Targeted Inactivation of the mecB Gene, Encoding Cystathionine-γ-Lyase, Shows that the Reverse Transsulfuration Pathway Is Required for High-Level Cephalosporin Biosynthesis inAcremonium chrysogenum C10 but Not for Methionine Induction of the Cephalosporin Genes

[EN] Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of themecB 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-γ-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 Shen's 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 (SH2) 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-γ-lyase.SIThis work was supported by a grant of the CICYT (BIO97-0289-C02-01). We thank A. Paszewski for providing Aspergillus nidulansstrains M63 and C47 and M. Mediavilla, B. Martı́n, R. Barrientos, and M. Corrales for excellent technical assistance

Modeling Dynamics of Human Gut Microbiota Derived from Gluten Metabolism: Obtention, Maintenance and Characterization of Complex Microbial Communities

[EN] Western diets are rich in gluten-containing products, which are frequently poorly digested. The human large intestine harbors microorganisms able to metabolize undigested gluten fragments that have escaped digestion by human enzymatic activities. The aim of this work was obtaining and culturing complex human gut microbial communities derived from gluten metabolism to model the dynamics of healthy human large intestine microbiota associated with different gluten forms. For this purpose, stool samples from six healthy volunteers were inoculated in media containing predigested gluten or predigested gluten plus non-digested gluten. Passages were carried out every 24 h for 15 days in the same medium and community composition along time was studied via V3–V4 16S rDNA sequencing. Diverse microbial communities were successfully obtained. Moreover, communities were shown to be maintained in culture with stable composition for 14 days. Under non-digested gluten presence, communities were enriched in members of Bacillota, such as Lachnospiraceae, Clostridiaceae, Streptococcaceae, Peptoniphilaceae, Selenomonadaceae or Erysipelotrichaceae, and members of Actinomycetota, such as Bifidobacteriaceae and Eggerthellaceae. Contrarily, communities exposed to digested gluten were enriched in Pseudomonadota. Hence, this study shows a method for culture and stable maintenance of gut communities derived from gluten metabolism. This method enables the analysis of microbial metabolism of gluten in the gut from a community perspectiveSIThis research was funded by Junta de Castilla y León, grant number LE015P20, and Ministerio de Ciencia e Innovación, grant PID2020-119044GB-I00. Y.C.M. received a grant from Conserjería de Educación of Junta de Castilla y León, co-funded by the European Social Fund (Orden de 12 de diciembre de 2019

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.Fil: Teves, Franco. Universidad de León; EspañaFil: Lamas Maceiras, Mónica. Universidad de León; EspañaFil: García Estrada, Carlos. Instituto de Biotecnología de León; EspañaFil: Casqueiro, Javier. Instituto de Biotecnología de León; España. Universidad de León; EspañaFil: Naranjo, Leopoldo. Universidad de León; EspañaFil: Ullán, Ricardo V.. Instituto de Biotecnología de León; EspañaFil: Scervino, Jose Martin. Instituto de Biotecnología de León; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Wu, Xiaobin. Instituto de Biotecnología de León; EspañaFil: Velasco Conde, Tania. Instituto de Biotecnología de León; EspañaFil: Martín, Juan F.. Instituto de Biotecnología de León; España. Universidad de León; Españ

Monitoring of gluten-free diet compliance in celiac patients by assessment of gliadin 33-mer equivalent epitopes in feces123

Background: Certain immunotoxic peptides from gluten are resistant to gastrointestinal digestion and can interact with celiac-patient factors to trigger an immunologic response. A gluten-free diet (GFD) is the only effective treatment for celiac disease (CD), and its compliance should be monitored to avoid cumulative damage. However, practical methods to monitor diet compliance and to detect the origin of an outbreak of celiac clinical symptoms are not available

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

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

Targeted Inactivation of the mecB Gene, Encoding Cystathionine-γ-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-γ-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 Shen's 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-γ-lyase

m.6267G > A: A recurrent mutation in the human mitochondrial DNA that reduces cytochrome C oxidase activity and is associated with tumors

41 p.-1 tab.-2 tab. supl.Complete sequencing of the mitochondrial genome of 13 cell lines derived from a variety of human cancers revealed nine novel mitochondrial DNA (mtDNA) variations. One of them, m.6267G > A, is a recurrent mutation that introduces the Ala122Thr substitution in the mitochondrially encoded cytochrome c oxidase I (MT-CO1): p.MT-CO1: Ala122Thr (GenBank: NP_536845.1). Biochemical analysis of the original cell lines and the transmitochondrial cybrids generated by transferring mitochondrial DNAs to a common nuclear background, indicate that cytochrome c oxidase (COX) activity, respiration, and growth in galactose are impaired by the m.6267G > A mutation. This mutation, found twice in the cancer cell lines included in this study, has been also encountered in one out of 63 breast cancer samples, one out of 64 colon cancer samples, one out of 260 prostate cancer samples, and in one out of 15 pancreatic cancer cell lines. In all instances the m.6267G > A mutation was associated to different mtDNA haplogroups. These findings, contrast with the extremely low frequency of the m.6267G > A mutation in the normal population (1:2264) and its apparent absence in other pathologies, strongly suggesting that the m.6267G > A missense mutation is a recurrent mutation specifically associated with cancer. © 2006 Wiley-Liss, Inc.C.L. and R.M.L-H. are supported by a predoctoral fellowship from the Spanish Ministerio de Educacio´n y Ciencia and M.E.G. is supported by a postdoctoral contract from the Comunidad Autonoma de MadridPeer Reviewe