Malic Acid Production Research Articles

13 C-MFA helps to identify metabolic bottlenecks for improving malic acid production in Myceliophthora thermophila

BackgroundMyceliophthora thermophila has been engineered as a significant cell factory for malic acid production, yet strategies to further enhance production remain unclear and lack rational guidance. 13C-MFA (13C metabolic flux analysis) offers a means to analyze cellular metabolic mechanisms and pinpoint critical nodes for improving product synthesis. Here, we employed 13C-MFA to investigate the metabolic flux distribution of a high-malic acid-producing strain of M. thermophila and attempted to decipher the crucial bottlenecks in the metabolic pathways.ResultsCompared with the wild-type strain, the high-Malic acid-producing strain M. thermophila JG207 exhibited greater glucose uptake and carbon dioxide evolution rates but lower oxygen uptake rates and biomass yields. Consistent with these phenotypes, the 13C-MFA results showed that JG207 displayed elevated flux through the EMP pathway and downstream TCA cycle, along with reduced oxidative phosphorylation flux, thereby providing more precursors and NADH for malic acid synthesis. Furthermore, based on the 13C-MFA results, we conducted oxygen-limited culture and nicotinamide nucleotide transhydrogenase (NNT) gene knockout experiments to increase the cytoplasmic NADH level, both of which were shown to be beneficial for malic acid accumulation.ConclusionsThis work elucidates and validates the key node for achieving high malic acid production in M. thermophila. We propose effective fermentation strategies and genetic modifications for enhancing malic acid production. These findings offer valuable guidance for the rational design of future cell factories aimed at improving malic acid yields.

Investigation on the influence of GPI-AP for the production of malic acid in Aspergillus niger

Based on the potential of microbial fermentation in the sustainable and environmentally friendly production of L-malic acid (L-MA), this study aims to investigate the effect of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) on enhancing the stability of Aspergillus niger (A. niger) cell wall to increase L-MA production. Through experiments, we discovered and screened GPI2, a GPI-anchored cell surface glycoprotein, and confirmed for the first time that it has a promoting effect on L-MA, a metabolite of A. niger. This recombinant strain RG0098 that overexpressed gpi2 showed an increase in cell wall thickness and robustness, a decrease in intracellular reactive oxygen species (ROS) levels, which improved the antioxidant capacity. After 108 h of fermentation, the production of L-MA reached 114.7 ± 2.12 g/L, and the productivity was 1.06 ± 0.025 g/L/h. The strain RG0098 has enhanced the productivity of L-MA by a notable margin of 49.7 %. This study provides a sustainable and environmentally friendly pathway for the production of L-MA through microbial fermentation.

PeWRKY20 represses PeMDH1 to modulate malic acid metabolism and flavor formation in postharvest passion fruit

Postharvest events enhance fruit palatability, fragrance and sweetness and reduce sourness. With passion fruit (Passiflora edulis) the underlying molecular events occurring during the postharvest period remain largely unknown. In this study we define how passion fruit flavor alters during the postharvest period using integrated transcriptome and metabolome analyses. Passion fruits were stored under three conditions: room temperature, low temperature, and room temperature with a pre-fumigation with 100 μM methyljasmonate (MeJA). During the postharvest period, organic acid content decreased but sugars and most volatiles increased. While both low temperature and MeJA treatment effectively delayed the aging process, MeJA better maintained fruit flavor. Transcriptomic and network modelling analyses targeted PeWRKY20 was a potential key regulator of the flavor of passion fruit. PeWRKY20 was shown to bind to the Malate Dehydrogenase 1 (PeMDH1) promoter to represses PeMDH1 expression. Transient assays in Nicotiana benthamiana leaves suggested that PeWRKY20 and PeMDH1 function oppositely to regulate the production of malic acid and volatiles, such as heptadecane, dimeththyl phthalate, and L-α-terpineol. Our work unravels a key regulatory hub in postharvest passion fruit development based on an association between malic acid and volatiles.

Phosphate limitation enhances malic acid production on nitrogen-rich molasses with Ustilago trichophora

BackgroundAn important step in replacing petrochemical products with sustainable, cost-effective alternatives is the use of feedstocks other than, e.g., pure glucose in the fermentative production of platform chemicals. Ustilaginaceae offer the advantages of a wide substrate spectrum and naturally produce a versatile range of value-added compounds under nitrogen limitation. A promising candidate is the dicarboxylic acid malic acid, which may be applied as an acidulant in the food industry, a chelating agent in pharmaceuticals, or in biobased polymer production. However, fermentable residue streams from the food and agricultural industry with high nitrogen content, e.g., sugar beet molasses, are unsuited for processes with Ustilaginaceae, as they result in low product yields due to high biomass and low product formation.ResultsThis study uncovers challenges in evaluating complex feedstock applicability for microbial production processes, highlighting the role of secondary substrate limitations, internal storage molecules, and incomplete assimilation of these substrates. A microliter-scale screening method with online monitoring of microbial respiration was developed using malic acid production with Ustilago trichophora on molasses as an application example. Investigation into nitrogen, phosphate, sulphate, and magnesium limitations on a defined minimal medium demonstrated successful malic acid production under nitrogen and phosphate limitation. Furthermore, a reduction of nitrogen and phosphate in the elemental composition of U. trichophora was revealed under the respective secondary substrate limitation. These adaptive changes in combination with the intricate metabolic response hinder mathematical prediction of product formation and make the presented screening methodology for complex feedstocks imperative. In the next step, the screening was transferred to a molasses-based complex medium. It was determined that the organism assimilated only 25% and 50% of the elemental nitrogen and phosphorus present in molasses, respectively. Due to the overall low content of bioavailable phosphorus in molasses, the replacement of the state-of-the-art nitrogen limitation was shown to increase malic acid production by 65%.ConclusionThe identification of phosphate as a superior secondary substrate limitation for enhanced malic acid production opens up new opportunities for the effective utilization of molasses as a more sustainable and cost-effective substrate than, e.g., pure glucose for biobased platform chemical production.

Unlocking the potentials of Ustilago trichophora for up-cycling polyurethane-derived monomer 1,4-butanediol.

Plastic usage by microbes as a carbon source is a promising strategy to increase the recycling quota. 1,4-butanediol (BDO) is a common monomer derived from polyesters and polyurethanes. In this study, Ustilago trichophora was found to be an efficient cell-factory to valorize BDO. To investigate product formation by U. trichophora, we refined the traditional ion exclusion liquid chromatography method by examining eluent, eluent concentrations, oven temperatures, and organic modifiers to make the chromatography compatible with mass spectrometry. An LC-UV/RI-MS2 method is presented here to identify and quantify extracellular metabolites in the cell cultures. With this method, we successfully identified that U. trichophora secreted malic acid, succinic acid, erythritol, and mannitol into the culture medium. Adaptive laboratory evolution followed by medium optimization significantly improved U. trichophora growth on BDO and especially malic acid production. Overall, the carbon yield on the BDO substrate was approximately 33% malic acid. This study marks the first report of a Ustilaginaceae fungus capable of converting BDO into versatile chemical building blocks. Since U. trichophora is not genetically engineered, it is a promising microbial host to produce malic acid from BDO, thereby contributing to the development of the envisaged sustainable bioeconomy.

Rewiring metabolic flux to simultaneously improve malate production and eliminate by-product succinate accumulation by Myceliophthora thermophila.

Although a high titre of malic acid is achieved by filamentous fungi, by-product succinic acid accumulation leads to a low yield of malic acid and is unfavourable for downstream processing. Herein, we conducted a series of metabolic rewiring strategies in a previously constructed Myceliophthora thermophila to successfully improve malate production and abolish succinic acid accumulation. First, a pyruvate carboxylase CgPYC variant with increased activity was obtained using a high-throughput system and introduced to improve malic acid synthesis. Subsequently, shifting metabolic flux to malate synthesis from mitochondrial metabolism by deleing mitochondrial carriers of pyruvate and malate, led to a 53.7% reduction in succinic acid accumulation. The acceleration of importing cytosolic succinic acid into the mitochondria for consumption further decreased succinic acid formation by 53.3%, to 2.12 g/L. Finally, the importer of succinic acid was discovered and used to eliminate by-product accumulation. In total, malic acid production was increased by 26.5%, relative to the start strain JG424, to 85.23 g/L and 89.02 g/L on glucose and Avicel, respectively, in the flasks. In a 5-L fermenter, the titre of malic acid reached 182.7 g/L using glucose and 115.8 g/L using raw corncob, without any by-product accumulation. This study would accelerate the industrial production of biobased malic acid from renewable plant biomass.

Efficient polymalic acid production from corn straw hydrolysate by detoxification of phenolic inhibitors.

Inhibitory compounds generated from lignocellulose pretreatment would inhibit Poly (malic acid) (PMA) production by Aureobasidium pullulans, but the tolerance mechanism of A. pullulans to lignocellulosic inhibitor is poorly understood. In this study, the cellular response of A. pullulans to lignocellulosic inhibitor stress was studied. Among the three groups of inhibitors (furans, weak acids and phenolic aldehydes), phenolic aldehyde was the dominant inhibitor for PMA production. Phenolic aldehyde was mainly converted into phenolic alcohol by A. pullulans, and phenolic alcohol also exhibited severe inhibition on PMA production. Furthermore, the effect of detoxification methods on inhibitor-removal and PMA fermentation was investigated, both CaCO3 and overliming presented poor detoxification effect, whereas resin H103 could remove both furan derivatives and phenolic compounds efficiently, thereby producing 26.27g/L of PMA with a yield of 0.30g/g in batch fermentation. This study will be beneficial for the development of PMA production from lignocellulosic biomass.

Role of the different catalytic sites in the H2O2-mediated aqueous-phase furfural partial oxidation

The potential of hydrogen peroxide as oxidizing agent for furfural partial oxidation is studied in this work. Hydrogen peroxide is proposed as an alternative to air, the most typical oxidant, to reduce the severity and complexity of the process, as the reaction occurs in the liquid phase at ambient pressure. Comparing the activity of different catalysts and the selectivity for different oxidation products, including furoic acid, 2(5 H)furanone, and maleic and malic acids, a comprehensive mechanistic kinetic model is presented. This model identifies the critical catalytic properties ruling the selectivity of the process for each target compound. The results of this study emphasize the role of acidity in promoting the oxidations, the function of basicity for stabilizing the intermediate acids, and the impact of redox properties and metal particle size, which synergistically influence selectivity. This study concludes that Au/TiO2 is the optimum catalyst to produce furoic acid (42% of selectivity). Additionally, Pt/MgZr selectively produces 2(5 H)furanone (100%), and Pd/TiO2 and Pt/CeO2 maximize the production of maleic acid (76.5%) and malic acid (78.5%), respectively.

Changes on physiochemical properties and volatile compounds of Chinese kombucha during fermentation

Kombucha is a traditional fermented beverage in China. In this study, six samples of kombucha from different regions of China were fermented, and the pH, total acidity, organic acid, carbohydrate and volatile flavor compounds at different fermentation time were determined. The results showed that the acidity of the samples increased as fermentation progressed, which was manifested as a decrease in pH and an increase in total acidity. During the fermentation period, the content of all organic acids showed an upward trend, and acetic acid was the most dominant organic acid in kombucha. The content of sucrose showed a downward trend, but remained stable in the middle and late stages. However, the contents of glucose and fructose were increasing throughout the fermentation. Twenty volatile compounds were identified by headspace solid-phase microextraction with gas chromatography-mass spectrometry (HS-SPME-GC-MS), among which the contents of 2-phenylethanol, 2,5-dimethylbenzaldehyde, acetic acid, ethyl acetate, ethyl hexanoate, ethyl 2-phenylacetate, and 2-phenylethyl acetate were greatly increased, and could be used as markers of the flavor quality of kombucha. Principal component analysis and correlation analysis showed that unique quality characteristics of kombucha were formed during the fermentation, and fructose and glucose were related to the production of tartaric acid, malic acid, acetic acid, and other volatile compounds. This study provides a theoretical reference for the quality evaluation of kombucha, and is expected to help in developing kombucha products with high quality.

ChsA, a Class Ⅱ Chitin Synthase, Contributes to Asexual Conidiation, Mycelial Morphology, Cell Wall Integrity, and the Production of Enzymes and Organic Acids in Aspergillus niger.

Chitin synthases (CHSs) are vital enzymes for the synthesis of chitin and play important and differential roles in fungal development, cell wall integrity, environmental adaptation, virulence, and metabolism in fungi. However, except for ChsC, a class III CHS, little is known about the functions of CHSs in Aspergillus niger, an important fungus that is widely applied in the fermentation industry and food processing, as well as a spoilage fungus of food and a human pathogen. This study showed the important functions of ChsA, a class II CHS, in A. niger using multi-phenotypic and transcriptional analyses under various conditions. The deletion of chsA led to severe defects in conidiation on different media and resulted in the formation of smaller and less compact pellets with less septa in hyphal cells during submerged fermentation. Compared with the WT, the ΔchsA mutants exhibited less chitin content, reduced growth under the stresses of cell wall-disturbing and oxidative agents, more released protoplasts, a thicker conidial wall, decreased production of amylases, pectinases, cellulases, and malic acid, and increased citric acid production. However, ΔchsA mutants displayed insignificant changes in their sensitivity to osmotic agents and infection ability on apple. These findings concurred with the alteration in the transcript levels and enzymatic activities of some phenotype-related genes. Conclusively, ChsA is important for cell wall integrity and mycelial morphology, and acts as a positive regulator of conidiation, cellular responses to oxidative stresses, and the production of malic acid and some enzymes, but negatively regulates the citric acid production in A. niger.

Recent advances in the production of malic acid by native fungi and engineered microbes.

Malic acid is mainly produced by chemical methods which lead to various environmental sustainability concerns associated with CO2 emissions and resulting global warming. Since malic acid is naturally synthesized, microorganisms offer an eco-friendly and cost-effective alternative for its production. An additional advantage of microbial production is the synthesis of pure L-form of malic acid. Due to its numerous applications, biotechnologically- produced L-malic acid is a much sought-after platform chemical. Malic acid can be produced by microbial fermentation via oxidative/reductive TCA and glyoxylate pathways. This article elaborates the potential and limitations of high malic acid producing native fungi belonging to Aspergillus, Penicillium, Ustilago and Aureobasidium spp. The utilization of industrial side streams and low value renewable substrates such as crude glycerol and lignocellulosic biomass is also discussed with a view to develop a competitive bio-based production process. The major impediments present in the form of toxic compounds from lignocellulosic residues or synthesized during fermentation along with their remedial measures are also described. The article also focuses on production of polymalic acid from renewable substrates which opens up a cost-cutting dimension in production of this biodegradable polymer. Finally, the recent strategies being employed for its production in recombinant organisms have also been covered.

New malic acid producer strains of Saccharomyces cerevisiae for preserving wine acidity during alcoholic fermentation.

In the context of climate change, the chemical composition of wines is characterized by a massive drop of malic acid concentration in grape berries. Then wine professionals have to find out physical and/or microbiological solutions to manage wine acidity. The aim of this study is to develop wine Saccharomyces cerevisiae strains able to produce significant amount of malic acid during the alcoholic fermentation. By applying a large phenotypic survey in small scale fermentations, the production level of malic acid in seven grape juices confirmed the importance of the grape juice in the production of malic acid during the alcoholic fermentation. Beside the grape juice effect, our results demonstrated that extreme individuals able to produce up to 3g/L of malic acid can be selected by crossing together appropriate parental strains. A multivariate analysis of the dataset generated illustrate that the initial the amount of malic acid produced by yeast is a determining exogenous factor for controlling the final pH of wine. Interestingly most of the acidifying strains selected are particularly enriched in alleles that have been previously reported for increasing the level of malic acid at the end of the alcoholic fermentation. A small set of acidifying strains were compared with strains able to consume a large amount of malic acid previously selected. The total acidity of resulting wines was statistically different and a panelist of 28 judges was able to discriminate the two groups of strains during a free sorting task analysis.

Metabolic Engineering of Trichoderma reesei for l-Malic Acid Production.

l-Malic acid has various applications in the chemical and food industries. The filamentous fungus Trichoderma reesei is known to be an efficient enzyme producer. Here, through metabolic engineering, T. reesei was constructed for the first time as an excellent cell factory for l-malic acid production. The heterologous overexpression of genes encoding the C4-dicarboxylate transporter from Aspergillus oryzae and Schizosaccharomyces pombe initiated l-malic acid production. The overexpression of pyruvate carboxylase from A. oryzae in the reductive tricarboxylic acid pathway further increased both the titer and yield of l-malic acid, resulting in the highest titer reported in a shake-flask culture. Furthermore, the deletion of malate thiokinase blocked l-malic acid degradation. Finally, the engineered T. reesei strain produced 220.5 g/L of l-malic acid in a 5 L fed-batch culture (productivity of 1.15 g/L/h). A T. reesei cell factory was created for the efficient production of l-malic acid.

Bipolar Membrane Electrodialysis for Cleaner Production of Diprotic Malic Acid: Separation Mechanism and Performance Evaluation.

Bipolar membrane electrodialysis (BMED) is a promising process for the cleaner production of organic acid. In this study, the separation mechanism of BMED with different cell configurations, i.e., BP-A, BP-A-C, and BP-C (BP, bipolar membrane; A, anion exchange membrane; C, cation exchange membrane), to produce diprotic malic acid from sodium malate was compared in consideration of the conversion ratio, current efficiency and energy consumption. Additionally, the current density and feed concentration were investigated to optimize the BMED performance. Results indicate that the conversion ratio follows BP-C > BP-A-C > BP-A, the current efficiency follows BP-A-C > BP-C > BP-A, and the energy consumption follows BP-C < BP-A-C < BP-A. For the optimized BP-C configuration, the current density was optimized as 40 mA/cm2 in consideration of low total process cost; high feed concentration (0.5-1.0 mol/L) is more feasible to produce diprotic malic acid due to the high conversion ratio (73.4-76.2%), high current efficiency (88.6-90.7%), low energy consumption (0.66-0.71 kWh/kg) and low process cost (0.58-0.59 USD/kg). Moreover, a high concentration of by-product NaOH (1.3497 mol/L) can be directly recycled to the upstream process. Therefore, BMED is a cleaner, high-efficient, low energy consumption and environmentally friendly process to produce diprotic malic acid.

Dissecting key residues of a C4-dicarboxylic acid transporter to accelerate malate export in Myceliophthora

Efficient transporters are necessary for high concentration and purity of desired products during industrial production. In this study, we explored the mechanism of substrate transport and preference of the C4-dicarboxylic acid transporter AoMAE in the fungus Myceliophthora thermophila, and investigated the roles of 18 critical amino acid residues within this process. Among them, the residue Arg78, forming a hydrogen bond network with Arg23, Phe25, Thr74, Leu81, His82, and Glu94 to stabilize the protein conformation, is irreplaceable for the export function of AoMAE. Furthermore, varying the residue at position 100 resulted in changes to the size and shape of the substrate binding pocket, leading to alterations in transport efficiencies of both malic acid and succinic acid. We found that the mutation T100S increased malate production by 68%. Using these insights, we successfully generated an AoMAE variant with mutation T100S and deubiquitination, exhibiting an 81% increase in the selective export activity of malic acid. Simply introducing this version of AoMAE into M. thermophila wild-type strain increased production of malic acid from 1.22 to 54.88g/L. These findings increase our understanding of the structure-function relationships of organic acid transporters and may accelerate the process of engineering dicarboxylic acid transporters with high efficiency. KEY POINTS: • This is the first systematical analysis of key residues of a malate transporter in fungi. • Protein engineering of AoMAE led to 81% increase of malate export activity. • Arg78 was essential for the normal function of AoMAE in M. thermophila. • Substitution of Thr100 affected export efficiency and substrate selectivity of AoMAE.

Engineering Escherichia coli for Efficient Aerobic Conversion of Glucose to Malic Acid through the Modified Oxidative TCA Cycle

Malic acid is a versatile building-block chemical that can serve as a precursor of numerous valuable products, including food additives, pharmaceuticals, and biodegradable plastics. Despite the present petrochemical synthesis, malic acid, being an intermediate of the TCA cycle of a variety of living organisms, can also be produced from renewable carbon sources using wild-type and engineered microbial strains. In the current study, Escherichia coli was engineered for efficient aerobic conversion of glucose to malic acid through the modified oxidative TCA cycle resembling that of myco- and cyanobacteria and implying channelling of 2-ketoglutarate towards succinic acid via succinate semialdehyde formation. The formation of succinate semialdehyde was enabled in the core strain MAL 0 (∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆ptsG, PL-glk, Ptac-galP, ∆aceBAK, ∆glcB) by the expression of Mycobacterium tuberculosis kgd gene. The secretion of malic acid by the strain was ensured, resulting from the deletion of the mdh, maeA, maeB, and mqo genes. The Bacillus subtilis pycA gene was expressed in the strain to allow pyruvate to oxaloacetate conversion. The corresponding recombinant was able to synthesise malic acid from glucose aerobically with a yield of 0.65 mol/mol. The yield was improved by the derepression in the strain of the electron transfer chain and succinate dehydrogenase due to the enforcement of ATP hydrolysis and reached 0.94 mol/mol, amounting to 94% of the theoretical maximum. The implemented strategy offers the potential for the development of highly efficient strains and processes of bio-based malic acid production.

Optimization of l-malic acid production from acetate with Aspergillus oryzae DSM 1863 using a pH-coupled feeding strategy

BackgroundMalic acid, a dicarboxylic acid mainly used in the food industry, is currently produced from fossil resources. The utilization of low-cost substrates derived from biomass could render microbial processes economic. Such feedstocks, like lignocellulosic hydrolysates or condensates of fast pyrolysis, can contain high concentrations of acetic acid. Acetate is a suitable substrate for l-malic acid production with the filamentous fungus Aspergillus oryzae DSM 1863, but concentrations obtained so far are low. An advantage of this carbon source is that it can be used for pH control and simultaneous substrate supply in the form of acetic acid. In this study, we therefore aimed to enhance l-malate production from acetate with A. oryzae by applying a pH-coupled feeding strategy.ResultsIn 2.5-L bioreactor fermentations, several feeding strategies were evaluated. Using a pH-coupled feed consisting of 10 M acetic acid, the malic acid concentration was increased about 5.3-fold compared to the batch process without pH control, resulting in a maximum titer of 29.53 ± 1.82 g/L after 264 h. However, it was not possible to keep both the pH and the substrate concentration constant during this fermentation. By using 10 M acetic acid set to a pH of 4.5, or with the repeated addition of NaOH, the substrate concentration could be maintained within a constant range, but these strategies did not prove beneficial as lower maximum titers and yields were obtained. Since cessation of malic acid production was observed in later fermentation stages despite carbon availability, a possible product inhibition was evaluated in shake flask cultivations. In these experiments, malate and succinate, which is a major by-product during malic acid production, were added at concentrations of up to 50 g/L, and it was found that A. oryzae is capable of organic acid production even at high product concentrations.ConclusionsThis study demonstrates that a suitable feeding strategy is necessary for efficient malic acid production from acetate. It illustrates the potential of acetate as carbon source for microbial production of the organic acid and provides useful insights which can serve as basis for further optimization.

Malic Acid Production by Aspergillus Oryzae: the Effect of Alkaline-earth Carbonate Buffer Identity

Malic Acid Production by Aspergillus Oryzae: the Effect of Alkaline-earth Carbonate Buffer Identity

Malic acid production by Aspergillus oryzae: the immobilized fungal fermentation route

AbstractIt has been proposed that the microbial bioproduction of chemical precursors could reduce societal overreliance on the petrochemical industry. However, economic implementation remains elusive due to a lack of understanding of the mechanisms involved. The current study evaluated malic acid production using a novel immobilized Aspergillus oryzae fungal bed bioreactor. The experimental procedures involved two different nitrogen sources (urea and (NH4)2SO4), two‐phase growth/production and simultaneous single‐step growth/production runs, the presence and absence of added CO2, the presence and absence of pH control, and the use of glucose and dl‐malate as the carbon sources. The experimental malic acid titers were remarkably poor (between 1.7 and 3.3 g.L−1) in comparison with CaCO3 buffered malic acid titers from literature (concentrations in excess of 100 g.L−1 have been reported reported). In contrast, citric acid was observed to be the main metabolite. The dl‐malate carbon source was consumed with concomitant growth, indicating A. oryzae′s ability to utilize both d and l enantiomers of malic acid. Detailed metabolic flux analyses predicted partial consumption of urea during growth, while (NH4)2SO4 was fully utilized. During the urea runs, significant amounts of glycogen were accumulated in the biomass – which were utilized as substrate during the production run. Nitrogen limitations induced significant lipid production, corresponding to citric acid accumulation in the media; the mechanism for fungal lipid biosynthesis involves citric acid excretion at the expense of malic acid. These results indicate a possible calcareous trigger for malic acid production as opposed to the traditionally accepted nitrogen starvation mechanism – nitrogen starvation resulted in lipid accumulation with corresponding malate loss. © 2022 The Authors. Biofuels, Bioproducts and Biorefining published by Society of Industrial Chemistry and John Wiley &amp; Sons Ltd.

Metabolic engineering of Mannheimia succiniciproducens for malic acid production using dimethylsulfoxide as an electron acceptor.

Microbial production of various TCA intermediates and related chemicals through the reductive TCA cycle has been of great interest. However, rumen bacteria that naturally possess strong reductive TCA cycle have been rarely studied to produce these chemicals, except for succinic acid, due to their dependence on fumarate reduction to transport electrons for ATP synthesis. In this study, malic acid (MA), a dicarboxylic acid of industrial importance, was selected as a target chemical for mass production using Mannheimia succiniciproducens, a rumen bacterium possessing a strong reductive branch of the TCA cycle. The metabolic pathway was reconstructed by eliminating fumarase to prevent MA conversion to fumarate. The respiration system of M. succiniciproducens was reconstructed by introducing the Actinobacillus succinogenes dimethylsulfoxide (DMSO) reductase to improve cell growth using DMSO as an electron acceptor. Also, the cell membrane was engineered by employing Pseudomonas aeruginosa cis-trans isomerase to enhance MA tolerance. High inoculum fed-batch fermentation of the final engineered strain produced 61 g/L of MA with an overall productivity of 2.27 g/L/h, which is the highest MA productivity reported to date. The systems metabolic engineering strategies reported in this study will be useful for developing anaerobic bioprocesses for the production of various industrially important chemicals.