The balance between fumarate and malate plays an important role in plant development and postharvest quality in tomato fruit

Organic acids, produced as intermediates of the tricarboxylic cycle, play a crucial role in the plant primary metabolism and are considered as being ones of the most important quality traits in edible fruits. Even if they are key metabolites in a multitude of cellular functions, little is known about their physiological relevance and regulation. Transgenic tomato (Solanum lycopersicum) plants expressing constitutively a bacterial maleate isomerase, which converts reversibly maleate to fumarate, were generated in order to improve our knowledge about the role of organic acids in the crop and fruit metabolism. Growth and reproduction were affected by the unbalance of tricarboxylic cycle intermediates, as a dwarf phenotype and a flowering delay were observed in the transgenic plants. In addition, a delay in chlorophyll synthesis, a decrease in the numbers of stomata and significant changes in some photosynthetic parameters indicated alterations in central primary metabolism. Postharvest was also impaired, as transgenic fruits showed increased water lost and deterioration, indicating a possible role of the organic acids in cell wall metabolism. Finally, preliminary metabolomics analysis pointed out important changes during fruit ripening in flavor-related metabolites, such as acids and sugars, revealing the importance of organic acids in fruit metabolism. Taken together, these data indicate a pivotal role of tricarboxylic cycle intermediates, such as malate or fumarate, as regulatory metabolites. Besides their role in quality fruit characteristics, they are involved in a multitude of functions including growth and photosynthesis.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Genetic engineering of Synechocystis PCC6803 for the photoautotrophic production of the sweetener erythritol

BACKGROUND: Erythritol is a polyol that is used in the food and beverage industry. Due to its non-caloric and non-cariogenic properties, the popularity of this sweetener is increasing. Large scale production of erythritol is currently based on conversion of glucose by selected fungi. In this study, we describe a biotechnological process to produce erythritol from light and CO2, using engineered Synechocystis sp. PCC6803. METHODS: By functionally expressing codon-optimized genes encoding the erythrose-4-phosphate phosphatase TM1254 and the erythrose reductase Gcy1p, or GLD1, this cyanobacterium can directly convert the Calvin cycle intermediate erythrose-4-phosphate into erythritol via a two-step process and release the polyol sugar in the extracellular medium. Further modifications targeted enzyme expression and pathway intermediates. CONCLUSIONS: After several optimization steps, the best strain, SEP024, produced up to 2.1 mM (256 mg/l) erythritol, excreted in the medium

Yeast Gcy1p Reduces Erythrose and Erythrose-4-phosphate

Erythrose reductase reduces D-erythrose into erythritol, a low calorie sweetner. Erythrose redutases have been identified in erythritol- producing fungi (Trichosporonoides megachiliensis), while yeast orthologues are still unknown. Gcy1p, known as a Saccharomyces cerevisiae aldose-reductase, possessed significant homology with the fungal erythrose reductases. The recombinant Gcy1p, expressed in Escherichia coli, showed similar substrate specificity towards D-erythrose and glycerol using NADPH as a reductant. Moreover, the Gcy1p reduced D-erythrose-4-phosphate as efficiently as D-erythrose. These facts suggest that S.cerevisiae has the potential to produce erythritol.酵母Gcy1pはNADPH依存性のアルドースリダクターゼである. 大腸菌で発現させた組換え型Gcy1pは, トリコスポロノイデスメガチリエンスのエリスロースリダクターゼと同様にC-4及びC-3アルデヒドに対する基質特異性を示した. さらに, 酵母Gcy1pはエリスロース-4-リン酸も還元した. このことは, エリスリトールを産生しないとされてきた酵母においても, Gcy1pとホスファターゼ存在下では, エリスリトールが産生できる可能性を示している

Molecular cloning and biochemical characterization of a novel erythrose reductase from Candida magnoliae JH110

<p>Abstract</p> <p>Background</p> <p>Erythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. <it>Candida magnoliae </it>JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from <it>C. magnoliae </it>JH110.</p> <p>Results</p> <p>The gene encoding a novel ER was isolated from an osmophilic yeast <it>C. magnoliae </it>JH110. The ER gene composed of 849 nucleotides encodes a polypeptide with a calculated molecular mass of 31.4 kDa. The deduced amino acid sequence of ER showed a high degree of similarity to other members of the aldo-keto reductase superfamily including three ER isozymes from <it>Trichosporonoides megachiliensis </it>SNG-42. The intact coding region of ER from <it>C. magnoliae </it>JH110 was cloned, functionally expressed in <it>Escherichia coli </it>using a combined approach of gene fusion and molecular chaperone co-expression, and subsequently purified to homogeneity. The enzyme displayed a temperature and pH optimum at 42°C and 5.5, respectively. Among various aldoses, the <it>C. magnoliae </it>JH110 ER showed high specific activity for reduction of erythrose to the corresponding alcohol, erythritol. To explore the molecular basis of the catalysis of erythrose reduction with NADPH, homology structural modeling was performed. The result suggested that NADPH binding partners are completely conserved in the <it>C. magnoliae </it>JH110 ER. Furthermore, NADPH interacts with the side chains Lys252, Thr255, and Arg258, which could account for the enzyme's absolute requirement of NADPH over NADH.</p> <p>Conclusions</p> <p>A novel ER enzyme and its corresponding gene were isolated from <it>C. magnoliae </it>JH110. The <it>C. magnoliae </it>JH110 ER with high activity and catalytic efficiency would be very useful for <it>in vitro </it>erythritol production and could be applied for the production of erythritol in other microorganisms, which do not produce erythritol.</p

Genomewide and Enzymatic Analysis Reveals Efficient d-Galacturonic Acid Metabolism in the Basidiomycete Yeast Rhodosporidium toruloides.

Biorefining of renewable feedstocks is one of the most promising routes to replace fossil-based products. Since many common fermentation hosts, such as Saccharomyces cerevisiae, are naturally unable to convert many component plant cell wall polysaccharides, the identification of organisms with broad catabolism capabilities represents an opportunity to expand the range of substrates used in fermentation biorefinery approaches. The red basidiomycete yeast Rhodosporidium toruloides is a promising and robust host for lipid- and terpene-derived chemicals. Previous studies demonstrated assimilation of a range of substrates, from C5/C6 sugars to aromatic molecules similar to lignin monomers. In the current study, we analyzed the potential of R. toruloides to assimilate d-galacturonic acid, a major sugar in many pectin-rich agricultural waste streams, including sugar beet pulp and citrus peels. d-Galacturonic acid is not a preferred substrate for many fungi, but its metabolism was found to be on par with those of d-glucose and d-xylose in R. toruloides A genomewide analysis by combined transcriptome sequencing (RNA-seq) and RB-TDNA-seq revealed those genes with high relevance for fitness on d-galacturonic acid. While R. toruloides was found to utilize the nonphosphorylative catabolic pathway known from ascomycetes, the maximal velocities of several enzymes exceeded those previously reported. In addition, an efficient downstream glycerol catabolism and a novel transcription factor were found to be important for d-galacturonic acid utilization. These results set the basis for use of R. toruloides as a potential host for pectin-rich waste conversions and demonstrate its suitability as a model for metabolic studies with basidiomycetes.IMPORTANCE The switch from the traditional fossil-based industry to a green and sustainable bioeconomy demands the complete utilization of renewable feedstocks. Many currently used bioconversion hosts are unable to utilize major components of plant biomass, warranting the identification of microorganisms with broader catabolic capacity and characterization of their unique biochemical pathways. d-Galacturonic acid is a plant component of bioconversion interest and is the major backbone sugar of pectin, a plant cell wall polysaccharide abundant in soft and young plant tissues. The red basidiomycete and oleaginous yeast Rhodosporidium toruloides has been previously shown to utilize a range of sugars and aromatic molecules. Using state-of-the-art functional genomic methods and physiological and biochemical assays, we elucidated the molecular basis underlying the efficient metabolism of d-galacturonic acid. This study identified an efficient pathway for uronic acid conversion to guide future engineering efforts and represents the first detailed metabolic analysis of pectin metabolism in a basidiomycete fungus

耐糖性酵母の特性解明及び生育制御に関する研究

京都大学0048新制・論文博士博士(農学)乙第8014号論農博第1791号新制||農||638(附属図書館)学位論文||H4||N2513(農学部図書室)UT51-92-U250(主査)教授 熊谷 英彦, 教授 小清水 弘一, 教授 鬼頭 誠学位規則第4条第2項該当Doctor of Agricultural ScienceKyoto UniversityDFA

Erythritol metabolism in a dome mutant of Schizophyllum commune

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department ([email protected])

Erythritol production from crude glycerol by Yarrowia species: strains comparison and oxygen influence

Dissertação de mestrado em BiotechnologyThe global emerge of metabolic disorders caused by excessive consumption of food with refined sugars triggered a demand for healthier options, such as low-calorie sweeteners. Erythritol is a natural sweetener with nearly 70 % of sucrose sweetness, that has no calories, is non-glycemic and is non-cariogenic. Industrially, this polyol is produced via fermentation from glucose by osmophilic/osmotolerant fungi. The production of erythritol by yeasts from Yarrowia genus can be affected by several parameters, such as yeast strain, operational conditions and dissolved oxygen concentration (DOC) in the medium. The main goal of the present work was the study of erythritol production by different yeast strains of Yarrowia genus, from crude glycerol derived from biodiesel production. The performance of three strains of Y. lipolytica and two of Y. divulgata was compared in flasks batch culture. Erythritol and mannitol production, as well as, cellular growth and glycerol consumption were monitored for 7 days. Y. lipolytica species proved to be the most suitable for the production of erythritol, under the tested conditions, being the strains Y. lipolytica W29 (ATCC 20460) and Y. lipolytica Ch 3/4 the ones that produced the highest concentration (34 and 25 g·L-1), leading to highest yield (38 % w/w) and productivity of erythritol (0.20 and 0.15 g·L-1h -1). In a stirred tank reactor (STR), the effect of different operational conditions on kLa values were analysed. The highest experimental kLa value (162 h-1) was observed in the experiment with the conditions of 3 vvm of aeration rate and 900 rpm of stirring rate. These conditions were found to be optimal for erythritol production and they also prevented the exhaustion of DOC in the medium. The scale-up of the bioprocess, with the strain W29 and the strain Ch 3/4 allowed a 2.4-fold and a 3.3-fold increase on productivity, respectively, reducing the operation time from 168 hours to only 72 hours. The erythritol concentration produced (35 g·L-1), the erythritol yield (40%, w/w) and the erythritol productivity (0.5 g·L-1·h-1) were approximately equal for both strains. A stepwise fed-batch experiment with the addition of glycerol pulses was also performed as alternative of using high initial glycerol concentration in batch mode experiments, that was not successful. The stepwise fed-batch strategy allowed to reach almost the double of erythritol concentration for both strains, without decreasing global productivity. The best results of erythritol concentration (64 g·L-1), yield (42 % w/w) and productivity (0.45 g·L-1·h-1) were obtained in the experiments with Y. lipolytica W29.O aparecimento global de distúrbios metabólicos causados pelo consumo excessivo de alimentos com açúcares refinados, levou a uma crescente procura por opções mais saudáveis, como é o caso dos adoçantes com baixas calorias. O eritritol é um adoçante natural com cerca de 70 % da doçura da sacarose, que não possui calorias, é não-glicémico e é não-cariogénico. Industrialmente, este poliól é produzido via fermentação a partir de glucose por fungos osmofílicos/osmotolerantes. A produção de eritritol por leveduras do género Yarrowia pode ser afetada por diversos parâmetros tais como a estirpe da levedura, as condições de operação e a concentração de oxigénio dissolvido no meio. O principal objetivo deste trabalho foi o estudo da produção de eritritol, por diferentes estirpes de levedura do género Yarrowia, a partir de glicerol bruto derivado da produção do biodiesel. Em cultura descontínua em matraz foi comparado o desempenho de três estirpes de Y. lipolytica e de duas de Y. divulgata. A produção de eritritol e manitol, bem como o crescimento celular e o consumo de glicerol foram monitorizados durante 7 dias. A espécie Y. lipolytica foi a mais adequada para a produção de eritritol nas condições testadas, sendo as estirpes Y. lipolytica W29 (ATCC 20460) e Y. lipolytica Ch 3/4 aquelas que produziram uma maior concentração (34 e 25 g/L), rendimento (38 % p/p) e produtividade em eritritol (0,20 e 0,15 g/L/h). Num reator do tipo tanque agitado foi analisado o efeito de diferentes condições de operação no valor experimental de kLa. O maior valor experimental de kLa (162 h-1) foi observado no ensaio com as condições de taxa de arejamento de 3 vvm e taxa de agitação de 900 rpm. Estas condições foram consideradas as ideais para a produção de eritritol, prevenindo ainda o esgotamento da concentração de oxigénio dissolvido no meio. Com o aumento de escala do processo observou-se um aumento de cerca de 2,4 vezes para a estirpe W29 e de 3,3 vezes para a estirpe Ch 3/4, na produtividade, sendo o tempo de ensaio reduzido de 168 horas para apenas 72 horas. A concentração de eritritol produzida (35 g/L), o rendimento em eritritol (40 % p/p) e a produtividade em eritritol (0,5 g/L/h) foram aproximadamente iguais para ambas as estirpes. Foram também realizados ensaios em modo semí-contínuo com a adição de um pulso de glicerol como alternativa ao uso de elevadas concentrações iniciais de glicerol bruto em ensaios em modo descontínuo, que não foi bem sucedido. A estratégia em modo semi-contínuo com pulso de glicerol permitiu que se atingisse quase o dobro da concentração de eritritol para ambas as estirpes, sem que se tenha observado uma diminuição da produtividade global. Os melhores resultados de concentração de eritritol (64 g/L), rendimento em eritritol (42 % p/p) e produtividade em eritritol (0,45 g/L/h) foram obtidos nos ensaios com a Y. lipolytica W29