Diosgenin Production Research Articles

Screening and Selection of a New Medium and Culture Conditions for Diosgenin Production via Microbial Biocatalysis of SYt1

Diosgenin (DSG) is a phytosterol saponin mainly found in Dioscorea zingiberensis C.H. Wright. It has shown promising results in treating various diseases such as cancer, diabetes, arthritis, asthma, and cardiovascular diseases. Diosgenin is also an important medicinal chemical for synthesizing various steroid medicines. The production of diosgenin by acid hydrolysis generates a large amount of wastewater, leading to severe environmental pollution. However, producing diosgenin through microbial fermentation can effectively reduce environmental pollution. Numerous studies have demonstrated that various microorganisms can produce diosgenin via solid-state fermentation. Nevertheless, due to the complexity, high maintenance costs, uneven heat production, and other characteristics of solid-state fermentation, it is not commonly used in the industrial production of diosgenin. In contrast, liquid fermentation offers advantages such as simple operation, easy maintenance, and stable fermentation, making it more suitable for the industrial production of diosgenin. However, few studies have focused on producing diosgenin using liquid fermentation. In this study, endophytic Bacillus licheniformis SYt1 was used to produce diosgenin via liquid fermentation, with Dioscorea tuber powder as a substrate. Soxhlet extraction and silica gel column chromatography were employed to identify the diosgenin from the liquid fermentation products. Suitable fermentation conditions were screened and identified. The environmental variables that significantly affect the diosgenin yield were determined by the Plackett–Burman design (P-BD) with eight factors. The three factors (peptone, yeast extract powder and inorganic salt) with the greatest influence on the diosgenin yield were selected and further optimized using a response surface methodology (RSM). The final culture conditions were determined to be 35.79 g/L of peptone, 14.56 g/L of yeast extract powder, and 1.44 g/L of inorganic salt. The yield of diosgenin under these conditions was 132.57 mg/L, which was 1.8 times greater than the yield under pre-optimization conditions. This effective, clean, and promising liquid fermentation method possesses the potential to replace the traditional acid hydrolysis method for the industrial production of diosgenin.

Diosgenin production from Dioscorea zingiberensis tubers by novel pressurized hydrolysis in acidic ionic liquids

Diosgenin production from Dioscorea zingiberensis tubers by novel pressurized hydrolysis in acidic ionic liquids

Hydrogen bond regulation to promote the direct production of diosgenin from Dioscorea zingiberensis rhizomes

Herein, a renewable system consisted of tartaric acid and isopropanol was developed to produce diosgenin efficiently from Dioscorea zingiberensis rhizomes. The yield of diosgenin reached 4.15% comparable to the highest value reported in previous researches. The combination of experiment and DFT calculation revealed that the acid-alcohol system could regulate the hydrogen bond interaction of each component in the rhizomes to disassemble the connection network and promote the release of saponin. Meanwhile, the intramolecular hydrogen bond of tartaric acid and the steric hindrance effect of isopropanol could prevent the esterification reaction and maintain the strong acidity of the system for the high yield of diosgenin. The scale-up experiment with a 100-fold feeding capacity was also conducted successfully. Furthermore, the technological process integrated with purification of diosgenin, recovery of organic acid and solvent and upgrading of residual solid to porous carbon materials has been established systematically to verify the prospective of industrialization.

Isolation of endophytes from Dioscorea nipponica Makino for stimulating diosgenin production and plant growth.

Both bacterial and fungal endophytes exhibited one or more plant growth-promoting (PGP) traits. Among these strains, the Paenibacillus peoriae SYbr421 strain demonstrated the greatest activity in the direct biotransformation of tuber powderfrom D. nipponica into diosgenin. Endophytes play crucial roles in shaping active metabolites within plants, significantly influencing both the quality and yield of host plants. Dioscorea nipponica Makino accumulates abundant steroidal saponins, which can be hydrolyzed to produce diosgenin.However, our understanding of the associated endophytes and their contributions to plant growth and diosgenin production is limited. The present study aimed to assess the PGP ability and potential of diosgenin biotransformation by endophytesisolatesassociated withD. nipponica for the efficient improvement of plant growth and development of a clean and effective approach for producing the valuable drug diosgenin. Eighteen bacterial endophytes were classified into six genera through sequencing and phylogenetic analysis of the 16S rDNA gene. Similarly, 12 fungal endophytes were categorized into 5 genera based on sequencing and phylogenetic analysis of the ITS rDNA gene. Pure culture experiments revealed that 30 isolated endophytic strains exhibited one or more PGP traits, such as nitrogen fixation, phosphate solubilization, siderophore synthesis, and IAA production. One strain of endophytic bacteria, P. peoriae SYbr421, effectively directly biotransformed the saponin components in D. nipponica. Moreover, a high yield of diosgenin (3.50%) was obtained at an inoculum size of 4% after 6days of fermentation. Thus, SYbr421 could be used for a cleaner and more eco-friendly diosgenin production process. In addition, based on the assessment of growth-promoting isolates and seed germination results, the strains SYbr421, SYfr1321, and SYfl221 were selected for greenhouse experiments. The results revealed that the inoculation of these promising isolates significantly increased the plant height and fresh weight of the leaves and roots compared to the control plants. These findings underscore the importance of preparing PGP bioinoculants from selected isolates as an additional option for sustainable diosgenin production.

Molecular characterization of diosgenin biosynthesis in different organs of Trigonella foenum-graecum L.

Fenugreek, an annual herb, is a significant source of diosgenin, a triterpene with bioactive properties. This study aimed to molecularly characterize diosgenin biosynthesis in various organs of fenugreek, including flowers, immature leaves, developing leaves, mature leaves, stems, roots, and pods. The biosynthetic pathway involves vital enzymes such as squalene synthase, squalene monooxygenase, cycloartenol synthase, and Sterol-3-ß-glucosyl transferase. The study found that flowers and roots exhibited the highest gene expression levels, indicating their potential significance in diosgenin biosynthesis. Diosgenin content was quantified using high-performance liquid chromatography, with seeds being the primary source. The findings suggest that fenugreek offers a cost-effective and rapid alternative to yam for diosgenin production, and the differential accumulation of diosgenin in different plant organs underscores the importance of understanding organ-specific biosynthesis. This comprehensive analysis provides valuable insights into fenugreek’s potential as a bioresource for diosgenin and its diverse applications in medicine and agriculture.

Cleaning Process of Diosgenin from Dioscorea nipponica

This investigation employed HPLC and LC-MS techniques to elucidate the enzymolysis and acid hydrolysis mechanisms of diosgenin obtained through a cleaning process. The findings revealed that the enzymolysis led to the cleavage and subsequent recombination of the glycosidic bond at the C-26 position of protodioscin, resulting in the formation of dioscin present in the enzymatic hydrolysis filter residue. Leveraging this observation, a streamlined and eco-friendly method for diosgenin extraction was devised. Incorporating the Box-Behnken response surface methodology alongside wastewater assessment, the optimal parameters for the cleaning process were established: a sulfuric acid concentration of 3 mol · L−1, a solid–liquid ratio of 1:10, an acid hydrolysis temperature of 100 °C, and an acid hydrolysis duration of 3 h. Under these parameters, the yield and purity of diosgenin were 31.07±0.56% and 72.30±0.24% respectively. When benchmarked against the direct acid hydrolysis approach, there was an increase of 133.08% in diosgenin yield, 44.08% enhancement in diosgenin purity, 50% reduction in wastewater generation and acid utilization, and an 83.57% decrease in wastewater’s chemical oxygen demand (COD). This optimized cleaning process is viable for large-scale production and offers a sustainable method for diosgenin production.

Synthesis and characterization of SO3H-functionalized silica solid acid catalyst and application for the production of diosgenin

Synthesis and characterization of SO3H-functionalized silica solid acid catalyst and application for the production of diosgenin

The Functional Characterization of DzCYP72A12-4 Related to Diosgenin Biosynthesis and Drought Adaptability in Dioscorea zingiberensis.

Dioscorea zingiberensis is a perennial herb famous for the production of diosgenin, which is a valuable initial material for the industrial synthesis of steroid drugs. Sterol C26-hydroxylases, such as TfCYP72A616 and PpCYP72A613, play an important role in the diosgenin biosynthesis pathway. In the present study, a novel gene, DzCYP72A12-4, was identified as C26-hydroxylase and was found to be involved in diosgenin biosynthesis, for the first time in D. zingiberensis, using comprehensive methods. Then, the diosgenin heterogenous biosynthesis pathway starting from cholesterol was created in stable transgenic tobacco (Nicotiana tabacum L.) harboring DzCYP90B71(QPZ88854), DzCYP90G6(QPZ88855) and DzCYP72A12-4. Meanwhile, diosgenin was detected in the transgenic tobacco using an ultra-performance liquid chromatography system (Vanquish UPLC 689, Thermo Fisher Scientific, Bremen, Germany) tandem MS (Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer, Thermo Fisher Scientific, Bremen, Germany). Further RT-qPCR analysis showed that DzCYP72A12-4 was highly expressed in both rhizomes and leaves and was upregulated under 15% polyethylene glycol (PEG) treatment, indicating that DzCYP72A12-4 may be related to drought resistance. In addition, the germination rate of the diosgenin-producing tobacco seeds was higher than that of the negative controls under 15% PEG pressure. In addition, the concentration of malonaldehyde (MDA) was lower in the diosgenin-producing tobacco seedlings than those of the control, indicating higher drought adaptability. The results of this study provide valuable information for further research on diosgenin biosynthesis in D. zingiberensis and its functions related to drought adaptability.

Deciphering the network of cholesterol biosynthesis in Paris polyphylla laid a base for efficient diosgenin production in plant chassis

Cholesterol serves as a key precursor for many high-value chemicals such as plant-derived steroidal saponins and steroidal alkaloids, but a plant chassis for effective biosynthesis of high levels of cholesterol has not been established. Plant chassis have significant advantages over microbial chassis in terms of membrane protein expression, precursor supply, product tolerance, and regionalization synthesis. Here, using Agrobacterium tumefaciens-mediated transient expression technology, Nicotiana benthamiana, and a step-by-step screening approach, we identified nine enzymes (SSR1-3, SMO1-3, CPI-5, CYP51G, SMO2-2, C14-R-2, 8,7SI-4, C5-SD1, and 7-DR1-1) from the medicinal plant Paris polyphylla and established detailed biosynthetic routes from cycloartenol to cholesterol. Specfically, we optimized HMGR, a key gene of the mevalonate pathway, and co-expressed it with the PpOSC1 gene to achieve a high level of cycloartenol (28.79 mg/g dry weight, which is a sufficient amount of precursor for cholesterol biosynthesis) synthesis in the leaves of N. benthamiana. Subsequently, using a one-by-one elimination method we found that six of these enzymes (SSR1-3, SMO1-3, CPI-5, CYP51G, SMO2-2, and C5-SD1) were crucial for cholesterol production in N. benthamiana, and we establihed a high-efficiency cholesterol synthesis system with a yield of 5.63 mg/g dry weight. Using this strategy, we also discovered the biosynthetic metabolic network responsible for the synthesis of a common aglycon of steroidal saponin, diosgenin, using cholesterol as a substrate, obtaining a yield of 2.12 mg/g dry weight in N. benthamiana. Our study provides an effective strategy to characterize the metabolic pathways of medicinal plants that lack a system for in vivo functional verification, and also lays a foundation for the synthesis of active steroid saponins in plant chassis.

Efficient alcoholysis of total saponins in D. zingiberensis C. H. Wright with novel magnetic solid acids through a cleaner approach

From the perspective of full utilization of biomass, a clean approach for the production of diosgenin from total saponins in Dioscorea zingiberensis C.H. Wright (DZW) was realized by preliminary separation and extraction. An alcoholysis approach was adopted to obtain diosgenin using a novel magnetic solid acid Fe3O4 @SiO2-Pr-S-Pr-SO3H Pr = -(CH2)3-) as the catalyst. The prepared solid acid has been fully characterized. The single factor methodology was used to optimize the process parameters. The optimum conditions were: 0.50 g Fe3O4 @SiO2-Pr-S-Pr-SO3H, 6.0 mL ethanol, the reaction temperature 120 ℃, reaction time 7.0 h and the maximum yield of diosgenin was 24.18% which are higher than conventional hydrolysis with 1.5 M sulfuric acid indicating the high catalytic activity of the solid acids. In addition, the solid acid had good reusability. The LC-MS technique was applied to analyze the main steroidal saponins in total saponins and possible mechanisms and pathways of hydrolysis and alcoholysis processes. Alcoholysis by an as-prepared magnetic solid acid is clearly excellent in high efficiency and reusability. It is a promising strategy for the green production of diosgenin through a cleaner approach.

Ultrasound-assisted deep eutectic solvent/petroleum ether biphase system for the green and efficient production of diosgenin

Diosgenin, as an important precursor of steroid hormone drugs, is distributed in the root of Dioscorea Zingiberensis C. H. Wright (DZW) in the form of its sugar ligand-dioscin. The traditional production method of diosgenin is using sulfuric acid to hydrolyze the DZW, during the hydrolysis process, all components of the raw materials are degraded, resulting in a large amount of acid-containing wastewater with high Chemical Oxygen Demand (COD) values, causing serious pollution of the environment. In this paper, we proposed a biphase system composed of B acid type deep eutectic solvent (DES) and petroleum ether which can combine the extraction and hydrolysis of dioscin, and the purification of diosgenin into one step. The yield of diosgenin obtained by this method can reach 99.5% of that of the traditional method, while the COD value in the wastewater are less than 1% of the traditional method. The results showed that the best composition of DES is benzyltrimethylammonium chloride and oxalic acid, and the optimized parameters were material-to-liquid ratio 1:6 (w/v), ultrasonic power 200W, temperature 40 °C, ultrasonic time 15min and DES/petroleum ether ratio 1:6(w/v). After repeated use of the bi-phase system for 6 times, the production efficiency of diosgenin can still reach more than 95%, and the COD value of wastewater were stable. Furthermore, in order to better understand the advantages of this method, this method was compared with several other diosgenin production methods. In summary, the DES/petroleum ether biphase system proposed in this paper is an efficient and cleaner production method of diosgenin. • DES is simple to prepare and easy to recycle. • The DES/petroleum ether biphase system used in this study couples the production and purification of diosgenin into one step. • The yield and purity of diosgenin obtained in this process is comparable to that of the traditional method, while the COD value in the wastewater is less than 1% of the value of the traditional method.

Metabolic engineering of Saccharomyces cerevisiae for gram-scale diosgenin production

Diosgenin (DSG) is a naturally occurring steroidal saponin with a variety of biological activities that is also an important precursor for the synthesis of various steroidal drugs. The traditional industrial production of DSG is based on natural plant extraction and chemical processing. However, the whole process is time-consuming, laborious, and accompanied by severe environmental pollution. Therefore, it is necessary to develop a more convenient and environmentally-friendly process to realize the green production of DSG. In our previous work, we achieved de novo synthesis of DSG in Saccharomyces cerevisiae using glucose as the carbon source. However, DSG production was only at the milligram level, which is too low for industrial production. In this work, we further developed yeast strains for DSG overproduction by optimizing the synthesis pathway, fine-tuning pathway gene expression, and eliminating competing pathways. Cholesterol 22-hydroxylase was used to construct the DSG biosynthesis pathway. The optimal ratio of cytochrome P450 (CYP) to cytochrome P450 reductase (CPR) associated with DSG synthesis was screened to increase DSG production. Weakening the expression of the ERG6 gene further increased DSG synthesis and reduced the formation of by-products. In addition, we investigated the impact of DSG accumulation on yeast cell physiology and growth by transcriptome analysis and found that the multidrug transporter PDR5 and the sterol-binding protein PRY1 contributed to DSG production. Finally, we obtained a DSG titer of 2.03 g/L after 288 h of high-cell-density fed-batch fermentation using the engineered strain LP118, which represents the highest DSG titer reported to date for a yeast de novo synthesis system.

Alcoholysis approach for an efficient and cleaner production of diosgenin by low cost and green carbon based solid acids from biomass residues

Aiming to achieve more efficient and cleaner production of diosgenin, the waste liquid and residue in the process should be minimized or utilized. An alcoholysis approach was adopted to obtain diosgenin from zingiberensis C. H. Wright (DZW) using carbon-based solid acids as catalysts, which were prepared from the residues of the process via in situ carbonization and sulfonation. Solid acids were also subjected to a full range of physicochemical characterizations. The single factor and response surface methods were used to optimize the production process parameters of diosgenin. The best yield of hydrolysis with glycolic acid is 22.40%, which is 18.55% higher than traditional hydrolysis with sulfuric acid. The solid acids can be reused for 5 cycles with less loss of activity. Importantly, after a simple regeneration procedure, it can be further recycled another 5 times and maintain the same activity as before. Possible alcoholysis routes and mechanisms were analyzed by liquid chromatograph-mass spectrometer, which has an important application foreground in diosgenin production.

In vitro tuberization, genetic, and phytochemical fidelity assessment of Dioscorea deltoidea

Dioscorea deltoidea Wall. ex Griseb. (family Dioscoreaceae) has been traditionally used as a remedy for various human ailments. In modern medicine, it is used as an efficacious remedy against diabetes and cardiovascular diseases. Dioscorea deltoidea tubers are known to be a rich reservoir of diosgenin. A consistent and reproducible protocol is needed for the production of tubers for its conservation as well as for the production of diosgenin in vitro. Therefore, the current investigation was aimed to develop an in vitro protocol for tuberization as well as for production of diosgenin with genetic and phytochemical fidelity assessments. Murashige and Skoog (MS) medium fortified with 6-benzyl amino purine (BAP) exhibited tuberization in D. deltoidea; however BAP at 0.9 mg L−1 significantly increased the tuber number (5.8), weight (3.4% FW) and size (9.3 mm). In combinations, BAP (0.15 mg L−1) + naphthalene acetic acid (NAA) (1.0 mg L−1) + kinetin (Kn) (0.25 mg L−1) favoured tuberization in D. deltoidea cultures. Increased concentrations of sucrose (4–8%) in the optimized culture medium with 0.9 mg L−1 BAP had significant effects on tuberization. However, MS medium fortified with 6% sucrose and 0.9 mg L−1 BAP was found to be optimum for tuberization producing maximum tubers (7.5). Genetic fidelity of the regenerated bulbs were analysed using six inter simple sequence repeats (ISSR) markers. In vitro raised D. deltoidea plants exhibited dissimilar banding pattern to the mother plant thus demonstrated the genetic variability in the newly regenerated plants. A phytochemical analysis by liquid chromatography-mass spectrometry (LC-MS) showed diosgenin content in the range of 0.000571% DW to 0.000732% dry weight (DW) in in vitro raised tubers. The developed protocol for in vitro tuberization of D. deltoidea is cost-effective and can be proficiently used for the germplasm conservation as well as for industrial production of diosgenin.

Polymer‐based solid acid catalyst for the green production of diosgenin

AbstractIn this study, a novel polymer‐based solid acid catalyst was prepared by free radical polymerization of N, N′‐methylenebisacrylamide (MBA) and allyl sodium sulfonate (SAS), followed by acidifying of diluted hydrochloric acid. The prepared solid acid was fully characterized by FTIR, solid‐state 13C‐NMR, TGA, XPS, element analysis, and SEM. In addition, the prepared solid acid poly(MBA‐SAA) was used as a catalyst to extract diosgenin from Dioscorea zingiberensis C.H.Wright (DZW) under solvethermal condition using ethanol as a solvent. And the optimal conditions of temperature (104.02°C), alcoholysis time (5.87 h) and solid acid dosage (0.39 g), were obtained by response surface analysis based on the results of single factor experiments. The yield of diosgenin (14.53%) obtained in the alcoholysis is higher than that of the traditional sulfuric acid hydrolysis method. The alcoholysis products were analyzed by high performance liquid chromatography‐mass spectrometry (LC–MS) and UV–vis, respectively. The results showed that the solid acid with higher catalytic activity and good reusability can replace sulfuric acid to extract diosgenin, which has a great potential for the clean production of diosgenin in industry.

Efficient biocatalysis of trillin through recombinant enzyme hydrolysis for clean diosgenin production

Diosgenin has been widely used as a precursor in the pharmaceutical industry. The conventional diosgenin production method, direct acid hydrolysis, can cause severe environmental pollution and has low production efficiency. In this study, the β-glucosidase FBG1 was successfully over-expressed in Pichia pastoris. The engineered yeast was grown in 5- and 50-L bioreactors to a high-cell-density, and the resulting recombinant FBG1 without purification was directly used for trillin biocatalysis. A novel enzymatic biocatalysis method was established through efficient biocatalysis of trillin by recombinant FBG1 and a practical diosgenin purification process. Diosgenin production reached ∼1.7 mg/mL under optimized biocatalysis conditions with a corresponding ∼94 % trillin conversion. The production process was further scaled up from 2 mL to 20, 200, and 2000 mL working volumes, producing equivalent efficiency. This recombinant enzyme biocatalysis process resulted in ∼680 mg diosgenin when 1 g trillin was processed, together with ∼90 % environmental impact elimination and ∼36 % cost reduction. Enzymatic biocatalysis can convert trillin into a high-value-added pharmaceutical precursor of diosgenin with high eco-efficiency, cost-effectiveness, and sustainability is presented here for the first time and is a promising method for future applications in industrial processes.

Synthesis and characterization of magnetic solid acid Fe3O4@PEI@SO3H and application for the production of diosgenin by alcoholysis of turmeric saponins

• Fe 3 O 4 @PEI@SO 3 H was synthesized and used for the first time to extract diosgenin. • The FTIR, TGA, XRD, XPS, SEM, EDS, VSM, NH 3 -TPD of characterization for Fe3O4@PEI@SO3H. • The optimal conditions are obtained by single factor test and response surface method. • Catalytic pathways of alcoholysis were studied by LC-MS. The magnetic solid acid, Fe 3 O 4 @PEI@SO 3 H, was synthesized and used as a catalyst to transfer steroid saponins into diosgenin in an alcoholysis approach. The results showed that the magnetic solid acid greatly improved the alcoholysis efficiency for extraction diosgenin from steriod saponins. To optimize the parameters for alcoholysis, such as the mass of the solid acid, solvent volume, the reaction temperature and time, single factor experiments as well as response surface experiment have been performed. Under the optimal conditions, the diosgenin yield obtained by alcoholysis is higher than that of the traditional inorganic acid hydrolysis. And it worth to mention that no waste liquid is discharged. The catalyst is very stable and can be used for three times without obvious mass loss and activity decrease, indicating that the technology of extracting diosgenin from Dioscorea zingiberensis C.H. Wright tuber has a broad prospect in industrial application.

Bio-production enhancement of Natural diosgenin from Balanites Aegyptiacae by callus culture

In vitro the production of natural diosgenin from callus culture became an alternative biotechnological approach to diosgenin production. This study describes the effect of plant growth regulators (PGRs) on callus induction and using different elicitors in vitro to induce the production of (Diosgenin) in callus culture from different parts of Balanites Aegyptiacae and study the effect of diosgenin extract on different cell lines. Root, stem, leaves callus calture of B. aegyptiacae, were raised on Murashing and skoog Medium (MS) supplemented with both NAA (Naphthaline Acetic acid) and 2,4D (2,4 – Dichlorophenoxy acetic acid) as plant growth regulators at different concentration. Then Elicitation process was applied by adding different amino acids and coconut milk at different concentrations to culture medium. Medium of root which supplemented with 300mg/Ltryptophane gave the maximum diosgenin content in callus extract using spectrophotometric method and HPLC analysis. Then the cytotoxic activity of the root callus extract supplemented with tryptophan was determined against HepG2 (human hepatocellular carcinoma cell line), A549 (Human Lung Cancer cell line) and the result were 51.26 μm/ml, 28.9 μm/ml respectively. The bio safety of extract was detected on the normal human cells and the result was 287.3 μm/ml.

Pathway design and key enzyme analysis of diosgenin biosynthesis

As a naturally occurring steroid sapogenin, diosgenin acts as the precursor of hundreds of steroid medicines, and thereby has important medicinal value. Currently, industrial production of diosgenin relies primarily on chemical extraction from plant materials. Clearly, this strategy shows drawbacks of excessive reliance on plant materials and farmland as well as environment pollution. Due to development of metabolic engineering and synthetic biology, bio-production of diosgenin has garnered plenty of attention. Although the biosynthetic pathways of diosgenin have not been completely identified, in this review, we outline the identified biosynthetic pathways and key enzymes. In particular, we suggest heterologous biosynthesis of diosgenin in Saccharomyces cerevisiae. Overall, this review aims to provide valuable insights for future complete biosynthesis of diosgenin.

Screening and Selection of a New Medium for Diosgenin Production via Microbial Biocatalysis of Fusarium sp.

Steroidal saponins are widely used as starting precursors and medical intermediates for the semi-/total-synthesis of hundreds of steroidal drugs. One such steroidal saponin is diosgenin, which has attracted significant attention due to the huge market demand in the pharmaceutical industry. Due to water waste and severe environmental pollution, the traditional diosgenin production process based on direct acid hydrolysis is no longer used. In this study, to develop a submerged fermentation (SmF) medium for clean diosgenin production via efficient microbial biocatalysis, the Box–Behnken design (BBD) in combination with the Plackett–Burman design (PBD) was used to determine the medium compositions for Fusarium strains. Three components (wheat bran, phosphate, and Tween-80) were determined as significant factors by the PBD. Using the BBD, the three significant factors were further optimized, and the optimum values were determined for maximal diosgenin production. With 21.16 g/L of wheat bran, 9.60 g/L of phosphate, and 1.97 g/L of Tween-80, the diosgenin yield was 2.28%, i.e., 3.17 mg/L/h. The experimental values agreed with the predicted values, representing a significant increase in diosgenin production compared to its production using the basic SmF medium. For the first time, we reported the development of a new medium for Fusarium strains to produce diosgenin via microbial biocatalysis of the root of Dioscorea zingiberensis C. H. Wright (DZW). A simple-composition, low-cost, and high-efficiency medium was developed for the first time for the SmF of Fusarium strains. The medium is considered useful for large-scale SmF and may be applicable to other fungi. This study lays a solid foundation for diosgenin production in an acid-free and wastewater-free way. It may also provide fundamental support for producing other value-added products via microbial biocatalysis of low-value materials by endophytic fungi.