α-Aminoadipate pool concentration and penicillin biosynthesis in strains of Penicillium chrysogenum

1986 ◽  
Vol 32 (6) ◽  
pp. 473-480 ◽  
Author(s):  
W. M. Jaklitsch ◽  
W. Hampel ◽  
M. Röhr ◽  
C. P. Kubicek ◽  
G. Gamerith
Keyword(s):  
Amino Acid ◽  
Penicillium Chrysogenum ◽  
Intracellular Concentration ◽  
Lysine Biosynthesis ◽  
Penicillin Biosynthesis ◽  
Penicillin Production ◽  
Saccharopine Dehydrogenase ◽  
Two Factors ◽  
Homocitrate Synthase ◽  
Rate Limiting

Intracellular amino acid pools in four Penicillium chrysogenum strains, which differed in their ability to produce penicillin, were determined under conditions supporting growth without penicillin production and under conditions supporting penicillin production. A significant correlation between the rate of pencillin production and the intracellular concentration of α-aminoadipate was observed, which was not shown with any other amino acid in the pool. In replacement cultivation, penicillin production was stimulated by α-aminoadipate, but not by valine or cysteine. Exogenously added α-aminoadipate (2 or 3 mM) maximally stimulated penicillin synthesis in two strains of different productivity. Under these conditions intracellular concentrations of α-aminoadipate were comparable in the two strains in spite of the higher rate of penicillin production in the more productive strain. Results suggest that the lower penicillin titre of strain Q 176 is due to at least two factors: (i) the intracellular concentration of α-aminoadipate is insufficient to allow saturation of any enzyme which is rate limiting in the conversion of α-aminoadipate to penicillin and (ii) the level of an enzyme, which is rate limiting in the conversion of α-aminoadipate to penicillin, is lower in Q 176 (relative to strain D6/1014/A). Results suggest that the intracellular concentration of α-aminoadipate in strain D6/1014/A is sufficiently high to allow saturation of the rate-limiting penicillin biosynthetic enzyme in that strain. The basis of further correlation of intracellular α-aminoadipate concentration and penicillin titre among strains D6/1014/A, P2, and 389/3, the three highest penicillin producers studied here, remains to be established. Preliminary studies which attempted to explain the differences in intracellular α-aminoadipate concentrations in strains Q 176, D6/1014/A, and P2 in terms of differences in activities or kinetics of two enzymes of lysine biosynthesis (homocitrate synthase and saccharopine dehydrogenase) did not reveal differences in those enzymes among the three strains.

Regulation of α-aminoadipate reductase from Penicillium chrysogenum in relation to the flux from α-aminoadipate into penicillin biosynthesis

1992 ◽  
Vol 38 (8) ◽  
pp. 758-763 ◽  
Author(s):  
Ying Lu ◽  
Robert L. Mach ◽  
Karin Affenzeller ◽  
Christian P. Kubicek
Keyword(s):  
Key Words ◽  
Penicillium Chrysogenum ◽  
Reductase Activity ◽  
Lysine Biosynthesis ◽  
Penicillin Biosynthesis ◽  
Penicillin Production ◽  
Producer Strain ◽  
Ph Dependent ◽  
Producer Strains

The activity and regulation of α-aminoadipate reductase in three Penicillium chrysogenum strains (Q176, D6/1014/A, and P2), producing different amounts of penicillin, were studied. The enzyme exhibited decreasing affinity for α-aminoadipate with increasing capacity of the respective strain to produce penicillin. The enzyme from all three strains was inhibited by L-lysine, and the enzyme from the lowest producer, Q176, was least sensitive. Between pH 7.5 and 6.5, inhibition of α-aminoadipate reductase by L-lysine was pH dependent, being more pronounced at lower pH. The highest producer strain, P2, displayed the lowest α-aminoadipate reductase activity at pH 7.0. In Q176, the addition of 0.5–1 mM of exogenous lysine stimulated penicillin formation, whereas the same concentration was ineffective or inhibitory with strains D6/1014/A and P2. The addition of higher (up to 5 mM) lysine concentrations inhibited penicillin production in all three strains. In mutants of P. chrysogenum D6/1014/A, selected for resistance to 20 mM α-aminoadipate, highest penicillin production was observed in those strains whose α-aminoadipate reductase was most strongly inhibited by L-lysine. The results support the conclusion that the in vivo activity of α-aminoadipate reductase from superior penicillin producer strains of P. chrysogenum is more strongly inhibited by lysine, and that this is related to their ability to accumulate increased amounts of α-aminoadipate, and hence penicillin. Key words: α-aminoadipate, α-aminoadipate reductase, regulation of lysine biosynthesis, penicillin biosynthesis, Penicillium chrysogenum.

scholarly journals Post-translational enzyme modification by the phosphopantetheinyl transferase is required for lysine and penicillin biosynthesis but not for roquefortine or fatty acid formation in Penicillium chrysogenum

2008 ◽  
Vol 415 (2) ◽  
pp. 317-324 ◽  
Author(s):  
Carlos García-Estrada ◽  
Ricardo V. Ullán ◽  
Tania Velasco-Conde ◽  
Ramiro P. Godio ◽  
Fernando Teijeira ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Penicillium Chrysogenum ◽  
Fatty Acid Synthase ◽  
Single Copy ◽  
Lysine Biosynthesis ◽  
Penicillin Biosynthesis ◽  
Penicillin Production ◽  
Phosphopantetheinyl Transferase ◽  
Wide Range ◽  
Multiple Copies

NRPSs (non-ribosomal peptide synthetases) and PKSs (polyketide synthases) require post-translational phosphopantetheinylation to become active. This reaction is catalysed by a PPTase (4′-phosphopantetheinyl transferase). The ppt gene of Penicillium chrysogenum, encoding a protein that shares 50% similarity with the stand-alone large PPTases, has been cloned. This gene is present as a single copy in the genome of the wild-type and high-penicillin-producing strains (containing multiple copies of the penicillin gene cluster). Amplification of the ppt gene produced increases in isopenicillin N and benzylpenicillin biosynthesis. A PPTase-defective mutant (Wis54-PPT−) was obtained. It required lysine and lacked pigment and penicillin production, but it still synthesized normal levels of roquefortine. The biosynthesis of roquefortine does not appear to involve PPTase-mediated modification of the synthesizing enzymes. The PPT− mutant did not require fatty acids, which indicates that activation of the fatty acid synthase is performed by a different PPTase. Complementation of Wis54-PPT− with the ppt gene restored lysine biosynthesis, pigmentation and penicillin production, which demonstrates the wide range of processes controlled by this gene.

scholarly journals Transcriptional upregulation of four genes of the lysine biosynthetic pathway by homocitrate accumulation in Penicillium chrysogenum: homocitrate as a sensor of lysine-pathway distress

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 3881-3892 ◽  
Author(s):  
Franco Teves ◽  
Mónica Lamas-Maceiras ◽  
Carlos García-Estrada ◽  
Javier Casqueiro ◽  
Leopoldo Naranjo ◽  
...  
Keyword(s):  
Point Mutation ◽  
Penicillium Chrysogenum ◽  
Biosynthetic Pathway ◽  
Genomic Library ◽  
Lysine Biosynthesis ◽  
Penicillin Biosynthesis ◽  
Iron Sulfur Cluster ◽  
High Identity ◽  
Iron Sulfur ◽  
Homocitrate Synthase

The lysine biosynthetic pathway has to supply large amounts of α-aminoadipic acid for penicillin biosynthesis in Penicillium chrysogenum. In this study, we have characterized the P. chrysogenum L2 mutant, a lysine auxotroph that shows highly increased expression of several lysine biosynthesis genes (lys1, lys2, lys3, lys7). The L2 mutant was found to be deficient in homoaconitase activity since it was complemented by the Aspergillus nidulans lysF gene. We have cloned a gene (named lys3) that complements the L2 mutation by transformation with a P. chrysogenum genomic library, constructed in an autonomous replicating plasmid. The lys3-encoded protein showed high identity to homoaconitases. In addition, we cloned the mutant lys3 allele from the L2 strain that showed a G1534 to A1534 point mutation resulting in a Gly495 to Asp495 substitution. This mutation is located in a highly conserved region adjacent to two of the three cysteine residues that act as ligands to bind the iron–sulfur cluster required for homoaconitase activity. The L2 mutant accumulates homocitrate. Deletion of the lys1 gene (homocitrate synthase) in the L2 strain prevented homocitrate accumulation and reverted expression levels of the four lysine biosynthesis genes tested to those of the parental prototrophic strain. Homocitrate accumulation seems to act as a sensor of lysine-pathway distress, triggering overexpression of four of the lysine biosynthesis genes.

scholarly journals Gene Targeting in Penicillium chrysogenum: Disruption of the lys2 Gene Leads to Penicillin Overproduction

1999 ◽  
Vol 181 (4) ◽  
pp. 1181-1188 ◽  
Author(s):  
Javier Casqueiro ◽  
Santiago Gutiérrez ◽  
Oscar Bañuelos ◽  
Maria Jose Hijarrubia ◽  
Juan Francisco Martín
Keyword(s):  
Penicillium Chrysogenum ◽  
Reductase Activity ◽  
Gene Replacement ◽  
Lysine Biosynthesis ◽  
Homologous Region ◽  
Crossing Over ◽  
Penicillin Biosynthesis ◽  
Single Crossing ◽  
Or Gene ◽  
Acid Precursor

ABSTRACT Two strategies have been used for targeted integration at thelys2 locus of Penicillium chrysogenum. In the first strategy the disruption of lys2 was obtained by a single crossing over between the endogenous lys2 and a fragment of the same gene located in an integrative plasmid.lys2-disrupted mutants were obtained with 1.6% efficiency when the lys2 homologous region was 4.9 kb, but no homologous integration was observed with constructions containing a shorter homologous region. Similarly,lys2-disrupted mutants were obtained by a double crossing over (gene replacement) with an efficiency of 0.14% by using two lys2 homologous regions of 4.3 and 3.0 kb flanking thepyrG marker. No homologous recombination was observed when the selectable marker was flanked by short lys2 homologous DNA fragments. The disruption of lys2 was confirmed by Southern blot analysis of three different lysine auxotrophs obtained by a single crossing over or gene replacement. Thelys2-disrupted mutants lacked α-aminoadipate reductase activity (encoded by lys2) and showed specific penicillin yields double those of the parental nondisrupted strain, Wis 54-1255. The α-aminoadipic acid precursor is channelled to penicillin biosynthesis by blocking the lysine biosynthesis branch at the α-aminoadipate reductase level.

scholarly journals Omics Approaches Applied to Penicillium chrysogenum and Penicillin Production: Revealing the Secrets of Improved Productivity

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 712
Author(s):  
Carlos García-Estrada ◽  
Juan F. Martín ◽  
Laura Cueto ◽  
Carlos Barreiro
Keyword(s):  
Synthetic Biology ◽  
Secondary Metabolites ◽  
Penicillium Chrysogenum ◽  
Molecular Mechanisms ◽  
Penicillin Biosynthesis ◽  
Biological Processes ◽  
Penicillin Production ◽  
Bioactive Secondary Metabolites ◽  
Industrial Strains ◽  
Points Of View

Penicillin biosynthesis by Penicillium chrysogenum is one of the best-characterized biological processes from the genetic, molecular, biochemical, and subcellular points of view. Several omics studies have been carried out in this filamentous fungus during the last decade, which have contributed to gathering a deep knowledge about the molecular mechanisms underlying improved productivity in industrial strains. The information provided by these studies is extremely useful for enhancing the production of penicillin or other bioactive secondary metabolites by means of Biotechnology or Synthetic Biology.

scholarly journals Uptake of the β-Lactam Precursor α-Aminoadipic Acid in Penicillium chrysogenum Is Mediated by the Acidic and the General Amino Acid Permease

2004 ◽  
Vol 70 (8) ◽  
pp. 4775-4783 ◽  
Author(s):  
Hein Trip ◽  
Melchior E. Evers ◽  
Jan A. K. W. Kiel ◽  
Arnold J. M. Driessen
Keyword(s):  
Amino Acid ◽  
Penicillium Chrysogenum ◽  
Acid Uptake ◽  
Penicillin Production ◽  
Acid Transport ◽  
Acidic Amino Acid ◽  
Amino Acid Permease ◽  
Relative Contribution ◽  
Amino Acid Permeases ◽  
General Amino Acid Permease

ABSTRACT External addition of the β-lactam precursor α-aminoadipic acid to the filamentous fungus Penicillium chrysogenum leads to an increased intracellular α-aminoadipic acid concentration and an increase in penicillin production. The exact route for α-aminoadipic acid uptake is not known, although the general amino acid and acidic amino acid permeases have been implicated in this process. Their corresponding genes, PcGAP1 and PcDIP5, of P. chrysogenum were cloned and functionally expressed in a mutant of Saccharomyces cerevisiae (M4276) in which the acidic amino acid and general amino acid permease genes (DIP5 and GAP1, respectively) are disrupted. Transport assays show that both PcGap1 and PcDip5 mediated the uptake of α-aminoadipic acid, although PcGap1 showed a higher affinity for α-aminoadipic acid than PcDip5 (Km values, 230 and 800 μM, respectively). Leucine strongly inhibits α-aminoadipic acid transport via PcGap1 but not via PcDip5. This difference was exploited to estimate the relative contribution of each transport system to the α-aminoadipic acid flux in β-lactam-producing P. chrysogenum. The transport measurements demonstrate that both PcGap1 and PcDip5 contribute to the α-aminoadipic acid flux.

scholarly journals Molecular Evolution of Lysine Biosynthesis in Agaricomycetes

2021 ◽  
Vol 8 (1) ◽  
pp. 37
Author(s):  
Zili Song ◽  
Maoqiang He ◽  
Ruilin Zhao ◽  
Landa Qi ◽  
Guocan Chen ◽  
...  
Keyword(s):  
Molecular Evolution ◽  
Evolutionary Relationship ◽  
Deep Understanding ◽  
Evolutionary Conservation ◽  
Fungal Species ◽  
Lysine Biosynthesis ◽  
Tertiary Structures ◽  
Saccharopine Dehydrogenase ◽  
Homocitrate Synthase ◽  
Substrate Binding Sites

As an indispensable essential amino acid in the human body, lysine is extremely rich in edible mushrooms. The α-aminoadipic acid (AAA) pathway is regarded as the biosynthetic pathway of lysine in higher fungal species in Agaricomycetes. However, there is no deep understanding about the molecular evolutionary relationship between lysine biosynthesis and species in Agaricomycetes. Herein, we analyzed the molecular evolution of lysine biosynthesis in Agaricomycetes. The phylogenetic relationships of 93 species in 34 families and nine orders in Agaricomycetes were constructed with six sequences of LSU, SSU, ITS (5.8 S), RPB1, RPB2, and EF1-α datasets, and then the phylogeny of enzymes involved in the AAA pathway were analyzed, especially homocitrate synthase (HCS), α-aminoadipate reductase (AAR), and saccharopine dehydrogenase (SDH). We found that the evolution of the AAA pathway of lysine biosynthesis is consistent with the evolution of species at the order level in Agaricomycetes. The conservation of primary, secondary, predicted tertiary structures, and substrate-binding sites of the enzymes of HCS, AAR, and SDH further exhibited the evolutionary conservation of lysine biosynthesis in Agaricomycetes. Our results provide a better understanding of the evolutionary conservation of the AAA pathway of lysine biosynthesis in Agaricomycetes.

scholarly journals Increased Penicillin Production in Penicillium chrysogenum Production Strains via Balanced Overexpression of Isopenicillin N Acyltransferase

2012 ◽  
Vol 78 (19) ◽  
pp. 7107-7113 ◽  
Author(s):  
Stefan S. Weber ◽  
Fabiola Polli ◽  
Rémon Boer ◽  
Roel A. L. Bovenberg ◽  
Arnold J. M. Driessen
Keyword(s):  
Penicillium Chrysogenum ◽  
Phenylacetic Acid ◽  
Strain Improvement ◽  
Single Copy ◽  
Limiting Factor ◽  
Penicillin Biosynthesis ◽  
Penicillin Production ◽  
Content Type ◽  
Coa Ligase ◽  
Isopenicillin N

ABSTRACTIntense classical strain improvement has yielded industrialPenicillium chrysogenumstrains that produce high titers of penicillin. These strains contain multiple copies of the penicillin biosynthesis cluster encoding the three key enzymes: δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS), isopenicillin N synthase (IPNS), and isopenicillin N acyltransferase (IAT). The phenylacetic acid coenzyme A (CoA) ligase (PCL) gene encoding the enzyme responsible for the activation of the side chain precursor phenylacetic acid is localized elsewhere in the genome in a single copy. Since the protein level of IAT already saturates at low cluster copy numbers, IAT might catalyze a limiting step in high-yielding strains. Here, we show that penicillin production in high-yielding strains can be further improved by the overexpression of IAT while at very high levels of IAT the precursor 6-aminopenicillic acid (6-APA) accumulates. Overproduction of PCL only marginally stimulates penicillin production. These data demonstrate that in high-yielding strains IAT is the limiting factor and that this limitation can be alleviated by a balanced overproduction of this enzyme.

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