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

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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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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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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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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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 ◽

10.1099/mic.0.031005-0 ◽

2009 ◽

Vol 155 (12) ◽

pp. 3881-3892 ◽

Cited By ~ 4

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.

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The Budding Yeast Msh4 Protein Functions in Chromosome Synapsis and the Regulation of Crossover Distribution

Genetics ◽

10.1093/genetics/158.3.1013 ◽

2001 ◽

Vol 158 (3) ◽

pp. 1013-1025 ◽

Cited By ~ 7

Author(s):

Janet E Novak ◽

Petra B Ross-Macdonald ◽

G Shirleen Roeder

Keyword(s):

Mismatch Repair ◽

Budding Yeast ◽

Null Mutation ◽

Crossing Over ◽

Wild Type ◽

Crossover Interference ◽

Meiotic Chromosomes ◽

Chromosome Synapsis ◽

Protein Functions ◽

Or Gene

AbstractThe budding yeast MSH4 gene encodes a MutS homolog produced specifically in meiotic cells. Msh4 is not required for meiotic mismatch repair or gene conversion, but it is required for wild-type levels of crossing over. Here, we show that a msh4 null mutation substantially decreases crossover interference. With respect to the defect in interference and the level of crossing over, msh4 is similar to the zip1 mutant, which lacks a structural component of the synaptonemal complex (SC). Furthermore, epistasis tests indicate that msh4 and zip1 affect the same subset of meiotic crossovers. In the msh4 mutant, SC formation is delayed compared to wild type, and full synapsis is achieved in only about half of all nuclei. The simultaneous defects in synapsis and interference observed in msh4 (and also zip1 and ndj1/tam1) suggest a role for the SC in mediating interference. The Msh4 protein localizes to discrete foci on meiotic chromosomes and colocalizes with Zip2, a protein involved in the initiation of chromosome synapsis. Both Zip2 and Zip1 are required for the normal localization of Msh4 to chromosomes, raising the possibility that the zip1 and zip2 defects in crossing over are indirect, resulting from the failure to localize Msh4 properly.

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A spontaneous chromosomal amplification of the ADH2 gene in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/130.2.263 ◽

1992 ◽

Vol 130 (2) ◽

pp. 263-271

Author(s):

C E Paquin ◽

M Dorsey ◽

S Crable ◽

K Sprinkel ◽

M Sondej ◽

...

Keyword(s):

Dna Sequences ◽

Sequence Similarity ◽

Resistant Mutant ◽

Yeast Genome ◽

Crossing Over ◽

Antimycin A ◽

Extra Copy ◽

Multiple Copies ◽

Or Gene ◽

Functional Copy

Abstract A spontaneous antimycin A-resistant mutant carrying approximately four extra copies of ADH2 on chromosome XII was isolated from yeast strain 315-1D which lacks a functional copy of ADH1 and thus is antimycin A-sensitive. The additional copies of the normally glucose-repressed ADH2 are expressed during growth on glucose accounting for the antimycin A resistance. These extra copies are inserted into nonadjacent ribosomal DNA sequences (rDNA) near the recombination stimulating sequence HOT1. Each extra copy of the ADH2 gene (1548 bp) replaces most of the 37S transcript (approximately 7400 bp) in one of the approximately 200 copies of the rDNA present in the yeast genome. All four extra copies of ADH2 are lost at a rate of approximately 1 x 10(-5) deletions per cell per generation. One of the joints between the rDNA and ADH2 DNA is located 7 nucleotides downstream from 20 adenine residues in the normal copy of ADH2. This joint occurs at the end of a stretch of 16-29 thymidines in the rDNA which has been expanded to 57-59 thymidines. The other novel joint is located in a short region of sequence similarity between ADH2 and the rDNA. These observations suggest that amplification of ADH2 was a two step process: first the ADH2 gene was inserted into the rDNA, then multiple copies were generated by unequal crossing over or gene conversion within the rDNA.

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Twenty-Five Years of Spinal Muscular Atrophy Research: From Phenotype to Genotype to Therapy, and What Comes Next

Annual Review of Genomics and Human Genetics ◽

10.1146/annurev-genom-102319-103602 ◽

2020 ◽

Vol 21 (1) ◽

pp. 231-261 ◽

Cited By ~ 21

Author(s):

Brunhilde Wirth ◽

Mert Karakaya ◽

Min Jeong Kye ◽

Natalia Mendoza-Ferreira

Keyword(s):

Spinal Muscular Atrophy ◽

Muscular Atrophy ◽

Gene Replacement ◽

Efficient Treatment ◽

The Road ◽

Cellular Pathways ◽

On The Road ◽

Or Gene ◽

Novel Therapeutic Strategies ◽

Homozygous Deletions

Twenty-five years ago, the underlying genetic cause for one of the most common and devastating inherited diseases in humans, spinal muscular atrophy (SMA), was identified. Homozygous deletions or, rarely, subtle mutations of SMN1 cause SMA, and the copy number of the nearly identical copy gene SMN2 inversely correlates with disease severity. SMA has become a paradigm and a prime example of a monogenic neurological disorder that can be efficiently ameliorated or nearly cured by novel therapeutic strategies, such as antisense oligonucleotide or gene replacement therapy. These therapies enable infants to survive who might otherwise have died before the age of two and allow individuals who have never been able to sit or walk to do both. The major milestones on the road to these therapies were to understand the genetic cause and splice regulation of SMN genes, the disease's phenotype–genotype variability, the function of the protein and the main affected cellular pathways and tissues, the disease's pathophysiology through research on animal models, the windows of opportunity for efficient treatment, and how and when to treat patients most effectively.This review aims to bridge our knowledge from phenotype to genotype to therapy, not only highlighting the significant advances so far but also speculating about the future of SMA screening and treatment.

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