An unusual ring—A opening and other reactions in steroid transformation by the thermophilic fungus Myceliophthora thermophila

A series of steroids (progesterone, testosterone acetate, 17β-acetoxy-5α-androstan-3-one, testosterone and androst-4-en-3,17-dione) have been incubated with the thermophilic ascomycete Myceliophthora thermophila CBS 117.65. A wide range of biocatalytic activity was observed with modification at all four rings of the steroid nucleus and the C-17β side-chain.This is the first thermophilic fungus to demonstrate the side-chain cleavage of progesterone. A unique fungal transformation was observed following incubation of the saturated steroid 17β-acetoxy-5α-androstan-3-one resulting in 4-hydroxy-3,4-seco-pregn-20-one-3-oic acid which was the product generated following the opening of an A-homo steroid, presumably by lactonohydrolase activity. Hydroxylation predominated at axial protons of the steroids containing 3-one-4-ene ring-functionality. This organism also demonstrated reversible acetylation and oxidation of the 17β-alcohol of testosterone.All steroidal metabolites were isolated by column chromatography and were identified by ¹H, ¹³C NMR, DEPT analysis and other spectroscopic data. The range of steroidal modification achieved with this fungus indicates that these organisms may be a rich source of novel steroid biocatalysis which deserve greater investigation in the future.     

Characterization of a new keratinolytic Trichoderma atroviride strain F6 that completely degrades native chicken feather

Aims:  To isolate novel nonpathogenic fungus that completely degrades native chicken feather and characterize its keratinases. Methods and Results:  Feather‐degrading fungi were isolated from decaying feathers using a novel method based on simulating decaying process in the environment. The isolate F6 with high keratinolytic activity was identified as Trichoderma atroviride based on morphological traits and ITS1‐5·8S‐ITS2 sequence analysis. The purified dominant component of keratinase had a molecular mass of 21 kDa. The purified keratinase belonged to serine protease. Its isoelectric point, molecular weight, optimum pH, optimum temperature, and substrate specificity are different from those of other serine proteases of Trichoderma species. The optimum pH and temperature values of purified keratinase were consistent with those of crude keratinase. However, the differences between crude and purified enzymes such as thermostability, resistance to Ba²⁺, Mn²⁺, Hg²⁺, Zn²⁺, Cu²⁺, 1,10‐phenanthroline, 2,2′‐bipyridyl, and PMSF (phenylmethylsulfonyl fluoride) were observed. Conclusions:  The results suggested the purified keratinase is predominantly extracellular proteins when strain F6 was grown on keratinous substrates. The protease, in combination with other components, is effective in feather degradation. The strain F6 is more suitable for feather degradation than its purified keratinase. Significance and Impact of the Study:  The novel nonpathogenic T. atroviride F6 with high feather‐degrading activity showed potentials in biotechnological process of converting feathers into economically useful feather meal.     

Degradation of Ficus elastica rubber latex by Aspergillus terreus,Aspergillus flavus and Myceliophthora thermophila

Since isolates recovered on medium containing-Ficus elastica latex showed good growth on the respective natural rubber than those recovered on Euphorbia pulcherrima or Ficus nitida, 16 of these isolates were selected for further growth experiments on natural rubber to determine their protein content as well as rubber viscosity. Of these, the mesophilic strains Aspergillus terreus AUMC 4682, Aspergillus flavus AUMC 4795 and the thermophilic strain Myceliophthora thermophila AUMC 4653 showed low rubber viscosity and high mycelia protein content indicating high biodegradation ability of rubber. The strains were subjected for further analysis. They showed high ability to degrade poly (cis-1, 4-isoprene) rubber fig. The ability was also determined by measuring the increase in protein content of each fungus (mg g⁻¹ dry wt), reduction in molecular weight (g mol⁻¹) and inherent viscosity (dl g⁻¹). Moreover the degradation was characterized by determining aldehyde or keto group by Schiff reagent and observing the growth using scanning electron microscopy (SEM).     

Metabolic engineering of the thermophilic filamentous fungus <Emphasis Type="Italic">Myceliophthora thermophila</Emphasis> to produce fumaric acid

Background

Fumaric acid is widely used in food and pharmaceutical industries and is recognized as a versatile industrial chemical feedstock. Increasing concerns about energy and environmental problems have resulted in a focus on fumaric acid production by microbial fermentation via bioconversion of renewable feedstocks. Filamentous fungi are the predominant microorganisms used to produce organic acids, including fumaric acid, and most studies to date have focused on Rhizopus species. Thermophilic filamentous fungi have many advantages for the production of compounds by industrial fermentation. However, no previous studies have focused on fumaric acid production by thermophilic fungi.

Results

We explored the feasibility of producing fumarate by metabolically engineering Myceliophthora thermophila using the CRISPR/Cas9 system. Screening of fumarases suggested that the fumarase from Candida krusei was the most suitable for efficient production of fumaric acid in M. thermophila. Introducing the C. krusei fumarase into M. thermophila increased the titer of fumaric acid by threefold. To further increase fumarate production, the intracellular fumarate digestion pathway was disrupted. After deletion of the two fumarate reductase and the mitochondrial fumarase genes of M. thermophila, the resulting strain exhibited a 2.33-fold increase in fumarate titer. Increasing the pool size of malate, the precursor of fumaric acid, significantly increased the final fumaric acid titer. Finally, disruption of the malate–aspartate shuttle increased the intracellular malate content by 2.16-fold and extracellular fumaric acid titer by 42%, compared with that of the parental strain. The strategic metabolic engineering of multiple genes resulted in a final strain that could produce up to 17 g/L fumaric acid from glucose in a fed-batch fermentation process.

Conclusions

This is the first metabolic engineering study on the production of fumaric acid by the thermophilic filamentous fungus M. thermophila. This cellulolytic fungal platform provides a promising method for the sustainable and efficient-cost production of fumaric acid from lignocellulose-derived carbon sources in the future.

     

Optimizing Xylanase Production by a Newly Isolated Strain CAU44 of the Thermophile Thermomyces lanuginosus

Summary Thermomyces lanuginosus CAU44, a newly isolated thermophilic fungus strain, was used for the production of extracellular xylanase on various lignocellulosic materials under shake flask conditions. High-level production of xylanase by the strain was enhanced by optimizing the type of carbon sources, substrate concentration, particle size and surfactants in the culture medium. The titre of xylanase activity obtained of up to 4156 U ml⁻¹ was the highest ever reported.     

Thermostable amylolytic enzymes from a cellulolytic fungusMyceliophthora thermophila D14 (ATCC 48 104)

Summary The production of amylolytic enzymes by a thermophilic cellulolytic fungus,Myceliophthora thermophila D14 was investigated by batch cultivation in Czapek-Dox medium at 45° C. Among various nitrogenous compounds used, NaNO₃ and KNO₃ were found to be the best for amylase production. Starch, cellobiose and maltose induced the synthesis of amylase while glucose, fructose, galactose, lactose, arabinose, xylose, sorbitol, mesoinositol and sucrose did not. Calcium ions had the most stimulating effect on enzyme formation amongst many ions investigated. The synthesis of amylolytic enzymes was dependent on growth and occurred predominantly in the mid-stationary phase. The enzyme was active in a broad temperature range (50° C–60° C) and displayed activity optima at 60° C and pH 5.6.     

Production of 3‐Fucosyllactose in Engineered Escherichia coli with α‐1,3‐Fucosyltransferase from Helicobacter pylori

3‐Fucosyllactose (3‐FL), one of the major oligosaccharides in human breast milk, is produced in engineered Escherichia coli. In order to search for a good α‐1,3‐fucosyltransferase, three bacterial α‐1,3‐fucosyltransferases are expressed in engineered E. coli deficient in β‐galactosidase activity and expressing the essential enzymes for the production of guanosine 5′‐diphosphate‐l ‐fucose, the donor of fucose for 3‐FL biosynthesis. Among the three enzymes tested, the fucT gene from Helicobacter pylori National Collection of Type Cultures 11637 gives the best 3‐FL production in a simple batch fermentation process using glycerol as a carbon source and lactose as an acceptor. In order to use glucose as a carbon source, the chromosomal ptsG gene, considered the main regulator of the glucose repression mechanism, is disrupted. The resulting E. coli strain of ?LP‐YA+FT shows a much lower performance of 3‐FL production (4.50 g L^?1) than the ?L‐YA+FT strain grown in a glycerol medium (10.7 g L^?1), suggesting that glycerol is a better carbon source than glucose. Finally, the engineered E. coli ?LW‐YA+FT expressing the essential genes for 3‐FL production and blocking the colanic acid biosynthetic pathway (?wcaJ) exhibits the highest concentration (11.5 g L^?1), yield (0.39 mol mol^?1), and productivity (0.22 g L^?1 h) of 3‐FL in glycerol‐limited fed‐batch fermentation.     

Synthesis,Characterization, and Anti‐Amoebic Activity of N‐(Pyrimidin‐2‐yl)benzenesulfonamide Derivatives

A new series of N‐(pyrimidin‐2‐yl)benzenesulfonamide derivatives, 3a – 3i and 4a – 4i , was synthesized from pyrimidin‐2‐amines, 2a – 2i , with the aim to explore their effects on in vitro growth of Entamoeba histolytica. The chemical structures of the compounds were elucidated by elemental analysis, FT‐IR, ¹H‐ and ¹³C‐NMR, and ESI mass‐spectral data. In vitro anti‐amoebic activity was evaluated against HM1 : IMSS strain of Entamoeba histolytica. The IC₅₀ values were calculated by using the double dilution method. The results were compared with the IC₅₀ value of the standard drug ‘metronidazole’. The selected compounds were tested for their cytotoxic activities by cell‐viability assay using H9C2 cardiac myoblasts cell line, and the results indicated that all the compounds displayed remarkable >80% viabilities to a concentration of 100 μg/ml.     

Efficient production of Al(OH)3‐immobilized laccase with a Heterobasidion annosum strain selected by microplate screening

Aims: Wild‐type white rot fungi are the most important production organisms for laccase, a promising oxidative biocatalyst with numerous applications. This study aimed at identifying novel highly productive strains, finding optimal cultivation conditions for laccase production and establishing a simple immobilization procedure. Methods and Results: By using a newly developed 96‐well microplate cultivation method, 23 species of white rot fungi, represented by 29 strains, were directly compared with regard to the amount of secreted laccase. Both, with glucose and spruce saw dust as growth substrate a Heterobasidion annosum strain and a Physisporinus vitreus strain were the most productive (730–2200 U l^?1 of secreted laccase). Cultivation conditions for laccase production with H. annosum were optimized in larger‐scale liquid cultures. Aeration with a sparger lead to a 3·8‐fold increase in laccase activity when compared to nonaerated flask cultures. More than 3000 U l^?1 laccase was produced in glucose medium supplemented with yeast extract and the inducer veratryl alcohol. Culture supernatant was incubated with short‐range ordered Al(OH)₃ particles to directly immobilize and concentrate laccase by adsorption. Active laccase was recovered in 40% yield and the Al(OH)₃‐adsorbed laccase was suitable for repeated decolourization of indigo carmine. Conclusions: Microplate cultivation allowed a large‐scale comparison of the capacity of different fungal species for laccase production. Laccase secretion of a highly productive H. annosum strain was found to vary strongly with different cultivation conditions. Adsorption to Al(OH)₃ proved to be suitable as direct immobilization technique. Significance and Impact of the Study: The microplate screening method simplifies strain and medium development for laccase production. Two novel fungal strains suitable for laccase production were identified. Procedures for simple and efficient production of immobilized H. annosum laccase were established.     

Production and characterization of keratinase from<Emphasis Type="Italic">Paracoccus</Emphasis> sp. WJ-98

A bacterial strain WJ-98 found to produce active extracellular keratinase was isolated from the soil of a poultry factory. It was identified asParacoccus sp. based on its 16S rRNA sequence analysis, morphological and physiological characteristics. The optimal culture conditions for the production of keratinase byParacoccus sp. WJ-98 were investigated. The optimal medium composition for keratinase production was determined to be 1.0% keratin, 0.05% urea and NaCl, 0.03% K₂HPO₄, 0.04% KH₂PO₄, and 0.01% MgCl₂·6H₂O. Optimal initial pH and temperature for the production of keratinase were 7.5 and 37°C, respectively. The maximum keratinase production of 90 U/mL was reached after 84 h of cultivation under the optimal culturing conditions. The keratinase fromParacoccus sp. WJ-98 was partially purified from a culture broth by using ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, followed by gel filtration chromatography on Sephadex G-75. Optimum pH and temperature for the enzyme reaction were pH 6.8 and 50°C, respectively and the enzymes were stable in the pH range from 6.0 to 8.0 and below 50°C. The enzyme activity was significantly inhibited by EDTA, Zn²⁺ and Hg²⁺. Inquiry into the characteristics of keratinase production from these bacteria may yield useful agricultural feed processing applications.     

Medium optimization for keratinase production in hair substrate by a new <Emphasis Type="Italic">Bacillus subtilis</Emphasis> KD-N2 using response surface methodology

Culture medium for keratinase production from hair substrate by a new Bacillus subtilis strain, KD-N2, was optimized. Effects of culture conditions on keratinase production were tested, and optimal results were obtained with 10% inocula (v/v), 16 g/L hair substrate, an initial pH value of 6.5 and a culture volume of 20 mL. Several carbon sources (sucrose, cornflour) and nitrogen sources (yeast extract, tryptone and peptone) had positive effects on keratinase production, with sucrose giving optimal results. To improve keratinase yield, statistically based experimental designs were applied to optimize the culture medium. Fractional factorial design (FFD) experiments showed that MgSO₄ and K₂HPO₄ were the most significant factors affecting keratinase production. Further central composite design (CCD) experiments indicated that the optimal MgSO₄ and K₂HPO₄ concentrations were 0.91 and 2.38 g/L, respectively. Using an optimized fermentation medium (g/L: NaCl 1.0, CaCl₂ 0.05, KH₂PO₄ 0.7, sucrose 3, MgSO₄ 0.91, K₂HPO₄ 2.38), keratinase activity increased to 125 U/mL, an approximate 1.7-fold increase over the previous activity (75 U/mL). Human hair was degraded during the submerged cultivation.     

Fermentation production of keratinase from Bacillus licheniformisPWD-1 and a recombinant B. subtilis FDB-29

Bacillus licheniformis PWD-1, the parent strain, and B. subtilis FDB-29, a recombinant strain. In both strains, keratinase was induced by proteinaceous media, and repressed by carbohydrates. A seed culture of B. licheniformis PWD-1 at early age, 6–10 h, is crucial to keratinase production during fermentation, but B. subtilis FDB-29 is insensitive to the seed culture age. During the batch fermentation by both strains, the pH changed from 7.0 to 8.5 while the keratinase activity and productivity stayed at high levels. Control of pH, therefore, is not necessary. The temperature for maximum keratinase production is 37°C for both strains, though B. licheniformis is thermophilic and grows best at 50°C. Optimal levels of dissolved oxygen are 10% and 20% for B. licheniformis and B. subtilis respectively. A scale-up procedure using constant temperature at 37°C was adopted for B. subtilis. On the other hand, a temperature-shift procedure by which an 8-h fermentation at 50°C for growth followed by a shift to 37°C for enzyme production was used for B. licheniformis to shorten the fermentation time and increase enzyme productivity. Production of keratinase by B. licheniformis increased by ten-fold following this new procedure. After respective optimization of fermentation conditions, keratinase production by B. licheniformis PWD-1 is approximately 40% higher than that by B. subtilis FDB-29. Received 16 July 1998/ Accepted in revised form 07 March 1999     

Myceliophthora thermophila syn. Sporotrichum thermophile: a thermophilic mould of biotechnological potential

Myceliophthora thermophila syn. Sporotrichum thermophile is a ubiquitous thermophilic mould with a strong ability to degrade organic matter during optimal growth at 45?°C. Both genome analysis and experimental data have suggested that the mould is capable of hydrolyzing all major polysaccharides found in biomass. The mould is able to secrete a large number of hydrolytic enzymes (cellulases, laccases, xylanases, pectinases, lipases, phytases and some other miscellaneous enzymes) employed in various biotechnological applications. Characterization of the biomass-hydrolyzing activity of wild and recombinant enzymes suggests that this mould is highly efficient in biomass decomposition at both moderate and high temperatures. The native enzymes produced by the mould are more efficient in activity than their mesophilic counterparts beside their low enzyme titers. The mould is able to synthesize various biomolecules, which are used in multifarious applications. Genome sequence data of M. thermophila also supported the physiological data. This review describes the biotechnological potential of thermophilic mould, M. thermophila supported by genomic and experimental evidences.     

Isolation and characterization of α‐(1→3)‐glucan‐degrading bacteria from the gut of Diaperis boleti feeding on Laetiporus sulphureus

Bacteria degrading α‐(1→3)‐glucan were sought in the gut of fungivorous insects feeding on fruiting bodies of a polypore fungus Laetiporus sulphureus, which are rich in this polymer. One isolate, from Diaperis boleti, was selected in an enrichment culture in the glucan‐containing medium. The bacterium was identified as Paenibacillus sp. based on the results of the ribosomal DNA analysis. The Paenibacillus showed enzyme activity of 4.97 mU/cm³ and effectively degraded fungal α‐(1→3)‐glucan, releasing nigerooligosaccharides and a trace amount of glucose. This strain is the first reported α‐(1→3)‐glucan‐degrading microorganism in the gut microbiome of insects inhabiting fruiting bodies of polypore fungi.     

A new thermophilic species of Myceliophthora from China

Myceliophthora guttulata sp. nov. is described and illustrated based on strains isolated from soil in China. This species is thermophilic with optimal growth temperature of 40–45 °C, and minimum growth temperature of 25 °C. Morphologically, this species is characterized by smooth, guttulate, pyriform to obovoid blastoconidia born directly on the side of hyphae, on long or short pedicels or in groups of 1–4 on ampulliform swellings. Phylogenetic analysis based on multi-locus alignment of internal transcribed spacer (ITS), elongation factor 1-alpha (EF1-α) and RNA polymerase II subunit (RPB2) regions showed that M. guttulata clustered within the genus Myceliophthora, and is closely related to four thermophilic species, i.e. M. fergusii, M. thermophila, M. heterothallic, and M. hinnulea.     

Loss‐of‐function of ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen‐fixing symbiosis with specific Mesorhizobium loti strains

The nitrogen‐fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix^– mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix^– mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix^– mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (LjAPN1), encodes a nepenthesin‐type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain‐specific Fix^– phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen‐fixing) symbiosis in a rhizobial strain‐dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.     

A novel endophytic Huperzine A–producing fungus,Shiraia sp. Slf14, isolated from Huperzia serrata

Aims: To characterize and identify a novel Huperzine A (HupA)‐producing fungal strain Slf14 isolated from Huperzia serrata (Thunb. ex Murray) Trev. in China. Methods and Results: The isolation, identification and characterization of a novel endophytic fungus producing HupA specifically and consistently from the leaves of H. serrata were investigated. The fungus was identified as Shiraia sp. Slf14 by molecular and morphological methods. The HupA produced by this endophytic fungus was shown to be identical to authentic HupA analysed by thin layer chromatographic, High‐performance liquid chromatography (HPLC), LC‐MS, ¹H NMR and acetylcholinesterase (AChE) inhibition activity in vitro. The amount of HupA produced by Shiraia sp. Slf14 was quantified to be 327·8 μg l^?1 by HPLC, which was far higher than that of the reported endophytic fungi, Acremonium sp., Blastomyces sp. and Botrytis sp. Conclusions: The production of HupA by endophyte Shiraia sp. Slf14 is an enigmatic observation. It would be interesting to further study the HupA production and regulation by the cultured endophyte in H. serrata and in axenic cultures. Significance and Impact of the Study: Although the current accumulation of HupA by the endophyte is not very high, it could provide a promising alterative approach for large‐scale production of HupA. However, further strain improvement and the fermentation process optimization are required to result in the consistent and dependable production.     

Sterols of Saccharomyces cerevisiae erg6 Knockout Mutant Expressing the Pneumocystis carinii S‐Adenosylmethionine:Sterol C‐24 Methyltransferase

The AIDS‐associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The Pneumocystis carinii S‐adenosylmethionine:sterol C24‐methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C‐24 position of the sterol side chain producing both C₂₈ and C₂₉ 24‐alkylsterols in approximately the same proportions, whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild‐type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy (¹H‐NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C₂₈ sterol ergosterol as the major sterol, and also resulted in low levels of C₂₉ sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ²⁴⁽²⁸⁾‐sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C₂₉ sterols as that found in P. carinii.     

Mutual benefit interactions between banded pine weevil Pissodes castaneus and blue‐stain fungus Leptographium serpens in maritime pine

• 1 Ecological interactions between banded pine weevil Pissodes castaneus and blue‐stain fungus Leptographium serpens, when simultaneously sharing the same host plant (maritime pine Pinus pinaster) in winter and spring, were investigated. Temporal components of the interaction were taken into account by either introducing the weevils and the pathogen simultaneously or sequentially, with the weevils being introduced 1 month after the fungal inoculation.
• 2 We measured larval mortality, development time, offspring number, sex ratio and body size of P. castaneus. Phloem phosphorus and nitrogen concentrations were also assessed. Furthermore, we tested whether: (i) emerging offspring transported propagules of the fungus; (ii) artificially‐contaminated weevils may transmit the disease to healthy trees; and (iii) field collected P. castaneus carry the fungus.
• 3 The fungus enhanced weevil colonization and brood production in both seasons. During winter and spring, adults from trees where the pathogen was inoculated prior to weevil introduction emerged earlier than weevils from trees where they had been introduced simultaneously with the fungus. During winter, weevils from pre‐inoculated trees were also larger. Sex ratio and larval mortality were not affected. Leptographium serpens did not affect phloem nitrogen content but phosphorus content was greater in plants inoculated with the pathogen, which may explain the findings on weevil growth.
• 4 Sixty‐five percent of the weevils that emerged from inoculated trees carried spores of L. serpens, although no successful isolation was made from field collected weevils. The fungus was recovered from 25% of the trees infested with artificially‐contaminated weevils.
• 5 These results suggest that P. castaneus benefits from the presence of L. serpens and may contribute to its spread.

     

Controllable Chain‐Length for Covalent Sulfur–Carbon Materials Enabling Stable and High‐Capacity Sodium Storage

Room temperature sodium–sulfur batteries have emerged as promising candidate for application in energy storage. However, the electrodes are usually obtained through infusing elemental sulfur into various carbon sources, and the precipitation of insoluble and irreversible sulfide species on the surface of carbon and sodium readily leads to continuous capacity degradation. Here, a novel strategy is demonstrated to prepare a covalent sulfur–carbon complex (SC‐BDSA) with high covalent‐sulfur concentration (40.1%) that relies on ? SO₃H (Benzenedisulfonic acid, BDSA) and SO₄^2? as the sulfur source rather than elemental sulfur. Most of the sulfur is exists in the form of O? S/C? S bridge‐bonds (short/long‐chain) whose features ensure sufficient interfacial contact and maintain high ionic/electronic conductivities of the sulfur–carbon cathode. Meanwhile, the carbon mesopores resulting from the thermal‐treated salt bath can confine a certain amount of sulfur and localize the diffluent polysulfides. Furthermore, the C? S_x? C bridges can be electrochemically broken at lower potential (<0.6 V vs Na/Na⁺) and then function as a capacity sponsor. And the R‐SO units can anchor the initially generated S_x^2? to form insoluble surface‐bound intermediates. Thus SC‐BDSA exhibits a specific capacity of 696 mAh g^?1 at 2500 mA g^?1 and excellent cycling stability for 1000 cycles with 0.035% capacity decay per cycle.