). This included two orthologs of proteins involved in cephalosporin biosynthesis, which we demonstrate to be bona fide microbody matrix constituents. Subsequently, we performed a proteomics based inventory of P. chrysogenum microbody matrix proteins using nano-LC-MS/MS analysis. We identified 89 microbody proteins, 79 with a PTS, including the two known microbody-borne penicillin biosynthesis enzymes, isopenicillin N:acyl CoA acyltransferase and phenylacetyl-CoA ligase. Comparative analysis revealed that 69 out of 79 PTS proteins identified experimentally were in the reference list. A prominent microbody protein was identified as a novel fumarate reductase-cytochrome b5 fusion protein, which contains an internal PTS2 between the two functional domains. We show that this protein indeed localizes to P. chrysogenum microbodies.