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Jens Nielsen - One of the best experts on this subject based on the ideXlab platform.
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physiological characterization of the high Malic Acid producing aspergillus oryzae strain 2103a 68
Applied Microbiology and Biotechnology, 2014Co-Authors: Christoph Knuf, Intawat Nookaew, Stephen H Brown, Alan Berry, Jens Nielsen, Ilse M Remmers, Sakda KhoomrungAbstract:Malic Acid is a C4 dicarboxylic Acid that is currently mainly used in the food and beverages industry as an Acidulant. Because of the versatility of the group of C4 dicarboxylic Acids, the chemical industry has a growing interest in this chemical compound. As Malic Acid will be considered as a bulk chemical, microbial production requires organisms that sustain high rates, yields, and titers. Aspergillus oryzae is mainly known as an industrial enzyme producer, but it was also shown that it has a very competitive natural production capacity for Malic Acid. Recently, an engineered A. oryzae strain, 2103a-68, was presented which overexpressed pyruvate carboxylase, malate dehydrogenase, and a Malic Acid transporter. In this work, we report a detailed characterization of this strain including detailed rates and yields under Malic Acid production conditions. Furthermore, transcript levels of the genes of interest and corresponding enzyme activities were measured. On glucose as carbon source, 2103a-68 was able to secrete Malic Acid at a maximum specific production rate during stationary phase of 1.87 mmol (g dry weight (DW))−1 h−1 and with a yield of 1.49 mol mol−1. Intracellular fluxes were obtained using 13C flux analysis during exponential growth, supporting the success of the metabolic engineering strategy of increasing flux through the reductive cytosolic tricarboxylic Acid (rTCA) branch. Additional cultivations using xylose and a glucose/xylose mixture demonstrated that A. oryzae is able to efficiently metabolize pentoses and hexoses to produce Malic Acid at high titers, rates, and yields.
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investigation of Malic Acid production in aspergillus oryzae under nitrogen starvation conditions
Applied and Environmental Microbiology, 2013Co-Authors: Christoph Knuf, Intawat Nookaew, Stephen H Brown, Michael Mcculloch, Alan Berry, Jens NielsenAbstract:Malic Acid has great potential for replacing petrochemical building blocks in the future. For this application, high yields, rates, and titers are essential in order to sustain a viable biotechnological production process. Natural high-capacity Malic Acid producers like the Malic Acid producer Aspergillus flavus have so far been disqualified because of special growth requirements or the production of mycotoxins. As A. oryzae is a very close relative or even an ecotype of A. flavus, it is likely that its high Malic Acid production capabilities with a generally regarded as safe (GRAS) status may be combined with already existing large-scale fermentation experience. In order to verify the Malic Acid production potential, two wild-type strains, NRRL3485 and NRRL3488, were compared in shake flasks. As NRRL3488 showed a volumetric production rate twice as high as that of NRRL3485, this strain was selected for further investigation of the influence of two different nitrogen sources on Malic Acid secretion. The cultivation in lab-scale fermentors resulted in a higher final titer, 30.27 +/- 1.05 g liter(-1), using peptone than the one of 22.27 +/- 0.46 g liter(-1) obtained when ammonium was used. Through transcriptome analysis, a binding site similar to the one of the Saccharomyces cerevisiae yeast transcription factor Msn2/4 was identified in the upstream regions of glycolytic genes and the cytosolic Malic Acid production pathway from pyruvate via oxaloacetate to malate, which suggests that Malic Acid production is a stress response. Furthermore, the pyruvate carboxylase reaction was identified as a target for metabolic engineering, after it was confirmed to be transcriptionally regulated through the correlation of intracellular fluxes and transcriptional changes.
I Goldberg - One of the best experts on this subject based on the ideXlab platform.
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the cytosolic pathway of l Malic Acid synthesis in saccharomyces cerevisiae the role of fumarase
Applied Microbiology and Biotechnology, 1996Co-Authors: Ophry Pines, Sharona Evenram, N Elnathan, Emil Battat, O Aharonov, D Gibson, I GoldbergAbstract:Saccharomyces cerevisiae accumulates l-Malic Acid but only minute amounts of fumaric Acid. A 13C-nuclear magnetic resonance study following the label from glucose to l-Malic Acid indicates that the l-Malic Acid is synthesized from pyruvic Acid via oxaloacetic Acid. From this, and from previously published studies, we conclude that a cytosolic reductive pathway leading from pyruvic Acid via oxaloacetic Acid to l-Malic Acid is responsible for the l-Malic Acid production in yeast. The non-production of fumaric Acid can be explained by the conclusion that, in the cell, cytosolic fumarase catalyzes the conversion of fumaric Acid to l-Malic Acid but not the reverse. This conclusion is based on the following findings. (a) The cytosolic enzyme exhibits a 17-fold higher affinity towards fumaric Acid than towards l-Malic Acid; the Km for l-Malic Acid is very high indicating that l-Malic Acid is not an in vivo substrate of the enzyme. (b) Overexpression of cytosolic fumarase does not cause accumulation of fumaric Acid (but rather more l-Malic Acid). (c) According to 13C NMR studies there is no interconversion of cytosolic l-Malic and fumaric Acids.
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l Malic Acid formation by immobilized saccharomyces cerevisiae amplified for fumarase
Enzyme and Microbial Technology, 1991Co-Authors: R J Neufeld, Ophry Pines, Y. Peleg, J S Rokem, I GoldbergAbstract:The yeast Saccharomyces cerevisiae was amplified for the enzyme fumarase by cloning the single nuclear gene downstream of a strong promoter. The overproducing strain converted fumaric Acid to l-Malic Acid at a rate of 65 mM g−1 h−1 in free cell experiments, and approximately 87% of the fumaric Acid was converted to l-Malic Acid within 45 min. Activity was dependent on the addition of surfactant to the medium, and minimal activity was seen with the wild-type yeast strain. The constructed strain was immobilized in agarose beads (2.4 mm mean diameter) and within agarose microspheres (193 and 871 μm mean diameter). The rate of bioconversion increased with decreasing bead diameter, with similar rates observed with the 193-μm diameter microspheres to that achieved with the free cells. The presence of surfactant was essential for initial activity of the immobilized cells; however, high activity was observed in subsequent experiments in the absence of surfactant. Stable activities over a 48-h period were maintained within the large-diameter agarose beads, while decreasing activities were observed within the agarose microspheres.
Christoph Knuf - One of the best experts on this subject based on the ideXlab platform.
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Malic Acid production by Aspergillus oryzae
2017Co-Authors: Christoph KnufAbstract:Malic Acid is a C4 dicarboxylic Acid which is used as an Acidulant in food and beverages. It is also considered as a bio-building block to replace petrochemically derived compounds in the post oil era. This organic Acid can be biotechnologically derived from fermentation using renewable feedstocks as carbon source. Aspergilli are among the best producers of organic Acid and A. flavus/oryzae is the best natural producer of Malic Acid. The mechanism of Malic Acid production in A. oryzae was first assessed by transcriptome analysis. A nitrogen starvation response, probably regulated by a transcription factor related to the S. cerevisiae Msn2/4 transcriptional activator of stress related genes, was found to result in high Malic Acid production. Furthermore the pyruvate carboxylase reaction was identified as a metabolic engineering target. This gene, together with the malate dehydrogenase and a Malic Acid exporter was overexpressed in the strain 2103a-68, which was characterized in a second project. The overexpression led to an 80% increase in yield during the starvation phase (1.49 mol (mol gluc)-1) and a triplication of the specific production rate. The increase in citric Acid production in the engineered strain and its evaluation through model simulations led to the curation of the A. oryzae GEM. The existing model was curated with special emphasis on the mitochondrial transport reactions and let to a more defined network around the production of organic Acids. Furthermore, the performance of the strain 2103a-68 on xylose as carbon source was evaluated as well and the good results led to the final project of manipulating the carbon source utilization by deleting the carbon catabolite repressor CreA. This work contributed to the understanding of the regulation of Malic Acid production. This knowledge was used for the development of A. oryzae as an organic Acid producer through metabolic engineering. Furthermore, the evaluation of xylose as an alternative carbon source paved the way towards the use of lignucellulosic feedstocks and showed the suitability of A. oryzae for the biorefinery of the future.
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physiological characterization of the high Malic Acid producing aspergillus oryzae strain 2103a 68
Applied Microbiology and Biotechnology, 2014Co-Authors: Christoph Knuf, Intawat Nookaew, Stephen H Brown, Alan Berry, Jens Nielsen, Ilse M Remmers, Sakda KhoomrungAbstract:Malic Acid is a C4 dicarboxylic Acid that is currently mainly used in the food and beverages industry as an Acidulant. Because of the versatility of the group of C4 dicarboxylic Acids, the chemical industry has a growing interest in this chemical compound. As Malic Acid will be considered as a bulk chemical, microbial production requires organisms that sustain high rates, yields, and titers. Aspergillus oryzae is mainly known as an industrial enzyme producer, but it was also shown that it has a very competitive natural production capacity for Malic Acid. Recently, an engineered A. oryzae strain, 2103a-68, was presented which overexpressed pyruvate carboxylase, malate dehydrogenase, and a Malic Acid transporter. In this work, we report a detailed characterization of this strain including detailed rates and yields under Malic Acid production conditions. Furthermore, transcript levels of the genes of interest and corresponding enzyme activities were measured. On glucose as carbon source, 2103a-68 was able to secrete Malic Acid at a maximum specific production rate during stationary phase of 1.87 mmol (g dry weight (DW))−1 h−1 and with a yield of 1.49 mol mol−1. Intracellular fluxes were obtained using 13C flux analysis during exponential growth, supporting the success of the metabolic engineering strategy of increasing flux through the reductive cytosolic tricarboxylic Acid (rTCA) branch. Additional cultivations using xylose and a glucose/xylose mixture demonstrated that A. oryzae is able to efficiently metabolize pentoses and hexoses to produce Malic Acid at high titers, rates, and yields.
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investigation of Malic Acid production in aspergillus oryzae under nitrogen starvation conditions
Applied and Environmental Microbiology, 2013Co-Authors: Christoph Knuf, Intawat Nookaew, Stephen H Brown, Michael Mcculloch, Alan Berry, Jens NielsenAbstract:Malic Acid has great potential for replacing petrochemical building blocks in the future. For this application, high yields, rates, and titers are essential in order to sustain a viable biotechnological production process. Natural high-capacity Malic Acid producers like the Malic Acid producer Aspergillus flavus have so far been disqualified because of special growth requirements or the production of mycotoxins. As A. oryzae is a very close relative or even an ecotype of A. flavus, it is likely that its high Malic Acid production capabilities with a generally regarded as safe (GRAS) status may be combined with already existing large-scale fermentation experience. In order to verify the Malic Acid production potential, two wild-type strains, NRRL3485 and NRRL3488, were compared in shake flasks. As NRRL3488 showed a volumetric production rate twice as high as that of NRRL3485, this strain was selected for further investigation of the influence of two different nitrogen sources on Malic Acid secretion. The cultivation in lab-scale fermentors resulted in a higher final titer, 30.27 +/- 1.05 g liter(-1), using peptone than the one of 22.27 +/- 0.46 g liter(-1) obtained when ammonium was used. Through transcriptome analysis, a binding site similar to the one of the Saccharomyces cerevisiae yeast transcription factor Msn2/4 was identified in the upstream regions of glycolytic genes and the cytosolic Malic Acid production pathway from pyruvate via oxaloacetate to malate, which suggests that Malic Acid production is a stress response. Furthermore, the pyruvate carboxylase reaction was identified as a target for metabolic engineering, after it was confirmed to be transcriptionally regulated through the correlation of intracellular fluxes and transcriptional changes.
Shangtian Yang - One of the best experts on this subject based on the ideXlab platform.
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polyMalic Acid fermentation by aureobasidium pullulans for Malic Acid production from soybean hull and soy molasses fermentation kinetics and economic analysis
Bioresource Technology, 2017Co-Authors: Chi Cheng, Yipin Zhou, Shangtian YangAbstract:Abstract PolyMalic Acid (PMA) production by Aureobasidium pullulans ZX-10 from soybean hull hydrolysate supplemented with corn steep liquor (CSL) gave a Malic Acid yield of ∼0.4 g/g at a productivity of ∼0.5 g/L·h. ZX-10 can also ferment soy molasses, converting all carbohydrates including the raffinose family oligosaccharides to PMA, giving a high titer (71.9 g/L) and yield (0.69 g/g) at a productivity of 0.29 g/L·h in fed-batch fermentation under nitrogen limitation. A higher productivity of 0.64 g/L·h was obtained in repeated batch fermentation with cell recycle and CSL supplementation. Cost analysis for a 5000 MT plant shows that Malic Acid can be produced at $1.10/kg from soy molasses, $1.37/kg from corn, and $1.74/kg from soybean hull. At the market price of $1.75/kg, Malic Acid production from soy molasses via PMA fermentation offers an economically competitive process for industrial production of bio-based Malic Acid.
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production of polyMalic Acid and Malic Acid by aureobasidium pullulans fermentation and Acid hydrolysis
Biotechnology and Bioengineering, 2013Co-Authors: Yipin Zhou, Shangtian YangAbstract:Malic Acid is a dicarboxylic Acid widely used in the food industry and also a potential C4 platform chemical that can be produced from biomass. However, microbial fermentation for direct Malic Acid production is limited by low product yield, titer, and productivity due to end-product inhibition. In this work, a novel process for Malic Acid production from polyMalic Acid (PMA) fermentation followed by Acid hydrolysis was developed. First, a PMA-producing Aureobasidium pullulans strain ZX-10 was screened and isolated. This microbe produced PMA as the major fermentation product at a high-titer equivalent to 87.6 g/L of Malic Acid and high-productivity of 0.61 g/L h in free-cell fermentation in a stirred-tank bioreactor. Fed-batch fermentations with cells immobilized in a fibrous-bed bioreactor (FBB) achieved the highest product titer of 144.2 g/L and productivity of 0.74 g/L h. The fermentation produced PMA was purified by adsorption with IRA-900 anion-exchange resins, achieving a ∼100% purity and a high recovery rate of 84%. Pure Malic Acid was then produced from PMA by hydrolysis with 2 M sulfuric Acid at 85°C, which followed the first-order reaction kinetics. This process provides an efficient and economical way for PMA and Malic Acid production, and is promising for industrial application. Biotechnol. Bioeng. 2013; 110: 2105–2113. © 2013 Wiley Periodicals, Inc.
Ophry Pines - One of the best experts on this subject based on the ideXlab platform.
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the cytosolic pathway of l Malic Acid synthesis in saccharomyces cerevisiae the role of fumarase
Applied Microbiology and Biotechnology, 1996Co-Authors: Ophry Pines, Sharona Evenram, N Elnathan, Emil Battat, O Aharonov, D Gibson, I GoldbergAbstract:Saccharomyces cerevisiae accumulates l-Malic Acid but only minute amounts of fumaric Acid. A 13C-nuclear magnetic resonance study following the label from glucose to l-Malic Acid indicates that the l-Malic Acid is synthesized from pyruvic Acid via oxaloacetic Acid. From this, and from previously published studies, we conclude that a cytosolic reductive pathway leading from pyruvic Acid via oxaloacetic Acid to l-Malic Acid is responsible for the l-Malic Acid production in yeast. The non-production of fumaric Acid can be explained by the conclusion that, in the cell, cytosolic fumarase catalyzes the conversion of fumaric Acid to l-Malic Acid but not the reverse. This conclusion is based on the following findings. (a) The cytosolic enzyme exhibits a 17-fold higher affinity towards fumaric Acid than towards l-Malic Acid; the Km for l-Malic Acid is very high indicating that l-Malic Acid is not an in vivo substrate of the enzyme. (b) Overexpression of cytosolic fumarase does not cause accumulation of fumaric Acid (but rather more l-Malic Acid). (c) According to 13C NMR studies there is no interconversion of cytosolic l-Malic and fumaric Acids.
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l Malic Acid formation by immobilized saccharomyces cerevisiae amplified for fumarase
Enzyme and Microbial Technology, 1991Co-Authors: R J Neufeld, Ophry Pines, Y. Peleg, J S Rokem, I GoldbergAbstract:The yeast Saccharomyces cerevisiae was amplified for the enzyme fumarase by cloning the single nuclear gene downstream of a strong promoter. The overproducing strain converted fumaric Acid to l-Malic Acid at a rate of 65 mM g−1 h−1 in free cell experiments, and approximately 87% of the fumaric Acid was converted to l-Malic Acid within 45 min. Activity was dependent on the addition of surfactant to the medium, and minimal activity was seen with the wild-type yeast strain. The constructed strain was immobilized in agarose beads (2.4 mm mean diameter) and within agarose microspheres (193 and 871 μm mean diameter). The rate of bioconversion increased with decreasing bead diameter, with similar rates observed with the 193-μm diameter microspheres to that achieved with the free cells. The presence of surfactant was essential for initial activity of the immobilized cells; however, high activity was observed in subsequent experiments in the absence of surfactant. Stable activities over a 48-h period were maintained within the large-diameter agarose beads, while decreasing activities were observed within the agarose microspheres.