Gabriele Romagnoli 🥽

Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely… Meer weergeven

Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of the deletion mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in γ-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1Δ mutant of a guanidinobutyrase (EC.3.5.3.7), a key enzyme in a new pathway for arginine degradation. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi. Minder weergeven

Phenylethanol has a characteristic rose-like aroma that makes it a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbour an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source, such as glucose, provides an economically attractive alternative for phenylalanine bioconversion. In this study, synthetic genetic array… Meer weergeven

Phenylethanol has a characteristic rose-like aroma that makes it a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbour an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source, such as glucose, provides an economically attractive alternative for phenylalanine bioconversion. In this study, synthetic genetic array (SGA) screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to underlie the transcriptional upregulation of ARO10 during growth, with ammonium sulphate as the sole nitrogen source. Physiological characterization revealed that the aro8Δ mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8Δ mutation also stimulated phenylethanol production when combined with other, previously documented, mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced >3 mm phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products Minder weergeven

The hybrid genomes of Saccharomyces pastorianus consist of subgenomes simi-
lar to those of S. cerevisiae and S. eubayanus, and impact of the genome struc-
ture on flavour production and its regulation is poorly understood. This study
focuses on ARO10, a 2-oxo-acid decarboxylase involved in production of
higher alcohols. In S. pastorianus CBS1483, four ARO10 copies were identified,
three resembled S. cerevisiae ARO10 and one S. eubayanus ARO10. Substrate
specificities of lager… Meer weergeven

The hybrid genomes of Saccharomyces pastorianus consist of subgenomes simi-
lar to those of S. cerevisiae and S. eubayanus, and impact of the genome struc-
ture on flavour production and its regulation is poorly understood. This study
focuses on ARO10, a 2-oxo-acid decarboxylase involved in production of
higher alcohols. In S. pastorianus CBS1483, four ARO10 copies were identified,
three resembled S. cerevisiae ARO10 and one S. eubayanus ARO10. Substrate
specificities of lager strain (Lg)ScAro10 and LgSeubAro10 were compared by
individually expressing them in a pdc1D-pdc5D-pdc6D-aro10D-thi3D S. cerevisiae
strain. Both isoenzymes catalysed decarboxylation of the 2-oxo-acids derived
from branched-chain, sulphur-containing amino acids isoleucine, and prefera-
bly phenylpyruvate. Expression of both alleles was induced by phenylalanine,
however in contrast to the S. cerevisiae strain, the two genes were not induced
by leucine. Additionally, LgSeubARO10 showed higher basal expression levels
during growth with ammonia. ARO80, which encodes ARO10 transcriptional
activator, is located on CHRIV and counts three Sc-like and one Seub-like cop-
ies. Deletion of LgSeubARO80 did not affect LgSeubARO10 phenylalanine
induction, revealing ‘trans’ regulation across the subgenomes. ARO10 transcript
levels showed a poor correlation with decarboxylase activities. These results
provide insights into flavour formation in S. pastorianus and illustrate the
complexity of functional characterization in aneuploid strains. Minder weergeven

Fusel alcohols are precursors and contributors to flavour and aroma compounds in fermented beverages and some are under investigation as biofuels. Decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine-pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs). PDC1, PDC5 and PDC6 encode differentially regulated pyruvate-decarboxylase isoenzymes, ARO10… Meer weergeven

Fusel alcohols are precursors and contributors to flavour and aroma compounds in fermented beverages and some are under investigation as biofuels. Decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine-pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs). PDC1, PDC5 and PDC6 encode differentially regulated pyruvate-decarboxylase isoenzymes, ARO10 encodes a 2-oxo-acid decarboxylase with broad substrate specificity and THI3 has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production in S. cerevisiae, substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in a pdc1∆ pdc5∆ pdc6∆ aro10∆ thi3∆ strain, only Pdc1, Pdc5 and Pdc6 catalyzed decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxo-butanoate and 2-oxo-pentanoate in cell extracts. Presence of a Pdc isoenzyme was also required for production of n-propanol and n-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production of n-propanol and n-butanol by S. cerevisiae. No decarboxylation activity was found for Thi3p with any of the substrates tested. Only Aro10p and Pdc5p catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10p showing superior kinetic properties. Aro10, Pdc1, Pdc5 and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested, but with markedly different decarboxylation kinetics. The high affinity of Aro10p identified it as key contributor to production of branched-chain and sulfur-containing fusel alcohols. Minder weergeven

Industrial production of penicillin G by Penicillium chrysogenum requires supplementation of the growth medium with the side-chain precursor phenylacetate. Growth of P.chrysogenum with phenylalanine as sole nitrogen source resulted in extracellular production of phenylacetate and penicillin G. To analyse this 'natural' pathway for penicillin G production, chemostat cultures were switch to (13)C(9)-phenylalanine as nitrogen source. Quantification and modelling of the dynamics of labelled… Meer weergeven

Industrial production of penicillin G by Penicillium chrysogenum requires supplementation of the growth medium with the side-chain precursor phenylacetate. Growth of P.chrysogenum with phenylalanine as sole nitrogen source resulted in extracellular production of phenylacetate and penicillin G. To analyse this 'natural' pathway for penicillin G production, chemostat cultures were switch to (13)C(9)-phenylalanine as nitrogen source. Quantification and modelling of the dynamics of labelled metabolites indicated that phenylalanine was: i) incorporated in nascent protein, ii) transaminated to phenylpyruvate and further converted by oxidation or by decarboxylation and iii) hydroxylated to tyrosine and subsequently metabolized via the homogentisate pathway. Involvement of the homogentisate pathway was supported by comparative transcriptome analysis of P. chrysogenum cultures grown with phenylalanine and (NH(4))(2)SO(4) as nitrogen source. This transcriptome analysis also enabled the identification of two putative 2-oxo acid decarboxylase genes (Pc13g9300 and Pc18g01490). cDNAs of both genes were cloned and expressed in the 2-oxo-acid-decarboxylase-free Saccharomyces cerevisiae strain CEN.PK711-7C (pdc1,5,6Δ aro10Δ thi3Δ). Introduction of Pc13g09300 restored growth of this S. cerevisiae mutant on glucose and phenylalanine, thereby demonstrating that Pc13g09300 encodes a dual substrate pyruvate and phenylpyruvate decarboxylase, which plays a key role in an Ehrlich-type pathway for production of phenylacetate in P. chrysogenum. These results provide a basis for metabolic engineering of P. chrysogenum for production of the penicillin G side-chain precursor phenylacetate Minder weergeven

The assumption that cells are temporally organized systems, i.e. showing relevant dynamics of their state variables such as gene expression or protein and metabolite concentration, while tacitly given for granted at the molecular level, is not explicitly taken into account when interpreting biological experimental data. This conundrum stems from the (undemonstrated) assumption that a cell culture, the actual object of biological experimentation, is a population of billions of independent… Meer weergeven

The assumption that cells are temporally organized systems, i.e. showing relevant dynamics of their state variables such as gene expression or protein and metabolite concentration, while tacitly given for granted at the molecular level, is not explicitly taken into account when interpreting biological experimental data. This conundrum stems from the (undemonstrated) assumption that a cell culture, the actual object of biological experimentation, is a population of billions of independent oscillators (cells) randomly experiencing different phases of their cycles and thus not producing relevant coordinated dynamics at the population level. Moreover the fact of considering reproductive cycle as by far the most important cyclic process in a cell resulted in lower attention given to other rhythmic processes. Here we demonstrate that growing yeast cells show a very repeatable and robust cyclic variation of the concentration of proteins with different cellular functions. We also report experimental evidence that the mechanism governing this basic oscillator and the cellular entrainment is resistant to external chemical constraints. Finally, cell growth is accompanied by cyclic dynamics of medium pH. These cycles are observed in batch cultures, different from the usual continuous cultures in which yeast metabolic cycles are known to occur, and suggest the existence of basic, spontaneous, collective and synchronous behaviors of the cell population as a whole. Minder weergeven

Laccases are multicopper oxidases of wide specificity that catalyze the oxidation of phenolic and related compounds using molecular oxygen as the electron acceptor. Here, we report the production of the Lcc1 laccase of the fungus Trametes trogii in strains of the yeast Kluyveromyces lactis, using the pyruvate decarboxylase promoter (KlPDC1) as an expression system. We assayed laccase production in various strains, with replicative and integrative transformants and with different cultivation… Meer weergeven

Laccases are multicopper oxidases of wide specificity that catalyze the oxidation of phenolic and related compounds using molecular oxygen as the electron acceptor. Here, we report the production of the Lcc1 laccase of the fungus Trametes trogii in strains of the yeast Kluyveromyces lactis, using the pyruvate decarboxylase promoter (KlPDC1) as an expression system. We assayed laccase production in various strains, with replicative and integrative transformants and with different cultivation parameters. A comparison with Lcc1 enzymes from other yeasts and from the original organism was also performed. The best production conditions were obtained with integrative transformants of an individual strain, whereas cultivation conditions were less stringent than the use of the regulated KlPDC1 promoter could anticipate. The secreted recombinant laccase showed better enzyme properties than the native enzyme or recombinant enzyme from other yeasts. We conclude that selected K. lactis strains, with opportune physiological properties and transcription regulation of the heterologous gene, could be optimal hosts for laccase isoenzyme production Minder weergeven