Improved vanillin production in baker's yeast through in silico design

<p>Abstract</p> <p>Background</p> <p>Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid <it>Vanilla planifolia</it>. Currently vanillin is mostly produced <it>via </it>chemical synthesis. A <it>de novo </it>synthetic pathway for heterologous vanillin production from glucose has recently been implemented in baker's yeast, <it>Saccharamyces cerevisiae</it>. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis.</p> <p>Results</p> <p>Expression of a glycosyltransferase from <it>Arabidopsis thaliana </it>in the vanillin producing <it>S. cerevisiae </it>strain served to decrease product toxicity. An <it>in silico </it>metabolic engineering strategy of this vanillin glucoside producing strain was designed using a set of stoichiometric modelling tools applied to the yeast genome-scale metabolic network. Two targets (<it>PDC1 </it>and <it>GDH1</it>) were selected for experimental verification resulting in four engineered strains. Three of the mutants showed up to 1.5 fold higher vanillin β-D-glucoside yield in batch mode, while continuous culture of the <it>Δpdc1 </it>mutant showed a 2-fold productivity improvement. This mutant presented a 5-fold improvement in free vanillin production compared to the previous work on <it>de novo </it>vanillin biosynthesis in baker's yeast.</p> <p>Conclusion</p> <p>Use of constraints corresponding to different physiological states was found to greatly influence the target predictions given minimization of metabolic adjustment (MOMA) as biological objective function. <it>In vivo </it>verification of the targets, selected based on their predicted metabolic adjustment, successfully led to overproducing strains. Overall, we propose and demonstrate a framework for <it>in silico </it>design and target selection for improving microbial cell factories.</p

A new class of IMP dehydrogenase with a role in self-resistance of mycophenolic acid producing fungi

<p>Abstract</p> <p>Background</p> <p>Many secondary metabolites produced by filamentous fungi have potent biological activities, to which the producer organism must be resistant. An example of pharmaceutical interest is mycophenolic acid (MPA), an immunosuppressant molecule produced by several <it>Penicillium </it>species. The target of MPA is inosine-5'-monophosphate dehydrogenase (IMPDH), which catalyses the rate limiting step in the synthesis of guanine nucleotides. The recent discovery of the MPA biosynthetic gene cluster from <it>Penicillium brevicompactum </it>revealed an extra copy of the IMPDH-encoding gene (<it>mpaF</it>) embedded within the cluster. This finding suggests that the key component of MPA self resistance is likely based on the IMPDH encoded by <it>mpaF</it>.</p> <p>Results</p> <p>In accordance with our hypothesis, heterologous expression of <it>mpaF </it>dramatically increased MPA resistance in a model fungus, <it>Aspergillus nidulans</it>, which does not produce MPA. The growth of an <it>A. nidulans </it>strain expressing <it>mpaF </it>was only marginally affected by MPA at concentrations as high as 200 μg/ml. To further substantiate the role of <it>mpaF </it>in MPA resistance, we searched for <it>mpaF </it>orthologs in six MPA producer/non-producer strains from <it>Penicillium </it>subgenus <it>Penicillium</it>. All six strains were found to hold two copies of IMPDH. A cladistic analysis based on the corresponding cDNA sequences revealed a novel group constituting <it>mpaF </it>homologs. Interestingly, a conserved tyrosine residue in the original class of IMPDHs is replaced by a phenylalanine residue in the new IMPDH class.</p> <p>Conclusions</p> <p>We identified a novel variant of the IMPDH-encoding gene in six different strains from <it>Penicillium </it>subgenus <it>Penicillium</it>. The novel IMPDH variant from MPA producer <it>P. brevicompactum </it>was shown to confer a high degree of MPA resistance when expressed in a non-producer fungus. Our study provides a basis for understanding the molecular mechanism of MPA resistance and has relevance for biotechnological and pharmaceutical applications.</p

Current challenges of research on filamentous fungi in relation to human welfare and a sustainable bio-economy: a white paper.

The EUROFUNG network is a virtual centre of multidisciplinary expertise in the field of fungal biotechnology. The first academic-industry Think Tank was hosted by EUROFUNG to summarise the state of the art and future challenges in fungal biology and biotechnology in the coming decade. Currently, fungal cell factories are important for bulk manufacturing of organic acids, proteins, enzymes, secondary metabolites and active pharmaceutical ingredients in white and red biotechnology. In contrast, fungal pathogens of humans kill more people than malaria or tuberculosis. Fungi are significantly impacting on global food security, damaging global crop production, causing disease in domesticated animals, and spoiling an estimated 10 % of harvested crops. A number of challenges now need to be addressed to improve our strategies to control fungal pathogenicity and to optimise the use of fungi as sources for novel compounds and as cell factories for large scale manufacture of bio-based products. This white paper reports on the discussions of the Think Tank meeting and the suggestions made for moving fungal bio(techno)logy forward

Adaptive evolution of drug targets in producer and non-producer organisms

Mycophenolic acid (MPA) is an immunosuppressive drug produced by several fungi in Penicillium subgenus Penicillium. This toxic metabolite is an inhibitor of IMP dehydrogenase (IMPDH). The MPA biosynthetic cluster of P. brevicompactum contains a gene encoding a B-type IMPDH, IMPDH-B, which confers MPA-resistance. Surprisingly, all members of subgenus Penicillium contain genes encoding IMPDHs of both the A and B type, regardless of their ability to produce MPA. Duplication of the IMPDH gene occurred prior to and independent of the acquisition of the MPA biosynthetic cluster. Both P. brevicompactum IMPDHs are MPA-resistant while the IMPDHs from a nonproducer are MPA-sensitive. Resistance comes with a catalytic cost: while P. brevicompactum IMPDH-B is >1000-fold more resistant to MPA than a typical eukaryotic IMPDH, its value of k(cat)/K(m) is 0.5% of “normal”. Curiously, IMPDH-B of Penicillium chrysogenum, which does not produce MPA, is also a very poor enzyme. The MPA binding site is completely conserved among sensitive and resistant IMPDHs. Mutational analysis shows that the C-terminal segment is a major structural determinant of resistance. These observations suggest that the duplication of the IMPDH gene in Pencillium subgenus Penicillium was permissive for MPA production and that MPA production created a selective pressure on IMPDH evolution. Perhaps MPA production rescued IMPDH-B from deleterious genetic drift

EasyClone: method for iterative chromosomal integration of multiple genes in <em>Saccharomyces cerevisiae</em>

Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log(10) mean +/- 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out

Molecular and Chemical Characterization of the Biosynthesis of the 6-MSA-Derived Meroterpenoid Yanuthone D in Aspergillus niger.

SummarySecondary metabolites in filamentous fungi constitute a rich source of bioactive molecules. We have deduced the genetic and biosynthetic pathway of the antibiotic yanuthone D from Aspergillus niger. Our analyses show that yanuthone D is a meroterpenoid derived from the polyketide 6-methylsalicylic acid (6-MSA). Yanuthone D formation depends on a cluster composed of ten genes including yanA and yanI, which encode a 6-MSA polyketide synthase and a previously undescribed O-mevalon transferase, respectively. In addition, several branching points in the pathway were discovered, revealing five yanuthones (F, G, H, I, and J). Furthermore, we have identified another compound (yanuthone X1) that defines a class of yanuthones that depend on several enzymatic activities encoded by genes in the yan cluster but that are not derived from 6-MSA

The Role of DNA Double-Strand Breaks in Spontaneous Homologous Recombination in S. cerevisiae

Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, we describe a class of rad52 mutants that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of our conclusions, we show that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells