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Metabolic engineering of Saccharomyces cerevisiae for the production of ergothioneine
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Khan Salman, Xiaoxiao LI, Qun YANG, Yunying ZHAO, Yu DENG
Acta Microbiologica Sinica | 2026, 66(8) : 4097 - 4118
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Acta Microbiologica Sinica | 2026, 66(8): 4097-4118
• 研究报告 •
Metabolic engineering of Saccharomyces cerevisiae for the production of ergothioneine
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Khan Salman, Xiaoxiao LI, Qun YANG, Yunying ZHAO, Yu DENG
Affiliations
  • Key Laboratory of Industrial Biotechnology of the Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China
About Author:

Credit authorship contribution statement

Salman Khan: Designed the study, drafted the manuscript, and performed the majority of the experiments; LI Xiaoxiao: Conducted data analysis; YANG Qun: Conducted data analysis; ZHAO Yunying: Supervised the project, revised the manuscript, and provided funding; DENG Yu: Supervised the project, revised the manuscript, and provided funding.

Published: 2026-08-04 doi: 10.13343/j.cnki.wsxb.20260134
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[Objective] Ergothioneine (EGT), a sulfur-rich derivative of histidine, is utilized in the food, pharmaceutical, and cosmetic industries. However, large-scale production of EGT faces challenges due to the high costs and inefficiency of conventional chemical synthesis and extraction techniques. This study aims to engineer Saccharomyces cerevisiae to provide a microbial platform for EGT biosynthesis. [Methods] The biosynthetic pathway for EGT was reconstructed in S. cerevisiae by heterologously expressing Egt1 from Neurospora crassa and Egt2 from Claviceps purpurea. To overcome the metabolic bottlenecks related to precursor supply, we optimized the upstream pathways for histidine, cysteine, methionine, and S-adenosylmethionine to enhance the flux toward EGT synthesis. Fermentation performance of the engineered strain was assessed in both shake flasks and a 5-L bioreactor. [Results] The engineered S. cerevisiae strain produced 312.8 mg/L of EGT in shake flask fermentation. In a 5-L bioreactor, the strain achieved the EGT titer of 1 312.2 mg/L after 168 h, with the productivity of 7.8 mg/(L·h). [Conclusion] This study presents a metabolic engineering strategy for producing EGT in S. cerevisiae. The approach not only significantly improves EGT biosynthesis but also serves as a reference for microbial production of other compounds.

ergothioneine  /  Saccharomyces cerevisiae  /  metabolic engineering  /  precursor supply  /  fermentation optimization
Khan Salman, Xiaoxiao LI, Qun YANG, Yunying ZHAO, Yu DENG. Metabolic engineering of Saccharomyces cerevisiae for the production of ergothioneine[J]. Acta Microbiologica Sinica, 2026 , 66 (8) : 4097 -4118 . DOI: 10.13343/j.cnki.wsxb.20260134
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Ergothioneine (EGT) is a unique sulfur-containing compound derived from histidine, produced from L-histidine and L-cysteine, with S-adenosyl-L-methionine (SAM) acting as the methyl donor in its biosynthesis[1]. The compound was initially discovered in the ergot fungus Claviceps purpurea in 1909[2]. Although EGT is commonly found in both plants and mammals[3], only specific microorganisms, such as certain bacteria and fungi—including Cyanobacteria, Actinobacteria, and Basidiomycete mushrooms have the ability to synthesize it[4-6]. In the solution, EGT interconverts between thiol and thione tautomers[7-8], with the thione form dominant under physiological conditions. This structural configuration underlies its remarkable resistance to autoxidation and contributes to its greater stability than that of classical thiol antioxidants such as glutathione[9-10]. Due to its robust redox buffering ability and protective effects on cells, EGT is gaining recognition as a bioactive metabolite with various physiological and therapeutic importance. Reported functions involve modulating inflammatory responses and reducing cellular aging processes[11-12], antidepressant-like effects, and protection from ultraviolet-induced oxidative stress[13-14].
In addition to its redox activity, EGT plays a vital role in cellular functions such as maintaining DNA synthesis, regulating cell growth, and supporting immune system health. It also offers radioprotective properties and is linked to skin-whitening and anti-aging effects, among various other physiological advantages[15]. Growing evidence suggests that EGT plays a significant role in chronic diseases, especially in neurodegenerative and cardiovascular conditions[16-17]. In a transgenic Caenorhabditis elegans model of Alzheimer’s disease that expresses human β-amyloid, supplementing with EGT improved both healthspan and lifespan[18]. In humans, circulating EGT levels have been identified as a strong biomarker linked to a decreased risk of cardiovascular disease and lower all-cause mortality in a Swedish population cohort study[19]. Due to its antioxidant and cytoprotective properties, EGT has gained significant interest for use in medical and cosmetic applications. However, low yields from plant-based extraction restrict the scalability of EGT production[20]. The chemical synthesis of EGT is challenging because of its chiral amino acid structure, making downstream separation and purification more complicated[21]. As a result, metabolic engineering and microbial fermentation have emerged as primary research areas to boost EGT production, given their advantages of higher yields, lower costs, and greater sustainability[22].
The biosynthesis pathway of EGT varies considerably among microorganisms, especially between bacteria and fungi. Bacterial species produce EGT through a five-enzyme process, from EgtA to EgtE. In Mycobacterium smegmatis, the pathway involves histidine, cysteine, glutamate, and methionine, ultimately leading to the formation of EGT[23]. In contrast, fungi like Neurospora crassa use a simpler two-enzyme pathway (Figure 1) involving Egt1 and Egt2[24]. In the bacterial pathway, L-histidine initially undergoes methylation by the SAM-dependent methyltransferase EgtD, resulting in the formation of hercynine (HER)[25]. The mononuclear non-heme iron enzyme EgtB then catalyzes the transformation of hercynine into γ-glutamyl-hercynylcysteine sulfoxide (γGC-HER), using γ-glutamylcysteine produced by EgtA as the sulfur donor[26]. The amidohydrolase EgtC then cleaves the glutamate part, forming hercynylcysteine sulfoxide (Cys-HER), which is subsequently transformed into EGT through the action of the pyridoxal-5′-phosphate (PLP)-dependent β-lyase EgtE[27]. In the N. crassa fungal pathway, the multifunctional enzyme Egt1 methylates L-histidine to produce hercynine and also catalyzes its conversion to hercynylcysteine sulfoxide[28]. The second step enzyme, Egt2, acts similarly to bacterial EgtE by cleaving the C-S bond of hercynylcysteine sulfoxide through a PLP-dependent cysteine desulfurase reaction to produce EGT[29-30].
To date, various organisms have been genetically modified to produce EGT. Osawa et al. initially overexpressed the five enzymes from M. smegmatis: EgtA, EgtB, EgtC, EgtD, and EgtE[31]. Supplements of thiosulfate, which provide sulfur for cysteine, boosted γ-glutamylcysteine production, leading to a 120-fold rise in EGT yield from 0.2 to 24 mg/L after optimizing fermentation conditions. Similarly, the N. crassaEgt1 and Egt2 genes were inserted into the Aspergillus oryzae genome in multiple copies, resulting in an EGT production of 231 mg/kg on solid media[32]. Another study showed that S. cerevisiae can be genetically engineered to produce significant amounts of EGT, reaching 598 mg/L in a fed-batch bioreactor process[33]. However, the expression of the Gfegt1 and Gfegt2 genes from Grifola frondosa in the engineered S. cerevisiae strain resulted in an EGT titer of only 20.61 mg/L[34]. Interestingly, recombinant S. cerevisiae was engineered to express Aspergillus fumigatus methyltransferase and sulfoxide synthase (egtA), which produced just 7.93 mg/L of EGT in shake-flask culture. Because S. cerevisiae does not naturally have EGT biosynthetic genes, the detected EGT formation through C-S bond cleavage, which is normally facilitated by Egt2 or EgtE, may be caused by the yeast’s own enzymes or abiotic chemical reactions[35]. An alternative approach involved adding an extra native egtBD copy and deleting the histidine ammonia-lyase gene hutH in Methylobacterium aquaticum, resulting in a strain that produced 7.0 mg/g (dry cell weight, DCW) of EGT over seven days[36]. Likewise, introducing the egtB gene from M. pseudosasicola into a cysteine-and methionine-overproducing E. coli strain led to the production of 657 mg/L EGT after 192 hours of cultivation with added histidine and methionine. However, fed-batch fermentation was not feasible due to the slow growth of the strain[37]. Meanwhile, an E. coli strain was engineered to enhance cysteine production by activating its synthesis and secretion pathways. This involved heterologous expression of M. smegmatis egtA, egtB, egtC, egtD, and egtE, along with knocking out the methionine repressor gene metJ. After 216 hours of fed-batch fermentation in a 3-L bioreactor, a yield of 1.3 g/L EGT was achieved[38]. In E. coli, boosting the SAM cycle and rebuilding the EGT biosynthesis pathway resulted in about 2.52 g/L of EGT in fed-batch culture, due to increased precursor and cofactor supplies[39]. Expression of Trichoderma reeseiTregt1 and Tregt2 in E. colivia whole-cell catalysis and fed-batch fermentation produced 4.34 g/L of extracellular EGT after 143 hours in a 2-L bioreactor[40].
Introducing N. crassaegt1 and M. smegmatisegtD and egtE into E. coli, along with mutagenesis of egt1 and egtD and fermentation optimization, led to an EGT production of 5.4 g/L[41]. By employing a multicopy genomic integration approach in Yarrowia lipolytica, the researchers achieved an ergothioneine production level of 7.3 g/L in fed-batch fermentation[42]. By combining enzyme engineering (Tregt1 mutations) in Y. lipolytica, systematic enhancement of the precursor pathway, and optimized fermentation conditions, production reached 9.3 g/L EGT in a 5-L bioreactor after 168 hours, with a productivity of 55.35 mg/(L·h)[43].
Although many metabolic engineering projects focus on prokaryotic hosts, S. cerevisiae has significant advantages as a Generally Recognized as Safe (GRAS) organism widely used in the food and nutraceutical industries. It provides high regulatory approval, reduced endotoxin risk, and easier downstream processing. Its eukaryotic architecture ensures correct folding of heterologous enzymes, while strong sulfur and amino acid metabolic pathways, including those for histidine, cysteine, methionine, and SAM, provide essential precursors for EGT production. Furthermore, yeast demonstrates significant stress tolerance and the ability to reach high cell densities in aerobic environments. This study presents a systematic metabolic engineering strategy to boost EGT production in S. cerevisiae by concurrently optimizing precursor supply, methyl donor availability, and enzyme capacity within the pathway. The precursor metabolic network was modularly engineered to enhance sulfur assimilation and the production of methionine, histidine, and cysteine. We particularly reinforced the methionine-SAM axis to raise intracellular SAM levels and reduced competing pathways that use SAM. Moreover, ATP-producing genes were upregulated to support the higher biosynthetic demand, and multiple copies of Egt1 and Egt2 were integrated to boost pathway capacity. This work develops a comprehensive metabolic engineering framework by coordinating improvements in precursor metabolism, methyl donor supply, and enzymatic steps, offering new insights into increasing EGT production in yeast.
1 Materials and methods
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1.1 Strains and media
The S. cerevisiae strain CEN.PK2, maintained in our laboratory, was used as the parental strain for all genetic modifications, whereas E. coli JM109 was used for plasmid construction and amplification (Table 1). E. coli cultures were grown in lysogeny broth (LB) and on LB agar containing 100 mg/L ampicillin at 37 ℃. Yeast cells were cultured in yeast extract-peptone-dextrose (YPD) medium, made with 20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract, at 30 ℃ with shaking at 200 r/min. For the Cas9 vector selection, we supplemented YPD media with G418 and maintained its concentration at 200 mg/L. For the gRNA vector, 100 mg/L hygromycin (HYG) was added to YPD media. To apply selective pressure, the yeast strains were grown in SD medium (6.7 g/L yeast nitrogen base without amino acids, 2% glucose) supplemented with the appropriate amino acids. The plasmid pRS423 contains the HIS3 marker, while the pHAC181 plasmid carries the LEU2 marker. Accordingly, the amino acids were supplemented in the SD medium during the transformation of the plasmids in the yeast strains. Minimal medium (MM) was formulated for controlled yeast cultivation, consisting of 7.5 g/L (NH4)2SO4, 14.4 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, 2 mL/L trace metal solution, and 1 mL/L vitamin solution. The trace metal solution contained 4.5 g/L CaCl2·2H2O, 4.5 g/L ZnSO4·7H2O, 3 g/L FeSO4·7H2O, 1 g/L H3BO3, 1 g/L MnCl2·4H2O, 0.4 g/L Na2MoO4·2H2O, 0.3 g/L CoCl2·6H2O, 0.1 g/L CuSO4·5H2O, 0.1 g/L KI, and 15 g/L EDTA. The vitamin solution comprised 50 mg/L biotin, 200 mg/L p-aminobenzoic acid, 1 g/L nicotinic acid, 1 g/L pyridoxine HCl, 1 g/L thiamine HCl, and 25 g/L myo-inositol. All media components were sterilized by autoclaving at 121 ℃ for 20 min or by filtration when heat-sensitive. Cultures were initiated under sterile conditions to ensure reproducibility, optimal growth, and suitability for downstream genetic and fermentation experiments.
1.2 Construction of plasmids and strains
For the production of EGT, the genes Egt1 and Egt2 were obtained from various organisms known for their EGT biosynthesis. These genes, along with their respective accession numbers, are as follows: Egt1 from N. crassa (accession No. XP_956324.3, Ncegt1), Egt2 from C. purpurea (accession No. CCE33140.1, Cpegt2), Egt1 from Schizosaccharomyces pombe (accession No. NP_596639.2, Spegt1), Egt2 from S. pombe (accession No. NP_595091.1, Spegt2), EgtE from M. smegmatis (accession No. ABK70212.1, MsegtE), Egt1 from Trichoderma reesei (accession No. XP_006968620, Tregt1), Egt2 from T. reesei (accession No. XP_006968735, Tregt2), and Egt1 from Rhodotorula toruloides (accession No. XP_016270018.1, Rtegt1), Egt2 from Rhodotorula toruloides (accession No. XP_016270654.1, Rtegt2). These genes were codon-optimized for S. cerevisiae to ensure high expression efficiency in the yeast host. Codon optimization was performed by Azenta Life Sciences (Suzhou, China).
The plasmids used in this study are listed in Table 2. The P TDH3 and P TEF1 promoter, along with the T CYC1 and T ADH1 terminator, were amplified from the BY4741 genome. The gene fragments of Egt1 and Egt2 were amplified directly from the synthesized DNA using high-fidelity Phusion DNA polymerase (Vazyme, Nanjing, China). These gene fragments were then fused in a series of PCR reactions, in which two gene fragments (P TDH3 -Egt1-T CYC1 and P TEF1 -Egt2-T ADH1 ) were joined to ensure proper gene expression and termination in yeast. The sequences of the Egt1 cassette were cloned into the pRS423 plasmid, and the sequences of the Egt2 cassette were cloned into the pHAC181 plasmid. The plasmids were assembled using the Gibson Assembly system, and Sanger sequencing verified the integrity of the constructs.
For genome editing in S. cerevisiae, we used two plasmids provided by our lab: pRS42H-gRNA, a plasmid for expressing the CRISPR guide RNA to generate sgRNAs, and p414-P TEF1 -Cas9-T CYC1 -KanMX, a plasmid for expressing the Cas9 nuclease. These plasmids were used together to enable precise genome modifications in S. cerevisiae by integrating the desired genes via a CRISPR-Cas9-based genome editing approach. To facilitate genome editing, single-guide RNAs (sgRNAs) were designed using the CHOPCHOP tool (https://chopchop.cbu.uib.no/) to target specific genomic loci for gene integration and deletion. The sgRNAs were cloned into a plasmid vector designed to insert into a Cas9-expressing cassette. These plasmids were then introduced into yeast cells to achieve precise genome modifications.
1.3 qPCR analysis
Firstly, the total RNA from the ST4021, ST4016, and ST4022 strains was isolated by using Ultrapure RNA Kit (CWBIO, Beijing) following the manufacturer’s instructions. Then, the first-strand cDNA synthesis was subsequently performed using the HIScriptIV 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing) according to the manufacturer’s instructions. Quantitative real-time PCR (qPCR) was conducted on a CFX96 Real-Time Thermal Cycler (Step OnePlus, American Applied Biosystems Corporation) using the Taq Pro Universal qPCR Master Mixture (Vazyme, Nanjing). Relative gene expression levels were calculated employing the 2 -ΔΔCt method, with the housekeeping gene PGK1 serving as the endogenous reference for normalization. The final data are presented as the mean relative fold change in expression compared to the control strain ST4022.
1.4 Shake flask fermentation
Strains preserved in glycerol stocks were first streaked onto SD agar plates and incubated at 30 ℃ for 2-3 days to obtain single colonies. A well-isolated colony was inoculated into a 50 mL shake tube containing 5 mL of sterilized YPD medium and cultured at 30 ℃ with shaking at 220 r/min for 14 to 16 hours to produce an actively growing pre-culture. To ensure consistent inoculation and strong growth, 5% (V/V) of this pre-culture was transferred into a 250 mL shake flask containing 50 mL of YPD medium (1:5 flask-to-medium volume ratio for optimal aeration) and incubated at 30 ℃ with shaking at 220 r/min for up to 120 hours. During cultivation, samples were periodically taken to monitor optical density at 600 (OD600). Culture supernatants were clarified via centrifugation and analyzed for EGT content with high-performance liquid chromatography (HPLC), ensuring precise measurement of metabolite production during the fermentation process.
To determine the optimal production conditions for EGT, the concentrations of L-methionine (Met), L-histidine (His), L-cysteine (Cys), and L-arginine (Arg) were varied across six experimental groups (A to F). Group A: 1 g/L Met, His, Cys, and Arg were added to the culture medium. Group B: 5 g/L of Met, 5 g/L of His, and 5 g/L of Cys and Arg were added to the culture medium. Group C: 10 g/L of Met, 4 g/L of His, and 10 g/L of Cys and Arg were added to the culture media. Group D: 20 g/L of Met, 8 g/L His, and 8 g/L Cys and Arg were added to the culture medium. Group E: 25 g/L Met, 12 g/L His, 12 g/L Cys, and Arg were added to the culture medium. Group F: 30 g/L Met, 16 g/L His, and 16 g/L Cys and Arg were added to the culture medium.
1.5 Fed-Batch fermentation
Glycerol stock strains were streaked on SD agar plates to isolate colonies. Ten well-isolated colonies were individually selected and inoculated into 50 mL shake flasks with 10 mL SD medium. These were incubated at 30 ℃ with orbital shaking at 220 r/min for 24 hours to generate seed cultures. Next, 5% (V/V) of each seed culture was transferred into 250 mL shake flasks containing 100 mL SD medium, then incubated at the same temperature and shaking speed for another 24 hours.
For large-scale production, a 5% (V/V) inoculum was added to a 5-L bioreactor containing 2.5 L of YPD medium with 20 g/L yeast extract, 40 g/L peptone, and an initial glucose concentration of 50 g/L. To increase the EGT precursor availability, L-methionine (20 g/L), L-histidine (8 g/L), L-cysteine (8 g/L), and L-arginine (8 g/L) were added directly to the fermentation medium before sterilization as a single initial supplement. The bioreactor was maintained at 30 ℃ with a maximum agitation speed of 500 r/min to ensure proper mixing and oxygen transfer. Dissolved oxygen was rigorously controlled at 20%, and pH was stabilized near 5.0 through automated addition of pure ammonia. At 24 hours, 0.05 g/L of butylated hydroxytoluene was added as an antioxidant. Additionally, an antifoam agent was added to reduce foam formation during fermentation and ensure a smooth, efficient process.
Glucose consumption was closely monitored throughout the fermentation using a controlled feeding strategy. A sterile glucose stock solution (450 g/L) served as the feed. Glucose was supplemented via pulse feeding at approximately 10 g/L every 24 hours. Samples were collected at defined intervals to determine optical density, residual glucose concentration, and EGT production.
1.6 Compound analysis
After the fermentation process, extracellular EGT was measured by taking 1 mL of fermentation broth, which was then immediately centrifuged at 3 000×g for 5 minutes at 4 ℃ to preserve metabolite stability. The clear supernatant was subsequently transferred to HPLC vials for analysis using high-performance liquid chromatography (HPLC). To measure intracellular EGT, the remaining cell pellet was washed twice with 1 mL of Milli-Q water and then resuspended in 1 mL of water. The extraction of intracellular EGT was carried out as described by Alamgir et al. (2015)[44]. For yeast cell optimization, the suspension was heated at 94 ℃ for 10 minutes, vortexed at 1 600 r/min for 30 minutes, and centrifuged at 10 000×g for 5 minutes. The supernatant was then collected into HPLC vials for analysis. All samples were filtered through 0.22 μm membranes immediately prior to injection to remove residual particulates.
EGT quantification was conducted using HPLC with a mobile phase of methanol and ultrapure water in a 1:99 ratio. Chromatography was performed at 30 ℃, with a flow rate of 1 mL/min, an injection volume of 10 μL, and UV detection at 257 nm. Each run lasted 18 min, providing accurate and reproducible measurements of both extracellular and intracellular EGT. All tests were performed in triplicate, and system suitability was verified with standard EGT solutions before analyzing samples to ensure consistency and precision.
2 Results and analysis
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2.1 Construction and optimization of the ergothioneine biosynthetic pathway in S. cerevisiae
To establish an effective EGT biosynthesis pathway in S. cerevisiae, we first evaluated the functional diversity of EGT-synthetase homologs from fungal and bacterial origins. A panel of Egt1 and Egt2 genes was chosen based on their phylogenetic diversity and previous reports of EGT production, including homologs from fungi N. crassa (Ncegt1), R. toruloides (Rtegt1 and Rtegt2), S. pombe (Spegt1 and Spegt2), T. reesei (Tregt1 and Tregt2), and C. purpurea (Cpegt2), as well as the bacterial EgtE gene from M. smegmatis (MsegtE). Each Egt1 homolog was paired separately with its corresponding Egt2 gene to evaluate the combined pathway efficiency in yeast for EGT production (Figure 2). Eight different EGT-producing strains were developed in S. cerevisiae to study how enzyme origin and pathway setup affect EGT production. Seven strains contained only fungal genes, whereas one hybrid strain combined fungal and bacterial homologs. These strains were grown under controlled conditions, and measurements were taken of both intracellular and extracellular EGT levels.
Among these eight strains, five shared Egt1 from T. reesei, each paired with diverse second-step enzymes (Cpegt2, MsegtE, Rtegt2, Spegt2, and Tregt2). Within this group, strain ST4001 reached a titer of 22.0 mg/L extracellularly and 17.0 mg/L extracellularly with total accumulation of 39.0 mg/L, reflecting moderate pathway efficiency (Figure 2A). Therefore, the remaining three strains all contained Cpegt2 from C. purpurea as the common second-step enzyme, combined with alternative first-step enzymes (Ncegt1, Rtegt1, and Spegt1). Notably, strain ST4006, carrying the Ncegt1 and Cpegt2 combination, achieved the highest EGT titer of 36.0 mg/L extracellular and 20.0 mg/L intracellularly with a total of 56.0 mg/L (Figure 2B), highlighting the critical impact of enzyme selection on pathway performance. ST4006, combining N. crassaEgt1 and C. purpureaEgt2, was selected for further engineering.
2.2 Reinforcing precursor amino acid biosynthesis boosts ergothioneine production
S-adenosyl-L-methionine is essential for EGT biosynthesis because it provides the methyl groups required for the three methylation steps carried out by Egt1. Thus, the level of SAM inside the cell directly influences the activity of the pathway. In S. cerevisiae, native methylation processes such as those involved in phospholipid, histone, and polyamine synthesis consume SAM, further limiting EGT production[45]. Among the two SAM synthetases in yeast, the one that is more responsive to metabolic demand and more effectively increases SAM pools when engineered is the preferred target[46]. SAM2, encoding SAM synthase, was overexpressed in strain ST4009 to boost EGT accumulation (Figure 3A and 3B). The results demonstrated that overexpressing the SAM2 gene led to increased EGT production, reaching 50.6 mg/L extracellularly and 31.1 mg/L intracellularly, with a total of 81.7 mg/L. This indicates that SAM2 enhances EGT synthesis (Figure 3B).
Histidine and cysteine are crucial precursors for EGT biosynthesis, where histidine offers the imidazole scaffold and cysteine provides the sulfur atom necessary for thiol or thione formation[47]. However, intracellular histidine levels remain a significant limitation because His1, the first committed enzyme in the pathway, is strongly inhibited by histidine itself[48]. To overcome these precursor bottlenecks, we systematically enhanced the supply of both sulfur and imidazole precursors. First, CYS3 was overexpressed in strain ST4009 to generate strain ST4010, thereby increasing cysteine availability for sulfur incorporation. Building on this, we enhanced histidine biosynthesis by co-expressing HIS1 under the strong constitutive TDH3 promoter, resulting in the creation of the ST4011 strain. Overexpression of SAM2, CYS3, and HIS1 led to a significant, synergistic increase in EGT production, with no apparent effect on cell growth. Strain ST4011 was found to produce 109.4 mg/L extracellular and 53.6 mg/L intracellular with a total of 163.0 mg/L EGT (Figure 3B). Compared to strain ST4009, the extracellular EGT increased by 116.3%, and the intracellular levels increased by 72.0%, respectively. The results demonstrate that simultaneously increasing histidine and cysteine supply effectively alleviates key metabolic bottlenecks in EGT biosynthesis.
Increasing L-methionine supply, the immediate precursor of SAM, can effectively boost SAM production via bacterial fermentation[47]. Therefore, our goal was to improve the expression of other crucial genes in the SAM biosynthesis pathway, such as MET6 and STR2, to raise intracellular SAM levels and thereby enhance EGT production (Figure 3A). The STR2 gene was overexpressed in strain ST4009 to generate ST4012. Subsequently, the MET6 gene was overexpressed in ST4012, yielding the new strain of ST4013, in which all target genes, SAM2, STR2, and MET6, were simultaneously overexpressed. Strain ST4013 produced 125.1 mg/L extracellular and 71.1 mg/L intracellular with a total of 196.2 mg/L of EGT. Compared with strain ST4011, extracellular EGT increased by 14.4%, while intracellular levels increased 32.6%. To further reinforce precursor and cofactor supply, we expanded the engineering strategy used in ST4011, which already overexpressed SAM2, CYS3, and HIS1. In ST4016, we additionally integrated the STR2 and MET6 expression cassettes at the same genomic locus used in strain ST4013, thereby enhancing methionine and SAM biosynthesis. Furthermore, ADK1 was overexpressed to improve adenylate balance and facilitate efficient ATP regeneration, thereby further strengthening SAM formation. Flask fermentation of the resulting strain ST4016 showed a substantial increase in EGT production. Strain ST4016 accumulated 151.8 mg/L extracellular and 83.3 mg/L intracellular with a total of 235.1 mg/L of EGT. Compared with strain ST4013, extracellular and intracellular levels increased by 21.3% and 17.1%, respectively. These results demonstrate that simultaneously increasing histidine, cysteine, methionine, and SAM, along with improved ATP regeneration, leads to a synergistic improvement in overall EGT flux (Figure 3B).
2.3 Enhancing SAM availability by downregulating SAM-consuming pathways and increasing the expression of Egt1 and Egt2
SAM serves as the primary methyl donor in sterol biosynthesis, including the conversion of zymosterol to ergosterol[49]. During this process, SAM is converted to S-adenosylhomocysteine (SAH) and then recycled to L-homocysteine viaSah1[50]. SAM is also diverted into polyamine spermidine synthesis, with intermediates capable of recycling back into L-methionine. These pathways are the main competing sinks that restrict SAM availability. Previous studies show that restricting transmethylation flux significantly increases intracellular SAM[51], and disruption of the polyamine pathway can further enhance SAM pools. Building on these insights, we targeted specific SAM-consuming pathways to increase SAM levels and decrease metabolic burden. This approach alleviates methionine feedback inhibition and enhances methylation capacity for downstream processes engineering. The synthesis of ergosterol is regulated by a coordinated set of ERG genes (ergX)[52], which are involved in cell growth and are generally classified as essential or nonessential. Essential genes are required for survival, while nonessential genes might be unnecessary during normal growth conditions. Both ERG4 and ERG6 are nonessential genes, but according to previous reports, deletion of ERG6 (Δerg6) increases cellular susceptibility to stress-induced death[53]. In this study, instead of deleting ERG6 and ERG4, we reduce their expression by replacing their native promoters with the weak promoter HXT1. This approach enabled a controlled reduction of ergosterol biosynthesis while avoiding the severe growth defects usually linked to complete gene deletions.
Another gene that may consume precursor amino acids is SPE2, which encodes S-adenosylmethionine decarboxylase, and can degrade SAM[54], a key enzyme in the polyamine biosynthesis pathway converts SAM into decarboxylated SAM, which is then used for spermidine and spermine production. This reaction is one of the major competing pathways for intracellular SAM. To reduce SAM diversion into polyamine production and increase its availability for EGT biosynthesis, we downregulated SPE2 by replacing its native promoter with the weak HXT1 promoter. In strain ST4016, ERG6, ERG4, and SPE2 were simultaneously downregulated by substituting their native promoters with the weak HXT1 promoter, thereby limiting SAM consumption through both sterol and polyamine pathways, and that strain was named ST4019. To further maximize the metabolic flux toward EGT, multiple copies of Nc-egt1 were integrated into the rDNA locus and Cp-egt2 into the δ-locus of the ST4019 strain to construct the ST4021 strain. Quantitative analysis of the transcriptional profiles revealed a significant correlation between integration strategy and expression levels. The relative expression levels of Egt1 and Egt2 in ST4016 were 10.9-fold and 8.6-fold of those in the reference single-copy integration strain (Figure 3C). In the further optimized strain ST4021, these levels were dramatically elevated to 22.5-fold for Egt1 and 27.2-fold for Egt2 relative to the single-copy control. Consequently, the expression levels in ST4021 were approximately 2.1-fold (Egt1) and 3.2-fold (Egt2) higher than those observed in ST4016. Based on these relative transcript abundances under shake-flask fermentation conditions, we estimated that the gene copy numbers for Egt1 and Egt2 were approximately 11 and 9 in ST4016, 23 and 28 in ST4021, respectively. Strain ST4021 accumulated 211.2 mg/L extracellular and 101.6 mg/L intracellular with a total of 312.8 mg/L of EGT (Figure 3B and 3D). Compared to the ST4016 strain, the ST4021 strain demonstrated a substantial enhancement in production performance, with extracellular EGT titers increasing by 39.1% and intracellular levels by 22.0%. These data underscore the efficiency of combining targeted metabolic sink attenuation with high-copy-number pathway integration to overcome SAM availability limitations and drive high-level EGT biosynthesis.
2.4 Medium optimization with supplementation of precursor amino acids
To increase the production of EGT, four different amino acids of Met, His, Cys, and Arg were supplemented to the culture medium as essential precursors for the synthesis of EGT (Figure 4). Group A acted as the low-concentration control to determine the baseline impact of precursor amino acid supplementation on EGT production. Group B was set up to assess whether a slight increase in precursor levels would result in a noticeable boost in EGT yield. Group C aimed to balance the levels of methionine and histidine with those of cysteine and arginine, thereby testing a moderate-to-high concentration range to determine whether elevated levels of specific amino acids could promote EGT biosynthesis. Group D was selected as a high-concentration test to determine whether the increasing supply of methionine would substantially boost EGT production. Group E involved the application of very high levels of all precursor amino acids in an attempt to achieve even greater EGT accumulation, although such elevated concentrations may impose metabolic stress or lead to nutrient imbalance. Group F tested the effects of high concentrations of all precursor amino acids, under the hypothesis that extreme supplementation could result in oversaturation or cytotoxicity, thereby limiting yeast growth and consequently constraining EGT production.
The best formulation for EGT production belonged to Group D (Figure 4), which had a final concentration of 20 g/L of Met and 8 g/L of His, Cys, and Arg, respectively. This combination of amino acids was optimal for enhancing metabolic flux toward EGT biosynthesis, but higher concentrations led to diminishing returns or metabolic limitations. These results provide valuable information for further optimizing fermentation conditions for large-scale, environmentally friendly production of EGT.
2.5 Fed batch fermentation for EGT production
To further improve the production of EGT, the strain ST4021, which produced the highest EGT titer in flask fermentation, was cultured in a 5-L bioreactor with the addition of precursor amino acids. The initial glucose concentration was 50 g/L. The concentrations for precursor amino acids are 20 g/L MET and 8 g/L His, Cys, and Arg, which were used in the fermentation process. Glucose was added every 24 hours to boost the concentration by about 10 g/L each cycle. The fermentation was performed for 168 hours, at which the maximum OD600 value of fermentation broth reached 93.9, and EGT titer reached 969 mg/L extracellular and 342 mg/L intracellular, with total production of 1 312.2 mg/L, and productivity was calculated as 7.8 mg/(L·h) (Figure 5). These results highlight that combining controlled oxygen flux, antioxidant supplementation, and precise precursor feeding at optimal temperature and pH synergistically enhances EGT biosynthesis, providing a robust strategy for scale-up production.
3 Discussion and conclusion
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In this study, a high-level EGT production in S. cerevisiae was achieved by implementing a multi-layered metabolic engineering framework. This strategy involved screening heterologous biosynthetic enzymes, improving the supply of sulfur and precursor amino acids, reducing the activity of competing SAM-consuming pathways, and optimizing gene dosage via multi-copy integration. The final engineered strain ST4021 produced 312.8 mg/L EGT in shake flask fermentation and 1 312.2 mg/L in fed-batch fermentation, demonstrating the effectiveness of the combined metabolic engineering strategies.
The initial screening of eight EGT pathway variants revealed that enzyme origin and pairing critically influence pathway performance. The bimodular combination of Ncegt1 from N. crassa with Cpegt2 from C. purpurea in strain ST4006 yielded the highest EGT titer of 56.0 mg/L. Conversely, the bacterial-fungal hybrid strain incorporating MsegtE showed only moderate productivity (19.9 mg/L), consistent with reports that cross-kingdom enzyme combinations may suffer from incompatible catalytic mechanisms[37].
EGT biosynthesis involves three methylation steps mediated by Egt1, making SAM availability a key bottleneck for metabolic flux. In S. cerevisiae, the cell’s internal SAM pool is carefully controlled and significantly affected by internal metabolic processes[45]. To overcome this limitation, methylenetetrahydrofolate reductase (MTHFR) engineering has been employed to enable continuous homocysteine remethylation through methyl tetrahydrofolate (CH3-THF)[49], while the utilization of cost-effective one-carbon compounds[55] and the overexpression of SAM2[56] have also been shown to boost intracellular SAM levels. Overexpression of SAM2 alone in strain ST4009 increased EGT production by 45.9% compared to ST4006 (from 56.0 to 81.7 mg/L), confirming that methyl donor availability is a primary constraint. The subsequent overexpression of CYS3 and HIS1 in strain ST4011 doubled the EGT production relative to ST4009, reaching 163.0 mg/L, demonstrating that histidine and cysteine limitations become exposed once the SAM supply is increased. Significantly, the most substantial improvement occurred when we concurrently strengthened the methionine-SAM axis (MET6, STR2, and SAM2) and ATP regeneration (ADK1) in strain ST4016, achieving 235.1 mg/L. This result is consistent with earlier co-expression approaches that involved MET6 and SAM2[57]. These findings indicate that SAM availability depends on a coordinated system involving precursor supply, sulfur flux, and bioenergetics, rather than being limited by a single enzymatic bottleneck. The improved performance of strain ST4016 compared to earlier strains shows that simultaneously enhancing methionine regeneration and adenylate homeostasis more effectively increases methylation capacity than just overexpressing SAM2. This is consistent with findings that maintaining a high SAM biosynthetic pool is essential to sustain high-flux EGT production[58].
The biosynthesis of ergosterol (viaERG4 and ERG6) and polyamines (viaSPE2) constitutes significant endogenous sinks for SAM. It has been demonstrated that the deletion of SPE2 in S. cerevisiae substantially enhanced EGT. However, this change led to spermidine and pantothenate auxotrophy, requiring careful attention to supplementation[58]. Similarly, the deletion of metJ in E. coli alleviated the repression of methionine biosynthesis, achieving an EGT yield of 1.3 g/L, although they observed growth defects in the knockout strain[38]. In contrast, our promoter attenuation strategy using the weak HXT1 promoter achieved partial flux redistribution without compromising vital functions. The strain ST4021 exhibited an increased EGT production without any apparent growth impairments, confirming this method as a practical alternative to gene knockout for industrial applications.
With a shake flask titer of 312.8 mg/L, strain ST4021 demonstrates competitive performance compared with previously reported yeast-based systems. This titer exceeds the EGT titer reported in Y. lipolytica, R. toruloides, and E. coli before fed-batch optimization[59-60]. It also compares favorably with the 106 mg/L achieved in the early-stage engineered S. cerevisiae strain before the comprehensive precursor engineering and medium optimization, which ultimately yielded 2.4 g/L in fed-batch[58]. Scaling up to 5-L fed-batch fermentation confirmed the industrial potential of strain ST4021, achieving 1 312.2 mg/L EGT over 168 hours with a productivity of 7.8 mg/(L·h). However, comparison with the most recent advancements highlights significant opportunities for optimizing the process. It was achieved an EGT concentration of 9.3 g/L in Y. lipolytica through a combination of metabolic and enzyme engineering, specifically utilizing TrEgt1 mutations (Y786A-A492V) alongside modular precursor pathway enhancement, resulting in a productivity of 55.4 mg/(L·h)[43]. 7.2 g/L EGT was achieved in E. coli by employing a betaine-driven methyl supply system and an inorganic sulfur module (Table 3)[61]. Similarly, it was attained 7.3 g/L in Y. lipolytica using multicopy integration tools (YaliCMulti/YaliHMulti)[42]. More recently, it was demonstrated that integrating membrane permeability engineering with Tregt2 enzyme engineering (E155C mutation) and copy number optimization resulted in a yield of 4.1 g/L in E. coli within 96 hours[62]. These comparisons suggest that although the strain design presented here is competitive at the shake flask level, the fermentation process itself necessitates systematic optimization to fully realize its production potential.
Throughout the engineering process, a substantial fraction of the produced EGT remained intracellular, with extracellular titers consistently 1.5-to 2-fold higher than intracellular levels. This distribution suggests active export; however, the persistent intracellular pool indicates that native yeast transporters have a limited capacity for EGT secretion. This phenomenon has been observed across various hosts. For instance, it was reported that only 5%–30% of the total EGT was secreted in their initial S. cerevisiae strains[33]. Similarly, it was found that recombinant Corynebacterium glutamicum strains required extended cultivation periods for EGT secretion, suggesting that export is a rate-limiting step[63]. Despite the absence of identified EGT-specific exporters in S. cerevisiae, the observed gradual extracellular accumulation during fermentation suggests the possibility of passive diffusion or low-affinity transport proteins. Recent findings indicate that the overexpression of mfsT1 from Mycolicibacterium neoaurum, a putative EGT transporter, significantly increased production in E. coli[61]. Similarly, it was demonstrated that co-augmentation with mfsT1 enhanced EGT production in M. neoaurum[64]. Future engineering strategies should consider the heterologous expression of well-characterized EGT transporters, potentially in conjunction with membrane permeability modifications. This approach is exemplified by the work that achieved improved EGT export through the deletion of ΔwaaF and ΔmsbB in E. coli[63].
In conclusion, this study provides a comprehensive framework for resolving the multifaceted metabolic constraints associated with EGT production in yeast. By addressing enzyme selection, precursor supply, methyl donor availability, competing pathways, and gene dosage, we improved yields from 56.0 mg/L to 312.8 mg/L in shake flasks and 1 312.2 mg/L in fed-batch fermentation. Our shake flask titer is competitive among yeast systems; optimizing fermentation—especially the feeding strategy, precursor dynamics, and process control—is crucial for further improvement. The engineering framework we developed, which includes pathway screening, precursor reinforcement, competition reduction, and multicopy integration, can be applied to optimize other SAM-dependent or amino acid-derived biosynthetic pathways in yeast.
Funding
Less
  • 国家重点研发计划(2022YFA0911800)
  • 江苏省前沿技术研发计划(BF2025080)
  • 中央引导地方科技发展资金(254Z2601G)
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Year 2026 volume 66 Issue 8
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doi: 10.13343/j.cnki.wsxb.20260134
  • Receive Date：2026-02-13
  • Online Date：2026-08-21
  • Published：2026-08-04
Article Data
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History
  • Received：2026-02-13
  • Accepted：2026-04-09
Funding
国家重点研发计划(2022YFA0911800)
江苏省前沿技术研发计划(BF2025080)
中央引导地方科技发展资金(254Z2601G)
Affiliations
    Key Laboratory of Industrial Biotechnology of the Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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