State-of-the-art strategies and research advances for the biosynthesis of D-amino acids.

D-amino acids (D-AAs) are the enantiomeric counterparts of L-amino acids (L-AAs) and important functional factors with a wide variety of physiological activities and applications in the food manufacture industry. Some D-AAs, such as D-Ala, D-Leu, and D-Phe, have been favored by consumers as sweeteners and fragrances because of their unique flavor. The biosynthesis of D-AAs has attracted much attention in recent years due to their unique advantages. In this review, we comprehensively analyze the structure-function relationships, biosynthesis pathways, multi-enzyme cascade and whole-cell catalysis for the production of D-AAs. The state-of-the-art strategies, including immobilization, protein engineering, and high-throughput screening, are summarized. Future challenges and perspectives of strategies-driven by bioinformatics technologies and smart computing technologies, as well as enzyme immobilization, are also discussed. These new approaches will promote the commercial production and application of D-AAs in the food industry by optimizing the key enzymes for industrial biocatalysts.

Substantial Improvement of an Epimerase for the Synthesis of D-Allulose by Biosensor-Based High-Throughput Microdroplet Screening.

Biosynthesis of D-allulose has been achieved using ketose 3-epimerases (KEases), but its application is limited by poor catalytic performance. In this study, we redesigned a genetically encoded biosensor based on a D-allulose-responsive transcriptional regulator for real-time monitoring of D-allulose. An ultrahigh-throughput droplet-based microfluidic screening platform was further constructed by coupling with this D-allulose-detecting biosensor for the directed evolution of the KEases. Structural analysis of Sinorhizobium fredii D-allulose 3-epimerase (SfDAE) revealed that a highly flexible helix/loop region exposes or occludes the catalytic center as an essential lid conformation regulating substrate recognition. We reprogrammed SfDAE using structure-guided rational design and directed evolution, in which a mutant M3-2 was identified with 17-fold enhanced catalytic efficiency. Our research offers a paradigm for the design and optimization of a biosensor-based microdroplet screening platform.

Engineering of 3-ketosteroid-∆1-dehydrogenase based site-directed saturation mutagenesis for efficient biotransformation of steroidal substrates.

BACKGROUND: Biosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor. RESULTS: Recombinant KsdD3 exhibited organic solvent tolerance. W117, F296, W299, et al., which were located in substrate-binding cavity, were predicted to form hydrophobic interaction with the substrate. Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. W299A showed the highest increase in catalytic efficiency (kcat/Km) compared with the wild-type enzyme. Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. Steered molecular dynamics simulations revealed that W299A/G decreased the potential energy barrier of association of substrates and dissociation of the corresponding products. The biotransformation of AD with enzymatic catalysis and resting cells harbouring KsdD3 WT/mutants revealed that W299A catalyzed the maximum ADD yields of 71 and 95% by enzymatic catalysis and resting cell conversion respectively, compared with the wild type (38 and 75%, respectively). CONCLUSIONS: The successful rational design of functional KsdD3 greatly advanced our understanding of KsdD family enzymes. Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity.

Identification and characterization of the steroid 15α-hydroxylase gene from Penicillium raistrickii.

Penicillium raistrickii ATCC 10490 is used for the commercial preparation of 15α-13-methy-estr-4-ene-3,17-dione, a key intermediate in the synthesis of gestodene, which is a major component of third-generation contraceptive pills. Although it was previously shown that a cytochrome P450 enzyme in P. raistrickii is involved in steroid 15α-hydroxylation, the gene encoding the steroid 15α-hydroxylase remained unknown. In this study, we report the cloning and characterization of the 15α-hydroxylase gene from P. raistrickii ATCC 10490 by combining transcriptomic profiling with functional heterologous expression in Saccharomyces cerevisiae. The full-length open reading frame (ORF) of the 15α-hydroxylase gene P450pra is 1563 bp and predicted to encode a cytochrome P450 protein of 520 amino acids. Targeted gene deletion revealed that P450pra is solely responsible for 15α-hydroxylation activity on 13-methy-estr-4-ene-3,17-dione in P. raistrickii ATCC 10490. The identification of the 15α-hydroxylase gene from P. raistrickii should help elucidate the molecular basis of regio- and stereo-specificity of steroid 15α-hydroxylation and aid in the engineering of more efficient industrial strains for useful steroid 15α-hydroxylation reactions.

[Biosynthesis of calcifediol and calcitriol: a review].

VD3 is a crucial vitamin for human health, as it enhances calcium absorption in the intestines and prevent rickets. Calcifediol (25(OH)VD3) and calcitriol (1α,25(OH)2VD3) are two derivatives of vitamin D3 that play an important role in preventing and treating osteoporosis, as well as regulating human physiological functions. Currently, the production of calcifediol, and calcitriol primarily relies on chemical synthesis, which has disadvantages such as low product yield, numerous by-products, and environmental unfriendliness. Therefore, developing a green, safe, and environmentally friendly biocatalytic synthesis pathway is of utmost importance. This article mainly reviews the biocatalytic synthesis pathways of calcifediol, and calcitriol. The P450 enzymes, including P450 monooxygenases (cytochrome P450 monooxygenases, CYPs) and P450 peroxygenases (unspecific peroxygenases, UPOs), are crucial for the production of calcifediol and calcitriol. The catalytic mechanism of the extensively studied P450 monooxygenases, the selection of suitable redox partners, and the key residues involved in the enzyme's catalytic activity are analyzed. In addition, the review explores H2O2-driven UPOs, including their catalytic mechanism, strategies for high heterologous expression, and in situ regeneration of H2O2. UPOs are regarded as highly promising biocatalysts because they can facilitate reactions without the need for expensive cofactors and redox partners. This review offers insights into the engineering of P450 for the efficient production of vitamin D3 derivatives.

Production of 17α-hydroxyprogesterone using an engineered biocatalyst with efficient electron transfer and improved 5-aminolevulinic acid synthesis coupled with a P450 hydroxylase.

17α-Hydroxyprogesterone (17α-OH-PROG) is an important intermediate with a wide range of applications in the pharmaceutical industry. Strategies based on efficient electron transfer and cofactor regeneration were used for the production of 17α-OH-PROG. Here, CYP260A1, Fpr and Adx were expressed using a double plasmid system, resulting in higher biotransformation efficiency. Further optimization of reaction conditions and addition of polymyxin B increased the production of 17α-OH-PROG from 12.52 mg/L to 102.37 mg/L after 12 h of biotransformation. To avoid the addition of external 5-aminolevulinic acid (ALA) as a heme precursor for the P450 enzyme, a modified C5 pathway was introduced into the engineered strain, further reducing the overall process cost. The resulting whole-cell biocatalyst achieved the highest biotransformation yield of 17α-OH-PROG reported to date, offering a promising strategy for commercial application of P450 enzymes in industrial production of hydroxylated intermediates.

Construction of engineered Arthrobacter simplex with improved performance for cortisone acetate biotransformation.

Arthrobacter simplex 156 is a microorganism that is used for steroid drug biotransformation of cortisone acetate (CA) to prednisone acetate (PA). The enzyme 3-ketosteroid-△(1)-dehydrogenase encoded by the ksdD gene plays an important role in the bioconversion process. To further improve the biotransformation efficiencies of the industrial strain, a genetic manipulation system for A. simplex 156 was developed. Additional copies of the ksdD gene under the control of the cat promoter (from pXMJ19) were transferred into the strain A. simplex 156 and integrated into the 16S rDNA sites, yielding a series of recombinant strains. One of these recombinant strains, designated A. simplex M158, exhibited superior properties for CA biotransformation. At the substrate concentration of 83.6 g/l, the highest PA production of the recombinant strain reached 66.7 g/l, which is approximately 32.9 % higher than that of wild-type strains, and the incubation time for CA to PA bioconversion was reduced by 20 h. Southern blotting analysis of the recombinant strain indicated two copies of deregulated ksdD genes were integrated into the 16S rDNA sites, which means two of five 16S rRNA operons were insertionally disrupted in the recombinant strain. However, the disruption of the two 16S rRNA operons did not affect the growth rate of the recombinant strain, which survived and thrived under desired conditions. In addition, the new strain was genetically stable for more than 100 generations without the use of antibiotics for selection. These superior characteristics make the new strain more suitable than the wild-type strain for PA production.

[Adaptation of the electron transport chain improves the biocatalytic efficiency of progesterone 17α hydroxylation].

17α hydroxylase is a key enzyme for the conversion of progesterone to prepare various progestational drug intermediates. To improve the specific hydroxylation capability of this enzyme in steroid biocatalysis, the CYP260A1 derived from cellulose-mucilaginous bacteria Sorangium cellulosum Soce56 and the Fpr and bovine adrenal-derived Adx4-108 derived from Escherichia coli str. K-12 were used to construct a new electron transfer system for the conversion of progesterone. Selective mutation of CYP260A1 resulted in a mutant S276I with significantly enhanced 17α hydroxylase activity, and the yield of 17α-OH progesterone reached 58% after optimization of the catalytic system in vitro. In addition, the effect of phosphorylation of the ferredoxin Adx4-108 on 17α hydroxyl activity was evaluated using a targeted mutation technique, and the results showed that the mutation Adx4-108T69E transferred electrons to S276I more efficiently, which further enhanced the catalytic specificity in the C17 position of progesterone, and the yield of 17α-OH progesterone was eventually increased to 74%. This study provides a new option for the production of 17α-OH progesterone by specific transformation of bacterial-derived 17α hydroxylase, and lays a theoretical foundation for the industrial production of progesterone analogs using biotransformation method.

Advanced Marine Antifouling Hydrogels Based on 7-Amino-4-methylcoumarin Fluorescence Driven by Rare-Earth Phosphorescence.

At present, there are very few reports on the combination of phosphorescence and fluorescence in the field of pollution prevention. A composite antibacterial agent was designed to store energy by using the phosphorescence effect of rare earth oxides, emit light at night, and stimulate 7-amino-4-methylcoumarin to produce fluorescence and prevent algae from adhering. When complexed with PVA, it exhibited excellent characteristics as an all-weather autocatalytic phosphorescence-fluorescence antifouling hydrogel. The rare earth phosphorescent powder was prepared in a high-temperature tube furnace, coated with SiO2 on the surface for waterproofing, and then grafted with 7-amino-4-methylcoumarin to obtain a composite antibacterial agent with a phosphorescence-fluorescence effect. The composite antibacterial agent was added with PVA to obtain a hydrogel, which exhibited bactericidal rates of more than 99.98% against both Gram-positive and Gram-negative bacteria after 48 h. The results of fluorescence staining showed that the coverage rate of dead bacteria reached 41.6% after 24 h. The tensile strength of the antifouling hydrogel is up to 1.49 MPa, which is strong enough for real marine environments. Moreover, the algae coverage area of the composite hydrogel under natural light was only 2.7%, representing a 10-fold reduction compared with the control. The antifouling hydrogel has good antipollution and algae suppression performance, which is due to the fact that the rare earth phosphorescent powder when exposed to sunlight can provide a light source to stimulate 7-amino-4-methylcoumarin fluorescence at night and thereby prevent algae adhesion. After testing in the marine field and the real sea test when wrapped in a fishing net, the excellent antifouling performance was demonstrated. The functional hydrogel has great application potential in the protection of seawater-exposed structures, such as bridges and bay ports.

Sequence- and Structure-Based Mining of Thermostable D-Allulose 3-Epimerase and Computer-Guided Protein Engineering To Improve Enzyme Activity.

D-Allulose, a functional sweetener, can be synthesized from fructose using D-allulose 3-epimerase (DAEase). Nevertheless, a majority of the reported DAEases have inadequate stability under harsh industrial reaction conditions, which greatly limits their practical applications. In this study, big data mining combined with a computer-guided free energy calculation strategy was employed to discover a novel DAEase with excellent thermostability. Consensus sequence analysis of flexible regions and comparison of binding energies after substrate docking were performed using phylogeny-guided big data analyses. TtDAE from Thermogutta terrifontis was the most thermostable among 358 candidate enzymes, with a half-life of 32 h at 70 °C. Subsequently, structure-guided virtual screening and a customized strategy based on a combinatorial active-site saturation test/iterative saturation mutagenesis were utilized to engineer TtDAE. Finally, the catalytic activity of the M4 variant (P105A/L14C/T63G/I65A) was increased by 5.12-fold. Steered molecular dynamics simulations indicated that M4 had an enlarged substrate-binding pocket, which enhanced the fit between the enzyme and the substrate. The approach presented here, combining DAEases mining with further rational modification, provides guidance for obtaining promising catalysts for industrial-scale production.

Dietary administration of chitooligosaccharides to enhance growth, innate immune response and disease resistance of Trachinotus ovatus.

The present study was conducted to investigate the effects of dietary chitooligosaccharides (COS) supplementation on the innate immune response and protection against Vibrio harveyi infection in Trachinotus ovatus. A basal diet was supplemented with 0.0 (control), 2.0, 4.0 and 6.0 g COS kg(-1) to formulate four experimental diets. Each diet was randomly allocated to triplicate groups of fish in floating sea cages (1.5 × 1.0 × 2.0 m), and each cage was stocked with 80 fish (initial average weight 10.8 ± 0.05 g). After 8 weeks of feeding trial, Both the final weight and specific growth rate (SGR) significantly increased with increasing dietary COS levels up to 4.0 g kg(-1), whereas there were no significant differences for COS levels from 4.0 to 6.0 g kg(-1). A decreased feed conversion ratio (FCR) was observed with increasing dietary COS levels. The total leukocyte counts (WBC), differential leukocyte counts, respiratory burst activity, lysozyme and superoxide dismutase (SOD) activity were significantly increased with the increased levels of dietary COS (P < 0.05), and reached a maximum at level of 4.0 g kg(-1) COS. There were no significant differences in those immunological parameters between 4.0 and 6.0 g kg(-1) COS. Moreover, the dietary COS supplementation groups also exhibited a decrease in the cumulative symptom rates compared to the controls when challenged with V. harveyi. These results indicated that dietary intake containing COS could enhance the immune responses of fish and improve its resistance to infection by V. harveyi. Especially supplementation with 4.0 g kg(-1) COS to the fish for 56 days showed considerable improvement in the growth, survival and immune response of the fish.

15α-Hydroxylation of a steroid (13-ethyl-gon-4-en-3,17-dione) by Penicillium raistrickii in an ionic liquid/aqueous biphasic system.

Biphasic processes are used in whole-cell biotransformation to overcome the low water solubility of substrates and products as well as their inhibitory effects on the biocatalyst. Commercially available [NTf(2)]- and [PF(6)]-based ionic liquids (ILs) were used in a biphasic system for the 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione by Penicillium raistrickii. With the substrate at 5 g l(-1) and a volume ratio of IL to buffer, buffer pH and cell density at, 1:9, 6.5, 16.8 g(DW) l(-1), respectively, the 15α-hydroxylation of 13-ethyl-gon-4-en-3,17-dione was achieved with a yield of 70 % after 72 h using [BMIm][NTf(2)] in a 50 ml biphasic system. This is compared to a 30 % yield in a monophasic aqueous system. This suggests the potential industrial application of ILs-based biphasic systems for steroid biotransformation.

Engineering of a fungal steroid 11α-hydroxylase and construction of recombinant yeast for improved production of 11α-hydroxyprogesterone.

Cytochrome P450 enzyme CYP68J5 from filamentous fungus Aspergillus ochraceus is industrially used for selective C11α-hydroxylation of canrenone and progesterone. To improve its selectivity of C11α-hydroxylation for relevant steroid substrates, a sequence-based targeted mutagenesis combined with saturation mutagenesis was conducted to search for variants with improved hydroxylation reaction specificity toward progesterone and D-ethylgonendione. Recombinant yeast expressing triple mutant V64F/E65G/N66T showed significantly increased C11α-hydroxylation selectivity (85 % VS WT 69.7 %). Saturation mutagenesis of V64, E65 and N66 resulted in the identification of single mutant V64K with greatly enhanced 11α-hydroxylation specificity toward progesterone (90.6 % VS WT 69.7 %). Furthermore, mutant N66D showed significant enhanced selectivity of C11α-hydroxylation toward D-ethylgonendione (70.8 % VS WT 58 %). Evaluation of recombinant yeast over-expressing V64K for progesterone transformation in 50 mL scale resulted in product 11α-OH progesterone concentrations of 432.5 mg/L, a 30.2 % increase compared with the CYP68J5 control. Our results also reveal that V64, E65 and N66 are key residues of CYP68J5 influencing its selectivity of C11α-hydroxylation, thus offering opportunities for further engineering of CYP68J5 for expanded industrial applications.

Fine-Tuning of Carbon Flux and Artificial Promoters in Bacillus subtilis Enables High-Level Biosynthesis of d-Allulose.

d-Allulose is an attractive rare sugar that can be used as a low-calorie sweetener with significant health benefits. To meet the increasing market demands, it is necessary to develop an efficient and extensive microbial fermentation platform for the synthesis of d-allulose. Here, we applied a comprehensive systematic engineering strategy in Bacillus subtilis WB600 by introducing d-allulose 3-epimerase (DAEase), combined with the deactivation of fruA, levDEFG, and gmuE, to balance the metabolic network for the efficient production of d-allulose. This resulting strain initially produced 3.24 g/L of d-allulose with a yield of 0.93 g of d-allulose/g d-fructose. We further screened and obtained a suitable dual promoter combination and performed fine-tuning of its spacer region. After 64 h of fed-batch fermentation, the optimized engineered B. subtilis produced d-allulose at titers of 74.2 g/L with a yield of 0.93 g/g and a conversion rate of 27.6%. This d-allulose production strain is a promising platform for the industrial production of rare sugar.

Engineering of Acid-Resistant d-Allulose 3-Epimerase for Functional Juice Production.

d-Allulose, a rare sugar and functional sweetener, can be biosynthesized by d-allulose 3-isomerase (DAE). However, most of the reported DAEs exhibit poor resistance under acidic conditions, which severely limited their application. Here, surface charge engineering and random mutagenesis were used to construct a mutant library of CcDAE from Clostridium cellulolyticum H10, combined with high-throughput screening to identify mutants with high activity and resistance under acidic conditions. The mutant M3 (I114R/K123E/H209R) exhibited high activity (3.36-fold of wild-type) and acid resistance (10.6-fold of wild-type) at pH 4.5. The structure-function relationship was further analyzed by molecular dynamics (MD) simulations, which indicated that M3 had a higher number of hydrogen bonds and negative surface charges than the wild type. A multienzyme cascade system including M3 was used to convert high-calorie sugars in acidic juices, and functional juices containing 7.8-15.4 g/L d-allulose were obtained. Our study broadens the manufacture of functional foods containing d-allulose.

Efficient secretion expression of phospholipase D in Bacillus subtilis and its application in synthesis of phosphatidylserine by enzyme immobilization.

Transphosphatidylation catalyzed by phospholipase D has gained increasing attention for producing phosphatidylserine (PS), which can be used in functional food and medicine. In this study, we investigated the effects of six signal peptides on the secretion of PLD (PLDsa) from Streptomyces antibioticus TCCC 21059 in the food-grade GRAS bacterium Bacillus subtilis. It indicated that the optimal signal peptide DacB with an Ala-X-Ala sequence motif at the C-terminus showed the highest secretory expression ability, resulting in increased production of 2.84 U/mL PLDsa. Then PLDsa was immobilized on the epoxy-based carriers, and one of these carriers allowed PLDsa loading of up to 2.7 mg/g. The immobilized PLDsa was more stable over a wide range of pH value (4.5-7.5) and temperature (16 °C-60 °C) than free PLDsa. Subsequently, the synthesis of PS from soybean phosphatidylcholine (PC) was carried out in purely aqueous solution using immobilized PLDsa, leading to a high yield of 65%. The immobilized PLDsa catalyst maintained a relative PS production of 60% after 5 recycles. Notably, the use of toxic solvent was completely eliminated in the whole process, which would be more profitable for the application of PS.

Continuous Spectrophotometric Assay for High-Throughput Screening of Predominant d-Allulose 3-Epimerases.

d-Allulose is an attractive noncaloric sugar substitute with numerous health benefits, which can be biosynthesized by d-allulose 3-epimerases (DAEases). However, enzyme instability under harsh industrial reaction conditions hampered its practical applications. Here, we developed a continuous spectrophotometric assay (CSA) for the efficient analysis of d-allulose in a mixture. Furthermore, a high-throughput screening strategy for DAEases was developed using CSA by coupling DAEase with a NADH-dependent ribitol dehydrogenase, enabling high-throughput screening of DAEase variants with desired properties. The variant M15S/P40N/S209N exhibited a half-life of 22 h at 60 °C and an 8.7 °C increase of the T5060 value, with a 1.2-fold increase of activity. Structural modeling and molecular dynamics simulations indicated that the improvement of thermostability and activity was due to some new hydrogen bonds between chains at the dimer interface and between the residue and the substrate d-fructose. This work offers a robust tool and theoretical basis for the improvement of DAEases, which will benefit the enzymatic biosynthesis of d-allulose and promote its industrial application.

Enhancing the sustainability of KsdD as a biocatalyst for steroid transformation by immobilization on epoxy support.

The Δ1-dehydrogenation of 3-ketosteroid substrates is a crucial reaction in the production of steroids. Although 3-ketosteroid Δ1-dehydrogenase (KsdD) catalyzes this reaction with high efficiency and selectivity, the low stability and high cost of the purified enzyme catalyst have limited its industrial application. In this study, an epoxy support was used to evaluate the covalent immobilization of KsdD from Pimelobacter simplex, and the best androsta-1,4-diene-317-dione (ADD) production was achieved after optimized immobilization of KsdD enzyme in 1.5 M NaH2PO4- Na2HPO4 buffer (pH 6.5) for 12 h at 25 °C. The immobilized KsdD exhibited higher tolerance toward 20 % methanol. The dehydrogenation reaction reached a conversion efficiency of up to 90.0 % in 2 h when using 0.6 mg/mL of 4-androstene-317-dione (AD). The W299A and W299 G mutants of KsdD were also immobilized, and both showed the better catalytic performance with higher kcat/KM values compared with the wild type (WT). The immobilized W299A, W299 G and WT KsdD respectively maintained 70.5, 65.7 and 38.7 % of their initial activity at the end of 15 reaction cycles. Furthermore, the W299A retained 66.3 % of the initial activity after 30 days of incubation at 4 °C, and was more stable than free KsdD, Thus, the immobilized W299A is a promising biocatalyst for steroid dehydrogenation. In this study, we investigated the application of immobilized enzymes for the dehydrogenation of steroids, which will be of great importance for improving the development of green technology and sustainable use of biocatalysts in the steroid manufacturing industry.

Improving the enzyme property of D-allulose 3-epimerase from a thermophilic organism of Halanaerobium congolense through rational design.

The rare sugar d-allulose is an attractive sucrose substitute due to its sweetness and ultra-low caloric value. It can be produced from D-fructose using d-allulose 3-epimerase (DAE) as the biocatalyst. However, most of the reported DAEs show low catalytic efficiency and poor thermostability, which limited their further use in food industrial. Here, a putative d-allulose 3-epimerase from a thermophilic organism of Halanaerobium congolense (HcDAE) was characterized, showing optimal activity at pH 8.0 and 70 °C in the presence of Mg2+. Saturation mutagenesis of Y7, C66, and I108, the putative residues responsible for substrate recognition at the O-4, -5, and -6 atoms of D-fructose was performed, and it yielded the triple mutant Y7H/C66L/I108A with improved activity toward D-fructose (345 % of wild-type enzyme). The combined mutant Y7H/C66L/I108A/R156C/K260C exhibited a half-half (t1/2) of 5.2 h at 70 °C and an increase of the Tm value by 6.5 °C due to the introduction of disulfide bridges between intersubunit with increased interface interactions. The results indicate that mutants could be used as industrial biocatalysts for d-allulose production.

Customized exogenous ferredoxin functions as an efficient electron carrier.

Ferredoxin (Fdx) is regarded as the main electron carrier in biological electron transfer and acts as an electron donor in metabolic pathways of many organisms. Here, we screened a self-sufficient P450-derived reductase PRF with promising production yield of 9OHAD (9α-hydroxy4-androstene-3,17-dione) from AD, and further proved the importance of [2Fe-2S] clusters of ferredoxin-oxidoreductase in transferring electrons in steroidal conversion. The results of truncated Fdx domain in all oxidoreductases and mutagenesis data elucidated the indispensable role of [2Fe-2S] clusters in the electron transfer process. By adding the independent plant-type Fdx to the reaction system, the AD (4-androstene-3,17-dione) conversion rate have been significantly improved. A novel efficient electron transfer pathway of PRF + Fdx + KshA (KshA, Rieske-type oxygenase of 3-ketosteroid-9-hydroxylase) in the reaction system rather than KshAB complex system was proposed based on analysis of protein-protein interactions and redox potential measurement. Adding free Fdx created a new conduit for electrons to travel from reductase to oxygenase. This electron transfer pathway provides new insight for the development of efficient exogenous Fdx as an electron carrier.