A multicenter, randomized, open-label, phase 2 clinical study of telitacicept in adult patients with generalized myasthenia gravis.

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.

etiBsu1209: A comprehensive multiscale metabolic model for Bacillus subtilis.

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.

Pyruvate-responsive genetic circuits for dynamic control of central metabolism.

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.

Assembly of pathway enzymes by engineering functional membrane microdomain components for improved N-acetylglucosamine synthesis in Bacillus subtilis.

Enzyme clustering can improve catalytic efficiency by facilitating the processing of intermediates. Functional membrane microdomains (FMMs) in bacteria can provide a platform for enzyme clustering. However, the amount of FMMs at the cell basal level is still facing great challenges in multi-enzyme immobilization. Here, using the nutraceutical N-acetylglucosamine (GlcNAc) synthesis in Bacillus subtilis as a model, we engineered FMM components to improve the enzyme assembly in FMMs. First, by overexpression of the SPFH (stomatin-prohibitin-flotillin-HflC/K) domain and YisP protein, an enzyme involved in the synthesis of squalene-derived polyisoprenoid, the membrane order of cells was increased, as verified using di-4-ANEPPDHQ staining. Then, two heterologous enzymes, GlcNAc-6-phosphate N-acetyltransferase (GNA1) and haloacid dehalogenase-like phosphatases (YqaB), required for GlcNAc synthesis were assembled into FMMs, and the GlcNAc titer in flask was increased to 8.30 ± 0.57 g/L, which was almost three times that of the control strains. Notably, FMM component modification can maintain the OD600 in stationary phase and reduce cell lysis in the later stage of fermentation. These results reveal that the improved plasma membrane ordering achieved by the engineering FMM components could not only promote the enzyme assembly into FMMs, but also improve the cell fitness.

Gender differences in quality of life among patients with myasthenia gravis in China.

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.

ToyA, a positive pathway-specific regulator for toyocamycin biosynthesis in Streptomyces diastatochromogenes 1628.

The nucleoside antibiotic toyocamycin (TM), which was produced by Streptomyces diastatochromogenes 1628, was found to be highly efficient against a broad range of plant pathogenic fungi. Despite its importance, little is known about the regulation TM biosynthesis. In this study, toyA, located in the TM biosynthetic gene cluster, was identified as a regulatory gene encoding a large ATP-binding regulator of the LuxR family (LAL-family). The role of toyA in TM biosynthesis in S. diastatochromogenes 1628 was investigated by gene deletion, complementation, and over-expression. Gene disruption of toyA resulted in almost loss of TM production. TM production in complemented strain was restored to the level comparable to that in the wild-type strain S. diastatochromogenes 1628. Over-expression of toyA separately controlled by promoter SPL57, SPL21, and permE* in wild-type strain S. diastatochromogenes 1628 led to a 2-fold, 1-fold, and 80% increase in TM production compared with wild-type strain S. diastatochromogenes 1628, respectively. Quantitative RT-PCR analysis revealed that the transcriptional level of toy structural genes was downregulated in the ΔtoyA mutant but restored in complemented strain and further upregulated in the toyA over-expression strain. The detection results from GFP reporter system in Escherichia coli and GUS reporter system and GUS activities in S. albus J1074 and S. diastatochromogenes 1628 showed that ToyA activated the expression of toyB and toyE operon directly and activated the expression of other toy structural genes indirectly. These results indicate that ToyA is essential for TM biosynthesis controlling the expression of structural genes.

Advances and prospects of Bacillus subtilis cellular factories: From rational design to industrial applications.

Bacillus subtilis is the most characterized gram-positive bacterium that has significant attributes, such as growing well on cheap carbon sources, possessing clear inherited backgrounds, having mature genetic manipulation methods, and exhibiting robustness in large-scale fermentations. Till date, B. subtilis has been identified as attractive hosts for the production of recombinant proteins and chemicals. By applying various systems and synthetic biology tools, the productivity features of B. subtilis can be thoroughly analyzed and further optimized via metabolic engineering. In the present review, we discussed why B. subtilis is the primary organisms used for metabolic engineering and industrial applications. Additionally, we summarized the recent advances in systems and synthetic biology, engineering strategies for improving cellular performances, and metabolic engineering applications of B. subtilis. In particular, we proposed emerging opportunities and essential strategies to enable the successful development of B. subtilis as microbial cell factories.

Selection of an efficient promoter and its application in toyocamycin production improvement in Streptomyces diastatochromogenes 1628.

The selection of efficient promoter is usually very crucial for gene expression and metabolic engineering in Streptomycetes. In this study, the synthetic promoters SPL-57and SPL-21, and the engineered promoter kasOp*were selected and their activities were examined by using a reporter gene assay based on GUS. All selected promoters which have been reported to be stronger than promoter permE*, which was used as control promoter. As host we were choosing S. diastatochromogenes 1628, the producer of toyocamycin (TM). Our results indicate that all tested promoters can be used to express genes in S. diastatochromogenes 1628. Interesting, promoter SPL-21 showed the strongest transcriptional and expression level and gave rise to a 5.2-fold increase in GUS activity compared with control. In order to improve TM production, the promoters were used to control expression of toyF. This gene encodes an adenylosuccinate lyase involved in TM biosynthesis. Among all different recombinant strains, the strain 1628-21F, in which over-expression of toyF gene was driven by SPL-21, exhibited the largest increase in TOYF activity and TM production. In a 5-l fermenter this strain produced more than two times more TM compared with the wild-type strain.

Characterization of representative rpoB gene mutations leading to a significant change in toyocamycin production of Streptomyces diastatochromogenes 1628.

Modification of enzymes involved in transcription- or translation-processes is an interesting way to increase secondary metabolite production in Streptomycetes. However, application of such methods has not been widely described for strains which produce nucleoside antibiotics. The nucleoside antibiotic toyocamycin (TM) is produced by Streptomyces diastatochromogenes 1628. For improving TM production in S. diastatochromogenes 1628, the strain was spread on rifamycin-resistant (Rif(r)) medium. Several spontaneous mutants were obtained with mutations in the rpoB gene which encodes a RNA polymerase ß-subunit. The mutants which showed increased TM production were detected at a frequency of 7.5 % among the total Rif(r) mutants. Mutant 1628-T15 harboring amino acid substitution His437Arg was the best TM producer with a 4.5-fold increase in comparison to that of the wild-type strain. The worst producer was mutant 1628-T62 which also showed a poor sporulation behavior. RT-PCR was performed to study the transcription levels of the TM biosynthetic gene toyG in the parental strain as well as in mutants 1628-T15 and 1628-T62. The transcriptional level of toyG was higher in mutant 1628-T15 than that in parental strain 1628, while much lower in mutant 1628-T62. In mutant strain 1628-T62 the expression of adpA sd gene, which is required for morphological differentiation, was also much lower. Our studies also indicate that the introduction of mutations into rpoB is an effective strategy to improve the production of TM which is an important nucleoside antibiotic.

Isolation and identification of biocontrol agent Streptomyces rimosus M527 against Fusarium oxysporum f. sp. cucumerinum.

Actinomycetes have received considerable attention as biocontrol agents against fungal plant pathogens and as plant growth promoters. In this study, a total of 320 actinomycetes were isolated from various habitats in China. Among which, 77 strains have been identified as antagonistic activities against Fusarium oxysporum f. sp. cucumerinum which usually caused fusarium wilt of cucumber. Of these, isolate actinomycete M527 not only displayed broad-spectrum antifungal activity but also showed the strongest antagonistic activity against the spore germination of F. oxysporum f. sp. cucumerinum. In pot experiments, the results indicated that isolate M527 could promote the shoot growth and prevent the development of the disease on cucumber caused by F. oxysporum f. sp. cucumerinum. The control efficacy against seedling fusarium wilt of cucumber after M527 fermentation broth root-irrigation was up to 72.1% as compared to control. Based on 16S rDNA sequence analysis, the isolate M527 was identified as Streptomyces rimosus.

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.

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.

Detection and analysis of triacylglycerol regioisomers via electron activated dissociation (EAD) tandem mass spectrometry.

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.

Genetically encoded biosensors for microbial synthetic biology: From conceptual frameworks to practical applications.

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.

Metabolic Engineering of Saccharomyces cerevisiae for Efficient Retinol Synthesis.

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.

Combinatorial Protein Engineering and Metabolic Engineering for Efficient Synthesis of l-Histidine in Corynebacterium glutamicum.

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.

Combinatorial Metabolic Engineering and Enzymatic Catalysis Enable Efficient Production of Colanic Acid.

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.

Construction and Optimization of the de novo Biosynthesis Pathway of Mogrol in Saccharomyces Cerevisiae.

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.

De novo biosynthesis of rubusoside and rebaudiosides in engineered yeasts.

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.

[Current status and future perspectives of metabolic network models of industrial microorganisms].

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.