pH-Dependent Uptake of Fumaric Acid in Saccharomyces cerevisiae under Anaerobic Conditions
01 Feb 2012-Applied and Environmental Microbiology (American Society for Microbiology)-Vol. 78, Iss: 3, pp 705-716
TL;DR: The experimental results showed that at a cultivation pH of 5.0 and an external fumaric acid concentration of approximately 0.8 mmol · liter−1, the f Kumaric acid uptake rate was unexpectedly high and could not be explained by diffusion of the undissociated form across the plasma membrane alone, which could indicate the presence of protein-mediated import.
Abstract: Microbial production of C4 dicarboxylic acids from renewable resources has gained renewed interest. The yeast Saccharomyces cerevisiae is known as a robust microorganism and is able to grow at low pH, which makes it a suitable candidate for biological production of organic acids. However, a successful metabolic engineering approach for overproduction of organic acids requires an incorporation of a proper exporter to increase the productivity. Moreover, low-pH fermentations, which are desirable for facilitating the downstream processing, may cause back diffusion of the undissociated acid into the cells with simultaneous active export, thereby creating an ATP-dissipating futile cycle. In this work, we have studied the uptake of fumaric acid in S. cerevisiae in carbon-limited chemostat cultures under anaerobic conditions. The effect of the presence of fumaric acid at different pH values (3 to 5) has been investigated in order to obtain more knowledge about possible uptake mechanisms. The experimental results showed that at a cultivation pH of 5.0 and an external fumaric acid concentration of approximately 0.8 mmol · liter-1, the fumaric acid uptake rate was unexpectedly high and could not be explained by diffusion of the undissociated form across the plasma membrane alone. This could indicate the presence of protein-mediated import. At decreasing pH levels, the fumaric acid uptake rate was found to increase asymptotically to a maximum level. Although this observation is in accordance with proteinmediated import, the presence of a metabolic bottleneck for fumaric acid conversion under anaerobic conditions could not be excluded.
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TL;DR: The key problems of the industrial production of microbial fumaric acid are reviewed, and various strategies, including strain improvement, morphology control, substrate choice, fermentation process and separation process, and their economical possibilities for industrial processes are discussed.
Abstract: The growing concern about the safety of food and dairy additives and the increasing costs of petroleum-based chemicals have rekindled the interest in the fermentation processes for fumaric acid production. The key problems of the industrial production of microbial fumaric acid are reviewed in this paper. Various strategies, including strain improvement, morphology control, substrate choice, fermentation process and separation process, are summarized and compared, and their economical possibilities for industrial processes are discussed. The market prospects and technological strategies for value-added fumaric acid derivatives are also addressed. The future prospects of microbial fumaric acid production are proposed at the end of this article.
127 citations
TL;DR: Combining these results reveals the potential of U.trichophora TZ1 to become an industrially applicable production host for malic acid from biodiesel-derived glycerol, thus making the overall biodiesel production process economically and ecologically more feasible.
Abstract: In order to establish a cost-efficient biodiesel biorefinery, valorization of its main by-product, crude glycerol, is imperative. Recently, Ustilago trichophora TZ1 was found to efficiently produce malic acid from glycerol. By adaptive laboratory evolution and medium optimization, titer and rate could be improved significantly. Here we report on the investigation of this strain in fed-batch bioreactors. With pH controlled at 6.5 (automatic NaOH addition), a titer of 142 ± 1 g L−1 produced at an overall rate of 0.54 ± 0.00 g L−1 h−1 was reached by optimizing the initial concentrations of ammonium and glycerol. Combining the potential of bioreactors and CaCO3 as buffer system, we were able to increase the overall production rate to 0.74 ± 0.06 g L−1 h−1 with a maximum production rate of 1.94 ± 0.32 g L−1 reaching a titer of 195 ± 15 g L−1. The initial purification strategy resulted in 90 % pure calcium malate as solid component. Notably, the fermentation is not influenced by an increased temperature of up to 37 °C, which reduces the energy required for cooling. However, direct acid production is not favored as at a lowered pH value of pH 4.5 the malic acid titer decreased to only 9 ± 1 g L−1. When using crude glycerol as substrate, only the product to substrate yield is decreased. The results are discussed in the context of valorizing glycerol with Ustilaginaceae. Combining these results reveals the potential of U. trichophora TZ1 to become an industrially applicable production host for malic acid from biodiesel-derived glycerol, thus making the overall biodiesel production process economically and ecologically more feasible.
58 citations
Cites background from "pH-Dependent Uptake of Fumaric Acid..."
...It was reported that the most likely mechanism for export of dicarboxylic acids at low pH is an antiport with protons [47]....
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01 Jan 2012
TL;DR: The aim of this work is to contribute to the development of a knowledge and understanding of metabolic engineering and its applications in the context of synthetic and systems biology.
Abstract: Contributors Part I: Emerging Methods for Redesigning Microorganisms 1. Towards Synthetic Gene Circuits with Enhancers: Biology's Multi-input Integrators Roee Amit 2. Elementary Mode Analysis: A Useful Metabolic Pathway Analysis Tool for Reprograming Microbial Metabolic Pathways Cong T. Trinh and R. Adam Thompson 3. Evolutionary Engineering for Industrial Microbiology Niti Vanee, Adam B. Fisher and Stephen S. Fong 4. Monitoring Microbial Diversity of Bioreactors Using Metagenomic Approaches Joshua T. Ellis, Ronald C. Sims and Charles D. Miller 5. Synthetic Biology Triggers New Era of Antibiotics Development Jianfeng Wang, Zhi-Qiang Xiong, Hailin Meng, Yiguang Wang and Yong Wang 6. Cascades and Networks of Regulatory Genes That Control Antibiotic Biosynthesis Juan F. Martin and Paloma Liras 7. Systems Analysis of Microbial Adaptations to Simultaneous Stresses Ross P. Carlson, Olusegun J. Oshota and Reed L. Taffs 8. Metabolic Reprogramming under Microaerobic and Anaerobic Conditions in Bacteria Yue Shan, Yong Lai and Aixin Yan 9. Tunable Promoters in Synthetic and Systems Biology Tore Dehli, Christian Solem and Peter Ruhdal Jensen10. Analysis of Corynebacterium glutamicum Promoters and Their Applications Jan Nesvera, Jiri Holatko and Miroslav Patek Part II: Metabolic Engineering for Overproducing Chemicals and Materials 11. Production of Fumaric Acid by Fermentation Adrie J.J. Straathof and Walter M. van Gulik 12. Metabolic Engineering of Microorganisms for Vitamin C Production Jingwen Zhou, Guocheng Du and Jian Chen 13. Molecular Mechanisms and Metabolic Engineering of Glutamate Overproduction in Corynebacterium glutamicum Takashi Hirasawa, Jongpill Kim, Tomokazu Shirai, Chikara Furusawa and Hiroshi Shimizu 14. Microbial Metabolic Engineering for L-threonine Production Xunyan Dong, Peter J. Quinn and Xiaoyuan Wang 15. The Production of Coenzyme Q10 in Microorganisms Corinne P. Cluis, Dominic Pinel and Vincent J. Martin 16. Genetic Modification and Bioprocess Optimization for S-adenosyl-L-methionine biosynthesis Xiaoqing Hu, Peter J. Quinn, Zhou Wang, Guoqiang Han and Xiaoyuan Wang 17. Manipulation of Ralstonia eutropha Carbon Storage Pathways to Produce Useful Bio-based Products Christopher J. Brigham, Natalia Zhila, Ekaterina Shishatskaya, Tatiana G. Volova and Anthony J. Sinskey 18. Metabolic Engineering of Inducer Formation for Cellulase and Hemicellulase Gene Expression in Trichoderma reesei Bernhard Seiboth, Silvia Herold and Christian P. Kubicek 19. Microbiologically Produced Carboxylic Acids Used as Building Blocks in Organic Synthesis Andreas Aurich, Robert Specht, Roland A. Muller, Ulrich Stottmeister, Venelina Yovkova, Christina Otto, Martina Holz, Gerold Barth, Philipp Heretsch, Franziska A. Thomas, Dieter Sicker and Athanassios Giannis Index Index
39 citations
TL;DR: Systematic identification of keto acids transporters would provide clues to further improve the accumulation of specific organic acids with higher efficiency in eukaryotic microorganisms.
Abstract: Production of organic acids by microorganisms is of great importance for obtaining building-block chemicals from sustainable biomass. Extracellular accumulation of organic acids involved a series of transporters, which play important roles in the accumulation of specific organic acid while lack of systematic demonstration in eukaryotic microorganisms. To circumvent accumulation of by-product, efforts have being orchestrated to carboxylate transport mechanism for potential clue in Yarrowia lipolytica WSH-Z06. Six endogenous putative transporter genes, YALI0B19470g, YALI0C15488g, YALI0C21406g, YALI0D24607g, YALI0D20108g and YALI0E32901g, were identified. Transport characteristics and substrate specificities were further investigated using a carboxylate-transport-deficient Saccharomyces cerevisiae strain. These transporters were expressed in Y. lipolytica WSH-Z06 to assess their roles in regulating extracellular keto acids accumulation. In a Y. lipolytica T1 line over expressing YALI0B19470g, α-ketoglutarate accumulated to 46.7 g·L−1, whereas the concentration of pyruvate decreased to 12.3 g·L−1. Systematic identification of these keto acids transporters would provide clues to further improve the accumulation of specific organic acids with higher efficiency in eukaryotic microorganisms.
30 citations
TL;DR: In this paper, mitochondrial engineering was used to construct the oxidative pathway for fumarate production starting from the TCA cycle intermediate α-ketoglutarate in Candida glabrata.
Abstract: Microbial fumarate production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here, mitochondrial engineering was used to construct the oxidative pathway for fumarate production starting from the TCA cycle intermediate α-ketoglutarate in Candida glabrata. Accordingly, α-ketoglutarate dehydrogenase complex (KGD), succinyl-CoA synthetase (SUCLG), and succinate dehydrogenase (SDH) were selected to be manipulated for strengthening the oxidative pathway, and the engineered strain T.G-K-S-S exhibited increased fumarate biosynthesis (1.81 g L(-1)). To further improve fumarate production, the oxidative route was optimized. First, three fusion proteins KGD2-SUCLG2, SUCLG2-SDH1 and KGD2-SDH1 were constructed, and KGD2-SUCLG2 led to improved fumarate production (4.24 g L(-1)). In addition, various strengths of KGD2-SUCLG2 and SDH1 expression cassettes were designed by combinations of promoter strengths and copy numbers, resulting in a large increase in fumarate production (from 4.24 g L(-1) to 8.24 g L(-1)). Then, through determining intracellular amino acids and its related gene expression levels, argininosuccinate lyase in the urea cycle was identified as the key factor for restricting higher fumarate production. Correspondingly, after overexpression of it, the fumarate production was further increased to 9.96 g L(-1). Next, two dicarboxylic acids transporters facilitated an improvement of fumarate production, and, as a result, the final strain T.G-KS(H)-S(M)-A-2S reached fumarate titer of 15.76 g L(-1). This strategy described here paves the way to the development of an efficient pathway for microbial production of fumarate.
29 citations
Cites background from "pH-Dependent Uptake of Fumaric Acid..."
...Consequently, a successful metabolic engineering approach for overproduction of organic acids (e.g. fumarate) requires an incorporation of a proper exporter to increase the productivity (Jamalzadeh et al., 2012)....
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...According to thermodynamic equilibrium calculations, the export mechanism for fumarate at a low cultivation pH is a proton antiporter, importing one proton for each fumarate anion exported (Jamalzadeh et al., 2012)....
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References
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TL;DR: The effect of benzoate on respiration was dependent on the dilution rate: at high dilution rates respiration increased proportionally with increasing Benzoate concentration as mentioned in this paper.
Abstract: Addition of benzoate to the medium reservoir of glucose-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 growing at a dilution rate (D) of 0.10 h-1 resulted in a decrease in the biomass yield, and an increase in the specific oxygen uptake rate (qO2) from 2.5 to as high as 19.5 mmol g-1 h-1. Above a critical concentration, the presence of benzoate led to alcoholic fermentation and a reduction in qO2 to 13 mmol g-1 h-1. The stimulatory effect of benzoate on respiration was dependent on the dilution rate: at high dilution rates respiration was not enhanced by benzoate. Cells could only gradually adapt to growth in the presence of benzoate: a pulse of benzoate given directly to the culture resulted in wash-out. As the presence of benzoate in cultures growing at low dilution rates resulted in large changes in the catabolic glucose flux, it was of interest to study the effect of benzoate on the residual glucose concentration in the fermenter as well as on the level of some selected enzymes. At D = 0.10 h-1, the residual glucose concentration increased proportionally with increasing benzoate concentration. This suggests that modulation of the glucose flux mainly occurs via a change in the extracellular glucose concentration rather than by synthesis of an additional amount of carriers. Also various intracellular enzyme levels were not positively correlated with the rate of respiration. A notable exception was citrate synthase: its level increased with increasing respiration rate. Growth of S. cerevisiae in ethanol-limited cultures in the presence of benzoate also led to very high qO2 levels of 19-21 mmol g-1 h-1. During growth on glucose as well as on ethanol, the presence of benzoate coincided with an increase in the mitochondrial volume up to one quarter of the total cellular volume. Also with the Crabtree-negative yeasts Candida utilis, Kluyveromyces marxianus and Hansenula polymorpha, growth in the presence of benzoate resulted in an increase in qO2 and, at high concentrations of benzoate, in aerobic fermentation. In contrast to S. cerevisiae, the highest qO2 of these yeasts when growing at D = 0.10 h-1 in the presence of benzoate was equal to, or lower than the qO2 attainable at mu(max) without benzoate. Enzyme activities that were repressed by glucose in S. cerevisiae also declined in K. marxianus when the glucose flux was increased by the presence of benzoate.(ABSTRACT TRUNCATED AT 400 WORDS)
1,444 citations
TL;DR: Through analysis of the current advances in production of citric, lactic and succinic acid production, guidelines for future developments in this fast-moving field are presented.
Abstract: Microbial production of organic acids is a promising approach for obtaining building-block chemicals from renewable carbon sources. Although some acids have been produced for some time and in-depth knowledge of these microbial production processes has been gained, further microbial production processes seem to be feasible, but large-scale production has not yet been possible. Citric, lactic and succinic acid production exemplify three processes in different stages of industrial development. Although the questions being addressed by current research on these processes are diverging, a comparison is helpful for understanding microbial organic acid production in general. In this article, through analysis of the current advances in production of these acids, we present guidelines for future developments in this fast-moving field.
750 citations
"pH-Dependent Uptake of Fumaric Acid..." refers background in this paper
...An important aspect of organic acid production in microbial cell factories is the export of the product over the cell membrane with a sufficient capacity (30)....
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...cerevisiae has been proposed as a cell factory for the production of bulk chemicals, such as malic, lactic, and citric acid from renewable substrates (1, 30, 36, 42, 43)....
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TL;DR: On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.
Abstract: To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.
583 citations
TL;DR: The physiology of Saccharomyces cerevisiae CBS 8066 was studied in anaerobic glucose-limited chemostat cultures in a mineral medium supplemented with ergosterol and Tween 80, suggesting that the observed difference in cell yield may be ascribed to an uncoupling effect of acetic acid.
Abstract: The physiology of Saccharomyces cerevisiae CBS 8066 was studied in anaerobic glucose-limited chemostat cultures in a mineral medium supplemented with ergosterol and Tween 80 The organism had a mu max of 031 h-1 and a Ks for glucose of 055 mM At a dilution rate of 010 h-1, a maximal yield of 010 g biomass (g glucose)-1 was observed The yield steadily declined with increasing dilution rates, so a maintenance coefficient for anaerobic growth could not be estimated At a dilution rate of 010 h-1, the yield of the S cerevisiae strain H1022 was considerably higher than for CBS 8066, despite a similar cell composition The major difference between the two yeast strains was that S cerevisiae H1022 did not produce acetate, suggesting that the observed difference in cell yield may be ascribed to an uncoupling effect of acetic acid The absence of acetate formation in H1022 correlated with a relatively high level of acetyl-CoA synthetase The uncoupling effect of weak acids on anaerobic growth was confirmed in experiments in which a weak acid (acetate or propionate) was added to the medium feed This resulted in a reduction in yield and an increase in specific ethanol production Both yeasts required approximately 35 mg oleic acid (g biomass)-1 for optimal growth Lower or higher concentrations of this fatty acid, supplied as Tween 80, resulted in uncoupling of dissimilatory and assimilatory processes
490 citations
"pH-Dependent Uptake of Fumaric Acid..." refers background in this paper
...The yeast Saccharomyces cerevisiae is an organism that is able to grow at low pH values (38), although it does not naturally produce fumaric acid....
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TL;DR: Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on 13C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production.
Abstract: Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO(2)-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)(-1). A previously engineered glucose-tolerant, C(2)-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter(-1) at a malate yield of 0.42 mol (mol glucose)(-1). Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on (13)C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.
398 citations
"pH-Dependent Uptake of Fumaric Acid..." refers background in this paper
...cerevisiae has been proposed as a cell factory for the production of bulk chemicals, such as malic, lactic, and citric acid from renewable substrates (1, 30, 36, 42, 43)....
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