Oscar Bañuelos

The cefT gene of Acremonium chrysogenum C10 encodes a putative multidrug efflux pump protein that significantly increases cephalosporin C production.

Abstract. Transcriptional analysis of the region downstream of the pcbAB gene (which encodes the α-aminoadipyl-cysteinyl-valine synthetase involved in cephalosporin synthesis) of Acremonium chrysogenum revealed the presence of two different transcripts corresponding to two new ORFs. ORF3 encodes a putative D-hydroxyacid dehydrogenase and cefT (for transmembrane protein) encodes a multidrug efflux pump belonging to the Major Faciltator Superfamily (MFS) of membrane proteins. The CefT protein has 12 transmembrane segments (TMS) and contains motifs A, B, C, D2 and G characteristic of the Drug:H+ antiporter 12-TMS group of the major facilitator superfamily. The CefT protein confers resistance to some toxic organic acids, including isovaleric acid and phenylacetic acid. Targeted inactivation of ORF3 and cefT by gene replacement showed that they are not essential for cephalosporin biosynthesis. However, amplification of the cefT gene results in increments of up to 100% in cephalosporin production in the A. chrysogenum C10 strain. Amplification of a truncated form of the cefT insert did not lead to cephalosporin overproduction. It seems that the CefT protein is involved in cephalosporin export from A. chrysogenum or in transmembrane signal transduction, and that there are redundant systems involved in cephalosporin export.

Characterization of the lys2 gene of Penicillium chrysogenum encoding α-aminoadipic acid reductase

Abstract A DNA fragment containing a gene homologous to LYS2 gene of Saccharomyces cerevisiae was cloned from a genomic DNA library of Penicillium chrysogenum AS-P-78. It encodes a protein of 1409 amino acids (Mr^ 154 859) with strong similarity to the S. cerevisiae (49.9% identity) Schizosaccharomycespombe (51.3% identity) and Candida albicans (48.12% identity) α-aminoadipate reductases and a lesser degree of identity to the amino acid-activating domains of the non-ribosomal peptide synthetases, including the α-aminoadipate-activating domain of the α-aminoadipyl-cysteinyl-valine synthetase of P. chrysogenum (12.4% identical amino acids). The lys2 gene contained one intron in the 5′-region and other in the 3′-region, as shown by comparing the nucleotide sequences of the cDNA and genomic DNA, and was transcribed as a 4.7-kb monocistronic mRNA. The lys2 gene was localized on chromosome III (7.5 Mb) in P. chrysogenum AS-P-78 and on chromosome IV (5.6 Mb) in strain P2, whereas the penicillin gene cluster is known to be located in chromosome I in both strains. The lys2-encoded protein is a member of the aminoacyladenylate-forming enzyme family with a reductase domain in its C-terminal region.

Targeted inactivation of the mecB gene, encoding cystathionine-gamma-lyase, shows that the reverse transsulfuration pathway is required for high-level cephalosporin biosynthesis in Acremonium chrysogenum C10 but not for methionine induction of the cephalosporin genes.

Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of the mecB gene by the double-marker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive mecB gene by Southern blot analysis and no cystathionine-gamma-lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain A. chrysogenum C10 and the mutants T6 and T24 in Shens defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in A. chrysogenum derives from the autotrophic (SH(2)) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB mutants, indicating that the induction effect is not mediated by cystathionine-gamma-lyase.

Intrachromosomal recombination between direct repeats in Penicillium chrysogenum: gene conversion and deletion events

Abstract Recombination between direct repeats has been studied in Penicillium chrysogenum using strain TD7-88 (lys− pyr+), which contains two inactive copies of the lys2 gene separated by 4.5 kb of DNA (including the pyrG gene) in its genome. Gene conversion leading to products with the lys+ pyr+ phenotype was observed at a frequency of 1 in 3.2 × 103 viable spores. Two types of deletion events giving rise to lys+ pyr− and lys− pyr− phenotypes were obtained with different frequencies. Southern analysis revealed that gene conversion occurs mainly as a result of crossing over events that remove the BamHI frameshift mutation present in one of the repeats. In lys− pyr− recombinants, the deletion events do not affect the frameshift mutation in the BamHI site, while lys+ pyr− recombinants showed repair of the BamHI frameshift mutation and the genotype of the parental non-disrupted strain was restored. In summary, deletion events in P. chrysogenum tend to favor the restoration of the phenotype and genotype characteristic of the parental non-disrupted strain.

CHARACTERIZATION AND LYSINE CONTROL OF EXPRESSION OF THE LYS1 GENE OF PENICILLIUM CHRYSOGENUM ENCODING HOMOCITRATE SYNTHASE

A 2071-bp DNA fragment, containing a gene (lys1) encoding a protein that showed 71.1% identical amino acids with the Yarrowia lipolytica homocitrate synthase and 71.7% identity with the Saccharomyces cerevisiae homologous enzyme, was cloned from a genomic library of Penicillium chrysogenum. The lys1 gene contained three introns and encoded a protein of 474 amino acids with a deduced molecular mass of 52kDa. lys1 was located in chromosome II (9.6Mb) in the wild-type P. chrysogenum NRRL 1951, whereas it hybridized with chromosome III (7.5Mb) in the high penicillin production strain AS-P-78. The lys1 gene is transcribed as a monocistronic transcript of 2.0kb. Levels of the lys1 transcript were high in P. chrysogenum Wis 54-1255 cultures in defined penicillin production medium at 24 and 48h, coinciding with the rapid growth phase, but clearly decreased during the penicillin production phase, suggesting that alpha-aminoadipic acid formation for penicillin biosynthesis may be limited at the homocitrate synthase level. Expression of lys1 was partially repressed by high concentrations of lysine in the culture medium, but lysine repression seems to be a weak mechanism of control of the lysine pathway as compared to lysine inhibition of homocitrate synthase.

Inactivation of the lys7 Gene, Encoding Saccharopine Reductase in Penicillium chrysogenum, Leads to Accumulation of the Secondary Metabolite Precursors Piperideine-6-Carboxylic Acid and Pipecolic Acid from α-Aminoadipic Acid

ABSTRACT Pipecolic acid serves as a precursor of the biosynthesis of the alkaloids slaframine and swainsonine (an antitumor agent) in some fungi. It is not known whether other fungi are able to synthesize pipecolic acid. Penicillium chrysogenum has a very active α-aminoadipic acid pathway that is used for the synthesis of this precursor of penicillin. The lys7 gene, encoding saccharopine reductase in P. chrysogenum, was target inactivated by the double-recombination method. Analysis of a disrupted strain (named P. chrysogenum SR1−) showed the presence of a mutant lys7 gene lacking about 1,000 bp in the 3′-end region. P. chrysogenum SR1− lacked saccharopine reductase activity, which was recovered after transformation of this mutant with the intact lys7 gene in an autonomously replicating plasmid. P. chrysogenum SR1− was a lysine auxotroph and accumulated piperideine-6-carboxylic acid. When mutant P. chrysogenum SR1− was grown with l-lysine as the sole nitrogen source and supplemented with dl-α-aminoadipic acid, a high level of pipecolic acid accumulated intracellularly. A comparison of strain SR1− with a lys2-defective mutant provided evidence showing that P. chrysogenum synthesizes pipecolic acid from α-aminoadipic acid and not from l-lysine catabolism.

Conversion of Pipecolic Acid into Lysine in Penicillium chrysogenum Requires Pipecolate Oxidase and Saccharopine Reductase: Characterization of the lys7 Gene Encoding Saccharopine Reductase

Pipecolic acid is a component of several secondary metabolites in plants and fungi. This compound is useful as a precursor of nonribosomal peptides with novel pharmacological activities. In Penicillium chrysogenum pipecolic acid is converted into lysine and complements the lysine requirement of three different lysine auxotrophs with mutations in the lys1, lys2, or lys3 genes allowing a slow growth of these auxotrophs. We have isolated two P. chrysogenum mutants, named 7.2 and 10.25, that are unable to convert pipecolic acid into lysine. These mutants lacked, respectively, the pipecolate oxidase that converts pipecolic acid into piperideine-6-carboxylic acid and the saccharopine reductase that catalyzes the transformation of piperideine-6-carboxylic acid into saccharopine. The 10.25 mutant was unable to grow in Czapek medium supplemented with alpha-aminoadipic acid. A DNA fragment complementing the 10.25 mutation has been cloned; sequence analysis of the cloned gene (named lys7) revealed that it encoded a protein with high similarity to the saccharopine reductase from Neurospora crassa, Magnaporthe grisea, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Complementation of the 10.25 mutant with the cloned gene restored saccharopine reductase activity, confirming that lys7 encodes a functional saccharopine reductase. Our data suggest that in P. chrysogenum the conversion of pipecolic acid into lysine proceeds through the transformation of pipecolic acid into piperideine-6-carboxylic acid, saccharopine, and lysine by the consecutive action of pipecolate oxidase, saccharopine reductase, and saccharopine dehydrogenase.

Overexpression of the lys1 gene in Penicillium chrysogenum: homocitrate synthase levels, α-aminoadipic acid pool and penicillin production

Abstract Homocitrate synthase activity (encoded by the lys1 gene) catalyzes the first step of the lysine and penicillin pathway and is highly sensitive to feedback regulation by l-lysine. The transcript levels of the lys1 gene and the homocitrate synthase activity are high during the growth phase and decrease during the antibiotic production phase, except in the high penicillin producer strain AS-P-99 which maintained high levels of homocitrate synthase activity in cultures at 96 h and 120 h. The lys1 gene was overexpressed in Penicillium chrysogenum using additional copies of lys1 with its own promoter or under the control of the pcbC promoter in either autonomously replicating or integrative vectors. Transformants containing 3 to 32 additional copies of the lys1 gene were selected. Some of these transformants, particularly Ti-c4 (integrative) and Tar-l9 (with autonomously replicating plasmids) showed very high levels of lys1 transcript and, in the case of Tar-l9, high levels of homocitrate synthase activity in cultures of 120 h. However, these transformants did not show increased α-aminoadipate or lysine pools. A mutant P. chrysogenum L−G− disrupted in the lys2 gene (therefore lacking the lysine branch of the pathway) showed increased α-aminoadipate levels and produced higher levels of penicillin than non-disrupted control strains. Overexpression of the lys1 gene in the L−G− mutant resulted in high homocitrate synthase levels but no additional increase of the α-aminoadipate pool or penicillin production levels. These results suggest that after amplification of the homocitrate synthase levels there are other limiting steps in the common stem of the lysine and penicillin pathways.

Subcellular localization of the homocitrate synthase in Penicillium chrysogenum

Abstract. There are conflicting reports regarding the cellular localization in Saccharomyces cerevisiae and filamentous fungi of homocitrate synthase, the first enzyme in the lysine biosynthetic pathway. The homocitrate synthase (HS) gene (lys1) of Penicillium chrysogenum was disrupted in three transformants (HS–) of the Wis 54-1255 pyrG strain. The three mutants named HS1–, HS2– and HS3– all lacked homocitrate synthase activity and showed lysine auxotrophy, indicating that there is a single gene for homocitrate synthase in P. chrysogenum. The lys1 ORF was fused in frame to the gene for the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria. Homocitrate synthase-deficient mutants transformed with a plasmid containing the lys1-GFP fusion recovered prototrophy and showed similar levels of homocitrate synthase activity to the parental strain Wis 54-1255, indicating that the hybrid protein retains the biological function of wild-type homocitrate synthase. Immunoblotting analysis revealed that the HS-GFP fusion protein is maintained intact and does not release the GFP moiety. Fluorescence microscopy analysis of the transformants showed that homocitrate synthase was mainly located in the cytoplasm in P. chrysogenum; in S. cerevisiae the enzyme is targeted to the nucleus. The control nuclear protein StuA was properly targeted to the nucleus when the StuA (targeting domain)-GFP hybrid protein was expressed in P. chrysogenum. The difference in localization of homocitrate synthase between P. chrysogenum and S. cerevisiae suggests that this protein may play a regulatory function, in addition to its catalytic function, in S. cerevisiae but not in P. chrysogenum.

Co-transformation with autonomous replicating and integrative plasmids in Penicillium chrysogenum is highly efficient and leads in some cases to rescue of the intact integrative plasmid

The efficiency of co-transformation in Penicillium chrysogenum Wisconsin 54-1255 pyrG(-) and the fate of the transforming DNA were studied using an integrative (pEF43) and an autonomous replicating plasmid (pAM9L). The results showed a co-transformation frequency of nearly 70% of all transformants tested. The total efficiency of transformation was shown to be dependent on the plasmid marker used as transformant selection (i.e., markers in the integrative or autonomous replicating vector). Analysis of the plasmids re-isolated from several co-transformants showed that different populations of plasmids co-exist in the fungal host. Interestingly, in all co-transformants studied, the integrative plasmid was found to be replicating autonomously without integrating into the host genome. In some cases, co-integrates were formed by recombination between autonomous replicating (pAM9L) and integrative (pEF43) plasmids. However, unexpectedly in some cases, the non-reorganised pEF43 integrative plasmid used in the co-transformation assays was rescued from some co-transformants.