The LOV-domain blue-light receptor LreA of the fungus Alternaria alternata binds predominantly FAD as chromophore and acts as a light and temperature sensor.

Light and temperature sensing are important features of many organisms. Light may provide energy but may also be used by non-photosynthetic organisms for orientation in the environment. Recent evidence suggests that plant and fungal phytochrome and plant phototropin serve dual functions as light and temperature sensors. Here we characterized the fungal LOV-domain blue-light receptor LreA of Alternaria alternata and show that it predominantly contains FAD as chromophore. Blue-light illumination induced ROS production followed by protein agglomeration in vitro. In vivo ROS may control LreA activity. LreA acts as a blue-light photoreceptor but also triggers temperature-shift-induced gene expression. Both responses required the conserved amino acid cysteine 421. We therefore propose that temperature mimics the photoresponse, which could be the ancient function of the chromoprotein. Temperature-dependent gene expression control with LreA was distinct from the response with phytochrome suggesting fine-tuned, photoreceptor-specific gene regulation.

Differential regulation of phosphorylation, structure, and stability of circadian clock protein FRQ isoforms.

Neurospora crassa is an important model organism for circadian clock research. The Neurospora core circadian component FRQ protein has two isoforms, large FRQ (l-FRQ) and small FRQ (s-FRQ), of which l-FRQ bears an additional N-terminal 99-amino acid fragment. However, how the FRQ isoforms operate differentially in regulating the circadian clock remains elusive. Here, we show l-FRQ and s-FRQ play different roles in regulating the circadian negative feedback loop. Compared to s-FRQ, l-FRQ is less stable and undergoes hypophosphorylation and faster degradation. The phosphorylation of the C-terminal l-FRQ 794-aa fragment was markedly higher than that of s-FRQ, suggesting the l-FRQ N-terminal 99-aa region may regulate the phosphorylation of the entire FRQ protein. Quantitative label-free LC/MS analysis identified several peptides that were differentially phosphorylated between l-FRQ and s-FRQ, which were distributed in FRQ in an interlaced fashion. Furthermore, we identified two novel phosphorylation sites, S765 and T781; mutations S765A and T781A showed no significant effects on conidiation rhythmicity, although T781 conferred FRQ stability. These findings demonstrate that FRQ isoforms play differential roles in the circadian negative feedback loop and undergo different regulations of phosphorylation, structure, and stability. The l-FRQ N-terminal 99-aa region plays an important role in regulating the phosphorylation, stability, conformation, and function of the FRQ protein. As the FRQ circadian clock counterparts in other species also have isoforms or paralogues, these findings will also further our understanding of the underlying regulatory mechanisms of the circadian clock in other organisms based on the high conservation of circadian clocks in eukaryotes.

Optimizing fungal extracellular vesicle proteomic profiling through combined analysis of in-solution and in-gel digestion.

Proteomics offers a powerful tool to identify proteins within diverse microbial organisms, environments, and organelles, including extracellular vesicles (EVs). Fungal EVs are of particular interest due to their roles in cellular development and communication. While several methods exist to isolate EVs from cells, a universally accepted approach for EV protein characterization is lacking. This study investigated in-solution digestion (SD) and in-gel digestion (GD), for characterizing proteins from Neurospora crassa EVs, followed by LC-MS/MS analysis. GD identified three to four-times more proteins than SD while using the same number of unique peptides. Although GD requires a higher amount of starting sample, it offers a more comprehensive protein identification for fungal EVs, potentially preventing the omission of crucial data.

Cloning and expression of Neurospora crassa cellobiohydrolase II in Pichia pastoris.

A major cellobiohydrolase of Neurospora crassa CBH2 was successfully expressed in Pichia pastoris. The maximum Avicelase activity in shake flask among seven transformants which selected on 4.0 g/L G418 plates was 0.61 U/mL. The optimal pH and temperature for Avicelase activity of the recombinant CBH2 were determined to be 4.8 and 60 °C, respectively. The new CBH2 maintained 63.5 % Avicelase activity in the range of pH 4.0-10.4, and 60.2 % Avicelase activity in the range of 30-90 °C. After incubation at 70-90 °C for 1 h, the Avicelase activity retained 60.5 % of its initial activity. The presence of Zn2+, Ca2+ or Cd2+ enhanced the Avicelase activity of the CBH2, of which Cd2+ at 10 mM causing the highest increase. The recombinant CBH2 was used to enhance the Avicel hydrolysis by improving the exo-exo-synergism between CBH2 and CBH1 in N.crassa cellulase. The enzymatic hydrolysis yield was increased by 38.1 % by adding recombinant CBH2 and CBH1, and the yield was increased by 215.4 % when the temperature is raised to 70 °C. This work provided a CBH2 with broader pH range and better heat resistance, which is a potential enzyme candidate in food, textile, pulp and paper industries, and other industrial fields.

Correlating sugar transporter expression and activities to identify transporters for an orphan sugar substrate.

Filamentous fungi like Neurospora crassa are able to take up and metabolize important sugars present, for example, in agricultural and human food wastes. However, only a fraction of all putative sugar transporters in filamentous fungi has been characterized to date, and for many sugar substrates, the corresponding transporters are unknown. In N. crassa, only 14 out of the 42 putative major facilitator superfamily (MFS)-type sugar transporters have been characterized so far. To uncover this hidden potential for biotechnology, it is therefore necessary to find new strategies. By correlation of the uptake profile of sugars of interest after different induction conditions with the expression profiles of all 44 genes encoding predicted sugar transporters in N. crassa, together with an exhaustive phylogenetic analysis using sequences of characterized fungal sugar transporters, we aimed to identify transporter candidates for the tested sugars. Following this approach, we found a high correlation of uptake rates and expression strengths for many sugars with dedicated transporters, like galacturonic acid and arabinose, while the correlation is loose for sugars that are transported by several transporters due to functional redundancy. Nevertheless, this combinatorial approach allowed us to elucidate the uptake system for the disaccharide lactose, a by-product of the dairy industry, which consists of the two main cellodextrin transporters CDT-1 and CDT-2 with a minor contribution of the related transporter NCU00809. Moreover, a non-MFS transporter involved in glycerol transport was also identified. Deorphanization of sugar transporters or identification of transporters for orphan sugar substrates by correlation of uptake kinetics with transporter expression and phylogenetic information can thus provide a way to optimize the reuse of food industry by-products and agricultural wastes by filamentous fungi in order to create economic value and reduce their environmental impact. KEY POINTS: • The Neurospora crassa genome contains 30 uncharacterized putative sugar transporter genes. • Correlation of transporter expression and sugar uptake profiles can help to identify transporters for orphan sugar substrates. • CDT-1, CDT-2, and NCU00809 are key players in the transport of the dairy by-product lactose in N. crassa.

The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi.

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein-encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain Δcre-1, while glucose repression was still mostly functional in Δexo-1 Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in Δexo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in Δexo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

Cellobionate production from sodium hydroxide pretreated wheat straw by engineered Neurospora crassa HL10.

This study investigated cellobionate production from a lignocellulosic substrate using Neurospora crassa HL10. Utilizing NaOH-pretreated wheat straw as the substrate obviated the need for an exogenous redox mediator addition, as lignin contained in the pretreated wheat served as a natural mediator. The low laccase production by N. crassa HL10 on pretreated wheat straw caused slow cellobionate production, and exogenous laccase addition accelerated the process. Cycloheximide induced substantial laccase production in N. crassa HL10, enabling the strain to yield approximately 57 mM cellobionate from pretreated wheat straw (equivalent to 20 g/L cellulose), shortening the conversion time from 8 to 6 days. About 92% of the cellulose contained in the pretreated wheat straw is converted to cellobionate. In contrast to existing methods requiring pure cellobiose or cellulase enzymes, this process efficiently converts a low-cost feedstock into cellobionate at a high yield without enzyme or redox mediator supplementation.

A rapid PCR-based method to determine the Neurospora crassa mating type.

So far mating type determination in Neurospora crassa requires test crosses with strains of known mating type. We present a simple, quick, and reliable polymerase chain reaction-based method for mating type determination in N. crassa.

Do mitochondria use efflux pumps to protect their ribosomes from antibiotics?

Fungal environments are rich in natural and engineered antimicrobials, and this, combined with the fact that fungal genomes are rich in coding sequences for transporters, suggests that fungi are an intriguing group in which to search for evidence of antimicrobial efflux pumps in mitochondria. Herein, the range of protective mechanisms used by fungi against antimicrobials is introduced, and it is hypothesized, based on the susceptibility of mitochondrial and bacterial ribosomes to the same antibiotics, that mitochondria might also contain pumps that efflux antibiotics from these organelles. Preliminary evidence of ethidium bromide efflux is presented and several candidate efflux pumps are identified in fungal mitochondrial proteomes.

The transcriptional factor Clr-5 is involved in cellulose degradation through regulation of amino acid metabolism in Neurospora crassa.

BACKGROUND: Filamentous fungi are efficient degraders of plant biomass and the primary producers of commercial cellulolytic enzymes. While the transcriptional regulation mechanisms of cellulases have been continuously explored in lignocellulolytic fungi, the induction of cellulase production remains a complex multifactorial system, with several aspects still largely elusive. RESULTS: In this study, we identified a Zn2Cys6 transcription factor, designated as Clr-5, which regulates the expression of cellulase genes by influencing amino acid metabolism in Neurospora crassa during growth on cellulose. The deletion of clr-5 caused a significant decrease in secreted protein and cellulolytic enzyme activity of N. crassa, which was partially alleviated by supplementing with yeast extract. Transcriptomic profiling revealed downregulation of not only the genes encoding main cellulases but also those related to nitrogen metabolism after disruption of Clr-5 under Avicel condition. Clr-5 played a crucial role in the utilization of multiple amino acids, especially leucine and histidine. When using leucine or histidine as the sole nitrogen source, the Δclr-5 mutant showed significant growth defects on both glucose and Avicel media. Comparative transcriptomic analysis revealed that the transcript levels of most genes encoding carbohydrate-active enzymes and those involved in the catabolism and uptake of histidine, branched-chain amino acids, and aromatic amino acids, were remarkably reduced in strain Δclr-5, compared with the wild-type N. crassa when grown in Avicel medium with leucine or histidine as the sole nitrogen source. These findings underscore the important role of amino acid metabolism in the regulation of cellulase production in N. crassa. Furthermore, the function of Clr-5 in regulating cellulose degradation is conserved among ascomycete fungi. CONCLUSIONS: These findings regarding the novel transcription factor Clr-5 enhance our comprehension of the regulatory connections between amino acid metabolism and cellulase production, offering fresh prospects for the development of fungal cell factories dedicated to cellulolytic enzyme production in bio-refineries.

DNA interstrand crosslink repair by XPF-ERCC1 homologue confers ultraviolet resistance in Neurospora crassa.

Ultraviolet (UV) light is a mutagen that causes DNA damage. Some UV-sensitive Neurospora crassa strains have been reported to exhibit a partial photoreactivation defect (PPD) phenotype, and the possible cause of this has been unknown for more than half a century. In this study, in the process of elucidating the possible causes of a PPD phenotype, we discovered that the XPF homologue MUS-38 is involved in repairing the UV-induced DNA interstrand crosslink (ICL) in N. crassa. Furthermore, the sensitivity of the Δmus-38 and Δmus-44 strains to ICL agents was significantly higher than that of other nucleotide excision repair (NER)-related gene knockout (KO) strains, indicating that the MUS-38/MUS-44 complex is involved in an NER-independent ICL repair mechanism. Based on reports concerning the mammalian homologues XPF and ERCC1 we obtained separation-of-function mutants defective only in NER in mus-38 and mus-44. Additionally, the photoreactivation ability of these mutants was significantly higher than that of the KO strains. These results indicate that the PPD phenotype is caused by a defect in the repair-ability of ICL induced by UV and that an NER-independent ICL repair by MUS-38 and MUS-44 confers resistance to UV in N. crassa.

Parental effects in a filamentous fungus: Phenotype, fitness and mechanism.

In nature, organisms have to cope with constantly changing environments. In certain conditions, it may be advantageous for the parents to pass on information about the environment, or resources to their offspring. Such transfers are known as parental effects, and they are well documented in plants and animals, but not in other eukaryotes, such as fungi. Many fungi disperse through spores, and fungal spores can potentially carry information or resources to the next generation. Understanding parental effects and their evolutionary consequences in fungi is of vital importance as they perform crucial ecosystem functions. In this study, we investigated whether parental effects are present in the filamentous fungus Neurospora crassa, how long do they last, whether the effects are adaptive, and what is their mechanism. We performed a fully factorial match/mismatch experiment for a good and a poor quality environment, in which we measured the initial growth of strains that experienced either a matched or mismatched environment in their previous generation. We found a strong silver-spoon effect in initial mycelium growth, which lasted for one generation, and increased fitness during competition experiments. By using deletion mutants that lacked key genes in epigenetic processes, we show that epigenetic mechanisms are not involved in this effect. Instead, we show that spore glycogen content, glucose availability and a radical transcription shift in spores are the main mechanisms behind this parental effect.

The cell functions of phospholipase C-1, Ca2+/H+ exchanger-1, and secretory phospholipase A2 in tolerance to stress conditions and cellulose degradation in Neurospora crassa.

We investigated the cell functions of the Ca2+ signaling genes phospholipase C-1 (plc-1), Ca2+/H+ exchanger (cpe-1), and secretory phospholipase A2 (splA2) for stress responses and cellulose utilization in Neurospora crassa. The Δplc-1, Δcpe-1, and ΔsplA2 mutants displayed increased sensitivity to the alkaline pH and reduced survival during induced thermotolerance. The ΔsplA2 mutant also exhibited hypersensitivity to the DTT-induced endoplasmic reticulum (ER) stress, increased microcrystalline cellulose utilization, increased protein secretion, and glucose accumulation in the culture supernatants. Moreover, the ΔsplA2 mutant could not grow on microcrystalline cellulose during ER stress. Furthermore, plc-1, cpe-1, and splA2 synthetically regulate the acquisition of thermotolerance induced by heat shock, responses to alkaline pH and ER stress, and utilization of cellulose and other alternate carbon sources in N. crassa. In addition, expression of the alkaline pH regulator, pac-3, and heat shock proteins, hsp60, and hsp80 was reduced in the Δplc-1, Δcpe-1, and ΔsplA2 single and double mutants. The expression of the unfolded protein response (UPR) markers grp-78 and pdi-1 was also significantly reduced in the mutants showing growth defect during ER stress. The increased cellulolytic activities of the ΔsplA2 and Δcpe-1; ΔsplA2 mutants were due to increased cbh-1, cbh-2, and endo-2 expression in N. crassa. Therefore, plc-1, cpe-1, and splA2 are involved in stress responses and cellulose utilization in N. crassa.

Effect of itaconic acid production on Neurospora crassa in consolidated bioprocessing of cellulose.

A system for itaconic acid synthesis from cellulose by Neurospora crassa was established, resulting in the highest yield of itaconic acid was 354.08 + 35.99 mg/L. Meanwhile, cellulase activity increased significantly, without any strain modifications for improved cellulase production. Multi-omics analyses showed that itaconic acid synthesis reduced energy production, leading to decreases in trehalose, cell wall, fatty acids synthesis and downregulations in MAPK signaling pathway, cell cycle and meiosis. More importantly, the low-energy environment enhanced the energy-efficient cellobionic acid/gluconic acid pathway, and the cellulase composition also changed significantly, manifested as the up-regulation of LPMOs and the down-regulation of ß-glucosidases. Enhancing LPMOs-cellobionic acid/gluconic acid system has the potential to reduce energy consumption of the consolidated bioprocessing. These findings offer an overview of resource allocations by N. crassa in response to itaconic acid synthesis and highlight a series of intriguing connections between itaconic acid synthesis and cellulase synthesis in consolidated bioprocessing.

Circadian clock control of eIF2α phosphorylation is necessary for rhythmic translation initiation.

The circadian clock in eukaryotes controls transcriptional and posttranscriptional events, including regulation of the levels and phosphorylation state of translation factors. However, the mechanisms underlying clock control of translation initiation, and the impact of this potential regulation on rhythmic protein synthesis, were not known. We show that inhibitory phosphorylation of eIF2α (P-eIF2α), a conserved translation initiation factor, is clock controlled in Neurospora crassa, peaking during the subjective day. Cycling P-eIF2α levels required rhythmic activation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2), and rhythmic activation of CPC-3 was abolished under conditions in which the levels of charged tRNAs were altered. Clock-controlled accumulation of P-eIF2α led to reduced translation during the day in vitro and was necessary for the rhythmic synthesis of select proteins in vivo. Finally, loss of rhythmic P-eIF2α levels led to reduced linear growth rates, supporting the idea that partitioning translation to specific times of day provides a growth advantage to the organism. Together, these results reveal a fundamental mechanism by which the clock regulates rhythmic protein production, and provide key insights into how rhythmic translation, cellular energy, stress, and nutrient metabolism are linked through the levels of charged versus uncharged tRNAs.

Phylogenetic and Structure-Function Analyses of ENA ATPases: A Case Study of the ENA1 Protein from the Fungus Neurospora crassa.

ENA transporters are a group of P-type ATPases that are characterized by actively moving Na+ or K+ out of the cell against their concentration gradient. The existence of these transporters was initially attributed to some fungi, although more recently they have also been identified in mosses, liverworts, and some protozoa. Given the current increase in the number of organisms whose genomes are completely sequenced, we set out to expand our knowledge about the existence of ENA in organisms belonging to other phylogenetic groups. For that, a hidden Markov model profile was constructed to identify homologous sequences to ENA proteins in protein databases. This analysis allowed us to identify the existence of ENA-type ATPases in the most primitive groups of fungi, as well as in other eukaryotic organisms not described so far. In addition, this study has allowed the identification of a possible new group of P-ATPases, initially proposed as ENA but which maintain phylogenetic distances with these proteins. Finally, this work has also addressed this study of the structure of ENA proteins, which remained unknown due to the lack of crystallographic data. For this purpose, a 3D structure prediction of the NcENA1 protein of the fungus Neurospora crassa was performed using AlphaFold2 software v2.3.1. From this structure, the electrostatic potential of the protein was analyzed. With all these data, the protein regions and the amino acids involved in the transport of Na+ or K+ ions across the membrane were proposed for the first time. Targeted mutagenesis of some of these residues has confirmed their relevant participation in the transport function of ENA proteins.

Intracellular Nitric Oxide and cAMP Are Involved in Cellulolytic Enzyme Production in Neurospora crassa.

Although molecular regulation of cellulolytic enzyme production in filamentous fungi has been actively explored, the underlying signaling processes in fungal cells are still not clearly understood. In this study, the molecular signaling mechanism regulating cellulase production in Neurospora crassa was investigated. We found that the transcription and extracellular cellulolytic activity of four cellulolytic enzymes (cbh1, gh6-2, gh5-1, and gh3-4) increased in Avicel (microcrystalline cellulose) medium. Intracellular nitric oxide (NO) and reactive oxygen species (ROS) detected by fluorescent dyes were observed in larger areas of fungal hyphae grown in Avicel medium compared to those grown in glucose medium. The transcription of the four cellulolytic enzyme genes in fungal hyphae grown in Avicel medium was significantly decreased and increased after NO was intracellularly removed and extracellularly added, respectively. Furthermore, we found that the cyclic AMP (cAMP) level in fungal cells was significantly decreased after intracellular NO removal, and the addition of cAMP could enhance cellulolytic enzyme activity. Taken together, our data suggest that the increase in intracellular NO in response to cellulose in media may have promoted the transcription of cellulolytic enzymes and participated in the elevation of intracellular cAMP, eventually leading to improved extracellular cellulolytic enzyme activity.

The Neurospora crassa molybdate transporter: Characterizing a novel transporter homologous to the plant MOT1 family.

Molybdenum (Mo) is an essential element for animals, plants, and fungi. To achieve biological activity in eukaryotes, Mo must be complexed into the molybdenum cofactor (Moco). Cells are known to take up Mo in the form of the oxyanion molybdate. However, molybdate transporters are scarcely characterized in the fungal kingdom. In plants and algae, molybdate is imported into the cell via two families of molybdate transporters (MOT), MOT1 and MOT2. For the filamentous fungus Neurospora crassa, a sequence homologous to the MOT1 family was previously annotated. Here we report a characterization of this molybdate-related transporter, encoded by the ncmot-1 gene. We found that the deletion of ncmot-1 leads to an accumulation of total Mo within the mycelium and a roughly 51 % higher tolerance against high molybdate levels when grown on ammonium medium. The localization of a GFP tagged NcMOT-1 was identified among the vacuolar membrane. Thereby, we propose NcMOT-1 as an exporter, transporting molybdate out of the vacuole into the cytoplasm. Lastly, the heterologous expression of NcMOT-1 in Saccharomyces cerevisiae verifies the functionality of this protein as a MOT. Our results open the way towards understanding molybdate transport as part of Mo homeostasis and Moco-biosynthesis in fungi.

The cytoplasmic microtubule array in Neurospora crassa depends on microtubule-organizing centers at spindle pole bodies and microtubule +end-depending pseudo-MTOCs at septa.

γ-Tubulin ring complexes (γ-TuRC) mediate nucleation and anchorage of microtubules (MTs) to microtubule organizing centers (MTOCs). In fungi, the spindle pole body (SPB) is the functional equivalent of the centrosome, which is the main MTOC. In addition, non-centrosomal MTOCs (ncMTOCs) contribute to MT formation in some fungi like Schizosaccharomyces pombe and Aspergillus nidulans. In A. nidulans, MTOCs are anchored at septa (sMTOC) and share components of the outer plaque of the SPB. Here we show that the Neurospora crassa SPB is embedded in the nuclear envelope, with the γ-TuRC targeting proteins PCP-1Pcp1/PcpA located at the inner plaque and APS-2Mto1/ApsB located at the outer plaque of the SPB. PCP-1 was a specific component of nuclear MTOCs, while APS-2 was also present at the septal pore. Although γ-tubulin was only detected at the nucleus, spontaneous MT nucleation occurred in the apical and subapical cytoplasm during recovery from benomyl-induced MT depolymerization experiments. There was no evidence for MT nucleation at septa. However, without benomyl treatment MT plus-ends were organized in the septal pore through MTB-3EB1. Those septal MT plus ends polymerized MTs from septa in interphase cells Thus we conclude that the SPB is the only MT nucleation site in N. crassa, but the septal pore aids the MT network arrangement through the anchorage of the MT plus-ends through a pseudo-MTOC.

Toward more efficient ergothioneine production using the fungal ergothioneine biosynthetic pathway.

BACKGROUND: Ergothioneine (ERG) is a potent histidine-derived antioxidant that confers health-promoting effects. Only certain bacteria and fungi can biosynthesize ERG, but the ERG productivity in natural producers is low. ERG overproduction through genetic engineering represents an efficient and cost-effective manufacturing strategy. RESULTS: Here, we showed that Trichoderma reesei can synthesize ERG during conidiogenesis and hyphal growth. Co-expression of two ERG biosynthesis genes (tregt1 and tregt2) from T. reesei enabled E. coli to generate 70.59 mg/L ERG at the shaking flask level after 48 h of whole-cell biocatalysis, whereas minor amounts of ERG were synthesized by the recombinant E. coli strain bearing only the tregt1 gene. By fed-batch fermentation, the extracellular ERG production reached 4.34 g/L after 143 h of cultivation in a 2-L jar fermenter, which is the highest level of ERG production reported thus far. Similarly, ERG synthesis also occurred in the E. coli strain engineered with the two well-characterized genes from N. crassa and the ERG productivity was up to 4.22 g/L after 143 h of cultivation under the above-mentioned conditions. CONCLUSIONS: Our results showed that the overproduction of ERG in E. coli could be achieved through two-enzymatic steps, demonstrating high efficiency of the fungal ERG biosynthetic pathway. Meanwhile, this work offers a more promising approach for the industrial production of ERG.