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In recent years, genetic circuit-based regulation of metabolic flux in microbial cell factories has received significant attention. In this review, we describe a pipeline for the design and construction of genetic circuits for metabolic flux optimization. In particular, we summarize the recent advances in computationally assisted prediction of critical metabolic nodes and genetic circuit design automation. Further, we introduce strategies for constructing high-performance genetic circuits. We also summarize the latest applications of genetic circuits in the dynamic regulation of metabolism and high-throughput screening. Finally, we discuss the challenges and prospects associated with the design and construction of sophisticated genetic circuits. Through this review, we aim to provide a theoretical basis for designing and constructing high-performance genetic circuits to optimize metabolic flux.
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Redes Reguladoras de Genes , Redes y Vías Metabólicas , Redes y Vías Metabólicas/genética , Ingeniería Metabólica/métodos , Biología Sintética/métodos , Bacterias/genética , Bacterias/metabolismoRESUMEN
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.
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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.
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Escherichia coli , Fucosiltransferasas , Ingeniería Metabólica , Trisacáridos , Escherichia coli/genética , Escherichia coli/metabolismo , Trisacáridos/metabolismo , Trisacáridos/biosíntesis , Ingeniería Metabólica/métodos , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Lactosa/metabolismo , Bacteroides/genética , Bacteroides/metabolismo , Fermentación , OligosacáridosRESUMEN
BACKGROUND AND PURPOSE: This study aimed to investigate the clinical efficacy and safety of telitacicept in patients with generalized myasthenia gravis (gMG) who tested positive for acetylcholine receptor antibodies or muscle-specific kinase antibodies and were receiving standard-of-care therapy. METHODS: Patients meeting the eligibility criteria were randomly assigned to receive telitacicept subcutaneously once a week for 24 weeks in addition to standard-of-care treatment. The primary efficacy endpoint was the mean change in the quantitative myasthenia gravis (QMG) score from baseline to week 24. Secondary efficacy endpoints included mean change in QMG score from baseline to week 12 and gMG clinical absolute score from baseline to week 24. Additionally, safety, tolerability and pharmacodynamics were assessed. RESULTS: Twenty-nine of the 41 patients screened were randomly selected and enrolled. The mean (± standard deviation [SD]) reduction in QMG score from baseline to week 24 was 7.7 (± 5.34) and 9.6 (± 4.29) in the 160 mg and 240 mg groups, respectively. At week 12, mean reductions in QMG scores for these two groups were 5.8 (± 5.85) and 9.5 (± 5.03), respectively, indicating rapid clinical improvement. Safety analysis revealed no adverse events leading to discontinuation or mortalities. All patients showed consistent reductions in serum immunoglobulin (Ig) A, IgG and IgM levels throughout the study. CONCLUSION: Telitacicept demonstrated safety, good tolerability and reduced clinical severity throughout the study period. Further validation of the clinical efficacy of telitacicept in gMG will be conducted in an upcoming phase 3 clinical trial.
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Miastenia Gravis , Humanos , Miastenia Gravis/tratamiento farmacológico , Femenino , Masculino , Persona de Mediana Edad , Adulto , Anciano , Resultado del Tratamiento , Receptores Colinérgicos/inmunologíaRESUMEN
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.
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Ácido Betulínico , Ingeniería Metabólica , NADP , Triterpenos Pentacíclicos , Saccharomyces cerevisiae , Triterpenos , Ingeniería Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Triterpenos Pentacíclicos/metabolismo , Triterpenos/metabolismo , NADP/metabolismo , Acetilcoenzima A/metabolismo , Fermentación , Vías Biosintéticas/genéticaRESUMEN
Triacylglycerols (TGs) are important components of human diet. The positional distribution of fatty acids (FAs) on the glycerol backbone affects the chemistry and physical properties of fats. Especially for infants, the structure of TGs plays an important role in the growth and development. However, limited by detecting technology, accurately identifying regioisomers of ABA/AAB and BAC/ABC/ACB type TGs is a significant challenge for human milk utilization and the development of infant formula. For this, we exploit a novel method for identifying the regioisomers of ABA/AAB and BAC/ABC/ACB type TGs within complex lipid mixtures, via used electron activated dissociation (EAD) tandem mass spectrometry. The distribution information of acyl chains at the sn-2 and sn-1/3 positions of glycerol backbone and double bonds in unsaturated FAs can be easily obtained by fragmenting TG ions with energetic electrons (15 eV). Then, the standard curve was established by correlating the peak area intensity of sn-2 characteristic product ion with the content of TG regioisomers standard. These analytical methods successfully enabled the identification and quantification of TG regioisomers in human milk, cow milk, infant formula, palm oil, and sunflower oil. Additionally, the distribution of the double-bond positions of unsaturated FAs in these samples was also identified. Compared to traditional methods, this approach eliminates the need for complex processing and analysis procedures, enabling rapid structural characterization of ABA/AAB and BAC/ABC/ACB type TGs within 17 min. Hence, we provide a rapid and convenient methodology for detecting and analyzing ABA/AAB and BAC/ABC/ACB type TG regioisomers, thereby offering valuable assistance in the development of specialized formulations and facilitating effective process control for ensuring the quality of edible oils and fats.
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Electrones , Espectrometría de Masas en Tándem , Humanos , Triglicéridos/química , Glicerol , Aceites , GrasasRESUMEN
Retinol, the main active form of vitamin A, plays a role in maintaining vision, immune function, growth, and development. It also inhibits tumor growth and alleviates anemia. Here, we developed a Saccharomyces cerevisiae strain capable of high retinol production. Firstly, the de novo synthesis pathway of retinol was constructed in S. cerevisiae to realize the production of retinol. Second, through modular optimization of the metabolic network of retinol, the retinol titer was increased from 3.6 to 153.6 mg/L. Then, we used transporter engineering to regulate and promote the accumulation of the intracellular precursor retinal to improve retinol production. Subsequently, we screened and semi-rationally designed the key enzyme retinol dehydrogenase to further increase the retinol titer to 387.4 mg/L. Lastly, we performed two-phase extraction fermentation using olive oil to obtain a final shaking flask retinol titer of 1.2 g/L, the highest titer reported at the shake flask level. This study laid the foundation for the industrial production of retinol.
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l-Histidine is an essential proteinogenic amino acid in food with extensive applications in the pharmaceutical field. Herein, we constructed a Corynebacterium glutamicum recombinant strain for efficient biosynthesis of l-histidine. First, to alleviate the l-histidine feedback inhibition, the ATP phosphoribosyltransferase mutant HisGT235P-Y56M was constructed based on molecular docking and high-throughput screening, resulting in the accumulation of 0.83 g/L of l-histidine. Next, we overexpressed rate-limiting enzymes including HisGT235P-Y56M and PRPP synthetase and knocked out the pgi gene in the competing pathway, which increased the l-histidine production to 1.21 g/L. Furthermore, the energy status was optimized by decreasing the reactive oxygen species level and enhancing the supply of adenosine triphosphate, reaching a titer of 3.10 g/L in a shake flask. The final recombinant strain produced 5.07 g/L of l-histidine in a 3 L bioreactor, without the addition of antibiotics and chemical inducers. Overall, this study developed an efficient cell factory for l-histidine biosynthesis by combinatorial protein engineering and metabolic engineering.
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Corynebacterium glutamicum , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Ingeniería de Proteínas/métodos , Ingeniería Metabólica/métodos , Histidina/biosíntesis , Simulación por Computador , Biocatálisis , Mutación , Reactores BiológicosRESUMEN
Genome-scale metabolic models (GEMs) have been widely used to guide the computational design of microbial cell factories, and to date, seven GEMs have been reported for Bacillus subtilis, a model gram-positive microorganism widely used in bioproduction of functional nutraceuticals and food ingredients. However, none of them are widely used because they often lead to erroneous predictions due to their low predictive power and lack of information on regulatory mechanisms. In this work, we constructed a new version of GEM for B. subtilis (iBsu1209), which contains 1209 genes, 1595 metabolites, and 1948 reactions. We applied machine learning to fill gaps, which formed a relatively complete metabolic network able to predict with high accuracy (89.3%) the growth of 1209 mutants under 12 different culture conditions. In addition, we developed a visualization and code-free software, Model Tool, for multiconstraints model reconstruction and analysis. We used this software to construct etiBsu1209, a multiscale model that integrates enzymatic constraints, thermodynamic constraints, and transcriptional regulatory networks. Furthermore, we used etiBsu1209 to guide a metabolic engineering strategy (knocking out fabI and yfkN genes) for the overproduction of nutraceutical menaquinone-7, and the titer increased to 153.94 mg/L, 2.2-times that of the parental strain. To the best of our knowledge, etiBsu1209 is the first comprehensive multiscale model for B. subtilis and can serve as a solid basis for rational computational design of B. subtilis cell factories for bioproduction.
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Bacillus subtilis , Ingeniería Metabólica , Bacillus subtilis/metabolismoRESUMEN
Genetically encoded biosensors are the vital components of synthetic biology and metabolic engineering, as they are regarded as powerful devices for the dynamic control of genotype metabolism and evolution/screening of desirable phenotypes. This review summarized the recent advances in the construction and applications of different genetically encoded biosensors, including fluorescent protein-based biosensors, nucleic acid-based biosensors, allosteric transcription factor-based biosensors and two-component system-based biosensors. First, the construction frameworks of these biosensors were outlined. Then, the recent progress of biosensor applications in creating versatile microbial cell factories for the bioproduction of high-value chemicals was summarized. Finally, the challenges and prospects for constructing robust and sophisticated biosensors were discussed. This review provided theoretical guidance for constructing genetically encoded biosensors to create desirable microbial cell factories for sustainable bioproduction.
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Técnicas Biosensibles , Biología Sintética , Factores de Transcripción/genética , Regulación de la Expresión Génica , Ingeniería MetabólicaRESUMEN
High-sugar diet causes health problems, many of which can be addressed with the use of sugar substitutes. Rubusoside and rebaudiosides are interesting molecules, considered the next generation of sugar substitutes due to their low-calorie, superior sweetness and organoleptic properties. However, their low abundance in nature makes the traditional plant extraction process neither economical nor environmental-friendly. Here we engineer baker's yeast Saccharomyces cerevisiae as a chassis for the de novo production of rubusoside and rebaudiosides. In this process, we identify multiple issues that limit the production, including rate-liming steps, product stress on cellular fitness and unbalanced metabolic networks. We carry out a systematic engineering strategy to solve these issues, which produces rubusoside and rebaudiosides at titers of 1368.6 mg/L and 132.7 mg/L, respectively. The rubusoside chassis strain here constructed paves the way towards a sustainable, large-scale fermentation-based manufacturing of diverse rebaudiosides.
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Diterpenos de Tipo Kaurano , Ingeniería Metabólica , Diterpenos de Tipo Kaurano/metabolismo , Glucósidos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , EdulcorantesRESUMEN
Mogrol plays important roles in antihyperglycemic and antilipidemic through activating the AMP-activated protein kinase pathway. Although the synthesis pathway of mogrol in Siraitia grosvenorii has been clarified, few studies have focused on improving mogrol production. This study employed a modular engineerin g strategy to improve mogrol production in a yeast chassis cell. First, a de novo synthesis pathway of mogrol in Saccharomyces cerevisiae was constructed. Then, the metabolic flux of each synthetic module in mogrol metabolism was systematically optimized, including the enhancement of the precursor supply, inhibition of the sterol synthesis pathway using the Clustered Regularly Interspaced Short Palindromic Repeats Interference system (CRISPRi), and optimization of the expression and reduction system of P450 enzymes. Finally, the mogrol titer was increased to 9.1 µg/L, which was 455-fold higher than that of the original strain. The yeast strains engineered in this work can serve as the basis for creating an alternative way for mogrol production in place of extraction from S. grosvenorii.
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Colanic acid can promote the lifespan of humans by regulating mitochondrial homeostasis, and it has widespread applications in the field of health. However, colanic acid is produced at a low temperature (20 °C) with low titer. Using Escherichia coli K-12 MG1655, we constructed the SRP-4 strain with high colanic acid production at 30 °C by enhancing the precursor supply and relieving the regulation of transcription for colanic acid synthesis genes by the RCS system. After media optimization, the colanic acid titer increased by 579.9-fold and reached 12.2 g/L. Subsequently, we successfully purified the colanic acid hydrolase and reduced the molecular weight of colanic acid (106.854 kDa), thereby eliminating the inhibition of high-molecular-weight colanic acid on strain growth. Finally, after adding the colanic acid hydrolase (4000 U/L), the colanic acid with low molecular weight reached 24.99 g/L in 3-L bioreactor, the highest titer reported so far. This high-producing strain of colanic acid will promote the application of low-molecular-weight colanic acid in the field of health.
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The dynamic regulation of metabolic pathways is based on changes in external signals and endogenous changes in gene expression levels and has extensive applications in the field of synthetic biology and metabolic engineering. However, achieving dynamic control is not trivial, and dynamic control is difficult to obtain using simple, single-level, control strategies because they are often affected by native regulatory networks. Therefore, synthetic biologists usually apply the concept of logic gates to build more complex and multilayer genetic circuits that can process various signals and direct the metabolic flux toward the synthesis of the molecules of interest. In this review, we first summarize the applications of dynamic regulatory systems and genetic circuits and then discuss how to design multilayer genetic circuits to achieve the optimal control of metabolic fluxes in living cells.
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Redes y Vías Metabólicas , Ingeniería Metabólica , Biología SintéticaRESUMEN
As a typical food safety industrial model strain, Bacillus subtilis has been widely used in the field of metabolic engineering due to its non-pathogenicity, strong ability of extracellular protein secretion and no obvious codon preference. In recent years, with the rapid development of molecular biology and genetic engineering technology, a variety of research strategies and tools have been used to construct B. subtilis chassis cells for efficient synthesis of biological products. This review introduces the research progress of B. subtilis from the aspects of promoter engineering, gene editing, genetic circuit, cofactor engineering and pathway enzyme assembly. Then, we also summarized the application of B. subtilis in the production of biological products. Finally, the future research directions of B. subtilis are prospected.
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Bacillus subtilis , Ingeniería Metabólica , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Edición Génica , Regiones Promotoras GenéticasRESUMEN
Genome-scale metabolic network model (GSMM) is an extremely important guiding tool in the targeted modification of industrial microbial strains, which helps researchers to quickly obtain industrial microbes with specific traits and has attracted increasing attention. Here we reviewe the development history of GSMM and summarized the construction method of GSMM. Furthermore, the development and application of GSMM in industrial microorganisms are elaborated by using four typical industrial microorganisms (Bacillus subtilis, Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae) as examples. In addition, prospects in the development trend of GSMM are proposed.
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Corynebacterium glutamicum , Redes y Vías Metabólicas , Corynebacterium glutamicum/genética , Escherichia coli/genética , Ingeniería Metabólica , Redes y Vías Metabólicas/genéticaRESUMEN
The nucleoside antibiotic toyocamycin (TM) is a potential fungicide that can control plant diseases, and it has become an attractive target for research. Streptomyces diastatochromogenes 1628, a TM-producing strain, was isolated by our laboratory and was considered to be a potent industrial producer of TM. Recently, the putative TM biosynthetic gene cluster (toy cluster) in S. diastatochromogenes 1628 was found by genome sequencing. In this study, the role of toy cluster for TM biosynthesis in S. diastatochromogenes 1628 was investigated by heterologous expression, deletion, and complementation. The extract of the recombinant strain S. albusJ1074-TC harboring a copy of toy cluster produced TM as shown by HPLC analysis. The Δcluster mutant completely lost its ability to produce TM. TM production in the complemented strain was restored to a level comparable to that of the wild-type strain. These results confirmed that the toy cluster is responsible for TM biosynthesis. Moreover, the introduction of an extra copy of the toy cluster into S. diastatochromogenes 1628 led to onefold increase in TM production (312.9 mg/l vs. 152.1 mg/l) as well as the transcription of all toy genes. The toy gene cluster was engineered in which the native promoter of toyA gene, toyM gene, toyBD operon, and toyEI operon was, respectively, replaced by permE ∗ or SPL57. To further improve TM production, the engineered toy gene cluster was, respectively, introduced and overexpressed in S. diastatochromogenes 1628 to generate recombinant strains S. diastatochromogenes 1628-EC and 1628-SC. After 84 h, S. diastatochromogenes 1628-EC and 1628-SC produced 456.5 mg/l and 638.9 mg/l TM, respectively, which is an increase of 2- and 3.2-fold compared with the wild-type strain.
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Dynamic regulation is a promising strategy for fine-tuning metabolic fluxes in microbial cell factories. However, few of these synthetic regulatory systems have been developed for central carbon metabolites. Here we created a set of programmable and bifunctional pyruvate-responsive genetic circuits for dynamic dual control (activation and inhibition) of central metabolism in Bacillus subtilis. We used these genetic circuits to design a feedback loop control system that relies on the intracellular concentration of pyruvate to fine-tune the target metabolic modules, leading to the glucaric acid titer increasing from 207 to 527 mg l-1. The designed logic gate-based circuits were enabled by the characterization of a new antisense transcription mechanism in B. subtilis. In addition, a further increase to 802 mg l-1 was achieved by blocking the formation of by-products. Here, the constructed pyruvate-responsive genetic circuits are presented as effective tools for the dynamic control of central metabolism of microbial cell factories.
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Proteínas Bacterianas/genética , Redes Reguladoras de Genes/efectos de los fármacos , Ácido Pirúvico/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Escherichia coli , Regulación Bacteriana de la Expresión Génica , Biblioteca Genómica , Ácido Glucárico/metabolismo , Glucosa/metabolismo , Histidina/química , Inositol/metabolismo , Lógica , Ingeniería Metabólica/métodos , Metaboloma/genética , Modelos Genéticos , Oligopéptidos/química , Factores de Transcripción , Transcripción GenéticaRESUMEN
BACKGROUND: In the phase 3 TOWER core study (NCT00751881), the efficacy and safety of teriflunomide compared with placebo were demonstrated in patients with relapsing forms of multiple sclerosis (RMS). Here, the long-term safety and efficacy outcomes from the TOWER extension study (NCT00751881) are reported. METHODS: All patients who entered the extension (N = 751) were assigned to teriflunomide 14 mg and assessed for long-term safety and efficacy. RESULTS: Of 751 patients in the TOWER extension study, 253, 265, and 233 patients received placebo/teriflunomide 14 mg, teriflunomide 7 mg/14 mg, and teriflunomide 14 mg/14 mg, respectively. Median teriflunomide exposure was 4.25 years (maximum 6.3 years). The overall frequency of adverse events (AEs) was comparable across treatment groups, but a higher proportion of patients in the teriflunomide 7 mg/14 mg (12.4%) and 14 mg/14 mg (12.4%) groups had serious AEs compared with the placebo/teriflunomide 14 mg group (6.4%). Alanine aminotransferase increase and hair thinning occurred at a higher frequency in the placebo/teriflunomide 14 mg group (11.2% and 14.3%, respectively) compared with the teriflunomide 7 mg/14 mg (3.0% and 4.5%, respectively) and 14 mg/14 mg groups (5.2% and 4.3%, respectively). The incidences of AEs of interest (hematologic and hepatic effects, peripheral neuropathy, hypertension, and malignancy) were low and comparable across treatment arms. Disability worsening and adjusted annualized relapse rates were low and stable over time, and mean Expanded Disability Status Scale scores were unchanged over time, for all treatment groups. CONCLUSION: In the TOWER extension study, the efficacy of teriflunomide 14 mg was maintained in patients with RMS. No new or unexpected AEs were observed with teriflunomide treatment, supporting a safety profile in the extension that was consistent with the core trial. These findings support the positive benefit:risk profile of teriflunomide as a long-term immunomodulatory therapy.
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Esclerosis Múltiple Recurrente-Remitente , Esclerosis Múltiple , Crotonatos/efectos adversos , Humanos , Hidroxibutiratos , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Nitrilos , Recurrencia , Toluidinas/efectos adversosRESUMEN
BACKGROUND: Myasthenia gravis (MG), a chronic neuromuscular disorder, can adversely affect patients' health-related quality of life (HRQoL), especially in women. The study aimed to evaluate the difference in HRQoL of women and men MG patients and explore the factors that mediate the relationship between gender and HRQoL. METHODS: A cross-sectional study was conducted among 1815 patients with MG in China. The revised 15-item MG quality of life scale (MG-QOL15r) was used to access patients' HRQoL in overall, physical, social and emotional domains. Socio-demographic information, diagnosis and treatment history, comorbidities, social support, active lifestyle and the MG activities of daily living scale (MG-ADL) were recorded and compared between women and men using the Student's t-test and Pearson's Chi-square test. Multivariable regression analyses were conducted to identify independent contributors to HRQoL, especially those affecting different gender. RESULTS: On average, female patients with MG reported a lower MG-QOL15r score than the males (44.49 ± 29.10 vs 49.32 ± 29.18). The association between gender and patients' HRQoL interacted with the number of comorbidities across the overall, physical and social domains of patients. As the number of comorbidities increased, the scores of HRQoL decreased and it was faster among females than the males (p < 0.05). Moreover, unemployment, exacerbation of the disease, and active lifestyle contributed to the patients' HRQoL across all domains. Unemployment (ß = - 4.99 [95%CI, - 7.80 to - 2.18], p < 0.001) and exacerbations (ß = - 8.49 [95%CI, - 11.43 to - 5.54], p < 0.001) were correlated with poorer HRQoL; while an active lifestyle had a positive impact on HRQoL (ß = 0.28 [95%CI, 0.16 to 0.40], p < 0.001). CONCLUSIONS: The results indicate that the HRQoL of women MG patients was lower than that of men. The relationship between gender and HRQoL is modulated by the number of comorbidities. Thus, to improve the HRQoL of women MG patients, symptomatic treatments might not be enough, their comorbid conditions should be considered as well. Additionally, employment status, MG exacerbations, and an active lifestyle have been found as determining factors of the patients' HRQoL, which suggests future interventions should cope with these factors to improve their quality of life.