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

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.

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

Canadian Journal of Microbiology ◽

10.1139/m86-087 ◽

1986 ◽

Vol 32 (6) ◽

pp. 473-480 ◽

Cited By ~ 40

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.

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Dependence of heme biosynthesis on iron-sulfur cluster biogenesis: human ALAD is an unrecognized iron-sulfur protein

10.21203/rs.3.rs-37702/v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Gang Liu ◽

Debangsu Sil ◽

Wing-Hang Tong ◽

Nunziata Maio ◽

J. Martin Bollinger ◽

...

Keyword(s):

Metabolic Pathways ◽

Biosynthetic Pathway ◽

Aminolevulinic Acid ◽

Heme Biosynthesis ◽

Second Step ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Sulfur Protein ◽

Iron Sulfur Protein

Abstract Heme biosynthesis and iron-sulfur cluster (ISC) biogenesis are two major mammalian metabolic pathways that require iron. It has long been known that these two pathways interconnect, but the previously described interactions do not fully explain why heme biosynthesis depends on intact ISC biogenesis. Herein we have identified a previously unrecognized connection between these two pathways through our discovery that human aminolevulinic acid dehydratase (ALAD), which catalyzes the second step of heme biosynthesis, is an Fe-S protein. We found that several highly conserved cysteines and an Ala306-Phe307-Arg308 motif of human ALAD are important for [Fe4S4] cluster acquisition and coordination. The enzymatic activity of human ALAD was greatly reduced upon loss of its Fe-S cluster, which resulted in reduced heme biosynthesis in human cells. Our findings explain why heme biosynthesis depends on intact ISC biogenesis, as ALAD provides an early Fe-S-dependent checkpoint in the heme biosynthetic pathway.

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

Journal of Bacteriology ◽

10.1128/jb.181.4.1181-1188.1999 ◽

1999 ◽

Vol 181 (4) ◽

pp. 1181-1188 ◽

Cited By ~ 52

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.

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The Monothiol Single-Domain Glutaredoxin Is Conserved in the Highly Reduced Mitochondria of Giardia intestinalis

Eukaryotic Cell ◽

10.1128/ec.00181-09 ◽

2009 ◽

Vol 8 (10) ◽

pp. 1584-1591 ◽

Cited By ~ 15

Author(s):

Petr Rada ◽

Ondřej Šmíd ◽

Robert Sutak ◽

Pavel Doležal ◽

Jan Pyrih ◽

...

Keyword(s):

Biosynthetic Pathway ◽

Immunofluorescence Microscopy ◽

Giardia Intestinalis ◽

Cluster Assembly ◽

Mature Protein ◽

Iron Sulfur Cluster ◽

Iron Sulfur ◽

Processing Peptidase ◽

Mitochondrial Origin ◽

Active Site Sequence

ABSTRACT The highly reduced mitochondria (mitosomes) of Giardia intestinalis are recently discovered organelles for which, it was suggested, iron-sulfur cluster assembly was their only conserved function. However, only an incomplete set of the components required for FeS cluster biogenesis was localized to the mitosomes. Via proteomic analysis of a mitosome-rich cellular fraction together with immunofluorescence microscopy, we identified a novel mitosomal protein homologous to monothiol glutaredoxins containing a CGFS motif at the active site. Sequence analysis revealed the presence of long nonconserved N-terminal extension of 77 amino acids, which was absent in the mature protein. Expression of the complete and N-terminally truncated forms of the glutaredoxin indicated that the extension is involved in glutaredoxin import into mitosomes. However, the mechanism of preprotein processing is unclear, as the mitosomal processing peptidase is unable to cleave this type of extension. The recombinant mature protein was shown to form a homodimeric structure, which binds a labile FeS cluster. The cluster is stabilized by glutathione and dithiothreitol. Phylogenetic analysis showed that giardial glutaredoxin is related to the mitochondrial monothiol glutaredoxins involved in FeS cluster assembly. The identification of a mitochondrial-type monothiol glutaredoxin in the mitosomes of G. intestinalis thus completes the mitosomal FeS cluster biosynthetic pathway and provides further evidence for the mitochondrial origin of these organelles.

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Lysine control of penicillin biosynthesis

Canadian Journal of Microbiology ◽

10.1139/m72-162 ◽

1972 ◽

Vol 18 (7) ◽

pp. 1045-1048 ◽

Cited By ~ 15

Author(s):

Prakash S. Masurekar ◽

Arnold L. Demain

Keyword(s):

Penicillium Chrysogenum ◽

Biosynthetic Pathway ◽

Feedback Regulation ◽

Enzyme Synthesis ◽

Suspension System ◽

The Other ◽

Penicillin Biosynthesis ◽

Enzyme Action ◽

Other Hand ◽

Lysine Concentration

The known diminution in penicillin formation by added lysine is thought to occur via feedback regulation of the lysine biosynthetic pathway, thus depleting the cells of α-aminoadipic acid, a precursor of penicillin. Studies were conducted to determine whether feedback repression or inhibition is the key mechanism involved. Lysine at 20 mM prevented net penicillin formation by starved mycelia of Penicillium chrysogenum Wis. Q-176 and Wis. 54-1255 until the extracellular lysine concentration fell by 90%. Growth in lysine did not reduce incorporation of L-valine-U-14C into penicillin by washed suspensions of either strain. On the other hand, the presence of lysine in the suspension system did lower valine incorporation. The data show that lysine acts by inhibition of enzyme action rather than by repression of enzyme synthesis.

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

Canadian Journal of Microbiology ◽

10.1139/m92-123 ◽

1992 ◽

Vol 38 (8) ◽

pp. 758-763 ◽

Cited By ~ 10

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.

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Heme biosynthesis depends on previously unrecognized acquisition of iron-sulfur cofactors in human amino-levulinic acid dehydratase

Nature Communications ◽

10.1038/s41467-020-20145-9 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Gang Liu ◽

Debangsu Sil ◽

Nunziata Maio ◽

Wing-Hang Tong ◽

J. Martin Bollinger ◽

...

Keyword(s):

Metabolic Pathways ◽

Levulinic Acid ◽

Biosynthetic Pathway ◽

Aminolevulinic Acid ◽

Heme Biosynthesis ◽

Second Step ◽

Iron Sulfur Cluster ◽

Iron Sulfur ◽

Acid Dehydratase ◽

Amino Levulinic Acid

AbstractHeme biosynthesis and iron-sulfur cluster (ISC) biogenesis are two major mammalian metabolic pathways that require iron. It has long been known that these two pathways interconnect, but the previously described interactions do not fully explain why heme biosynthesis depends on intact ISC biogenesis. Herein we identify a previously unrecognized connection between these two pathways through our discovery that human aminolevulinic acid dehydratase (ALAD), which catalyzes the second step of heme biosynthesis, is an Fe-S protein. We find that several highly conserved cysteines and an Ala306-Phe307-Arg308 motif of human ALAD are important for [Fe4S4] cluster acquisition and coordination. The enzymatic activity of human ALAD is greatly reduced upon loss of its Fe-S cluster, which results in reduced heme biosynthesis in human cells. As ALAD provides an early Fe-S-dependent checkpoint in the heme biosynthetic pathway, our findings help explain why heme biosynthesis depends on intact ISC biogenesis.

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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

Biochemical Journal ◽

10.1042/bj20080369 ◽

2008 ◽

Vol 415 (2) ◽

pp. 317-324 ◽

Cited By ~ 28

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.

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