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Article

Improving Organic Acid Secretion of Aspergillus niger by Overexpression C4-Dicarboxylic Acid Transporters

by
Yiyang Tan
1,†,
Shutong Liu
1,†,
Sheng Wu
1,
Xiaolu Wang
2,
Depei Wang
1,3,* and
Xianli Xue
1,3,*
1
Key Laboratory of Industrial Microbiology & Engineering Research Center of Food Biotechnology of Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
2
State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
3
Tianjin Key Laboratory of Industrial Fermentation Microbiology, Tianjin 300457, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fermentation 2025, 11(3), 156; https://doi.org/10.3390/fermentation11030156
Submission received: 2 March 2025 / Revised: 18 March 2025 / Accepted: 18 March 2025 / Published: 20 March 2025
(This article belongs to the Special Issue Metabolic Engineering in Microbial Synthesis)
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Abstract

C4-dicarboxylic acids are essential organic compounds characterized by a four-carbon structure and two carboxyl groups. Their export from cells is mediated by specialized transporter proteins known as C4-dicarboxylic acid transporters (DCTs). The objective of this study was to investigate the specificity of six DCTs (DCT1-5 and C4t318) from Aspergillus niger or Aspergillus oryzae, focusing on their role in different production strategies for C4-dicarboxylic acids. The results indicate that compared to the WT strain, overexpressing dct2 or dct3 in A. niger CGMCC NO. 40550 specifically enhances the production of succinic acid, increasing its yield from 5.69 g/L to 6.28 g/L, and L-malic acid, increasing its yield from 11.02 g/L to 12.11 g/L. Additionally, dct5 appears to be involved in the transport of both succinic acid (6.19 g/L) and L-malic acid (16.33 g/L). The total acid yields of T-D3-7, which lacks the oxaloacetate hydrolase gene, were improved to 27.75 g/L, compared to 25.19 g/L for T-D3-26, due to blocking the branch of oxaloacetate metabolism. Furthermore, the heterologous expression of A. oryzae C4T318 in A. niger increased the production of L-malic acid by approximately 22.5%. Furthermore, the best results were observed when the strains T-D3-7 and T-D5-16 were scaled up in a 30 L bioreactor for 84 h. The succinic acid and L-malic acid yields of T-D3-7 and T-D5-16 reached 14.51 g/L and 70.79 g/L or 41.59 g/L and 81.08 g/L, respectively. Moreover, the purity of L-malic acid produced by T-D3-7 reached 71%. This work further clarifies the specificity of C4-dicarboxylic acid transporters and provides valuable insights for optimizing organic acid production.

1. Introduction

C4-dicarboxylic acids are organic compounds containing four carbon atoms and one or two carboxylic functional groups, including succinic acid, malic acid, and fumaric acid. These acids are key intermediates in the tricarboxylic acid (TCA) cycle and play vital roles in both aerobic and anaerobic metabolic processes in living organisms [1,2,3]. Succinic acid, malic acid, and fumaric acid are significant and commercially important C4-dicarboxylic acids. The United States Department of Energy has identified these compounds among the 12 most valuable bulk chemicals due to their high potential for added value. These acids are widely used in the food, pharmaceutical, and animal feed industries, with demand steadily rising [4,5,6]. Traditionally, their production has relied on chemical synthesis using fossil fuels as feedstocks. In this process, fossil-derived materials are catalyzed to produce fumaric and maleic acids, which are then converted into succinic or malic acid [4].
Conventional production methods for C4-dicarboxylic acids face challenges due to concerns about global warming, crude oil shortages, and environmental sustainability. To address these limitations and meet increasing demand, alternative production methods are being actively explored. Biotechnology, in particular, offers promising solutions to tackle global challenges such as energy security and environmental protection. To date, advances in genetic engineering have enabled the production of C4-dicarboxylic acids in microbial hosts like Escherichia coli [7], Corynebacterium glutamicum [8], and Saccharomyces cerevisiae [9,10]. The major metabolic engineering strategies concentrate on the efficiency of energy generation and the improvement of the central metabolic fluxes. These efforts focus on optimizing the anabolic pathways for these acids mostly via knocking out and overexpressing key genes in upstream pathways or inhibiting competing downstream pathways. For example, overexpression of genes such as mdh (malate dehydrogenase), fuh (fumarate hydratase), frd (fumarate reductase), or pyc (pyruvate carboxylase), either individually or in combination, has been shown to significantly enhance succinic acid and L-malic acid production in Aspergillus carbonarius and Saccharomyces cerevisiae [11,12]. Recent advances in the metabolic engineering of Aspergillus have revealed promising strategies for enhancing L-malic acid production. One approach involves redirecting metabolic flux by disrupting transporters such as cexA and overexpressing glycolytic genes including mstC, hxkA, pfkA, and pkiA [13]. Another strategy focuses on optimizing redox cofactor availability through the expression of NAD(H)-NADP(H) reductase (sthA) [14] and by enhancing NADPH regeneration systems [15]. In addition, the overexpression of the GPI-anchored protein GPI2 has been employed to mitigate oxidative stress [16], while the promoter engineering of fumarase FumA has improved production efficiency and reduced byproduct formation [17]. Targeted modifications of membrane transporters have further proven essential for enhancing product export and facilitating industrial scalability [18]. Collectively, these integrated metabolic and cellular engineering strategies address key bottlenecks in biosynthesis, redox balance, and stress tolerance, leading to significant improvements in L-malic acid titers (up to 201 g/L) [17], yield, and productivity. As the most direct transporters of metabolic products, controlling and coordinating cell membrane transporter proteins can increase metabolic product delivery capacity by some targeted manner, making the strains more suitable for industrial fermentation. For example, the overexpression of the dct1 gene in Aspergillus niger and Aspergillus carbonarius led to a substantial increase in L-malic acid production [19]. In filamentous fungi, multiple homologous dct genes have been identified, with A. niger containing five and A. nidulans and A. oryzae each containing four [20,21]. Transporter proteins are essential for various cellular metabolic activities, including nutrient uptake [22], metabolite release, and signaling transitions [23,24]. However, the specificity and functionality of many C4-dicarboxylic acid transporters remain unclear. Understanding their roles will be vital for improving the efficiency of four-carbon dicarboxylic acid production in genetically engineered strains.
Recent studies on both model and non-model strains have advanced our understanding of organic acid production. However, only a few genetically modified mutants have proven suitable for industrial applications. This has shifted focus toward the modification of industrial strains to enhance the efficiency of organic acid production [25,26,27]. Since 1987, when the U.S. Food and Drug Administration (FDA) classified A. niger as GRAS (Generally Recognized as Safe), it has been widely employed in industrial processes for the production of gluconic acid, citric acid, and antibiotics. As a well-established industrial producer of citric acid [28], A. niger serves as an excellent chassis organism for genetic engineering. By optimizing its metabolic pathways, A. niger holds great promise for the production of other organic acids at industrial scales.
A. niger CGMCC NO. 40550, an industrial citric acid-producing strain conserved in our laboratory, exhibits robust organic acid metabolism. In this study, we performed the following tasks: (1) We focused on screening and identifying DCTs in this strain. (2) We successfully constructed the overexpressing strains by overexpressing DCTs under a strong promoter. (3) We evaluated the fermentation performance of the selected transformants in shake flasks and assessed the expression levels of key enzyme genes involved in malic, succinic, and fumaric acid production. (4) For the top-performing strains, we proceeded with a fermentation trial in a 30 L bioreactor to further validate their performance. All in all, we aimed to investigate their roles in the transport of C4-dicarboxylic acids.

2. Materials and Methods

2.1. Strains and Culture Condition

All strains and plasmids employed in this study were either sourced from the Tianjin Key Laboratory of Industrial Fermentation Microbiology in China or constructed in-house, as detailed in Table 1. The wild-type (WT) strain used in this study was A. niger CGMCC NO. 40550, which was conserved in the General Microbiology Center of the China General Microbiological Culture Collection Center (CMSMC). All A. niger strains were cultured on complete medium (CM) at 37 °C, with hygromycin B added as needed. And minimal medium (MM) containing 100 μg/mL Ampicillin and 150 μg/mL Cephalexin was used for transformants screening. Escherichia coli DH5α, used for constructing and amplifying plasmids, was grown at 37 °C in Luria Bertani medium (LB) supplemented with 100 μg/mL kanamycin (kana) sulfate as needed. Agrobacterium tumefaciens AGL-1, used for Agrobacterium tumefaciens-mediated transformation (ATMT) of A. niger, was grown on LB supplemented with 100 μg/mL kana at 28 °C. The seed medium for shake flask fermentation was composed of glucose 60 g/L and 10× trace elements (composed of (NH4)2SO4 49.5 g/L, KH2PO4 5 g/L, MgSO4-7H2O 0.6 g/L, and CuSO4-5H2O 0.012 g/L), and 100 mL/L and 500 mL triangular flasks were filled with 30 mL. The fermentation medium was composed of glucose 180 g/L, 10× trace elements 100 mL/L, and CaCO3 8%, and 500 mL triangular flasks were filled with 60 mL.
For shake flask fermentation, seed fermentation conditions were 37 °C at 220 rpm, and after 24 h incubation, 60 mL fermentation medium was added and incubated at 37 °C at 200 rpm for 72 h.

2.2. Phylogenetic Tree and Multiple Sequence Alignments of DCTs

Phylogenetic trees of homologous amino acid sequences of DCTs in different species were constructed by the neighbor-joining method using MEGA11 software, which included A. niger 513.88, A. niger CBS 101883, Aspergillus nidulans, Aspergillus oryzae, and Penicllium alfredii, respectively. The value in brackets preceding the amino acid sequence number indicates the similarity of the sequence to the amino acid sequence of the corresponding dct-series genes.

2.3. Plasmid Construction

The glycosylase promoter (pX), identified for its high transcriptional activity in A. niger, was selected to drive the expression of target genes. To achieve this, plasmids p-C, p-D2, p-D3, p-D4, and p-D5 were constructed. These plasmids involved the knockout of the oah (encoding oxaloacetate hydrolase) and the heterologous expression or overexpression of C4-dicarboxylic acid transporters (c4t318, dct2, dct3, dct4, and dct5). Thiamphenicol resistance (hyg) was used as the selectable marker. A schematic representation of plasmids p-D4 and p-D5 is provided in Figure 1. The p44 plasmid, conserved in our laboratory, served as the starting plasmid for these constructions. The A. niger genome was used as a template to amplify the following sequences: the oah-L (the upstream sequence of oah), the pX, the dct2–5, and the oah-R. Additionally, the A. oryzae genome was used to amplify the c4t318, while the Thaumatin B was amplified from the p44 plasmid.
All the primers used in this work are listed in Table S1, all restriction endonucleases used were purchased from NEB, and the plasmid mini-preparation kit, PCR (Polymerase Chain Reaction) recovery kit, and gel recovery kit were purchased from OMEGA (Norcross, GA, USA). The physical mapping of the plasmids is shown in Supplementary Material Figure S1.

2.4. Transformation and Screening of Positive Strains

The treated and incubated Agrobacterium rhizogenes at a concentration of 2 × 109 CFU/mL were mixed 1:1 with WT (A. niger CGMCC NO. 40550) suspension at a concentration of 2 × 107 individuals/mL, incubated on IM solid medium for 48 h, and then transferred to CM medium for 48 h, i.e., 100 μg/mL Amp, 80 μg/mL Cefo, 150 mg/mL chaotropic acid. Single colonies were then selected and grown for 72 h in CM medium, 250 mg/mL chaotropic acid was added to the CM medium, and the growth status of the colonies was observed and genotyped, and when no errors were found, the bacterial juice of the 72 h fermentation was collected and RNA was extracted and subjected to real-time fluorescence quantitative PCR (qRT-PCR) for further analysis.

2.5. Shake Flask Cultivation

After culturing A. niger WT on CM for 5 d, spore suspension was prepared with 0.9% NaCl, and 1 × 106 A. niger spores were inoculated into 30 mL of seed medium and cultured at 37 °C and 220 rpm for 24 h, and then 60 mL of fermentation medium was replenished, and samples were taken at 36 h, 48 h, 60 h, and 72 h of fermentation, respectively, and used for analyses of acid production, biomass, residual sugar, and acid production analysis and biomass, residual sugar, and qRT-PCR analysis.

2.6. Transformants Cultured on Plate with Different Carbon Sources

A 1 μL droplet of conidial suspension (containing 107 conidia per mL) was spotted onto complete medium (CM) supplemented with glucose, malic acid, citric acid, fumaric acid, or succinic acid as the carbon source. Growth changes were monitored at 24 h intervals.

2.7. RNA Extraction and cDNA Preparation for qRT-PCR

Bacteria in the fermentation broth were collected using a 200 mesh sieve and liquid nitrogen, snap frozen, and ground; RNA was extracted using an OMEGA Biotech Fungal RNA Kit and reverse-transcribed using a Vazme HiScripy RT SuperMix for qPCR (+gDNA wiper) Kit (Nanjing, China) to obtain cDNA, and then qRT-PCR was performed [28].

2.8. Scale-Up Experiments and Parameterisation of a 30 L Bioreactor

The methodology for the bioreactor scale-up experiment follows that of Xue et al. [29]. Spores were collected from a PDA plate incubated at 35 °C for 4 days and cultured in branquette (RZBC Co., Ltd., Rizhao, Shandong, China) to reach a spore concentration of 1 × 105 spores/mL. A volume of 1 × 105 spores/mL was then inoculated into a 30 L fermenter containing 20 L of culture medium. The culture medium used in the bioreactor fermentation experiment was the same as that used in the shake flask experiments mentioned earlier. During the 30 L fermentation, the morphology of the cocci was observed at regular intervals. Organic acid yields were determined by HPLC (high-performance liquid chromatography). Data are presented as the mean ± standard deviation of triplicate samples. Statistical significance was assessed using a threshold of p < 0.05.

2.9. Analytical Methods

The biomass during the fermentation process was measured by counting the number of bacterial spheres. The fermentation broth was mixed, diluted 100 times, vortexed, and mixed to ensure uniform suspension of the bacterial spheres. Then, 0.1 mL of the suspension was placed on a slide for counting. Three independent counts were performed for each group as parallel measurements.
The reducing sugar concentration was determined using the DNS method. For this, 2 mL of fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was diluted 120 times. The fermentation broth, distilled water, and DNS reagent were added to a cuvette to form a reaction solution. The mixture was boiled for 5 min, then distilled water was added to bring the volume up to 25 mL. The absorbance at 540 nm was measured using a spectrophotometer. The glucose concentration was calculated using a standard curve, as described in reference [30].
Organic acid yield was analyzed by HPLC. The fermentation broth was collected, diluted with 10 mmol/L sulfuric acid solution, and centrifuged. The supernatant was then filtered through a 0.22 μm filter membrane into a liquid vial for HPLC analysis. Detection was performed using a Waterse2695 chromatograph equipped with a Waterse2996 detector (Milford, MA, USA). The analysis conditions included an injection volume of 10 μL, a flow rate of 0.6 mL/min, and a mobile phase consisting of 5 mmol/L sulfuric acid solution. Standard solutions of L-malic acid and succinic acid were prepared at concentrations of 0.025, 0.05, 0.1, 0.25, 0.5, and 1 g/L, and corresponding standard curves were established. After acidifying the fermentation broth, HPLC analysis was conducted. The peak area for L-malic acid, recorded as y, was used with the standard curve to determine its concentration, X. The final concentration of L-malic acid (g/L) was then calculated by multiplying X by the dilution factor and adjusting for water loss.
For statistical analysis, GraphPad PRISM V8.03 software was used to calculate significance, and all data were obtained from at least three independent experiments.

3. Results and Discussion

3.1. Amino Acid Multiple Sequence Alignment and Phylogenetic Tree Analysis

There are five C4-dicarboxylic acid transport proteins in A. niger, and their multiple sequence alignment was performed with C4t318 from A. oryzae. The results are shown in Figure 2A, indicated that the amino acid sequences of C4t318 and DCT1 share the highest identity of 71.08%, suggesting that they may be isoenzymes and function similarly in different strains. Additionally, the amino acid sequence identities between DCT1 and DCT2~5 were 29.68%, 31.8%, 20.88%, and 21.78%, respectively. The identities of DCT2 with DCT3~5 were 20.72%, 23.18%, and 21.69%. The identities of DCT3 with DCT4 and DCT5 were 17.14% and 19.54%, and the identities of DCT4 with DCT5 were 22.68%.
The evolutionary tree analysis is shown in Figure 2B; DCT1 to DCT5 in A. niger CGMCC No. 40550 belong to five distinct branching clusters. All of these were most closely related to A. niger 513.88, which exhibited 100% amino acid sequence identity. This was followed by A. niger 101,883, which contains only three DCTs, corresponding to the DCT1, DCT2, and DCT3 evolutionary clusters, with amino acid sequence identities of 100%, 99.2%, and 87.5%, respectively. In contrast, A. oryzae, A. nidulans, and P. alfredii each contain four homologous DCTs, distributed across different evolutionary clusters. The amino acid sequence similarity between the homologous DCTs in A. oryzae and A. niger CGMCC No. 40550 shows that DCT1 and DCT2, as well as DCT4 and DCT5, are more closely related, while DCT3 occupies a separate evolutionary group. This suggests that DCT3 may have a distinct function in the evolutionary context.

3.2. Construction of Overexpression Plasmid and Screening of Positive Transformants

To improve the C4-dicarboxylic acid transport efficiency production, five recombinant plasmids named p-C, p-D2, p-D3, p-D4, and p-D5 were successfully constructed. These plasmids contained the right and left arm sequences of oah and a highly efficient pX promoter driving the overexpression cassettes of heterologous c4t318 and homologous dcts (Figure 1). The plasmids were transformed into the parent strain A. niger CGMCC No. 40550 via Agrobacterium-mediated transformation. A total of 300–400 positive transformants were screened by culturing on CM plates with a high concentration of hygromycin (20 g/L). The fastest-growing transformants were passaged for 15 generations to assess genetic stability, confirmed by PCR analysis (Figure S1).
Ultimately, two transformants from each overexpression group were selected for further experiments: T-C-2, T-C-11, T-D2-6, T-D2-42, T-D3-7, T-D3-26, T-D4-16, T-D4-24, T-D5-16, and T-D5-41. Notably, T-D3-7 and T-D4-16 both exhibited disruption of the oah through homologous recombination with the right and left arm fragments, which led to colonies on the plate showing a pale yellow color instead of the typical black conidia (Figure S2).

3.3. Analysis of Transcript Levels of Overexpressed Genes in Positive Transformants

Compared to the wild-type (WT) strain, the transcript levels of the heterologously expressed or overexpressed genes (c4t318, dct2~5) in the transformants were all significantly increased, more than two-fold, between 36 h and 60 h. The transcript levels of dct2 to dct5 in the WT strain exhibited a declining trend from 36 h to 60 h as shown in Figure 3A–E. In contrast, the transcript levels of dct3 in T-D3-7 continued to increase, reaching a 40-fold increase. The transcript levels of dct4 and dct5 in T-D4-16 and T-D5-16 also reached their highest levels at 60 h, despite a decrease at 48 h, with a 17-fold and 3-fold increase compared to the WT, respectively. The target genes promoted by the pX showed distinct transcription patterns and levels, which may be attributed to differences in the insertion sites or copy numbers of the inserted genes [31,32].
Furthermore, oxaloacetate dehydrogenase (oah) plays a role in the metabolic pathway that converts oxaloacetate to oxalic acid, influencing the TCA cycle [33].
Studies have demonstrated that deleting the oxaloacetate hydrolase (oah) gene in A. niger results in the cessation of oxalic acid synthesis [34]. This suggests that in the absence of oxalic acid production, carbon flux may be redirected to synthesize more four-carbon dicarboxylic acids. Our subsequent findings support this hypothesis, indicating that the deletion of oah indeed leads to an increased production of four-carbon dicarboxylic acids. In this study, T-D3-7 and T-D4-16 both successfully deleted the oah, with transcription levels of the oah not detected in these strains. In contrast, distinct transcription levels of the oah were detected in the other transformants, as shown in Figure 3F. This deletion likely affected the production of organic acids [35].

3.4. Activating C4-Dicarboxylate Transporter for Improving Organic Acid Production

Transporter proteins are essential for various cellular metabolic activities, including nutrient uptake, metabolite release, and signal transduction [36]. However, the specificity of C4-dicarboxylic acid transporter proteins in filamentous fungi remains insufficiently studied. To functionally characterize these transporters, all positive transformants were cultured in shake flasks for 72 h.
The total acid production in the wild-type (WT) strain was approximately 21.48 g/L, with products of L-malic acid (14.11 g/L), succinic acid (4.72 g/L), citric acid (2.27 g/L), and fumaric acid (0.38 g/L), constituting 66%, 22%, 11%, and 1% of total acid production, respectively (Figure 4A). Among the 10 transformants, the highest total acid production was observed in T-D5-16 (30.28 g/L), followed by T-D3-7 (28.11 g/L), T-D5-41 (27.24 g/L), T-C-11 (23.50 g/L), and T-D3-26 (23.11 g/L). The remaining transformants exhibited slightly reduced total acid production: T-C-2 (19.09 g/L), T-D2-6 (19.85 g/L), T-D2-42 (18.95 g/L), T-D4-16 (20.34 g/L), and T-D4-24 (19.19 g/L). The recombinant strain overexpressing the dct3 demonstrated a marked increase in the proportion of L-malic acid, rising to 79–81% (18.18–22.78 g/L) of the total acid production, while the succinic acid proportion decreased to 3–10% (0.72–2.27 g/L). Conversely, heterologous expression of the c4t318 from A. oryzae and overexpression of the dct4 gene in A. niger did not significantly enhance total acid production, although the L-malic acid proportion increased slightly to 73–74% compared to the WT strain. These results align with previous studies, which demonstrated that L-malic acid production significantly declined when the dct1 gene was knocked out in A. niger. Consequently, DCT1 is hypothesized to be the primary transporter for L-malic acid, and its overexpression resulted in a 36.8% increase in L-malic acid production compared to the initial strain. Similarly, overexpression of the c4t318 in A. oryzae enhanced L-malic acid production by 33.8% [37].
Moreover, as shown in Figure 4, overexpression of dct2 in transformants T-D2-6 and T-D2-42 and dct5 in transformants T-D5-16 and T-D5-41 resulted in significant increases in succinic acid yield, reaching 4.86–6.52 g/L and 5.39–9.26 g/L, respectively. Conversely, the yield of L-malic acid showed opposing trends, with dct2 transformants producing 9.88–14.34 g/L and dct5 transformants achieving 16.12–18.93 g/L (Figure 4A). This suggests that the dct2 transporter is more specific for succinic acid, while the dct5 transporter may efficiently transport both succinic and L-malic acids. DCT2 could potentially serve as a protein exclusively transporting succinic acid, and future genetic engineering efforts could focus on truncating the downstream metabolic pathway to enhance succinic acid accumulation.
Additionally, the L-malic acid production in the oah knockout transformants T-D3-7 and T-D4-16 increased by approximately 2.3–4.2 g/L compared to the transformants with random insertion of the expression frame (T-D3-26 and T-D4-24). This supports the hypothesis that deletion of the oah reduces the conversion of oxaloacetate to oxalic acid, thereby redirecting more oxaloacetate towards L-malic acid production via malate dehydrogenase (MdhB) in the reductive tricarboxylic acid (TCA) cycle [38]. These findings align with previous studies, which demonstrated that knockout of the oah gene during the genetic modification of A. terreus and A. niger significantly enhanced L-malic acid production [39,40].
The results of the residual sugar analysis showed significant differences between the WT strain and seven transformants, with residual glucose levels ranging from 41.51 g/L to 57.88 g/L. And the transformants T-D4-16, T-D3-26, and T-C-11 exhibited higher residual glucose amounts, with values of 57.88 g/L, 51.22 g/L, and 50.12 g/L, respectively. Additionally, the number of mycelial pellets in T-D2-6, T-D3-7, T-D3-26, T-D4-14, and T-D5-16 ranged from 1.32 × 10⁶ to 1.8 × 10⁶, which was lower than that observed in the WT strain. In contrast, T-D3-26 and T-D4-16 showed relatively higher glucose residues but lower mycelial pellet counts. This suggests that the glucose utilization efficiency of these strains during fermentation may not be optimal. Therefore, it is speculated that optimizing the fermentation medium or selecting a more suitable carbon source could enhance tetracarbodicarboxylic acid production in these strains.
Organic acid production in the wild-type (WT) strain increased significantly after 60 h of fermentation, reaching 14.11 g/L of L-malic acid, 4.72 g/L of succinic acid, 2.72 g/L of citric acid, and 0.38 g/L of fumaric acid after 72 h (Figure 4). In contrast, the recombinant strain T-C-11, which expressed the c4t318 gene from A. oryzae, showed a modest though not significant increase in L-malic acid production. However, the strain T-D2-42 exhibited a notable improvement in succinic acid production, yielding 1.35 times more than the WT strain. This suggests that the dct2 gene might be more closely associated with succinic acid transport in A. niger. The recombinant strain T-D3-7 demonstrated a 1.61-fold increase in L-malic acid production, reaching 22.78 g/L, although succinic acid production decreased to 0.72 g/L. This indicates that the dct3 gene may be more effective in transporting L-malic acid in A. niger. Strain T-D4-16, overexpressing the dct4 gene, showed a slight improvement in L-malic acid yield (1.15-fold increase), but succinic acid production dropped to 1.80 g/L. No significant changes were observed in the production of citric or fumaric acid. Finally, the strain T-D5-16, which expressed the dct5 gene, exhibited a substantial increase in both L-malic acid and succinic acid yields 1.33-fold and 1.96-fold higher than the WT strain, respectively. This suggests that the transporter protein encoded by dct5 may play a role in facilitating the transport of both acids.

3.5. Observation on the Morphology of Mycelium Pellets and Plate Colonies

Observation of mycelium pellet morphology during fermentation showed that the bacteriophage spheres of the recombinant strain T-C-11 were larger compared to other strains, while those of T-D2-42 were smaller and more uneven. This irregularity in pellet growth likely contributed to T-D2-42’s lower acid production in contrast to the higher acid-producing strains, T-D3-7 and T-D5-16. To assess the growth of the transformants and the WT strain on different carbon sources, the strains were cultured on plates containing glucose, malic acid, citric acid, fumaric acid, and succinic acid. As shown in Figure 5F, the WT strain grew slightly slower than the others from 24 to 72 h on all plates. The transformants grew fastest on CM plates, followed by plates with succinic acid (SA) and fumaric acid (FA), and slowest on plates with citric acid (CA). Notably, T-D3-7 grew significantly better on CM, SA, and malic acid (MA) plates compared to the other transformants, forming a distinct acid ring, indicating a stronger acid production capacity.

3.6. qRT-PCR Analysis of Key Genes in Metabolic Pathway

qRT-PCR analysis was performed on key genes involved in organic acid metabolism, including pyruvate carboxylase (pc), isocitrate lyase (icl), malate synthase (ms), fumarate hydratase (fuh), mitochondrial malate dehydrogenase (mdhA), mitochondrial succinate dehydrogenase (sdhA), and cytoplasmic malate dehydrogenase (mdhB). This analysis aimed to assess the impact of overexpressing four-carbon dicarboxylic acid transporter proteins on the expression of genes involved in three major metabolic pathways: the TCA cycle, the glyoxylate cycle, and the reductive tricarboxylic acid cycle. Additionally, cytoplasmic succinate dehydrogenase (sdhB) was also analyzed to evaluate changes in expression.
As shown in Figure 6, in the WT strain, the expression levels of the eight genes were generally high, indicating active overall metabolism. However, the organic acids produced could not be efficiently exported from the cell, leading to their reutilization in other metabolic pathways [41,42]. In the recombinant strain T-C-11, the expression of fuh was upregulated, with a 7.07-fold increase at 60 h compared to the WT strain. Conversely, the expression levels of mdhA, mdhB, and sdhA were downregulated, peaking at 36 h. Additionally, the transcript levels of icl and ms involved in the glyoxylate cycle were also downregulated (Figure 6). For the recombinant strain T-D3-7, the expression levels of both pc and mdhB were significantly higher, with the pc transcript level reaching 6.28-fold at 36 h and mdhB reaching 4.2-fold at 60 h compared to the WT strain. This suggests that the oxaloacetate reduction pathway was the dominant route for pyruvic acid, which is carboxylated into oxaloacetic acid and then converted into L-malic acid by mdhB. The low transcription of sdhB likely limited the involvement of L-malic acid in the subsequent cycle, allowing it to be efficiently transported out of the cell by the DCT3. This may explain the higher production of L-malic acid in T-D3-7. Notably, among the four pathways capable of producing L-malic acid, the oxaloacetate reduction pathway has the highest theoretical conversion efficiency and is non-oxidative, meaning it does not require ATP for carbon synthesis [43,44,45,46,47].
In strain T-D5-16, the expression levels of fuh and sdhB were significantly higher, with 5.41-fold and 12.76-fold increases compared to the WT strain at 36 h, respectively. It is speculated that L-malate and succinate produced in the mitochondria and glyoxylate cycle are transported to the cytoplasm via mitochondrial membrane transport proteins and further metabolized. Concurrently, some of the L-malic acid in the cytoplasm is converted to fumaric acid by fuh, which is then exported from the cell via the DCT5 transporter [48]. The downstream synthesis of succinic acid is facilitated by sdhB, which is also transported by the DCT5 transporter [49,50]. In the future, it may be possible to genetically engineer a method to truncate this downward metabolic pathway to enhance acid production [51,52].

3.7. The Acid Production Capacity of the Transformants in 30 L Bioreactor and Statistical Analysis

As shown in Figure 7, the mycelial spheres of both T-D3-7 and T-D5-16 were compact and dense, with no signs of aging cavitation or overgrowth, and maintained morphological stability until the end. In terms of L-malic acid production, T-D5-16 exhibited a slightly higher yield than T-D3-7 at 84 h of fermentation. However, the purity of L-malic acid in T-D3-7 was higher, reaching 71% with a total production of 70.8 g/L. Both strains accumulated L-malic acid gradually from 12 h to 72 h, with a rapid increase in production between 72 h and 84 h. The overall trend in acid production was similar for both strains, with no significant differences in the rate of increase. In contrast, T-D5-16 outperformed T-D3-7 in succinic acid production. After 84 h of fermentation, T-D5-16 produced 41.59 g/L of succinic acid, nearly 2.87 times higher than the 14.51 g/L produced by T-D3-7 (Figure 7C). The rate of succinic acid accumulation in T-D5-16 was much higher than in T-D3-7 from 24 h onward, though the accumulation rate slowed after 48 h. In comparison, T-D3-7 exhibited a consistently slower rate of succinic acid accumulation.
Both strains primarily produced citric acid and oxalic acid as byproducts. T-D5-16 accumulated citric acid the fastest and in the largest quantity, while T-D3-7 accumulated citric acid to a lesser extent. Both strains showed low levels of oxalic acid, with concentrations remaining under 6.5 g/L at the end of fermentation (84 h).

4. Conclusions

In this study, we investigated the four-carbon dicarboxylic acid transporter proteins’ function and the associated metabolic pathways in A. niger. Among these, heterologous expression of the A. oryzae C4T318 in A. niger slightly increased the production of L-malic acid. After overexpression of dct2, the main metabolic pathway of the strain was changed from TCA to the glyoxylate cycle, and the synthesis of succinic acid was significantly increased. The yield of L-malate increased more than in the WT strain when this pathway was enhanced by overexpression of the dct3. Both T-D3-7 and T-D4-16 were oah-deficient strains that blocked the oxaloacetate pathway to some extent, so that improving the efficiency of oxaloacetate caused it to convert to L-malic acid. Two strains of T-D5-16 and T-D3-7 were selected for fermentation in a 30 L bioreactor for 84 h. The yields of succinic acid and L-malic acid reached 41.59 and 70.8 g/L, respectively. To conclude, we have constructed relevant strains overexpressing tetracarboxylic acid transporter proteins to produce tetracarboxylic acid, which showed great potential for future industrial production.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11030156/s1, Figure S1: Verification of gene overexpression transformant strains; Figure S2: T-D3-7 (A) and T-D4-16 (B) both destroyed oah gene resulting in their colonies on the plate showed a pale yellow color rather than black conidia; Table S1: The primers used in this work; Table S2: Screening of high acid-producing strains by the ratio of acid ring to colony diameter.

Author Contributions

Y.T.: investigation, methodology, investigation, visualization, writing—editing. S.L.: investigation, formal analysis, data curation, validation, writing—original draft. S.W.: project administration, software, formal analysis, supervision. X.W.: methodology, software, validation, supervision, data curation. X.X.: conceptualization, supervision, funding acquisition, writing—review and editing. D.W.: resources, funding acquisition, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the National Natural Science Foundation of China (Grant No. 31902193), the National Key Research and Development Program of China (No. 2021YFC1808901), the Project Program of Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, and Tianjin Key Laboratory of Industrial Microbiology, China (No. 2023KF02).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data generated in this study are available within the article and its Supplementary Data Files or upon request from the corresponding authors.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Schematic diagram of p-D1-5 plasmid construction.
Figure 1. Schematic diagram of p-D1-5 plasmid construction.
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Figure 2. Multi-sequence alignment and phylogenetic tree analysis. (A) Amino acid sequence alignment of DCT1-5 and C4t318. (B) Phylogenetic analysis of dct1-5 and c4t318. The tree is constructed by maximum likelihood method.
Figure 2. Multi-sequence alignment and phylogenetic tree analysis. (A) Amino acid sequence alignment of DCT1-5 and C4t318. (B) Phylogenetic analysis of dct1-5 and c4t318. The tree is constructed by maximum likelihood method.
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Figure 3. Overexpression of C4-dicarboxylate transporter in A. niger and the relative transcript levels. (A) c4t318. (B) dct2. (C) dct3. (D) dct4. (E) dct5. (F) oah.
Figure 3. Overexpression of C4-dicarboxylate transporter in A. niger and the relative transcript levels. (A) c4t318. (B) dct2. (C) dct3. (D) dct4. (E) dct5. (F) oah.
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Figure 4. Shake flask fermentation of relevant transformants at 72 h. (A) Analysis of acid production of transformants and its proportion. (B) Analysis of bacteriophage number and residual sugar of transformants.
Figure 4. Shake flask fermentation of relevant transformants at 72 h. (A) Analysis of acid production of transformants and its proportion. (B) Analysis of bacteriophage number and residual sugar of transformants.
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Figure 5. Shake flask fermentation acid production analysis, morphological observation of bacteriophages, and observations on the growth status. (A) Yield changes and trends in the production of L-malic acid by shaker fermentation of transformers. (B) Yield changes and trends in the production of succinic acid by shaker fermentation of transformers. (C) Yield changes and trends in the production of fumaric acid by shaker fermentation of transformers. (D) Changes and trends in production of citric acid by shaker fermentation of converters. (E) Analysis of bacteriophage status at 24 h, 48 h, and 72 h of fermentation. (F) Observations on the growth status of bacteria for 24 h, 48 h, and 72 h with glucose, malic acid, citric acid, fumaric acid, and succinic acid as carbon sources.
Figure 5. Shake flask fermentation acid production analysis, morphological observation of bacteriophages, and observations on the growth status. (A) Yield changes and trends in the production of L-malic acid by shaker fermentation of transformers. (B) Yield changes and trends in the production of succinic acid by shaker fermentation of transformers. (C) Yield changes and trends in the production of fumaric acid by shaker fermentation of transformers. (D) Changes and trends in production of citric acid by shaker fermentation of converters. (E) Analysis of bacteriophage status at 24 h, 48 h, and 72 h of fermentation. (F) Observations on the growth status of bacteria for 24 h, 48 h, and 72 h with glucose, malic acid, citric acid, fumaric acid, and succinic acid as carbon sources.
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Figure 6. Real-time qPCR analysis of the expression profile of the genes at 36 h, 48 h, and 72 h in different strains. Expression level of gene was evaluated using qRT-PCR. The strains were grown for 36 h, 48 h, and 72 h. (A) pc. (B) icl. (C) ms. (D) fuh. (E) mdhA. (F) mdhB. (G) sdhA. (H) sdhB.
Figure 6. Real-time qPCR analysis of the expression profile of the genes at 36 h, 48 h, and 72 h in different strains. Expression level of gene was evaluated using qRT-PCR. The strains were grown for 36 h, 48 h, and 72 h. (A) pc. (B) icl. (C) ms. (D) fuh. (E) mdhA. (F) mdhB. (G) sdhA. (H) sdhB.
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Figure 7. Acid production and morphological analysis of bacteriophages. (A) Observation of the morphology of the spheres of T-D5-16 and T-D3-7 at different times of fermentation; microscope magnification is objective lens magnification * eyepiece magnification. (B) L-malic of T-D5-16 and T-D3-7 at different times. (C) Succinic acid production of T-D5-16 and T-D3-7 versus the time. (D) Citric acid and oxalic acid of T-D5-16 and T-D3-7 versus the time.
Figure 7. Acid production and morphological analysis of bacteriophages. (A) Observation of the morphology of the spheres of T-D5-16 and T-D3-7 at different times of fermentation; microscope magnification is objective lens magnification * eyepiece magnification. (B) L-malic of T-D5-16 and T-D3-7 at different times. (C) Succinic acid production of T-D5-16 and T-D3-7 versus the time. (D) Citric acid and oxalic acid of T-D5-16 and T-D3-7 versus the time.
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Table 1. Accession numbers and characteristics of dct genes.
Table 1. Accession numbers and characteristics of dct genes.
Gene ID Gene NameLength (aa)EnzymeFPKM
MA48MA72MA120
4989224ANI_1_2040144dct1416C4-dicarboxylate transporter/malic acid transport protein687.92 164.78 206.71
4988409ANI_1_1316144dct2374C4-dicarboxylate transporter/malic acid transport protein223.13 268.80 545.86
4986875ANI_1_54124dct3479C4-dicarboxylate/malic acid transporter185.13 289.40 206.71
4980153ANI_1_1070034dct4403Malic acid transport protein65.28 20.60 39.84
4985437ANI_1_130104dct5395Sulfite efflux pump SSU1297.19 86.51 708.43
--AO090023000318c4t318380C4-dicarboxylate transporter/malic acid transport
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Tan, Y.; Liu, S.; Wu, S.; Wang, X.; Wang, D.; Xue, X. Improving Organic Acid Secretion of Aspergillus niger by Overexpression C4-Dicarboxylic Acid Transporters. Fermentation 2025, 11, 156. https://doi.org/10.3390/fermentation11030156

AMA Style

Tan Y, Liu S, Wu S, Wang X, Wang D, Xue X. Improving Organic Acid Secretion of Aspergillus niger by Overexpression C4-Dicarboxylic Acid Transporters. Fermentation. 2025; 11(3):156. https://doi.org/10.3390/fermentation11030156

Chicago/Turabian Style

Tan, Yiyang, Shutong Liu, Sheng Wu, Xiaolu Wang, Depei Wang, and Xianli Xue. 2025. "Improving Organic Acid Secretion of Aspergillus niger by Overexpression C4-Dicarboxylic Acid Transporters" Fermentation 11, no. 3: 156. https://doi.org/10.3390/fermentation11030156

APA Style

Tan, Y., Liu, S., Wu, S., Wang, X., Wang, D., & Xue, X. (2025). Improving Organic Acid Secretion of Aspergillus niger by Overexpression C4-Dicarboxylic Acid Transporters. Fermentation, 11(3), 156. https://doi.org/10.3390/fermentation11030156

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