Semi-rational design based on the interaction between SmFMO and FAD isoalloxazine ring to enhance the enzyme activity.

Flavin monooxygenases (FMOs) have been widely used in the biosynthesis of natural compounds due to their excellent stereoselectivity, regioselectivity and chemoselectivity. Stenotrophomonas maltophilia flavin monooxygenase (SmFMO) has been reported to catalyze the oxidation of various thiols to corresponding sulfoxides, but its activity is relatively low. Herein, we obtained a mutant SmFMOF52G which showed 4.35-fold increase in kcat/Km (4.96 mM-1s-1) and 6.84-fold increase in enzyme activity (81.76 U/g) compared to the SmFMOWT (1.14 mM-1s-1 and 11.95 U/g) through semi-rational design guided by structural analysis and catalytic mechanism combined with high-throughput screening. By forming hydrogen bond with O4 atom of FAD isoalloxazine ring and reducing steric hindrance, the conformation of FAD isoalloxazine ring in SmFMOF52G is more stable, and NADPH and substrate are closer to FAD isoalloxazine ring, shortening the distances of hydrogen transfer and substrate oxygenation, thereby increasing the rate of reduction and oxidation reactions and enhancing enzyme activity. Additionally, the overall structural stability and substrate binding capacity of the SmFMOF52G have significant improved than that of SmFMOWT. The strategy used in this study to improve the enzyme activity of FMOs may have generality, providing important references for the rational and semi-rational engineering of FMOs.

A customized self-assembled synergistic biocatalyst for plastic depolymerization.

The enzymatic degradation of plastic offers a green, sustainable strategy and scalable circular carbon route for solving polyester waste. Among the earlies discovered plastic-degrading enzymes are PET hydrolase (PETase) and MHET hydrolase (MHETase), which act synergistically. To promote the adsorption of enzymes on PET surfaces, increase their robustness, and enable directly depolymerization, we designed hydrophobin HFBI fused-PETase and MHETase. A customized self-assembled synergistic biocatalyst (MC@CaZn-MOF) was further developed to promote the two-step depolymerization process. The tailored catalysts showed better adhesion to the PET surface and desirable durability, retaining over 70% relative activity after incubation at pH 8.0 and 60 °C for 120 h. Importantly, MC@CaZn-MOF could directly decompose untreated AGf-PET to generate 9.5 mM TPA with weight loss over 90%. The successful implementation of a bifunctional customized catalyst makes the large-scale biocatalytic degradation of PET feasible, contributing to polymer upcycling and environmental sustainability.

[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.

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.

[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.

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 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.

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 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.

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.

Structural Basis of Salicylic Acid Decarboxylase Reveals a Unique Substrate Recognition Mode and Access Channel.

Salicylic acid (SA) decarboxylase from Trichosporon moniliiforme (TmSdc), which reversibly catalyses the decarboxylation of SA to yield phenol, is of significant interest because of its potential for the production of benzoic acid derivatives under environmentally friendly reaction conditions. TmSdc showed a preference for C2 hydroxybenzoate derivatives, with kcat/Km of SA being 3.2 × 103 M-1 s-1. Here, we presented the first crystal structures of TmSdc, including a complex with SA. The three conserved residues of Glu8, His169, and Asp298 are the catalytic residues within the TIM-barrel domain of TmSdc. Trp239 forms a unique hydrophobic recognition site by interacting with the phenyl ring of SA, while Arg235 is responsible for recognizing the hydroxyl group at the C2 of SA via hydrogen bond interactions. Using a semi-rational combinatorial active-site saturation test, we obtained the TmSdc mutant MT3 (Y64T/P191G/F195V/E302D), which exhibited a 26.4-fold increase in kcat/Km with SA, reaching 8.4 × 104 M-1 s-1. Steered molecular dynamics simulations suggested that the structural changes in MT3 relieved the steric hindrance within the substrate access channel and enlarged the substrate-binding pocket, leading to the increased activity by improving substrate access. Our data elucidate the unique substrate recognition mode and the substrate entrance tunnel of SA decarboxylase.

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.

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.

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.

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.

Expression, Purification, Refolding, and Characterization of a Neverland Protein From Caenorhabditis elegans.

Steroid hormones that serve as vital compounds are necessary for the development and metabolism of a variety of organisms. The neverland (NVD) family genes encode the conserved Rieske-type oxygenases, which are accountable for the dehydrogenation during the synthesis and regulation of steroid hormones. However, the His-tagged NVD protein from Caenorhabditis elegans expresses as inclusion bodies in Escherichia coli BL21 (DE3). This bottleneck can be solved through refolding by urea or the introduction of a maltose-binding protein (MBP) tag at the N-terminus. Through further research on purification after the introduction of a MBP tag at the N-terminus, the CD measurement and fluorescence-based thermal shift assay indicated that MBP was favorable for the NVD proteins' solubility and stability, which may be beneficial for the large-scale manufacture of NVD protein for further research. The structural model contained the Rieske [2Fe-2S] domain and non-heme iron-binding motif, which were similar to 3-ketosteroid 9 α-hydroxylase.

Design of an efficient whole-cell biocatalyst for the production of hydroxyarginine based on a multi-enzyme cascade.

3-Hydroxyarginine (3-OH-Arg) is an important intermediate for the synthesis of viomycin, an important antibiotic for the clinical treatment of tuberculosis. An efficient strategy for 3-OH-Arg production based on protein engineering and recombinant whole-cell biocatalysis was demonstrated for the first time. To avoid challenging product separation due to the generation of α-ketoglutarate (α-KG) in the system, the molar ratio of the substrates L-Arg and L-Glu was optimized to ensure the efficient production of 3-OH-Arg as well as the complete consumption of α-KG. Through the establishment of a fed-batch process, 3-OH-Arg and succinic acid (SA) production reached to 9.9 g/L and 5.98 g/L after 36 h of reaction under the optimized conditions. This is the highest biosynthetic yield of 3-OH-Arg achieved to date, potentially offering a promising strategy for commercial production of hydroxylated amino acids.