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Ali H.,Zernike Institute for Advanced Materials | Ali H.,Kluyver Center for Genomics of Industrial Fermentations | Ali H.,University of Swat | Ries M.I.,Leiden University | And 11 more authors.
PLoS ONE | Year: 2014

The filamentous fungus Penicillium chrysogenum harbors an astonishing variety of nonribosomal peptide synthetase genes, which encode proteins known to produce complex bioactive metabolites from simple building blocks. Here we report a novel non-canonical tetra-modular nonribosomal peptide synthetase (NRPS) with microheterogenicity of all involved adenylation domains towards their respective substrates. By deleting the putative gene in combination with comparative metabolite profiling various unique cyclic and derived linear tetrapeptides were identified which were associated with this NRPS, including fungisporin. In combination with substrate predictions for each module, we propose a mechanism for a 'trans-acting' adenylation domain. © 2014 Ali et al.


Munoz-Tamayo R.,Top Institute Food and Nutrition | Munoz-Tamayo R.,Wageningen University | Munoz-Tamayo R.,Kluyver Center for Genomics of Industrial Fermentations | De Groot J.,Top Institute Food and Nutrition | And 8 more authors.
Process Biochemistry | Year: 2012

Hydrolysis of milk proteins by lactic acid bacteria leads to the formation of a large number of peptides. In this work, the hydrolysis of β-casein by the protease PrtP I of Lactococcus lactis was studied. Experiments were carried out at different initial enzyme/substrate ratios. Identification and quantification of peptides were performed by MS and RP-UHPLC analyses. Nine low molecular weight (LMW) peptides were quantified absolutely. Additionally, semi-quantification of six high molecular weight peptides (HMW) was provided. To describe the dynamics of peptides concentrations, an aggregated model was developed. This model links peptide formation to the breakdown of intact protein by introducing the concept of virtual intermediate peptides (VIP). The model represented the experimental data with an average error of 14% (comparable with the experimental error). By using the model, three dynamic pools of peptides were identified. The model suggests that LMW peptides have similar dynamic characteristics as their counterpart HMW peptides in the β-casein sequence. This study indicates that the presence and structure of micelles affect the hydrolysis dynamics and that, for some peptides, the enzyme/substrate ratio appears to affect the hydrolysis stoichiometry. The model developed is parsimonious and has a basic mechanistic component. It allows for a rational study of protein hydrolysis. © 2011 Elsevier Ltd. All rights reserved.


Teusink B.,VU University Amsterdam | Teusink B.,Kluyver Center for Genomics of Industrial Fermentations | Bachmann H.,VU University Amsterdam | Bachmann H.,Kluyver Center for Genomics of Industrial Fermentations | And 2 more authors.
Microbial Cell Factories | Year: 2011

Understanding the properties of a system as emerging from the interaction of well described parts is the most important goal of Systems Biology. Although in the practice of Lactic Acid Bacteria (LAB) physiology we most often think of the parts as the proteins and metabolites, a wider interpretation of what a part is can be useful. For example, different strains or species can be the parts of a community, or we could study only the chemical reactions as the parts of metabolism (and forgetting about the enzymes that catalyze them), as is done in flux balance analysis. As long as we have some understanding of the properties of these parts, we can investigate whether their interaction leads to novel or unanticipated behaviour of the system that they constitute.There has been a tendency in the Systems Biology community to think that the collection and integration of data should continue ad infinitum, or that we will otherwise not be able to understand the systems that we study in their details. However, it may sometimes be useful to take a step back and consider whether the knowledge that we already have may not explain the system behaviour that we find so intriguing. Reasoning about systems can be difficult, and may require the application of mathematical techniques. The reward is sometimes the realization of unexpected conclusions, or in the worst case, that we still do not know enough details of the parts, or of the interactions between them.We will discuss a number of cases, with a focus on LAB-related work, where a typical systems approach has brought new knowledge or perspective, often counterintuitive, and clashing with conclusions from simpler approaches. Also novel types of testable hypotheses may be generated by the systems approach, which we will illustrate. Finally we will give an outlook on the fields of research where the systems approach may point the way for the near future. © 2011 Teusink et al; licensee BioMed Central Ltd.


Pudlik A.M.,Top Institute Food and Nutrition | Pudlik A.M.,University of Groningen | Pudlik A.M.,Kluyver Center for Genomics of Industrial Fermentations | Lolkema J.S.,University of Groningen
Applied and Environmental Microbiology | Year: 2012

Oxaloacetate is an intermediate of the citrate fermentation pathway that accumulates in the cytoplasm of Lactococcus lactis ILCitM(pFL3) at a high concentration due to the inactivation of oxaloacetate decarboxylase. An excess of toxic oxaloacetate is excreted into the medium in exchange for citrate by the citrate transporter CitP (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:4049-4056, 2011). In this study, transamination of amino acids with oxaloacetate as the keto donor is described as an additional mechanism to relieve toxic stress. Redirection of the citrate metabolic pathway into the transamination route in the presence of the branched-chain amino acids Ile, Leu, and Val; the aromatic amino acids Phe, Trp, and Tyr; and Met resulted in he formation of aspartate and the corresponding α-keto acids. Cells grown in the presence of citrate showed 3.5 to 7 times higher transaminase activity in the cytoplasm than cells grown in the absence of citrate. The study demonstrates that transaminases of L. lactis accept oxaloacetate as a keto donor. A significant fraction of 2-keto-4-methylthiobutyrate formed from methionine by citratedriven transamination in vivo was further metabolized, yielding the cheese aroma compounds 2-hydroxy-4-methylthiobutyrate and methyl-3-methylthiopropionate. Reducing equivalents required for the former compound were produced in the citrate fermentation pathway as NADH. Similarly, phenylpyruvate, the transamination product of phenylalanine, was reduced to phenyllactate, while the dehydrogenase activity was not observed for the branched-chain keto acids. Both α-keto acids and α-hydroxy acids are known substrates of CitP and may be excreted from the cell in exchange for citrate or oxaloacetate. © 2012, American Society for Microbiology.


Polli F.,University of Groningen | Meijrink B.,Royal DSM | Bovenberg R.A.L.,Royal DSM | Bovenberg R.A.L.,University of Groningen | And 2 more authors.
Fungal Genetics and Biology | Year: 2016

Filamentous fungi such as Aspergillus and Penicillium are widely used as hosts for the industrial products such as proteins and secondary metabolites. Although filamentous fungi are versatile in recognizing transcriptional and translational elements present in genes from other filamentous fungal species, only few promoters have been applied and compared in performance so far in Penicillium chrysogenum. Therefore, a set of homologous and heterologous promoters were tested in a reporter system to obtain a set of potential different strengths. Through in vivo homologous recombination in Saccharomyces cerevisiae, twelve Aspergillus niger and P. chrysogenum promoter-reporter pathways were constructed that drive the expression of green fluorescent protein while concurrent expression of the red fluorescent protein was used as an internal standard and placed under control of the PcPAF promoter. The pathways were integrated into the genome of P. chrysogenum and tested using the BioLector system for fermentation. Reporter gene expression was monitored during growth and classified according to promoter strength and expression profile. A set of novel promoters was obtained that can be used to tune the expression of target genes in future strain engineering programs. © 2015 Elsevier Inc.


Pudlik A.M.,Top Institute Food and Nutrition | Pudlik A.M.,University of Groningen | Pudlik A.M.,Kluyver Center for Genomics of Industrial Fermentations | Lolkema J.S.,University of Groningen
Journal of Bacteriology | Year: 2011

Citrate metabolism in resting cells of Lactococcus lactis IL1403(pFL3) results in the formation of two end products from the intermediate pyruvate, acetoin and acetate (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:706-714, 2011). Pyruvate is formed from citrate following uptake by the transporter CitP through the subsequent actions of citrate lyase and oxaloacetate decarboxylase. The present study describes the metabolic response of L. lactis when oxaloacetate accumulates in the cytoplasm. The oxaloacetate decarboxylase-deficient mutant ILCitM(pFL3) showed nearly identical rates of citrate consumption, but the end product profile in the presence of glucose shifted from mainly acetoin to only acetate. In addition, in contrast to the parental strain, the mutant strain did not generate proton motive force. Citrate consumption by the mutant strain was coupled to the excretion of oxaloacetate, with a yield of 80 to 85%. Following citrate consumption, oxaloacetate was slowly taken up by the cells and converted to pyruvate by a cryptic decarboxylase and, subsequently, to acetate. The transport of oxaloacetate is catalyzed by CitP. The parental strain IL1403(pFL3) containing CitP consumed oxaloacetate, while the original strain, IL1403, not containing CitP, did not. Moreover, oxaloacetate consumption was enhanced in the presence of L-lactate, indicating exchange between oxaloacetate and L-lactate catalyzed by CitP. Hence, when oxaloacetate inadvertently accumulates in the cytoplasm, the physiological response of L. lactis is to excrete oxaloacetate in exchange with citrate by an electroneutral mechanism catalyzed by CitP. Subsequently, in a second step, oxaloacetate is taken up by CitP and metabolized to pyruvate and acetate. © 2011, American Society for Microbiology.


Ali H.,Zernike Institute for Advanced Materials | Ali H.,Kluyver Center for Genomics of Industrial Fermentations | Ries M.I.,Leiden University | Nijland J.G.,Zernike Institute for Advanced Materials | And 8 more authors.
PLoS ONE | Year: 2013

Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopiperazine (DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO) which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway. © 2013 Ali et al.


Goffin P.,Kluyver Center for Genomics of Industrial Fermentations | Goffin P.,Top Institute Food and Nutrition | Goffin P.,NIZO Food Research | Goffin P.,Glaxosmithkline | And 14 more authors.
Molecular Systems Biology | Year: 2010

Situations of extremely low substrate availability, resulting in slow growth, are common in natural environments. To mimic these conditions, Lactobacillus plantarum was grown in a carbon-limited retentostat with complete biomass retention. The physiology of extremely slow-growing L. plantarumĝ€"as studied by genome-scale modeling and transcriptomicsĝ€"was fundamentally different from that of stationary-phase cells. Stress resistance mechanisms were not massively induced during transition to extremely slow growth. The energy-generating metabolism was remarkably stable and remained largely based on the conversion of glucose to lactate. The combination of metabolic and transcriptomic analyses revealed behaviors involved in interactions with the environment, more particularly with plants: production of plant hormones or precursors thereof, and preparedness for the utilization of plant-derived substrates. Accordingly, the production of compounds interfering with plant root development was demonstrated in slow-growing L. plantarum. Thus, conditions of slow growth and limited substrate availability seem to trigger a plant environment-like response, even in the absence of plant-derived material, suggesting that this might constitute an intrinsic behavior in L. plantarum. © 2010 EMBO and Macmillan Publishers Limited.


Gombert A.K.,Technical University of Delft | Gombert A.K.,Kluyver Center for Genomics of Industrial Fermentations | Gombert A.K.,University of Sao Paulo | Veiga T.,Technical University of Delft | And 13 more authors.
Fungal Genetics and Biology | Year: 2011

Penicillium chrysogenum is widely used as an industrial antibiotic producer, in particular in the synthesis of ß-lactam antibiotics such as penicillins and cephalosporins. In industrial processes, oxalic acid formation leads to reduced product yields. Moreover, precipitation of calcium oxalate complicates product recovery. We observed oxalate production in glucose-limited chemostat cultures of P. chrysogenum grown with or without addition of adipic acid, side-chain of the cephalosporin precursor adipoyl-6-aminopenicillinic acid (ad-6-APA). Oxalate accounted for up to 5% of the consumed carbon source. In filamentous fungi, oxaloacetate hydrolase (OAH; EC3.7.1.1) is generally responsible for oxalate production. The P. chrysogenum genome harbours four orthologs of the A. niger oahA gene. Chemostat-based transcriptome analyses revealed a significant correlation between extracellular oxalate titers and expression level of the genes Pc18g05100 and Pc22g24830. To assess their possible involvement in oxalate production, both genes were cloned in Saccharomyces cerevisiae, yeast that does not produce oxalate. Only the expression of Pc22g24830 led to production of oxalic acid in S. cerevisiae. Subsequent deletion of Pc22g28430 in P. chrysogenum led to complete elimination of oxalate production, whilst improving yields of the cephalosporin precursor ad-6-APA. © 2011 Elsevier Inc.


Branco dos Santos F.,VU University Amsterdam | Branco dos Santos F.,Kluyver Center for Genomics of Industrial Fermentations | de Vos W.M.,Wageningen University | de Vos W.M.,University of Helsinki | And 2 more authors.
Current Opinion in Biotechnology | Year: 2013

We review the uses and limitations of modelling approaches that are in use in the field of Lactic Acid Bacteria (LAB). We describe recent developments in model construction and computational methods, starting from application of such models to monocultures. However, since most applications in food biotechnology involve complex nutrient environments and mixed cultures, we extend the scope to discuss developments in modelling such complex systems. With metagenomics and meta-functional genomics data becoming available, the developments in genome-scale community models are discussed. We conclude that exploratory tools are available and useful, but truly predictive mechanistic models will remain a major challenge in the field. © 2012 Elsevier Ltd.

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