Fumaric Acid Titer Research Articles

Semi-rational evolution of pyruvate carboxylase from Rhizopus oryzae for elevated fumaric acid synthesis in Saccharomyces cerevisiae

Dicarboxylic acids are widely used in food, pharmaceutical, and chemical industries. Pyruvate carboxylase (PYC) plays a pivotal role in the production of dicarboxylic acids in microbial fermentation process. Our previous work showed that heterologous expression of pyruvate carboxylase (RoPYC) from Rhizopus oryzae resulted in an increase in fumaric acid titer to 226.0 ± 2.2 mg/L from 194.0 ± 4.0 mg/L in the S. cerevisiae pdc1adh1fum1 strain. However, PYC still remained the metabolic step limiting the production of target carboxylic acids. In this study, semi-rational evolution of pyruvate carboxylase by site-saturation mutagenesis combined with codon optimization was conducted to further improve fumaric acid synthesis. We demonstrated that each of three mutations (N315F, R485P and N1078F) or codon optimization of RoPYC significantly increased the production of fumaric acid. A maximal titer of 465.5 ± 6.5 mg/L was achieved in flasks by the strain expressing codon-optimized RoPYC mutant (R485P). Enzyme assays of these mutants showed higher PYC activities, while homology modeling indicated that the increased PYC activities could be attributed to the modulation of the allosteric domain and the biotin carboxylation domain. In addition, both calcium ion and carbon dioxide displayed positive effects on the fumaric acid production by this mutant. Overall, the strategy described here demonstrated an effective way for elevating PYC activity and further enhance the synthesis of dicarboxylic acids.

Fumaric acid production using alternate fermentation mode by immobilized Rhizopus oryzae-a greener production strategy

The current work investigates the impact of using immobilized Rhizopus oryzae NRRL 1526 for bioproduction of fumaric acid using agro-industrial residues as feedstock. This use of agro-industrial residues, a renewable feedstock, for the production of bio-based platform chemical makes the process cost-competitive as well as greener by preventing the release of assimilable organic carbon to the environment, thereby reducing the generation of greenhouse gases. Immobilization of R. oryzae has been proposed previously to alleviate operational difficulties confronted during free mycelial fungal fermentation. To this effect, three synthetic refuse materials namely polystyrene foam, polyester sponge and polyurethane foam were investigated for their suitability towards fumaric acid bioproduction. Polystyrene foam was identified as the most suitable support material for immobilization as well as fumaric acid production. In addition to the considerable reduction in the lag-phase (from 48 to 24 h) the reduction in the size of the support material from cubes of 1 cm to beads of 0.1–0.3 cm led to a 42% improvement in fumaric acid production (27 g/L against 19 g/L). Growing the polystyrene foam bead immobilized R. oryzae on apple pomace ultrafiltration sludge as sole feedstock yielded a final fumaric acid titer of 7.9 g/L whereas free mycelial fermentation yielded 6.3 g/L. Moreover, upon operating the fermentation with intermittent feeding, a three-fold increase (1.7 g/L to 5.1 g/L) in fumaric acid production was obtained upon supplementation of the apple pomace sludge media with molasses, an agro-industrial residue, as feed.

Influence analysis of glycerol in fumaric acid co-fermentation process by Rhizopus arrhizus

Fumaric acid production from industrial waste by microorganism was regarded as an available method. Glycerol as the by-product of biodiesel was a suitable feed stock for fumaric acid production co-fermented with glucose. In this work, the influence of glycerol on the co-fermentation process was explored on the basis of fermentation optimization. It was indicated that the optimum addition level of glycerol was 40 g/L, under which the efficiency of glucose conversion and fumaric acid titer was certainly preferable. After comparing the permeability and fatty acids of the cell membrane, Rhizopus arrhizus cells membrane in co-fermentation process showed a better permeability and a reasonable fatty acids composition, which caused the improvement of fumaric acid emission, leading to the promotion of glucose conversion. Identification of key metabolites combining with feeding strategies was further illustrated the superiority of co-fermentation, which had a potential development advantages in organic acid production process.

Metabolic engineering of the thermophilic filamentous fungus Myceliophthora thermophila to produce fumaric acid

BackgroundFumaric acid is widely used in food and pharmaceutical industries and is recognized as a versatile industrial chemical feedstock. Increasing concerns about energy and environmental problems have resulted in a focus on fumaric acid production by microbial fermentation via bioconversion of renewable feedstocks. Filamentous fungi are the predominant microorganisms used to produce organic acids, including fumaric acid, and most studies to date have focused on Rhizopus species. Thermophilic filamentous fungi have many advantages for the production of compounds by industrial fermentation. However, no previous studies have focused on fumaric acid production by thermophilic fungi.ResultsWe explored the feasibility of producing fumarate by metabolically engineering Myceliophthora thermophila using the CRISPR/Cas9 system. Screening of fumarases suggested that the fumarase from Candida krusei was the most suitable for efficient production of fumaric acid in M. thermophila. Introducing the C. krusei fumarase into M. thermophila increased the titer of fumaric acid by threefold. To further increase fumarate production, the intracellular fumarate digestion pathway was disrupted. After deletion of the two fumarate reductase and the mitochondrial fumarase genes of M. thermophila, the resulting strain exhibited a 2.33-fold increase in fumarate titer. Increasing the pool size of malate, the precursor of fumaric acid, significantly increased the final fumaric acid titer. Finally, disruption of the malate–aspartate shuttle increased the intracellular malate content by 2.16-fold and extracellular fumaric acid titer by 42%, compared with that of the parental strain. The strategic metabolic engineering of multiple genes resulted in a final strain that could produce up to 17 g/L fumaric acid from glucose in a fed-batch fermentation process.ConclusionsThis is the first metabolic engineering study on the production of fumaric acid by the thermophilic filamentous fungus M. thermophila. This cellulolytic fungal platform provides a promising method for the sustainable and efficient-cost production of fumaric acid from lignocellulose-derived carbon sources in the future.

Production of Fumaric Acid by Bioconversion of Corncob Hydrolytes Using an Improved Rhizopus oryzae Strain.

The use of microorganism fermentation for production of fumaric acid (FA), which is widely used in food, medicine, and other fields, can provide technical support for the FA industry. In this study, we aimed to increase the titer of FA production by using an improved Rhizopus oryzae WHT5, which was domesticated to obtain a furfural-resistant strain in corncob hydrolytes. The metabolic pathways and metabolic network of this strain were investigated, and the related enzymes and metabolic flux were analyzed. Metabolic pathway analysis showed that the R. oryzae WHT5 strain produced FA mainly through two pathways. One occurred in the cytoplasm and the other was a mitochondrial pathway. The key parameters of the fermentation process were analyzed. The FA titer was 49.05g/L from corncob hydrolytes using R. oryzae WHT5 in a 7-L bioreactor. The use of a furfural-resistant strain developed through domestication effectively increased the titer of FA. This capacity of the microorganisms to produce high amounts of FA by bioconverting corncob hydrolyte can be further applied for industrial production of FA.

Omics-based approaches reveal phospholipids remodeling of Rhizopus oryzae responding to furfural stress for fumaric acid-production from xylose

In order to relieve the toxicity of furfural on Rhizopus oryzae fermentation, the molecular mechanism of R. oryzae responding to furfural stress for fumaric acid-production was investigated by omics-based approaches. In metabolomics analysis, 29 metabolites including amino acid, sugars, polyols and fatty acids showed significant changes for maintaining the basic cell metabolism at the cost of lowering fumaric acid production. To further uncover the survival mechanism, lipidomics was carried out, revealing that phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol and polyunsaturated acyl chains might be closely correlated with R. oryzae’s adapting to furfural stress. Based on the above omics analysis, lecithin, inositol and soybean oil were exogenously supplemented separately with an optimized concentration in the presence of furfural, which increased fumaric acid titer from 5.78g/L to 10.03g/L, 10.05g/L and 12.13g/L (increased by 73.5%, 73.8% and 110%, respectively). These findings provide a methodological guidance for hemicellulose-fumaric acid development.

Study of metabolic profile of Rhizopus oryzae to enhance fumaric acid production under low pH condition.

Ensuring a suitable pH is a major problem in industrial organic acid fermentation. To circumvent this problem, we used a metabolic profiling approach to analyze metabolite changes in Rhizopus oryzae under different pH conditions. A correlation between fumaric acid production and intracellular metabolic characteristics of R. oryzae was revealed by principal component analysis. The results showed that to help cell survival in the presence of low pH, R. oryzae altered amino acid and fatty acid metabolism and promoted sugar or sugar alcohol synthesis, corresponding with a suppressing of energy metabolism, phenylalanine, and tyrosine synthesis and finally resulting in the low performance of fumaric acid production. Based on this observation, 1% linoleic acid was added to the culture medium in pH 3.0 to decrease the carbon demand for cell survival, and the fumaric acid titer was enhanced by 39.7% compared with the control (pH3.0 without linoleic acid addition), reaching 18.3g/L after 84h of fermentation. These findings provide new insights into the mechanism by which R. oryzae responds to acidic stress and would be helpful for the development of efficient strategies for fumaric acid production at low pH.

Combining rational metabolic engineering and flux optimization strategies for efficient production of fumaric acid.

Fumaric acid is an important C4-dicarboxylic acid widely used in chemical, food, and pharmaceutical industries. Rational metabolic engineering together with flux optimization were performed for the development of an Escherichia coli strain capable of efficiently producing fumaric acid. The initial engineered strain, CWF4N overexpressing phosphoenolpyruvate carboxylase (PPC), produced 5.30 g/L of fumaric acid. Optimization of PPC flux by examining 24 types of synthetic PPC expression vectors further increased the titer up to 5.72 g/L with a yield of 0.432 g/g·glucose. Overexpression of the succinate dehydrogenase complex (sdhCDAB) led to an increase in carbon yield up to 0.493 g/g·glucose. Based on this mutant strain, citrate synthase (CS) was combinatorially overexpressed and balanced with PPC using 48 types of synthetic expression vectors. As a result, 6.24 g/L of fumaric acid was produced with a yield of 0.500 g/g·glucose. Fed-batch culture of this final strain allowed production of 25.5 g/L of fumaric acid with a yield of 0.366 g/g·glucose. Deletion of the aspA gene encoding aspartase and supplementation of aspartic acid further increased the fumaric acid titer to 35.1 g/L with a yield of 0.490 g/g·glucose.

Engineering Scheffersomyces stipitis for fumaric acid production from xylose

In this work, Scheffersomyces stipitis, the yeast with excellent xylose-utilizing ability, was firstly engineered for fumaric acid production from xylose with the heterologous reductive pathway from Rhizopus oryzae FM19, and 1.86g/L fumaric acid was produced by the initial strain PSRPMF under the oxygen-limited condition. Furthermore, three strategies were performed to improve the fumaric acid production, including increasing the reductive pathway activity by codon optimization, blocking the fumaric acid conversion in tricarboxylic acid cycle by knocking out the native fumarases, and improving the fumaric acid transportation by overexpressing heterologous transporter. Finally, the strain PSYPMFfS was obtained and the fumaric acid titer reached to 4.67g/L, significantly increased by 37.92-fold than that of the control strain PSPYSS. It was indicated that the S. stipitis was a promising platform for fumaric acid production from xylose.

Effects of soybean meal hydrolysate as the nitrogen source on seed culture morphology and fumaric acid production by Rhizopus oryzae

Fumaric acid production by fungal fermentation from renewable resources has received much attention, but suffers from low productivity and yield largely because of poor cell morphology limiting mass transfer. In this study, a simple seed culture medium was developed with soybean meal hydrolysate (SMH) as the nitrogen source for controlling the morphology of Rhizopus oryzae ATCC 20344. With the optimal seed culture conditions (pH 3.0, SMH concentration: 30% (v/v), and inoculation spore concentration: 8×107/L), uniformly dispersed mycelial clumps with a diameter of ∼0.1mm were formed, which gave high fumaric acid production from glucose in shake flasks, reaching a fumaric acid titer of 50.2g/L and yield of 0.72g/g glucose. Compared to the fermentation with cell pellets, the fermentation with mycelial clumps generated with SMH as nitrogen source increased fumaric acid production because of improved mass transfer. This process can lower the cost for bio-based fumaric acid and provide a novel way of using soybean meal besides its current use as animal feed supplement.

Fumaric acid production by Torulopsis glabrata: engineering the urea cycle and the purine nucleotide cycle.

A multi-vitamin auxotrophic Torulopsis glabrata strain, a pyruvate producer, was further engineered to produce fumaric acid. Using the genome-scale metabolic model iNX804 of T. glabrata, four fumaric acid biosynthetic pathways, involving the four cytosolic enzymes, argininosuccinate lyase (ASL), adenylosuccinate lyase (ADSL), fumarylacetoacetase (FAA), and fumarase (FUM1), were found. Athough single overexpression of each of the four enzymes in the cytosol improved fumaric acid production, the highest fumaric acid titer (5.62 g L(-1) ) was obtained with strain T.G-ASL(H) -ADSL(L) by controlling the strength of ASL at a high level and ADSL at a low level. In order to further improve the production of fumaric acid, the SpMAE1 gene encoding the C4 -dicarboxylic acids transporter was overexpressed in strain T.G-ASL(H) -ADSL(L) -SpMAE1 and the final fumaric acid titer increased to 8.83 g L(-1) . This study provides a novel strategy for fumaric acid biosynthesis by utilizing the urea cycle and the purine nucleotide cycle to enhance the bridge between carbon metabolism and nitrogen metabolism.

Enhanced acid tolerance of Rhizopus oryzae during fumaric acid production.

Ensuring a suitable pH in the culture broth is a major problem in microorganism-assisted industrial fermentation of organic acids. To address this issue, we investigated the physiological changes in Rhizopus oryzae at different extracellular pH levels and attempted to solve the issue of cell shortage under low pH conditions. We compared various parameters, such as membrane fatty acids' composition, intracellular pH, and adenosine triphosphate (ATP) concentration. It was found that the shortage of intracellular ATP might be the main reason for the low rate of fumaric acid production by R. oryzae under low pH conditions. When 1 g/l citrate was added to the culture medium at pH 3.0, the intracellular ATP concentration increased from 0.4 to 0.7 µmol/mg, and the fumaric acid titer was enhanced by 63% compared with the control (pH 3.0 without citrate addition). The final fumaric acid concentration at pH 3.0 reached 21.9 g/l after 96 h of fermentation. This strategy is simple and feasible for industrial fumaric acid production under low pH conditions.

Alternative respiration and fumaric acid production of Rhizopus oryzae

Under the conditions of fumaric acid fermentation, Rhizopus oryzae ME-F14 possessed at least two respiratory systems. The respiration of mycelia was partially inhibited by the cytochrome respiration inhibitor antimycin A or the alternative respiration inhibitor salicylhydroxamic acid and was completely inhibited in the presence of both antimycin A and salicylhydroxamic acid. During fumaric acid fermentation process, the activity of alternative respiration had a great correlation with fumaric acid productivity; both of them reached peak at the same time. The alternative oxidase gene, which encoded the mitochondrial alternative oxidase responsible for alternative respiration in R. oryzae ME-F14, was cloned and characterized in Escherichia coli. The activity of alternative respiration, the alternative oxidase gene transcription level, as well as the fumaric acid titer were measured under different carbon sources and different carbon-nitrogen ratios. The activity of alternative respiration was found to be comparable to the transcription level of the alternative oxidase gene and the fumaric acid titer. These results indicated that the activity of the alternative oxidase was regulated at the transcription stage under the conditions tested for R. oryzae ME-F14.

Fumaric acid production in Saccharomyces cerevisiae by simultaneous use of oxidative and reductive routes

In this study, the simultaneous use of reductive and oxidative routes to produce fumaric acid was explored. The strain FMME003 (Saccharomyces cerevisiae CEN.PK2-1CΔTHI2) exhibited capability to accumulate pyruvate and was used for fumaric acid production. The fum1 mutant FMME004 could produce fumaric acid via oxidative route, but the introduction of reductive route derived from Rhizopus oryzae NRRL 1526 led to lower fumaric acid production. Analysis of the key factors associated with fumaric acid production revealed that pyruvate carboxylase had a low degree of control over the carbon flow to malic acid. The fumaric acid titer was improved dramatically when the heterologous gene RoPYC was overexpressed and 32 μg/L of biotin was added. Furthermore, under the optimal carbon/nitrogen ratio, the engineered strain FMME004-6 could produce up to 5.64 ± 0.16 g/L of fumaric acid. These results demonstrated that the proposed fermentative method is efficient for fumaric acid production.

Development of a low pH fermentation strategy for fumaric acid production by Rhizopus oryzae

Dicarboxylic acids that are produced from renewable resources are becoming attractive building blocks for the polymers industry. In this respect, fumaric acid is very interesting. Its low aqueous solubility facilitates product recovery. To avoid excessive waste salt production during downstream processing, a low pH for fumaric acid fermentation will be beneficial. Studying the influence of pH, working volume and shaking frequency on cell cultivation helped us to identify the best conditions to obtain appropriate pellet morphologies of a wild type strain of Rhizopus oryzae. Using these pellets, the effects of pH and CO 2 addition were studied to determine the best conditions to produce fumaric acid in batch fermentations under nitrogen-limited conditions with glucose as carbon source. Decreasing either the fermentation pH below 5 or increasing the CO 2 content of the inlet air above 10% was unfavourable for the cell-specific productivity, fumaric acid yield, and fumaric acid titer. However, switching off the pH control late in the batch phase did not affect these performance parameters and allowed achieving pH of 3.6. A concentration of 20 g L −1 of fumaric acid was obtained at pH 3.6 while the average cell mass specific productivity and fumaric acid yield were the same as at pH 5.0. Consequently, relatively modest amounts of inorganic base were required for pH control, while recovery of the acid should be relatively easy at pH 3.6.