Alberto Jiménez

Phosphoribosyl pyrophosphate synthetase activity affects growth and riboflavin production in Ashbya gossypii

BackgroundPhosphoribosyl pyrophosphate (PRPP) is a central compound for cellular metabolism and may be considered as a link between carbon and nitrogen metabolism. PRPP is directly involved in the de novo and salvage biosynthesis of GTP, which is the immediate precursor of riboflavin. The industrial production of this vitamin using the fungus Ashbya gossypii is an important biotechnological process that is strongly influenced by substrate availability.ResultsHere we describe the characterization and manipulation of two genes of A. gossypii encoding PRPP synthetase (AGR371C and AGL080C). We show that the AGR371C and AGL080C gene products participate in PRPP synthesis and exhibit inhibition by ADP. We also observed a major contribution of AGL080C to total PRPP synthetase activity, which was confirmed by an evident growth defect of the Δagl080c strain. Moreover, we report the overexpression of wild-type and mutant deregulated isoforms of Agr371cp and Agl080cp that significantly enhanced the production of riboflavin in the engineered A. gossypii strains.ConclusionIt is shown that alterations in PRPP synthetase activity have pleiotropic effects on the fungal growth pattern and that an increase in PRPP synthetase enzymatic activity can be used to enhance riboflavin production in A. gossypii.

Improved AlGaN/GaN high electron mobility transistor using AlN interlayers

This work reports on the effects of AlN interlayers embedded into the GaN semi-insulating buffer of AlGaN/GaN high electron mobility transistors, in comparison with standard heterostructures without AlN interlayers. Detailed optical and structural characterization data are presented, along with computer simulation results. The AlN interlayers generate a compressive strain in the GaN topmost layer, which slightly reduces the total polarization field, but most important, it prevents the AlGaN barrier from plastic relaxation. The final result is an enhanced polarization field with respect to standard heterostructures, providing an increased channel carrier density and pinch-off voltage. Electrical characterization confirms the advantages of using AlN interlayers, reaching maximum drain current density and extrinsic transconductance as high as 1.4 A/mm and 266 mS/mm, respectively, for 0.2-μm gate length.

Strain design of Ashbya gossypii for single-cell oil production

ABSTRACT Single-cell oil (SCO) represents a sustainable alternative for the oil industry. Accordingly, the identification of microorganisms with either higher lipidogenic ability or novel capacities for the transformation of raw materials constitutes a major challenge for the field of oil biotechnology. With this in mind, here, we were prompted to address the lipidogenic profile of the filamentous hemiascomycete Ashbya gossypii, which is currently used for the microbial production of vitamins. We found that A. gossypii mostly accumulates unsaturated fatty acids (FAs), with more than 50% of the total FA content corresponding to oleic acid. In addition, we engineered A. gossypii strains both lacking the beta-oxidation pathway and also providing ATP-citrate lyase (ACL) activity to block the degradation of FA and to increase the cytosolic acetyl-coenzyme A (CoA) content, respectively. The lipidogenic profile of the newly developed strains demonstrates that the mere elimination of the beta-oxidation pathway in A. gossypii triggers a significant increase in lipid accumulation that can reach 70% of cell dry weight. The use of A. gossypii as a novel and robust tool for the production of added-value oils is further discussed.

Tuning single‐cell oil production in Ashbya gossypii by engineering the elongation and desaturation systems

Microbial oils represent a sustainable alternative to vegetable oils and animal fats as feedstock for both the chemical and biofuel industries. The applications of microbial oils depend on their fatty acid composition, which is defined by the relative amount of each fatty acid, also considering the length and unsaturations of the acyl chain. These two properties are determined by elongases and desaturases. In the present study, we characterized the elongase and desaturase systems in the filamentous fungus Ashbya gossypii, which is able to accumulate high amounts of lipids. Additionally, both the elongation and desaturation systems were engineered in order to broaden the potential applications of A. gossypii oils. Finally, the properties of the strains engineered for biodiesel production were analyzed, with the observation that A. gossypii is a good candidate for the microbial production of renewable biofuels. Biotechnol. Bioeng. 2014;111: 1782–1791.

Bioproduction of riboflavin: a bright yellow history

Riboflavin (vitamin B2) is an essential nutrient for humans and animals that must be obtained from the diet. To ensure an optimal supply, riboflavin is used on a large scale as additive in the food and feed industries. Here, we describe a historical overview of the industrial process of riboflavin production starting from its discovery and the need to produce the vitamin in bulk at prices that would allow for their use in human and animal nutrition. Riboflavin was produced industrially by chemical synthesis for many decades. At present, the development of economical and eco-efficient fermentation processes, which are mainly based on Bacillus subtilis and Ashbya gossypii strains, has replaced the synthetic process at industrial scale. A detailed account is given of the development of the riboflavin overproducer strains as well as future prospects for its improvement.

Microbial production of vitamins

Abstract: This chapter is a short review of the production of vitamins with the main focus on the microbial fermentation processes that are currently being employed in the production of vitamins. The state of the art of the industry of vitamins is extensively analysed with a description of both chemical and biotechnological systems that have been developed for the production of each vitamin. Finally, future trends in the microbial production of vitamins are analysed and the recent advances in strain design for whole-cell production of vitamins are discussed.

The Protein Factor-arrest 11 (Far11) Is Essential for the Toxicity of Human Caspase-10 in Yeast and Participates in the Regulation of Autophagy and the DNA Damage Signaling

Background: Autophagy is associated to human caspase-10-induced cell death in yeast. Results: Caspase-10-induced cell death in yeast is a specific process that depends on an intact MAPK pathway, the Factor-arrest (Far) protein family, and the autophagy machinery. Conclusion: Far11 coordinates a death-promoting signal and regulates both autophagy and the DNA damage response. Significance: New insights into the function of Far11 in both autophagy and cell-cycle regulation. The heterologous expression of human caspase-10 in Saccharomyces cerevisiae induces a lethal phenotype, which includes some hallmarks of apoptosis and autophagy, alterations in the intra-S checkpoint, and cell death. To determine the cellular processes and pathways that are responsible of the caspase-10-induced cell death we have designed a loss-of-function screening system to identify genes that are essential for the lethal phenotype. We observed that the ER-Golgi-localized family of proteins Far, MAPK signaling, the autophagy machinery, and several kinases and phosphatases are essential for caspase-10 toxicity. We also found that the expression of caspase-10 elicits a simultaneous activation of the MAP kinases Fus3, Kss1, and Slt2. Furthermore, the protein Far11, which is a target of MAP kinases, is essential for the dephosphorylation of Atg13 and, consequently, for the induction of autophagy. In addition, Far11 participates in the regulation of the DNA damage response through the dephosphorylation of Rad53. Finally, we have also demonstrated that Far11 is able to physically interact with the phosphatases Pph21, Pph22, and Pph3. Overall, our results indicate that the expression of human caspase-10 in S. cerevisiae activates an intracellular death signal that depends on the Far protein complex and that Far11 may function as a regulator subunit of phosphatases in different processes, thus representing a mechanistic link between them.

Mitochondria and lipid raft-located FOF1-ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine

Background Leishmaniasis is the world’s second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs) are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine) for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated. Methodology/Principal findings Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast. Conclusions/Significance The present study shows that the antileishmanial and anticancer actions of edelfosine share some common signaling processes, with mitochondria and raft-located FOF1-ATP synthase being critical in the killing process, thus identifying novel druggable targets for the treatment of leishmaniasis.

Folic Acid Production by Engineered Ashbya gossypii.

Folic acid (vitamin B9) is the common name of a number of chemically related compounds (folates), which play a central role as cofactors in one-carbon transfer reactions. Folates are involved in the biosynthesis and metabolism of nucleotides and amino acids, as well as supplying methyl groups to a broad range of substrates, such as hormones, DNA, proteins, and lipids, as part of the methyl cycle. Humans and animals cannot synthesize folic acid and, therefore, need them in the diet. Folic acid deficiency is an important and underestimated problem of micronutrient malnutrition affecting billions of people worldwide. Therefore, the addition of folic acid as food additive has become mandatory in many countries thus contributing to a growing demand of the vitamin. At present, folic acid is exclusively produced by chemical synthesis despite its associated environmental burdens. In this work, we have metabolically engineered the industrial fungus Ashbya gossypii in order to explore its potential as a natural producer of folic acid. Overexpression of FOL genes greatly enhanced the synthesis of folates and identified GTP cyclohydrolase I as the limiting step. Metabolic flux redirection from competing pathways also stimulated folic acid production. Finally, combinatorial engineering synergistically increased the production of different bioactive forms of the folic vitamin. Overall, strains were constructed which produce 146-fold (6595µg/L) more vitamin than the wild-type and by far represents the highest yield reported.

Engineering Ashbya gossypii strains for de novo lipid production using industrial by-products

Ashbya gossypii is a filamentous fungus that naturally overproduces riboflavin, and it is currently exploited for the industrial production of this vitamin. The utilization of A. gossypii for biotechnological applications presents important advantages such as the utilization of low‐cost culture media, inexpensive downstream processing and a wide range of molecular tools for genetic manipulation, thus making A. gossypii a valuable biotechnological chassis for metabolic engineering. A. gossypii has been shown to accumulate high levels of lipids in oil‐based culture media; however, the lipid biosynthesis capacity is rather limited when grown in sugar‐based culture media. In this study, by altering the fatty acyl‐CoA pool and manipulating the regulation of the main ∆9 desaturase gene, we have obtained A. gossypii strains with significantly increased (up to fourfold) de novo lipid biosynthesis using glucose as the only carbon source in the fermentation broth. Moreover, these strains were efficient biocatalysts for the conversion of carbohydrates from sugarcane molasses to biolipids, able to accumulate lipids up to 25% of its cell dry weight. Our results represent a proof of principle showing the promising potential of A. gossypii as a competitive microorganism for industrial biolipid production using cost‐effective feed stocks.