Combinatorial metabolic engineering of Bacillus subtilis for menaquinone-7 biosynthesis.

Menaquinone-7 (MK-7), a form of vitamin K2, supports bone health and prevents arterial calcification. Microbial fermentation for MK-7 production has attracted widespread attention because of its low cost and short production cycles. However, insufficient substrate supply, unbalanced precursor synthesis, and low catalytic efficiency of key enzymes severely limited the efficiency of MK-7 synthesis. In this study, utilizing Bacillus subtilis BSAT01 (with an initial MK-7 titer of 231.0 mg/L) obtained in our previous study, the glycerol metabolism pathway was first enhanced to increase the 3-deoxy-arabino-heptulonate 7-phosphate (DHAP) supply, which led to an increase in MK-7 titer to 259.7 mg/L. Subsequently, a combination of knockout strategies predicted by the genome-scale metabolic model etiBsu1209 was employed to optimize the central carbon metabolism pathway, and the resulting strain showed an increase in MK-7 production from 259.7 to 318.3 mg/L. Finally, model predictions revealed the methylerythritol phosphate pathway as the major restriction pathway, and the pathway flux was increased by heterologous introduction (Introduction of Dxs derived from Escherichia coli) and fusion expression (End-to-end fusion of two enzymes by a linker peptide), resulting in a strain with a titer of 451.0 mg/L in a shake flask and 474.0 mg/L in a 50-L bioreactor. This study achieved efficient MK-7 synthesis in B. subtilis, laying the foundation for large-scale MK-7 bioproduction.

Nicotinamide N-Methyltransferase (NNMT): A New Hope for Treating Aging and Age-Related Conditions.

The complex process of aging leads to a gradual deterioration in the function of cells, tissues, and the entire organism, thereby increasing the risk of disease and death. Nicotinamide N-methyltransferase (NNMT) has attracted attention as a potential target for combating aging and its related pathologies. Studies have shown that NNMT activity increases over time, which is closely associated with the onset and progression of age-related diseases. NNMT uses S-adenosylmethionine (SAM) as a methyl donor to facilitate the methylation of nicotinamide (NAM), converting NAM into S-adenosyl-L-homocysteine (SAH) and methylnicotinamide (MNA). This enzymatic action depletes NAM, a precursor of nicotinamide adenine dinucleotide (NAD+), and generates SAH, a precursor of homocysteine (Hcy). The reduction in the NAD+ levels and the increase in the Hcy levels are considered important factors in the aging process and age-related diseases. The efficacy of RNA interference (RNAi) therapies and small-molecule inhibitors targeting NNMT demonstrates the potential of NNMT as a therapeutic target. Despite these advances, the exact mechanisms by which NNMT influences aging and age-related diseases remain unclear, and there is a lack of clinical trials involving NNMT inhibitors and RNAi drugs. Therefore, more in-depth research is needed to elucidate the precise functions of NNMT in aging and promote the development of targeted pharmaceutical interventions. This paper aims to explore the specific role of NNMT in aging, and to evaluate its potential as a therapeutic target.

Nicotinamide N-methyltransferase (NNMT): a novel therapeutic target for metabolic syndrome.

Metabolic syndrome (MetS) represents a constellation of metabolic abnormalities, typified by obesity, hypertension, hyperglycemia, and hyperlipidemia. It stems from intricate dysregulations in metabolic pathways governing energy and substrate metabolism. While comprehending the precise etiological mechanisms of MetS remains challenging, evidence underscores the pivotal roles of aberrations in lipid metabolism and insulin resistance (IR) in its pathogenesis. Notably, nicotinamide N-methyltransferase (NNMT) has recently surfaced as a promising therapeutic target for addressing MetS. Single nucleotide variants in the NNMT gene are significantly correlated with disturbances in energy metabolism, obesity, type 2 diabetes (T2D), hyperlipidemia, and hypertension. Elevated NNMT gene expression is notably observed in the liver and white adipose tissue (WAT) of individuals with diabetic mice, obesity, and rats afflicted with MetS. Knockdown of NNMT elicits heightened energy expenditure in adipose and hepatic tissues, mitigates lipid accumulation, and enhances insulin sensitivity. NNMT catalyzes the methylation of nicotinamide (NAM) using S-adenosyl-methionine (SAM) as the donor methyl group, resulting in the formation of S-adenosyl-l-homocysteine (SAH) and methylnicotinamide (MNAM). This enzymatic process results in the depletion of NAM, a precursor of nicotinamide adenine dinucleotide (NAD+), and the generation of SAH, a precursor of homocysteine (Hcy). Consequently, this cascade leads to reduced NAD+ levels and elevated Hcy levels, implicating NNMT in the pathogenesis of MetS. Moreover, experimental studies employing RNA interference (RNAi) strategies and small molecule inhibitors targeting NNMT have underscored its potential as a therapeutic target for preventing or treating MetS-related diseases. Nonetheless, the precise mechanistic underpinnings remain elusive, and as of yet, clinical trials focusing on NNMT have not been documented. Therefore, further investigations are warranted to elucidate the intricate roles of NNMT in MetS and to develop targeted therapeutic interventions.

[The expression and secretion of human lactoferrin in Bacillus subtilis].

Human lactoferrin (HLF), an essential nutrient found in breast milk, possesses antibacterial, anti-inflammatory, and immune-enhancing properties. In this study, the effects of three constitutive promoters (P21, P43, and Pveg) and three inducible promoters (Pgrac100, PxylA, and Ptet*) on the expression of HLF were compared using Bacillus subtilis G601 as the host strain. The results showed that the highest expression of HLF, reaching 651.57 µg/L, was achieved when regulated by the Ptet* promoter. Furthermore, the combinational optimization of ribosome binding site (RBS) and signal peptides was investigated, and the optimal combination of RBS6 and SPyycP resulted in increased HLF expression to 1 099.87 µg/L, with 498.68 µg/L being secreted extracellularly. To further enhance HLF secretion, the metal cations-related gene dltD was knocked out, leading to an extracellular HLF level of 637.28 µg/L. This study successfully demonstrated the secretory expression of HLF in B. subtilis through the selection and optimization of expression elements, laying the foundation for the development of efficient B. subtilis cell factories for lactoprotein synthesis.

Combinatorial Engineering of Escherichia coli for Enhancing 3-Fucosyllactose Production.

3-Fucosyllactose (3-FL) is an important fucosylated human milk oligosaccharide (HMO) with biological functions such as promoting immunity and brain development. Therefore, the construction of microbial cell factories is a promising approach to synthesizing 3-FL from renewable feedstocks. In this study, a combinatorial engineering strategy was used to achieve efficient de novo 3-FL production in Escherichia coli. α-1,3-Fucosyltransferase (futM2) from Bacteroides gallinaceum was introduced into E. coli and optimized to create a 3-FL-producing chassis strain. Subsequently, the 3-FL titer increased to 5.2 g/L by improving the utilization of the precursor lactose and down-regulating the endogenous competitive pathways. Furthermore, a synthetic membraneless organelle system based on intrinsically disordered proteins was designed to spatially regulate the pathway enzymes, producing 7.3 g/L 3-FL. The supply of the cofactors NADPH and GTP was also enhanced, after which the 3-FL titer of engineered strain E26 was improved to 8.2 g/L in a shake flask and 10.8 g/L in a 3 L fermenter. In this study, we developed a valuable approach for constructing an efficient 3-FL-producing cell factory and provided a versatile workflow for other chassis cells and HMOs.

Developing a novel heme biosensor to produce high-active hemoproteins in Pichia pastoris through comparative transcriptomics.

The development of a heme-responsive biosensor for dynamic pathway regulation in eukaryotes has never been reported, posing a challenge for achieving the efficient synthesis of multifunctional hemoproteins and maintaining intracellular heme homeostasis. Herein, a biosensor containing a newly identified heme-responsive promoter, CRISPR/dCas9, and a degradation tag N-degron was designed and optimized to fine-tune heme biosynthesis in the efficient heme-supplying Pichia pastoris P1H9 chassis. After identifying literature-reported promoters insensitive to heme, the endogenous heme-responsive promoters were mined by transcriptomics, and an optimal biosensor was screened from different combinations of regulatory elements. The dynamic regulation pattern of the biosensor was validated by the transcriptional fluctuations of the HEM2 gene involved in heme biosynthesis and the subsequent responsive changes in intracellular heme titers. We demonstrate the efficiency of this regulatory system by improving the production of high-active porcine myoglobin and soy hemoglobin, which can be used to develop artificial meat and artificial metalloenzymes. Moreover, these findings can offer valuable strategies for the synthesis of other hemoproteins.

Efficient biosynthesis of active hemoglobins through enhancing the import of heme in Saccharomyces cerevisiae.

Hemoglobins, with heme as a cofactor, are functional proteins that have extensive applications in the fields of artificial oxygen carriers and foods. Although Saccharomyces cerevisiae is an ideal host for hemoglobin synthesis, it lacks a suitable transport system to utilize additional heme for active expression of hemoglobins, resulting in the cellular aggregation and degradation of the latter. Here, an effective heme importer, heme-responsive gene 4 (Hrg-4), was selected from six candidates through the comparison of effects on the growth rates of Δhem1 S. cerevisiae strain and the activities of various hemoglobins when supplemented with 5 mg·L-1 exogenous heme. Additionally, to counter the instability of plasmid-based expression and the metabolic burden introduced from overexpressing Hrg-4, a series of hrg-4 integrated strains were constructed and the best engineered strain with five copies of hrg-4 was chosen. We found that this engineered strain was associated with an increased binding rate of heme in monomeric leghemoglobin and multimeric human hemoglobin (76.3% and 16.5%, respectively), as well as an enhanced expression of both hemoglobins (52.8% and 17.0%, respectively). Thus, the engineered strain with improved heme uptake can be used to efficiently synthesize other heme-binding proteins and enzymes in S. cerevisiae.

CRISPR/Cas9-Based Genome Editing for Protein Expression and Secretion in Kluyveromyces lactis.

The budding yeast Kluyveromyces lactis has emerged as a promising microbial chassis in industrial biotechnology. However, a lack of efficient molecular genetic manipulation tools and strategies has hindered the development of K. lactis as a biomanufacturing platform. In this study, we developed and applied a CRISPR/Cas9-based genome editing method to K. lactis. Single-gene editing efficiency was increased to 80% by disrupting the nonhomologous end-joining-related gene KU80 and performing a series of process optimizations. Subsequently, the CRISPR/Cas9 system was explored based on different sgRNA delivery modes for simultaneous multigene editing. With the aid of the color indicator, the editing efficiencies of two and three genes reached 73.3 and 36%, respectively, in the KlΔKU80 strain. Furthermore, the CRISPR/Cas9 system was used for multisite integration to enhance lactase production and combinatorial knockout of TMED10 and HSP90 to characterize the extracellular secretion of lactase in K. lactis. Generally, genome editing is a powerful tool for constructing K. lactis cell factories for protein and chemical production.

The modification of heme special importer to improve the production of active hemoglobins in Escherichia coli.

To enhance the import of heme for the production of active hemoproteins in Escherichia coli C41 (DE3) lacking the special heme import system, heme receptor ChuA from E. coli Nissle 1917 was modified through molecular docking and the other components (ChuTUV) for heme import was overexpressed, while heme import was tested through growth assay and heme sensor HS1 detection. A ChuA mutant G360K was selected, which could import 3.91 nM heme, compared with 2.92 nM of the wild-type ChuA. In addition, it presented that the expression of heme transporters ChuTUV was not necessary for heme import. Based on the modification of ChuA (G360K), the titer of human hemoglobin and the peroxidase activity of leghemoglobin reached 1.19 µg g-1 DCW and 24.16 103 U g-1 DCW, compared with 1.09 µg g-1 DCW and 21.56 103 U g-1 DCW of the wild-type ChuA, respectively. Heme import can be improved through the modification of heme receptor and the engineered strain with improved heme import has a potential to efficiently produce high-active hemoproteins.

High-activity recombinant human carboxypeptidase B expression in Pichia pastoris through rational protein engineering and enhancing secretion from the Golgi apparatus to the plasma membrane.

Human carboxypeptidase B1 (hCPB1) is vital for recombinant insulin production, holding substantial value in the pharmaceutical industry. Current challenges include limited hCPB1 enzyme activity. In this study, recombinant hCPB1 efficient expression in Pichia pastoris was achieved. To enhance hCPB1 secretion, we conducted signal peptides screening and deleted the Vps10 sortilin domain, reducing vacuolar mis-sorting. Overexpression of Sec4p increased the fusion of secretory vesicles with the plasma membrane and improved hCPB1 secretion by 20%. Rational protein engineering generated twenty-two single-mutation mutants and identified the A178L mutation resulted in a 30% increase in hCPB1 specific activity. However, all combinational mutations that increased specific activities decreased protein expression levels. Therefore, computer-aided global protein design with PROSS was employed for the aim of improving specific activities and preserving good protein expression. Among the six designed mutants, hCPB1-P6 showed a remarkable 114% increase in the catalytic rate constant (kcat), a 137% decrease in the Michaelis constant (Km), and a 490% increase in catalytic efficiency. Most mutations occurred on the surface of hCPB1-P6, with eight sites mutated to proline. In a 5 L fermenter, hCPB1-P6 was produced by the secretion-enhanced P. pastoris chassis to 199.6 ± 20 mg L-1 with a specific activity of 96 ± 0.32 U mg-1, resulting in a total enzyme activity of 19137 ± 1131 U L-1, demonstrating significant potential for industrial applications.

Combinatorial Metabolic Engineering for Improving Betulinic Acid Biosynthesis in Saccharomyces cerevisiae.

Betulinic acid (BA) is a lupane-type triterpenoid with potent anticancer and anti-HIV activities. Its great potential in clinical applications necessitates the development of an efficient strategy for BA synthesis. This study attempted to achieve efficient BA biosynthesis in Saccharomyces cerevisiae using systematic metabolic engineering strategies. First, a de novo BA biosynthesis pathway in S. cerevisiae was constructed, which yielded a titer of 14.01 ± 0.21 mg/L. Then, by enhancing the BA synthesis pathway and dynamic inhibition of the competitive pathway, a greater proportion of the metabolic flow was directed toward BA synthesis, achieving a titer of 88.07 ± 5.83 mg/L. Next, acetyl-CoA and NADPH supply was enhanced, which increased the BA titer to 166.43 ± 1.83 mg/L. Finally, another BA synthesis pathway in the peroxisome was constructed. Dual regulation of the peroxisome and cytoplasmic metabolism increased the BA titer to 210.88 ± 4.76 mg/L. Following fed-batch fermentation process modification, the BA titer reached 682.29 ± 8.16 mg/L. Overall, this work offers a guide for building microbial cell factories that are capable of producing terpenoids with efficiency.

Computer-Aided Semi-Rational Design to Enhance the Activity of l-Sorbosone Dehydrogenase from Gluconobacter oxidans WSH-004.

The titer of the microbial fermentation products can be increased by enzyme engineering. l-Sorbosone dehydrogenase (SNDH) is a key enzyme in the production of 2-keto-l-gulonic acid (2-KLG), which is the precursor of vitamin C. Enhancing the activity of SNDH may have a positive impact on 2-KLG production. In this study, a computer-aided semirational design of SNDH was conducted. Based on the analysis of SNDH's substrate pocket and multiple sequence alignment, three modification strategies were established: (1) expanding the entrance of SNDH's substrate pocket, (2) engineering the residues within the substrate pocket, and (3) enhancing the electron transfer of SNDH. Finally, mutants S453A, L460V, and E471D were obtained, whose specific activity was increased by 20, 100, and 10%, respectively. In addition, the ability of Gluconobacter oxidans WSH-004 to synthesize 2-KLG was improved by eliminating H2O2. This study provides mutant enzymes and metabolic engineering strategies for the microbial-fermentation-based production of 2-KLG.

Facilitating stable gene integration expression and copy number amplification in Bacillus subtilis through a reversible homologous recombination switch.

Strengthening the expression level of integrated genes on the genome is crucial for consistently expressing key enzymes in microbial cell factories for efficient bioproduction in synthetic biology. In comparison to plasmid-based multi-copy expression, the utilization of chromosomal multi-copy genes offers increased stability of expression level, diminishes the metabolic burden on host cells, and enhances overall genetic stability. In this study, we developed the "BacAmp", a stabilized gene integration expression and copy number amplification system for high-level expression in Bacillus subtilis, which was achieved by employing a combination of repressor and non-natural amino acids (ncAA)-dependent expression system to create a reversible switch to control the key gene recA for homologous recombination. When the reversible switch is turned on, genome editing and gene amplification can be achieved. Subsequently, the reversible switch was turned off therefore stabilizing the gene copy number. The stabilized gene amplification system marked by green fluorescent protein, achieved a 3-fold increase in gene expression by gene amplification and maintained the average gene copy number at 10 after 110 generations. When we implemented the gene amplification system for the regulation of N-acetylneuraminic acid (NeuAc) synthesis, the copy number of the critical gene increased to an average of 7.7, which yielded a 1.3-fold NeuAc titer. Our research provides a new avenue for gene expression in synthetic biology and can be applied in metabolic engineering in B. subtilis.

Enhancing Acid Resistance of Aspergillus niger Pectin Lyase through Surface Charge Design for Improved Application in Juice Clarification.

Pectin lyases (PNLs) can enhance juice clarity and flavor by degrading pectin in highly esterified fruits, but their inadequate acid resistance leads to rapid activity loss in juice. This study aimed to improve the acid resistance of Aspergillus niger PNL pelA through surface charge design. A modification platform was established by fusing pelA with a protein tag and expressing the fusion enzyme in Escherichia coli. Four single-point mutants were identified to increase the surface charge using computational tools. Moreover, the combined mutant M6 (S514D/S538E) exhibited 99.8% residual activity at pH 3.0. The M6 gene was then integrated into the A. niger genome using a multigene integration system to obtain the recombinant PNL AM6. Notably, AM6 improved the light transmittance of orange juice to 45.3%, which was 8.39 times higher than that of pelA. In conclusion, AM6 demonstrated the best-reported acid resistance, making it a promising candidate for industrial juice clarification.

Constructing an Antibiotic-Free Protein Expression System for Ovalbumin Biosynthesis in Probiotic Escherichia coli Nissle 1917.

Ovalbumin (OVA) is the principal protein constituent of eggs. As an alternative to eggs, cell-cultured OVA can reduce the environmental impact of global warming and land use. Escherichia coli Nissle 1917 (EcN), a probiotic with specific endogenous cryptic plasmids that stably exist in cells without the addition of antibiotics, was chosen as the host for the efficient heterologous expression of the OVA. OVA yield reached 20 mg·L-1 in shake flasks using the OVA expression cassette containing a tac promoter (Ptac) upstream of the OVA-coding sequences on the endogenous plasmid pMUT2. Subsequently, we improved the level of the expression of the OVA by employing a dual promoter (PP5 combined with Ptac via a sigma factor binding site 24) and ribosome binding site (RBS) substitution. These enhancements increased the level of production of OVA in shake flasks to 30 and 42 mg·L-1, respectively. OVA by EcNP-P28 harboring plasmid L28 equipped with both dual promoter and the strong RBS8 reached 3.70 g·L-1 in a 3 L bioreactor. Recombinant OVA and natural OVA showed similar biochemical characteristics, including secondary structure, isoelectric point, amino acid composition, and thermal stability. This is currently the highest OVA production reported among prokaryotes. We successfully constructed an antibiotic-free heterologous protein expression system for EcN.

Engineering Redox Cofactor Balance for Improved 5-Methyltetrahydrofolate Production in Escherichia coli.

5-Methyltetrahydrofolate (5-MTHF) is the sole active form of folate functioning in the human body and is widely used as a nutraceutical. Unlike the pollution from chemical synthesis, microbial synthesis enables green production of 5-MTHF. In this study, Escherichia coli BL21 (DE3) was selected as the host. Initially, by deleting 6-phosphofructokinase 1 and overexpressing glucose-6-phosphate 1-dehydrogenase and 6-phosphogluconate dehydrogenase, the glycolysis pathway flux decreased, while the pentose phosphate pathway flux enhanced. The ratios of NADH/NAD+ and NADPH/NADP+ increased, indicating elevated NAD(P)H supply. This led to more folate being reduced and the successful accumulation of 5-MTHF to 44.57 µg/L. Subsequently, formate dehydrogenases from Candida boidinii and Candida dubliniensis were expressed, which were capable of catalyzing the reaction of sodium formate oxidation for NAD(P)H regeneration. This further increased the NAD(P)H supply, leading to a rise in 5-MTHF production to 247.36 µg/L. Moreover, to maintain the balance between NADH and NADPH, pntAB and sthA, encoding transhydrogenase, were overexpressed. Finally, by overexpressing six key enzymes in the folate to 5-MTHF pathway and employing fed-batch cultivation in a 3 L fermenter, strain Z13 attained a peak 5-MTHF titer of 3009.03 µg/L, the highest level reported in E. coli so far. This research is a significant step toward industrial-scale microbial 5-MTHF production.

Metabonomics analysis of the flavor characteristics of Wuyi Rock Tea (Rougui) with "rock flavor" and microbial contributions to the flavor.

Wuyi Rock Tea (WRT) has different characteristics of "rock flavor" due to different production areas. In this study, we investigated the flavor characteristics and key components of "rock flavor" and the influence of microorganisms on the substances by combining metabolomics and microbiomics with the Rougui WRTs from the Zhengyan, Banyan, and Waishan production areas. The results showed that Rougui has a strong floral and fruity aroma, which is mainly brought by hotrienol, and the sweet, smooth, and fresh taste is composed of epicatechin gallate, epigallocatechin, epigallocatechin gallate, caffeine, theanine, soluble sugar, and sweet and bitter amino acids. Bacteria Chryseobacterium, Pedobacter, Bosea, Agrobacterium, Stenotrophomonas, and Actinoplanes mainly influence the production of hotrienol, epicatechin gallate, and theanine. Fungi Pestalotiopsis, Fusarium, Elsinoe, Teichospora and Tetracladium mainly influence the production of non-volatile compounds. This study provides a reference for the biological formation mechanism of the characteristic aroma of WRT's "rock falvor".

Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor.

Mutagenesis driving genetic diversity is vital for understanding and engineering biological systems. However, the lack of effective methods to generate in-situ mutagenesis in multiple genomic loci combinatorially limits the study of complex biological functions. Here, we design and construct MultiduBE, a dCas12a-based multiplexed dual-function base editor, in an all-in-one plasmid for performing combinatorial in-situ mutagenesis. Two synthetic effectors, duBE-1a and duBE-2b, are created by amalgamating the functionalities of cytosine deaminase (from hAPOBEC3A or hAID*Δ ), adenine deaminase (from TadA9), and crRNA array processing (from dCas12a). Furthermore, introducing the synthetic separator Sp4 minimizes interference in the crRNA array, thereby facilitating multiplexed in-situ mutagenesis in both Escherichia coli and Bacillus subtilis. Guided by the corresponding crRNA arrays, MultiduBE is successfully employed for cell physiology reprogramming and metabolic regulation. A novel mutation conferring streptomycin resistance has been identified in B. subtilis and incorporated into the mutant strains with multiple antibiotic resistance. Moreover, surfactin and riboflavin titers of the combinatorially mutant strains improved by 42% and 15-fold, respectively, compared with the control strains with single gene mutation. Overall, MultiduBE provides a convenient and efficient way to perform multiplexed in-situ mutagenesis.

Reshaping Phosphatase Substrate Preference for Controlled Biosynthesis Using a "Design-Build-Test-Learn" Framework.

Biosynthesis is the application of enzymes in microbial cell factories and has emerged as a promising alternative to chemical synthesis. However, natural enzymes with limited catalytic performance often need to be engineered to meet specific needs through a time-consuming trial-and-error process. This study presents a quantum mechanics (QM)-incorporated design-build-test-learn (DBTL) framework to rationally design phosphatase BT4131, an enzyme with an ambiguous substrate spectrum involved in N-acetylglucosamine (GlcNAc) biosynthesis. First, mutant M1 (L129Q) is designed using force field-based methods, resulting in a 1.4-fold increase in substrate preference (kcat/Km) toward GlcNAc-6-phosphate (GlcNAc6P). QM calculations indicate that the shift in substrate preference is caused by a 13.59 kcal mol-1 reduction in activation energy. Furthermore, an iterative computer-aided design is conducted to stabilize the transition state. As a result, mutant M4 (I49Q/L129Q/G172L) with a 9.5-fold increase in kcat-GlcNAc6P/Km-GlcNAc6P and a 59% decrease in kcat-Glc6P/Km-Glc6P is highly desirable compared to the wild type in the GlcNAc-producing chassis. The GlcNAc titer increases to 217.3 g L-1 with a yield of 0.597 g (g glucose)-1 in a 50-L bioreactor, representing the highest reported level. Collectively, this DBTL framework provides an easy yet fascinating approach to the rational design of enzymes for industrially viable biocatalysts.

Development and validation of a risk prediction model for valve regurgitation in Behçet's disease.

BACKGROUND: In Behçet's disease (BD), mild-to-severe valvular regurgitation (VR) poses a serious complication that contributes significantly to heart failure and eventually death. The accurate prediction of VR is crucial in the early stages of BD subjects for improved prognosis. Accordingly, this study aimed to develop a nomogram that can detect VR early in the course of BD. METHODS: One hundred seventy-two patients diagnosed with Behçet's disease (BD) were conducted to assess cardiac valve regurgitation as the primary outcome. The severity of regurgitation was classified as mild, moderate, or severe. The parameters related to the diagnostic criteria were used to develop model 1. The combination of stepAIC, best subset, and random forest approaches was employed to identify the independent predictors of VR and thus establish model 2 and create a nomogram for predicting the probability of VR in BD. Receiver operating characteristics (ROC) and decision curve analysis (DCA) were used to evaluate the model performance. RESULTS: Thirty-four patients experienced mild-to-severe VR events. Model 2 was established using five variables, including arterial involvement, sex, age at hospitalization, mean arterial pressure, and skin lesions. In comparison with model 1 (0.635, 95% CI: 0.512-0.757), the ROC of model 2 (0.879, 95% CI: 0.793-0.966) was improved significantly. DCA suggested that model 2 was more feasible and clinically applicable than model 1. CONCLUSION: A predictive model and a nomogram for predicting the VR of patients with Behçet's disease were developed. The good performance of this model can help us identify potential high-risk groups for heart failure. Key Points • In this study, the predictors of VR in BD were evaluated, and a risk prediction model was developed for the early prediction of the occurrence of VR in patients with BD. • The VR prediction model included the following indexes: arterial involvement, sex, age at hospitalization, mean arterial pressure, and skin lesions. • The risk model that we developed was better and more optimized than the models built with diagnostic criteria parameters, and visualizing and personalizing the model, a nomogram, provided clinicians with an easy and intuitive tool for practical prediction.