Redox engineering of thermophilic fungus Myceliophthora thermophila enhances production of L‑malic acid by consolidated bioprocessing

ABSTRACT

Consolidated bioprocessing (CBP) is a promising strategy for low-cost malic acid production from plant biomass. In this study, we report a comprehensive redox engineering strategy to redirect reducing equivalents towards the reductive tricarboxylic acid (rTCA) pathway for enhanced malic acid production directly from cellulose in the thermophilic fungus Myceliophthora thermophila JG207. Firstly, deletion of key NADH-consuming pathways, including glycerol-3-phosphate dehydrogenase (gpdh) and mitochondrial external NADH dehydrogenases (nde1 and nde2), resulted in a significant increase in the NADH/NAD⁺ ratio and a corresponding 17% increase in malic acid titre. Subsequent removal of alternative oxidase (AOX) further heightened the NADH pool, although it induced oxidative stress and slowed growth. To alleviate redox imbalance, we overexpressed the native transhydrogenase SthA, which effectively converted NADPH to NADH and improved malate production by an additional 6%. Fine-tuning NAD⁺ kinase (UTR1) expression further optimised cofactor homoeostasis, bringing about the highest malate titre of 89.2 g/L, a 38.9% improvement over the starting strain JG207. Finally, overexpression of lytic polysaccharide monooxygenase gene (lpmo) and cellobiose dehydrogenase gene (cdh) enhanced cellulose degradation, shortening the fermentation cycle by two days on Avicel and crushed corncob. This work not only advances the fundamental understanding of redox metabolism in filamentous fungi but also provides a proof-of-concept for sustainable organic acid production from inexpensive lignocellulosic feedstocks.

Graphical abstract

1. Introduction

Malic acid is a C4-dicarboxylic acid broadly used as a food acidulant, flavor enhancer, and as a building block for biopolymers and specialty chemicals (Wei et al. 2021). It is selected as one of the “Top 12” value-added chemicals from biomass identified by the U.S. Department of Energy, underscoring its significance as a platform chemical. Traditionally, malic acid is produced from petrochemical precursor maleic anhydride or enzymatic conversion processes. In recent years, intensive research efforts have focused on engineering microorganisms to efficiently synthesize malic acid from renewable feedstocks under mild process conditions (Wu et al. 2025). Multiple metabolic routes for malic acid biosynthesis have been identified in microbes. Among these, the cytoplasmic reductive TCA (rTCA) pathway is the most efficient route introduced in both fungi and bacteria for high-yield malate production. In rTCA pathway, pyruvate (from glycolysis) is carboxylated to oxaloacetate (OAA), which is then reduced to L-malate by malate dehydrogenase (Zelle et al. 2008). The highest theoretical carbon yield reached 2 mol malate per mol glucose (Glucose + CO₂ = 2 malate). During the synthesis of malate, NADH derived from glycolysis is consumed by malate dehydrogenase to reduce OAA to malate. Multiple engineered microbial chassis have been developed for efficient malic acid biosynthesis (Dai et al. 2018; Liu et al. 2018; Sun et al. 2023; Xi et al. 2023a).

While pathway engineering provides the necessary biochemical reactions for malate synthesis, intracellular redox balance often becomes a key limiting factor. The malate-forming step in the rTCA pathway requires NADH as electron donor to reduce OAA to malate. Thus, an adequate supply of NADH must be maintained to sustain malic acid production. Generally, cellular metabolism relies on two major pyridine nucleotide couples: NADH/NAD⁺ and NADPH/NADP⁺ (Xiao et al. 2018). NADH predominantly serves catabolic processes and energy generation, such as respiration and fermentation, whereas NADPH provides reducing power for anabolic biosynthesis and stress relief. These cofactors are not interchangeable under normal physiology and their pools and ratios are independently regulated – typically a high NAD⁺/NADH ratio to favour oxidative catabolism and a high NADPH/NADP⁺ ratio to drive reductive biosynthesis (Ying 2008).

Engineering microbial production of chemicals often necessitates tweaking these redox pools so that the driving force (NADH or NADPH) matches the target pathway’s needs. Redox cofactor engineering has been considered an important strategy to optimise metabolite production in cell factories. One approach is to eliminate or re-route competing pathways that consume the needed redox equivalents. In S. cerevisiae, two target genes encoding external NADH dehydrogenase (NDE1 and NDE2) were deleted to reduce oxidization of cytosolic NADH, resulting in 2.6% increase in final L-lactic acid titer (Lee et al. 2015). Another prominent strategy is to modulate conversion between NAD(H) and NADP(H). Escherichia coli sthA responsible for the transfer of NADPH to NADH was introduced into Pichia kudriavzevii and Aspergillus niger to maintain adequate supply of NADH for malate production (Xi et al. 2023b; Yang et al. 2024). In addition, an extra NAD⁺ kinase gene (nadK) led to higher NADPH availability, and improved yields of L-ornithine and L-isoleucine in Corynebacterium glutamicum (Shi et al. 2012), and polyhydroxybutyrate in E. coli (Jiang et al. 2013; Zhang et al. 2015). These studies demonstrate the efficacy of reallocating reducing power to favor the desired product.

One promising strategy for low-cost malic acid production from biomass is consolidated bioprocessing (CBP). In a CBP strategy, a single microorganism (or microbial consortium) is responsible for producing appropriate amount of cellulase, deconstructing lignocellulose, and fermenting the released sugars into the target product, simplifying the workflow and potentially reducing costs (Singhania et al. 2022). CBP is widely regarded as the ultimate goal for economical biorefinery. Myceliophthora thermophila (also known as Thermothelomyces thermophilus) is a thermophilic filamentous fungus known for its high capacity to deconstruct plant biomass (Berka et al. 2011; Huang et al. 2024). Another key advantage of M. thermophila is that it grows optimally around 45–50 °C, offering significant advantages for industrial biotechnology processes, including reduced microbial contamination risk due to higher operating temperatures, and optimal functionality of biomass-degrading enzymes under thermophilic conditions. With the development of genetic systems, researchers have started to reprogram M. thermophila from a mere enzyme producer into a microbial cell factory for fuels and chemicals from plant lignocellulose (Zhang et al. 2023; Gu et al. 2024). We previously constructed M. thermophila strains capable of producing C4-dicarboxylic acids directly from raw biomass (Li et al. 2019b). In fed-batch fermentations on Avicel and unpretreated corncob, the engineered M. thermophila produced malate efficiently without adding any external enzymes. In this study, we perturbed the intracellular redox cofactor balance to enhance malic acid production in our previous M. thermophila strain JG207 (Li et al. 2019b). We hypothesized that redirecting the flow of reducing equivalents (NADH and NADPH) in M. thermophila would channel more carbon into malate and accelerate fermentation on cellulose. To test this, we implemented a multifaceted redox engineering strategy, aimed at increasing cytosolic NADH availability for the rTCA pathway while preventing excessive NADH depletion. Key components of this strategy involved reducing NADH consumption, modulating conversion of NADPH to NADH, as well as improving NADP⁺ regeneration in tandem. By integrating these redox interventions with enhancement of the ability of cellulose hydrolysis, the higher malic acid titer by M. thermophila strain was achieved from cellulose in a shorter fermentation time. This study represents improvement of a fungal CBP platform by intracellular NADH/NAD⁺ redox modulation and opens new avenues for enhancing bioconversion of renewable biomass into organic acids.

2. Materials and methods

2.1. Strains and growth conditions

Myceliophthora thermophila JG207 (Li et al. 2019b) and its mutants were cultured on Vogel’s minimal medium (VMM) with 2% glucose at 35 °C for 12 days to produce mature conidia. Antibiotics were added as needed for transformant selection. Escherichia coli DH5α was used for plasmid construction and amplification, and cultured at 37 °C in Luria-Bertani (LB) broth with kanamycin (50 μg/mL) or ampicillin (100 μg/mL) for plasmid selection.

2.2. Vector construction and strain engineering

For the construction of the vectors for overexpressing target genes in M. thermophila, the amplicon of sthA (Mycth_2309210) was ligated between the SpeI and BamHI sites of pAN52-PgpdA-bar carrying the bar selectable marker, generating the plasmid PgpdA-sthA-bar with the aid of the NEB Gibson Assembly Kit. Lytic polysaccharide monooxygenase gene (lpmo, Mycth_85556) amplified from the M. thermophila genome, under control of the strong constitutive promoter of eif (Mycth_2297659), was inserted into the BglIII and BamHI sites of plasmid pAN52-PtrpC-neo-PMtgpdA (Li et al. 2019b) carrying the neo selectable marker to form the overexpression plasmid Peif-lpmo-neo. Similarly, the fragment of cellobiose dehydrogenase gene (cdh, Mycth_111388) was amplified and inserted between BglII and BamHI of pAN52-PtrpC-neo-PMtgpdA plasmid together with promoter of pgk (Mycth_2316240) to generate the corresponding plasmid Ppgk-cdh-bar. The promoters of hxk1 (Mycth_2295756), pdc (Mycth_112121), and tef1 (MYCTH_2298136) were employed to overexpress NAD⁺ kinase gene utr1. The promoter fragments were separately inserted between the BglII and BamHI sites of pAN52-PgpdA-bar, together with utr1 amplified from the S. cerevisiae genome, to generate the corresponding plasmids Phxk1-utr1-bar, Ppdc-utr1-bar, and Ptef1-utr1-bar.

The deletion of genes, including gpdh (Mycth_2313529), nde1 (Mycth_230426), nde2 (Mycth_2304512), and aox (Mycth_109542) was performed using the CRISPR/Cas9 system. The plasmids for sgRNA expression and the vectors carrying the donor DNA sequences were constructed as described previously (Liu et al. 2017).

Polyethylene glycol (PEG)-mediated transformation of M. thermophila protoplasts was carried out following the protocol described by Gu et al. (2024). For gene overexpression, 10 μg of the target gene expression cassette (generated by PCR) was introduced into the protoplasts. For gene deletion, a mixture containing 10 μg each of the Cas9 expression cassette, donor DNA cassette, and sgRNA cassette—in equal molar amounts—was co-transformed into the protoplasts. All strains constructed and used in this study, including their detailed genotypes and relevant references, are listed in Table 1.

Table 1.

Myceliophthora thermophila strains constructed and used in this study.

Strain ID	Relevant genotype/modification	Description
JG207	Parental strain for malate production	Previous strain (Li et al. 2019b)
N301	JG207Δgpdh (glycerol-3-P dehydrogenase knockout)	Eliminating glycerol NADH sink
N302	JG207Δnde1Δnde2 (mitochondrial external NADH dehydrogenases KO)	Blocking cytosolic NADH shuttle to ETC
N303	JG207ΔgpdhΔnde1Δnde2	Combined NADH shuttle knockouts
N303Δaox	N303Δaox (alternative oxidase knockout)	Removing alternative respiratory NADH sink
N401	N303Δaox+PgpdA-sthA (transhydrogenase)	Enhancing conversion of NADPH to NADH
N511	N401+Phxk-utr1 (low-level NAD+ kinase overexpression)	Slightly increased NADP+ pool
N525	N401+Ppdc-utr1 (medium-level NAD+ kinase overexpression)	Moderately increased NADP+ pool
N531	N401+Ptef-utr1 (high-level NAD+ kinase overexpression)	Greatly increased NADP+ pool
N6PC	Overexpression of lpmo and cdh in N525	Enhanced cellulose degradation capacity

2.3. Medium for malate production

Malic acid production by M. thermophila was carried out in 50 mL of medium inoculated with 2.5 × 10⁵ spores per milliliter in a 250 mL Erlenmeyer flask. The culture was incubated at 45 °C and 150 r/min (aerobic condition) on a rotary shaker. The medium consisted of 75 g/L carbon source (Avicel or crushed corncob), 0.15 g/L KH₂PO₄, 0.15 g/L K₂HPO₄, 0.1 g/L MgSO₄·7H₂O, 0.1 g/L CaCl₂·2H₂O, 8 g/L Bacto peptone, 0.1 mg/L biotin, and 1 mL trace elements from Vogel’s salt solution, and was sterilised by autoclaving. After sterilisation, CaCO₃ was added to a final concentration of 80 g/L.

2.4. Fungal biomass assay

To determine the fungal cell dry weight, 10 mL of culture was mixed with 2 mol/L HCl to dissolve any undissolved CaCO₃ and centrifuged to obtain a mixture of mycelia and residual corncob. The mixture was washed twice with distilled water and dried at 80 °C to a constant weight (recorded as W0). Subsequently, the sample was hydrolyzed by adding 6 mL acid hydrolysis solution (acetic acid:nitric acid:H₂O = 8:1:1) and boiling in a water bath for 1 h. After centrifugation, the precipitate was washed twice with distilled water and dried at 80 °C to a constant weight (recorded as W1). The difference between W0 and W1 was recorded as the mycelial weight in 10 mL cultures.

2.5. Enzyme activity assay

For the assay of lignocellulolytic enzyme activities, M. thermophila strains were inoculated into the medium of malic acid production with 75 g/L Avicel and cultured for 4 d. To measure extracellular enzyme activities, 1 mL cultures were centrifuged (14,000 r/min for 10 min at 4 °C) and the supernatant of each sample was retained. To protect proteins from proteases, PMSF was added to the samples (1 mmol/L final concentration). The protein concentration in the supernatant was determined using the Bio-Rad Protein Assay Kit (absorbance of 595 nm, Bio-Rad, Hercules, CA, USA).

Exoglucanase (PNPCase) activity in the culture supernatant was determined using p-nitrophenyl β-D-cellobioside as the substrate. All reagents were preheated at 45 °C for 5 min and then 200 μL 1 mg/mL PNPC was added to 200 μL supernatant, after which the solution was incubated at 45 °C for 10 min. The reaction was terminated by adding 500 μL 1 mol/L Na₂CO₃. The mixtures were centrifuged (12,000 r/min for 3 min at 4 °C) and the absorbance (420 nm) of the supernatants was measured. One unit (U) of enzyme activity was defined as the amount of enzyme required to catalyse the formation of 1 μmol p-nitrophenol (pNP) per minute using PNPC as the substrate at 45 °C and pH 4.8.

Cellobiose dehydrogenase (CDH) activity was assayed in culture supernatants at 4 d. CDH activity was measured by the reduction of 2,6-dichlorophenol indophenol (DCIP) in the presence of excess cellobiose. Reduction of DCIP was monitored by the decrease in absorbance at 530 nm. The reaction system included 1.0 mmol/L cellobiose, 200 μmol/L DCIP, and the condensed culture filtrate in 100 mmol/L sodium acetate pH 5.0 (Phillips et al. 2011). One unit of CDH activity was defined as the amount of enzyme reducing 1 μmol DCIP per minute.

Cellulose degradation capability of M. thermophila culture was determined by the release of soluble sugars from Avicel. The cellulose hydrolysis reaction system included the condensed culture (50 μg protein), 2% Avicel, and 1 μg/mL sodium azide in 1 mL Tris-HCl buffer (10 mmol/L, pH 5.6). The mixtures were incubated at 45 °C for 24 h with shaking at 150 r/min and then centrifuged (14,000 r/min for 15 min at 45 °C). The supernatants were filtered through a 0.22 μm membrane and analysed using an HPLC system (Liu et al. 2024).

For intracellular enzyme activity assays, M. thermophila strains were cultivated in 250 mL Erlenmeyer flasks containing 50 mL of fermentation medium supplemented with 20 g/L CaCO₃. Cultures were incubated at 150 r/min in an orbital shaker at 45 °C for 2 days. Mycelial samples were harvested, rapidly frozen in liquid nitrogen, and ground into a fine powder using a pre-chilled mortar and pestle. The homogenised mycelial powder was suspended in 1 mL of phosphate-buffered saline (pH 7.4) and centrifuged at 4 °C for 10 min. The resulting clear supernatant was collected for protein quantification and subsequent enzymatic activity assays. Malate dehydrogenase (MDHase) activity was determined by monitoring NADH oxidation at 340 nm. The reaction mixture contained 100 mmol/L Tris (pH 7.8), 5 mmol/L MgCl₂, 10 mmol/L NaHCO₃, 0.2 mmol/L NADH, and 1 mmol/L oxaloacetate. The assay was started by the addition of extracted crude enzymes at 37 °C. One unit of activity represents the amount of enzyme required to oxidize or reduce 1 μmol of substrate per min.

Glutathione reductase (GR) activity was assayed in clarified cell extracts by following NADPH oxidation at 340 nm. Each 1 mL reaction contained 100 mmol/L potassium phosphate (pH 7.5), 1 mmol/L EDTA, 0.2 mmol/L NADPH, 1 mmol/L oxidized glutathione (GSSG), and crude cell extract. The decrease of absorbance at 340 nm was recorded, and one unit of GR activity was defined as 1 µmol NADPH oxidized per minute.

2.6. Metabolite analysis

To determine the titer of malic acid in culture broth, 1 mL of 2 mol/L H₂SO₄ was added to a 1-mL well-mixed sample in a 15-mL tube. The mixture was incubated at 80 °C for 30 min and vortexed at intervals to adequately resolve the organic acid. Afterward, 2 mL distilled water was supplemented, and an aliquot was determined by HPLC system (Li et al. 2019b).

For intracellular cofactor assays, M. thermophila strains were cultivated in 250 mL Erlenmeyer flasks containing 50 mL of fermentation medium supplemented with 20 g/L CaCO₃. Cultures were incubated at 150 r/min in an orbital shaker at 45 °C for 2 days. The mycelial medium was poured into a Buchner funnel fitted with four pieces of gauze. The residue was washed with distilled water until most CaCO₃ was removed, and then immediately homogenised in liquid nitrogen and stored at −80 °C. The mitochondria were isolated following the manufacturer’s instructions provided with the Mitochondria Isolation Kit (Sigma-Aldrich, MITOISO1). The intracellular ATP concentration was determined via an ATP Assay Kit (Sigma-Aldrich, MAK190-1KT). The intracellular concentration of NADH was evaluated according to the manufacturer’s instructions using an NAD/NADH Quantitation Kit (Sigma-Aldrich, MAK037). A High Sensitivity NADPH Assay Kit (Sigma-Aldrich, MAK216) was used to detect the NADPH concentration in M. thermophila.

2.7. Quantitative real time-PCR (RT-qPCR) analysis

The expression level of target gene was determined using quantitative real-time PCR (qRT-PCR). Total RNA was extracted and purified as previously described by Li et al. (2019a). cDNA synthesis and qRT-PCR were performed using SYBR Green Realtime PCR Master Mix (Toyobo, Osaka, Japan) on a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Negative control reactions were prepared by substituting template RNA with an equal volume of nuclease-free water. The actin gene (MYCTH_2314852) was used as an internal reference to normalise gene expression. Relative transcript levels were calculated using the 2^–ΔΔCt method. The primer sequences used were as follows: Actin (Forward: 5′-TGCCCATCTACGAGGGTTTC-3′, Reverse: 5′-TTGATGTCACGGACAATTTCAC-3′); utr1 (Forward: 5′-AGTTTCAGACCCATCATCTTAC-3′, Reverse: 5′-AGCATATGGGCTGGCGCATATG-3′); mdh (Forward: 5′-TTCAGCAAGATCGCTCCGTC-3′, Reverse: 5′-GTATGCTGGTTACAGGTTCGC-3′).

3. Results

3.1. NADH shuttle pathway knockouts increase cytosolic NADH availability and malate production

With the goal of redirecting more NADH toward malic acid synthesis by cytosolic rTCA pathway, we first eliminated three native pathways that compete for cytosolic NADH (Figure 1a). The pathways targeted included glycerol formation (via NADH-dependent glycerol-3-phosphate dehydrogenase, GPDH) and two respiratory NADH dehydrogenases (NED1 and NED2) located on the mitochondrial outer membrane. In filamentous fungi, GAPH normally acts as a redox sink by converting NADH to NAD⁺ while producing glycerol. Meanwhile, Ned proteins transfer cytosolic NADH electrons to the mitochondrial electron transport chain, oxidizing NADH without yielding ATP. Deleting these should conserve NADH in the cytosol. Starting from parent strain JG207 (Li et al. 2019b), we created mutants N301 (Δgpdh), N302 (Δned1Δned2), and N303 (ΔgpdhΔned1Δned2).

Figure 1.

Eliminating NADH-consuming pathways increases NADH availability and malate production in Myceliophthora thermophila. (a) Schematic overview of the metabolic engineering in Myceliophthora thermophila. The parent strain JG207 was sequentially engineered to delete genes encoding key NADH reoxidation enzymes – glycerol-3-phosphate dehydrogenase gene (gpdh) and the mitochondrial external NADH dehydrogenase genes (ned1 and ned2). This yielded strains N301 (JG207Δgpdh), N302 (JG207Δned1Δned2), and N303 (JG207ΔgpdhΔned1Δned2). (b, c) Intracellular redox metrics measured on day 2 of fermentation in shake-flask cultures with 75 g/L Avicel as carbon source. (b) Intracellular NADH concentration and (c) NADH/NAD⁺ ratio was shown for the parent and each mutant strain. (d, e) Transcription level of malate dehydrogenase gene mdh (d) and malate dehydrogenase activity (e) were in the indicated strains when grown on Avicel for 2 days. (f) Malic acid titer in the culture broth after 8 days of fermentation on 75 g/L Avicel, measured by HPLC. (g, h) Total secreted protein concentration (g) and endoglucanase activity (h) of culture supernatant from the strains grown on Avicel for 4 days. Error bars indicate the s.d. of biological triplicates.

All strains were cultivated on Avicel (75 g/L) under identical conditions. We assessed intracellular NADH and NAD⁺ levels in early exponential phase (day 2) as well as malic acid accumulation at later stages. The NADH/NAD⁺ ratio was markedly elevated in the knockout strains relative to JG207. In particular, the triple knockout N303 showed the highest NADH content and NADH/NAD⁺ ratio, approximately 1.62-fold and 1.34-fold higher than the parent, respectively (Figure 1b,c). Deletion of gpdh alone (N301) led to a moderate increase in NADH/NAD⁺ (about 1.1-fold). Knocking out the external NADH dehydrogenases (N302) resulted in a greater NADH/NAD⁺ rise (~1.2-fold vs. JG207) and an observably increased NADH accumulation (Figure 1b). When both modifications were combined (N303), the effects synergised: N303 cells had significantly higher intracellular NADH concentration (Figure 1b) and a higher NADH fraction of the total NAD pool (reflected in NADH/NAD⁺). This indicates that blocking NADH-consuming pathways (glycerol synthesis and mitochondrial NADH shuttling) successfully redirected reducing equivalents into the cytosolic NADH pool.

Next, we examined the impact of this perturbed redox state on the rTCA malate synthesis pathway. Notably, the transcription level of the malate dehydrogenase gene mdh was upregulated in the engineered strains. At 48 h, transcription level of mdh in N303 was significantly higher than in JG207 (Figure 1d), suggesting that an elevated NADH/NAD⁺ ratio could enhance mdh gene expression. In agreement with the mRNA data, the MDH enzyme activity in cell-free lysates was markedly enhanced. The triple mutant N303 displayed the highest MDH specific activity (Figure 1e), approximately 50.3% greater than the parent strain JG207. Strains N301 and N302 showed moderate increases in MDH activity as well. Concomitantly, malic acid production was enhanced in the NADH-engineered strains. After 8 days of fermentation on Avicel, strain N303 produced 17.1% more malic acid than JG207 (Figure 1f). The final malate titer for N303 was 75.2 g/L, compared to 64.2 g/L for the parent under the same conditions. N301 and N302 showed intermediate malate titers (68.5 g/L and 69.4 g/L, respectively), consistent with their intermediate NADH availability. Consistent with the increased yield of target product, the secreted protein and endoglucanase activity in the supernatant of three engineered strains showed a significant increase compared to JG207 (Figure 1g,h). In summary, these data demonstrated that rerouting NADH from side pathways to malate synthesis is an effective strategy to boost malic acid production in M. thermophila.

3.2. Alternative oxidase deletion further increases NADH/NAD⁺ ratio

Filamentous fungi generally possess an alternative oxidase, which provides a bypass for the mitochondrial respiratory chain by directly oxidising ubiquinol and reducing oxygen to water without proton pumping (Ebiloma et al. 2019). AOX is non-energy-conserving and is typically upregulated under conditions of high NADH or respiratory stress to maintain redox balance. We hypothesised that deleting AOX in the NADH-rich N303 background might force more NADH through the primary respiratory chain or into product formation (Figure 2a). Thus, we constructed N303Δaox (N303 background with the aox gene knocked out) and then characterised the impact of AOX loss on cellular redox state, growth, and malate titer.

Figure 2.

Deletion of alternative oxidase (AOX) alters the redox balance. Strain N303 (JG207ΔgpdhΔned1Δned2) was further engineered to knock out the alternative oxidase gene (aox), yielding strain N303Δaox. (a) Schematic depicting the potential impact of alternative oxidase. (b, c) Whole-cell NADH pool and redox state on day 2 of 75 g/L Avicel fermentation. (b) Intracellular NADH concentration and (c) NADH/NAD⁺ ratio. (d, e) Mitochondrial redox state and energy status on day 2. (d) Mitochondrial NADH level and (e) ATP content. (f, g) Mitochondrial oxidative stress indicators measured on day 2 under 75 g/L Avicel conditions. (f) Glutathione reductase (GR) activity and (g) reduced glutathione (GSH) concentration, for N303 vs. N303Δaox. (h) Colony morphology on agar plates: N303 (left) and N303Δaox (right) after 3 days of incubation at 35 °C. (i) Biomass growth curve over the 4-day fermentation, illustrating the impact of aox deletion on growth. (j) Malic acid concentration in the culture after 8 days on 75 g/L Avicel. Error bars indicate the s.d. of biological triplicates.

Whole-cell NADH content was increased by 15% in N303Δaox and the NADH/NAD⁺ ratio rose correspondingly (Figure 2b,c). This confirms that AOX normally acts to maintain a lower NADH/NAD⁺ ratio by burning off excess NADH; without AOX, the cell’s NADH pool became more reduced. Notably, we found that the mitochondrial NADH pool in N303Δaox was especially elevated (Figure 2d). N303Δaox had 1.5-fold higher mitochondrial NADH concentration than N303 on day 2. This suggests that NADH was accumulating in the mitochondria, likely because electrons had to funnel through the cytochrome-mediated ETC only, which may have a limited throughput. We measured the ATP levels in mitochondria as an indicator of respiratory flux. Mitochondrial ATP in N303Δaox was higher than in N303 (Figure 2e). Blocking AOX led to increase ATP yield per NADH if the main ETC takes over. However, the absence of AOX, possibly leading to inhibition of respiration or a bottleneck at the cytochrome chain. This condition often leads to increased reactive oxygen species (ROS) generation, as a highly reduced ubiquinone pool can leak electrons to form superoxide. Consistent with this, we observed signs of oxidative stress in N303Δaox. The mitochondrial glutathione system was activated. The strain N303Δaox had 88% higher glutathione reductase activity compared to N303, and the content of reduced glutathione (GSH) in N303Δaox was 2.1-fold of that in N303 (Figure 2f,g). These data confirm that AOX deletion led to elevated ROS stress, presumably because the cells could not fully cope with the excess NADH electrons via the standard ETC alone.

The physiological consequences of this oxidative imbalance were evident in growth morphology. N303Δaox grew noticeably slower on solid and liquid medium. On agar plate, N303Δaox colonies were smaller and sparser than N303 colonies after 5 days (Figure 2h). In shake flasks, the biomass accumulation of strain N303Δaox lagged behind that of strain N303 (Figure 2i). This growth impairment is likely due to cell damage or metabolic burden from redox stress. Despite these drawbacks, malic acid production in N303Δaox was slightly improved relative to N303. By day 8 of culture on Avicel, N303Δaox produced 76.6 g/L malic acid, about 4.6% higher than N303 (73.2 g/L) (Figure 2j), and malic acid productivity per unit of mycelial biomass increased by 32.5%. The malate yield on cellulose reached 1.02 g/g for N303Δaox. In summary, AOX deletion pushed the NADH/NAD⁺ ratio even higher and boosted malate titer further, but at the cost of some growth reduction and mild oxidative stress. The N303Δaox strain provided a more NADH-reduced cytosol for our next engineering steps.

3.3. Overexpression of transhydrogenase (SthA) relieves redox stress and boosts malate production

With the strain N303Δaox providing a high-NADH background, we next addressed the NADPH/NADH balance. In filamentous fungi, primary metabolic pathways consume NADPH, while our target malate pathway consumes NADH (Chandel 2021). We speculated that converting some NADPH into NADH could further drive malate formation. We overexpressed native sthA (Mycth_2309210), encoding a soluble pyridine nucleotide transhydrogenase catalyzing the reaction of NADPH to NADH, in N303Δaox under a constitutive promoter. In resultant strain N401, the transcription level of sthA was increased by 3.86-fold. In fermentation, strain N401 outperformed N303Δaox. On Avicel, N401 reached 81.3 g/L malic acid, a 6% increase over N303Δaox (Figure 3a). Intracellular measurements supported this phenomenon: the strain N401 showed notable changes in cofactor levels compared to its parent N303Δaox. When we measured NADPH levels, NADPH content in N401 dropped by 29% relative to N303Δaox (Figure 3b). On the contrary, The NADH content in N401 was 36% higher than in N303Δaox (Figure 3c). The strain N401 also had roughly a 42.9% higher NADH/NAD⁺ (Figure 3d), indicating that sthA successfully converted additional NADH from NADPH. These results demonstrated that harnessing NADPH via a transhydrogenase could effectively bolster the NADH-dependent production of malic acid in M. thermophila.

Figure 3.

Introducing a transhydrogenase (SthA) in the AOX-deficient strain to boost NADH for malate synthesis. Strain N401 was constructed by overexpressing the native transhydrogenase gene sthA in strain N303Δaox. SthA catalyzes the transfer of reducing equivalents from NADPH to NAD⁺, increasing NADH at the expense of NADPH. (a) Relative expression levels of sthA in the engineered strain N401, determined by qRT-PCR. (b) Malic acid titer in the culture after 8 days on 75 g/L Avicel. (c–e) NADPH concentration (c), NADH concentration (d), NADH/NAD⁺ ratio (e), and in N303Δaox vs. N401 on day 2 of fermentation with 75 g/L Avicel. Error bars indicate the s.d. of biological triplicates.

3.4. Tuning NAD⁺ kinase expression optimizes cofactor balance and malate production

While SthA successfully leveraged NADPH to boost NADH and malate, we speculated that further adjustments to NADPH metabolism could maximise the benefit. If NADPH supply is too low, SthA may become substrate-limited. We therefore modulated the NADPH pool by overexpressing the S. cerevisiae NAD⁺ kinase gene utr1 (Kawai et al. 2001), which increases conversion of NAD⁺ to NADP⁺. Any additional NADP⁺ can be reduced to NADPH by the cell’s metabolism, and then sthA will convert it to NADH (Figure 4a). Additionally, an elevated intracellular NADPH level facilitates cellular resistance to oxidative stress by providing essential reducing power to the antioxidant defense system (Ying 2008; Jiang et al. 2024; Wu et al. 2024). However, excessively strong utr1 expression might deplete NAD⁺ or excessively divert flux into NADPH at the expense of NADH generation in glycolysis. Therefore, we created strains N511, N525, and N531 with low, medium, and high utr1 expression (driven by Phxk, Ppdc, Ptef promoters, respectively, in the N401 background, Figure 4b).

Figure 4.

Enhancing NADH/NAD⁺ ratio via NAD⁺ kinase (UTR1) overexpression under varied promoters. Building on strain N401, an NAD⁺ kinase gene (utr1) was overexpressed under promoters of different strengths to modulate the balance between NAD⁺, NADH, and NADPH. Three utr1-expressing strains were created: N511, N525, and N531, carrying utr1 driven by a weak promoter (Phxk), medium-strength promoter (Ppdc), and strong promoter (Ptef1), respectively. (a) Schematic illustration of the strategy: overexpression of NAD⁺ kinase (UTR1) diverts NAD⁺ to NADP⁺, influencing cellular redox pools (NADH vs. NADPH). (b) Design of the three constructs with different promoter strengths controlling utr1 expression, and the resulting strains: N531 (Strong promoter: Ptef–utr1), N525 (Medium: Ppdc–utr1), and N511 (Weak: Phxk–utr1). (c) Relative expression levels of utr1 in the three engineered strains (N531, N525, N511), determined by qRT-PCR. (d) Malic acid titer after 8 days of fermentation on 75 g/L Avicel. (e–i) Intracellular redox parameters measured on day 2 in 75 g/L Avicel fermentations of the control (N401) versus the utr1-expressing strains. (e) NADP⁺ concentration, (f) NAD⁺ concentration, (g) NADPH concentration, (h) NADPH/NADP⁺ ratio, and (i) NADH/NAD⁺ ratio in cell extracts of the culture when grown on Avicel for 2 days. (j) Fungal biomass (dry cell weight) achieved by day 2 for each strain. Error bars indicate the s.d. of biological triplicates.

We first confirmed that utr1 transcription levels varied as intended. Figure 4c showed N531 (Ptef-utr1) had the highest expression level of utr1 (approximately 2.1-fold higher than N525, and 3.9-fold higher than N511). N525 (Ppdc-utr1) had moderate expression, about 1.9-fold above the basal level in N401, whereas N511 (Phxk-utr1) was only slightly above basal. However, when grown on Avicel, the highest overexpression (N531) did not improve malate production beyond N401. In fact, malate titer (40.3 g/L) in N531 was 50.5% lower than N401 (Figure 4d), suggesting excessive NAD⁺ kinase might divert too much NAD⁺ to NADP⁺ (Figure 4e,f), which deprived the rTCA of NAD⁺. The lowest overexpression (N511) showed minimal change from N401. The strain N525 with medium utr1 performed best: on 75 g/L Avicel, N525 produced 89.2 g/L malic acid by day 8, the highest of all strains constructed so far, and 9.7% higher than N401 (Figure 4d). We inferred that moderate elevation of NAD⁺ kinase strikes the optimal balance – increasing NADPH for cellular needs, while still leaving ample NAD⁺ to accept electrons for NADH generation. Intracellular cofactor measurements at 48 h indicated that in strain N525, the NADP⁺ content and NADPH content showed an observable increase (Figure 4e,g), but the ratio of NADPH/NADP⁺ was reduced by 33.2% (Figure 4h), compared to that in strain N401. Accordingly, the NAD⁺ content showed a 28.9% decrease (Figure 4f), while NADH/NAD⁺ ratio in N525 was increased by 73.8% (Figure 4i), exhibiting a slightly more reducing environment in NADH pool. Notably, N525’s biomass accumulation was 30% higher than N401 (Figure 4j). These data indicated that fine-tuning the NADP(H) pool via NAD⁺ kinase expression is crucial. An optimal increase in NADPH supply allowed SthA to operate effectively without detracting from primary metabolism, thereby maximizing malic acid production. Although overexpression of NAD⁺ kinase elevated NADPH levels, it resulted in diminishing returns in malate titer and imposed a growth burden. These results clearly demonstrate the importance of balanced redox cofactor engineering, as more is not always better, and there exists an optimal level of NADPH regeneration for malate biosynthesis in this system.

3.5. Overexpression of LPMO and CDH accelerates cellulose degradation and enhances malate productivity

One potential bottleneck in CBP is the rate of biomass hydrolysis. Although M. thermophila secretes an arsenal of cellulases and even LPMOs naturally, the demands of high-level product formation might require enhanced enzyme activity to fully utilise the substrate. We therefore augmented strain N525 with additional copies of a lytic polysaccharide monooxygenase (LPMO) and cellobiose dehydrogenase (CDH). LPMOs are copper-dependent enzymes that oxidatively cleave crystalline cellulose, creating new chain ends for cellulases, and CDH can generate hydrogen peroxide and provide electrons to fuel LPMO action. The resulting strain N6PC was tested on Avicel and on a more complex substrate (crushed corncob).

In Avicel flask culture, the extracellular CDH enzymatic activity in N6PC was measured at 0.86 U/mL, which is about 1.87-fold higher than that of N525 (0.46 U/mL) (Figure 5a). To assess the cellulolytic power of the culture supernatants, we incubated equal volumes of filtered supernatant from N6PC vs. N525 (day 4) with fresh Avicel for 24 h and measured the release of reducing sugars (Figure 5b). The N6PC supernatant released more reducing sugars, including glucose and cellobiose, from Avicel than the N525 supernatant in this test, indicating enhanced cellulose-degrading enzyme activity. These results demonstrate that N6PC has a higher capacity to break down crystalline cellulose. The strain N6PC was cultured on Avicel and corn stover to evaluate cellulose conversion and malate production. As shown in Figure 5c, N6PC displayed faster malic acid production than N525 on Avicel. By day 4, N6PC had produced 69.3 g/L malate, compared to 44.8 g/L in N525, a 54.7% increase in early titer. This indicates a more rapid release of fermentable sugar from Avicel when CDH/LPMO were overexpressed. The malate production peaked by day 6 in N6PC, whereas N525 continued to produce malate up to day 8. The final malate titer in N6PC (89.8 g/L) was similar to N525 (89.2 g/L), but more significantly, the fermentation time to reach > 80 g/L was shortened by 2 days, which was an important improvement in productivity. Consistently, when we tested the performance of N6PC on real lignocellulosic biomass (Crushed corncob) to evaluate its CBP capability on a complex substrate, N6PC again outperformed N525. As shown in Figure 5d, N6PC reached about 11.1 g/L-day during the peak production phase (In the first 4 days), whereas N525 peaked at 8.6 g/L-day. By the end of 6 days, N6PC produced 48.1 g/L malate from 75 g/L corn cob, indicating 9.3% higher than that of N525 (44 g/L). These results confirm that augmenting M. thermophila with additional LPMO/CDH can accelerate substrate utilization and enhance malic acid productivity in CBP.

Figure 5.

Introducing cellulolytic enzymes (LPMO and CDH) to boost cellulose degradation and malate yield in consolidated bioprocessing. To enhance the conversion of solid biomass to product, strain N525 was further engineered to overexpress a lytic polysaccharide monooxygenase gene (lpmo) and a cellobiose dehydrogenase gene (cdh), yielding strain N6PC. (a) CDH activity: Cellobiose dehydrogenase enzymatic activity in the culture supernatant at day 4 of fermentation on 75 g/L Avicel. CDH activity was assayed by monitoring the oxidation of cellobiose (one unit is defined as the amount of enzyme catalyzing 1 μmol of substrate per minute under assay conditions). (b) Total cellulolytic activity: The cellulose-degrading capability of day-4 culture supernatants, assessed by incubating supernatant with crystalline cellulose (Avicel) and measuring the released sugars. (c, d) Time profiles of malic acid production using 75 g/L Avicel (c) and 75 g/L corn cob (d) as the carbon source. Error bars indicate the s.d. of biological triplicates.

4. Discussion

Filamentous fungi offer great potential advantages in the utilization of complex carbon sources and production of organic acids with high concentrations and impressive yields near the theoretical maximum (Peleg et al. 1988; Xu et al. 2010; Knuf et al. 2014; Dhiman et al. 2024). This study demonstrates that redirecting redox cofactor pathways in M. thermophila can substantially enhance malic acid production under CBP conditions. By deleting competing NADH-utilizing routes (glycerol synthesis via GPDH and two external mitochondrial membrane-bound NADH dehydrogenases Nde1/Nde2), we increased cytosolic NADH availability for the reductive TCA (rTCA) pathway. Consistent with reports in yeast and bacteria, removing NADH sinks can drive more carbon into reduced products, including bioethanol, lactic acid, and isobutanol (Lee et al. 2015; Zhang et al. 2023; Schulz-Mirbach et al. 2024). In our fungus, the ΔgpdhΔnde1Δnde2 mutant similarly showed a ~1.34-fold higher NADH/NAD⁺ ratio and 17% higher malate titer than the parent, reaching 75.2 g/L (vs. 64.2 g/L) on Avicel. Notably, the elevated NADH level was accompanied by ~50% higher malate dehydrogenase activity, suggesting a redox-driven upregulation of the rTCA flux. This aligns with observations in A. niger, where increasing cytosolic NADH availability (by limiting alternative NADH sink) enhanced malate-forming MDH activity (Yang et al. 2024).

Eliminating the alternative oxidase (AOX) provided a further boost to malate production, albeit with trade-offs. AOX is a ubiquitous “safety valve” in fungi, dissipating excess NADH by reducing O₂ to H₂O without ATP generation (Del-Saz et al. 2018; Flipphi et al. 2023). It is often upregulated during overflow metabolism to regenerate NAD⁺ during high organic acid production (e.g., citric or itaconic acid). In A. niger citric acid fermentation, AOX uncouples NADH oxidation from ATP synthesis, allowing glycolysis to continue despite high energy status (Kirimura et al. 2000). By deleting aox in M. thermophila, we forced NADH into the malate pathway. However, the ∆aox strain showed slower growth and signs of oxidative stress, likely due to NADH accumulation and reactive oxygen species (ROS) formation when the standard electron transport chain was overloaded (Zhao et al. 2019). Thus, while removing AOX increases the intracellular reducing power for product synthesis, it underscores the need to manage redox stress.

To further manipulate the intracellular redox balance, we overexpressed the native soluble transhydrogenase SthA. SthA catalyzes transfer of reducing equivalents from NADPH to NAD⁺, thus generating additional NADH (Tan et al. 2016). This strategy significantly boosted the NADH pool in M. thermophila (NADH/NAD⁺ increased by 42.9%), leading to a 6% higher malate titer (81.3 g/L). Increased NADH supply via transhydrogenase is a known tactic in other hosts to drive reductive product synthesis. In E. coli, combining an enhanced pentose phosphate pathway (to overproduce NADPH) with sthA overexpression converted the excess NADPH into NADH, enabling a succinate yield of ~94% of theoretical maximum (Tan et al. 2016). Likewise, Yang et al. (2024) showed that expressing E. coli sthA in A. niger elevated cytosolic NADH and improved malate titers, although constitutive expression caused developmental defects (Yang et al. 2024). These studies and our results highlight the power of transhydrogenases to redirect NADPH toward NADH-dependent pathways.

Moderate overexpression of NAD⁺ kinase (UTR1) proved crucial in restoring redox balance. By phosphorylating NAD⁺ to NADP⁺, Utr1 increases the NADP(H) pool, effectively buffering some excess reducing power into NADPH. In strain N525, a medium-level UTR1 expression raised NADPH levels and slightly lowered NAD⁺, creating a more favorable NADH/NAD⁺ ratio without crippling NAD⁺ availability for glycolysis. This strain achieved the highest malate titer (89.2 g/L, a 9.7% improvement over its parental strain). In contrast, high UTR1 overexpression (N531) overshot the balance – NAD⁺ was too depleted and NADPH too high, leading to 50% lower malate. Such an effect of redox cofactor modulation is echoed in other systems. Wu et al. (2024) found that boosting NADPH supply in A. niger by overexpressing its NAD⁺ kinases enhanced malic acid titer by ~24% and alleviated oxidative stress. Interestingly, A. niger exhibited increased NADPH/NADP⁺, whereas the reduction of NADPH/NADP⁺ was observed in M. thermophila, which might contribute to attributed to SthA overexpression channeling NADPH into NADH. NADPH plays an important role in cellular redox homeostasis and metabolism (Sun et al. 2024). An optimal increase in NADPH supply in strain N525 resulted in increased biomass accumulation, indicating the cells tolerated the redox intervention well. However, excessive NAD⁺ kinase activity in N531 deprived the rTCA of NAD⁺ acceptors. Overall, our results underline that fine-tuning redox cofactor ratios is more effective than brute-force alteration.

Finally, integrating redox engineering with enhanced cellulolytic capacity yielded synergistic benefits for CBP. Overexpressing a lytic polysaccharide monooxygenase (LPMO) and cellobiose dehydrogenase (CDH) in strain N6PC accelerated cellulose deconstruction, thus supplying fermentable sugars more rapidly. LPMOs and CDHs are known to work in tandem to boost cellulose saccharification (Phillips et al. 2011; Hemsworth et al. 2015). In our work, after overexpression of native lpmo and cdh, strain N6PC achieved 54.7% higher malate titer by day 4 than its progenitor (69.3 vs. 44.8 g/L), and finished the fermentation in 6 days instead of 8 with the similar final titer. The improvement was even more pronounced on real biomass: on untreated corncob. This illustrates the importance of synchronising fermentation metabolism with substrate liberation in CBP systems. Our previous study showed that the transcriptional levels of cellulase genes were gradually increased, along with improved production of malic acid, but increased secreted enhancement of cellulase by overexpressing cellulase activator Clr-1 did not increase the system efficiency of malic acid synthesis from Avicel (Li et al. 2019b). There are several plausible explanations for this outcome. First, over-activating the cellulase regulator Clr-1 may have produced a non-ideal cellulase cocktail, upregulating all cellulase components can lead to an imbalance in enzyme proportions. Second, the high-level cellulase expression likely imposed a metabolic burden on the fungus, diverting energy and resources towards enzyme production at the expense of malic acid biosynthesis (Zhao et al. 2023). However, the specific reasons need to be further confirmed by experimental validation.

In summary, we successfully regulated the intracellular NADH/NAD⁺ balance in M. thermophila to dramatically improve malic acid production from cellulose. Through strategic redox engineering – specifically, deleting NADH-draining pathways (glycerol synthesis and external NADH dehydrogenases) and removing the alternative oxidase – we channeled more reducing equivalents into the malate synthesis pathway. This led to higher malate flux and titers, albeit with a more reduced cytosolic environment. By overexpressing the transhydrogenase SthA and fine-tuning NAD⁺ kinase expression, we achieved an optimal redox state that maximised malate output while avoiding excessive NADH accumulation or NADPH depletion. The engineered strain attained a 38.9% improvement of malic acid production from Avicel over the starting strain JG207. Equally important, the addition of LPMO and CDH enzymes accelerated cellulose depolymerisation, shortening the fermentation time by 2 days and boosting productivity. The final titer of malic acid achieved 89.8 g/L and 48.1 g/L on Avicel and crushed corncob, respectively. This study demonstrated that redox adjustments can be synergistically combined with improvements in substrate utilisation, resulting in a fungus that both generates fermentable sugars faster and converts them more efficiently into the desired product.

Funding Statement

This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences [XDC0110301], the National Natural Science Foundation of China [31601013, 32271481, 32200064], and the Guangxi Science and Technology Major Program [Guike-AA22117013].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The datasets generated during the current study are available from the corresponding author upon request.

Abbreviations

GPDH

glycerol-3-phosphate dehydrogenase

Nde

external NADH dehydrogenase

AOX

alternative oxidase

MDH

malate dehydrogenase

rTCA

reductive TCA pathway

NADK

NAD⁺ kinase

CDH

cellobiose dehydrogenase

LPMO

lytic polysaccharide monooxygenase

ROS

reactive oxygen species