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
. 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
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.
Full text
Available for:
FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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.
•The product portfolio of B. subtilis is expanding from proteins to bio-chemicals.•Reason of B. subtilis as a primary strain for metabolic engineering was discussed.•Recent advances in basic studies and applications of B. subtilis were summarized.•Essential strategies of developing B. subtilis to be cell factories were proposed.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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.
Repeat buyer prediction is crucial for e-commerce companies to enhance their customer services and product sales. In particular, being aware of which factors or rules drive repeat purchases is as ...significant as knowing the outcomes of predictions in the business field. Therefore, an interpretable model with excellent prediction performance is required. Many classifiers, such as the multilayer perceptron, have exceptional predictive abilities but lack model interpretability. Tree-based models possess interpretability; however, their predictive performances usually cannot achieve high levels. Based on these observations, we design an approach to balance the predictive and interpretable performance of a decision tree with model distillation and heterogeneous classifier fusion. Specifically, we first train multiple heterogeneous classifiers and integrate them through diverse combination operators. Then, classifier combination plays the role of teacher model. Subsequently, soft targets are obtained from the teacher and guide training of the decision tree. A real-world repeat buyer prediction dataset is utilized in this paper, and we adopt features with respect to three aspects: users, merchants, and user–merchant pairs. Our experimental results show that the accuracy and AUC of the decision tree are both improved, and we provide model interpretations of three aspects.
Full text
Available for:
DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, ODKLJ, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Abstract
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 (β = − 4.99 95%CI, − 7.80 to − 2.18,
p
< 0.001) and exacerbations (β = − 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 (β = 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.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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. albus
J1074-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.
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 (Rifʳ) medium. Several spontaneous mutants were obtained with mutations in the rpoB gene which encodes a RNA polymerase β-subunit. The mutants which showed increased TM production were detected at a frequency of 7.5 % among the total Rifʳ 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.
Full text
Available for:
CEKLJ, DOBA, FZAB, GEOZS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
With the increasing demand for the biodegradable polymer material polylactic acid and its advantage of being metabolized by the human body, L-lactic acid (L-LA) is becoming increasingly attractive in ...environmental protection and food industry applications. However, the supply of L-LA is not satisfied, and the price is still high. Compared to enzymatic and chemical synthesis methods, L-LA production by microbial fermentation has the advantages of low cost, large yield, simple operation, and environmental protection. This review summarizes the advances in engineering microbial cell factories to produce L-LA. First, the synthetic pathways and microorganisms for L-LA production are outlined. Then, the metabolic engineering strategies for constructing cell factories to overproduce L-LA are summarized and fermentation modes for L-LA production are also given. Finally, the challenges and prospects of the microbial production of L-LA are discussed. This review provides theoretical guidance for researchers engaged in L-LA production.
•The promoters and signal peptides were optimized to enhance the extracellular expression of hOPN.•Overexpressing transcription and translation factors elevated hOPN expression level.•The ...co-expression of molecular chaperone resolved the intracellular accumulation of hOPN.
Osteopontin (OPN), a bioactive milk protein in breast milk, has diverse applications in food and medicine. OPN is predominantly extracted from fresh bovine milk; however, the concentration of OPN in bovine milk is exceedingly low. To address these issues, in the present study, we used Komagataella phaffii for the recombinant expression of human OPN (hOPN). The results of host cell screening revealed that X33 cells were more suitable than other K. phaffii hosts for hOPN expression, yielding a titer of 340.5 μg/L. Subsequently, it was observed that the extracellular production of hOPN significantly increased to 8.02 mg/L from strain XPSA01 using the PAOX1 promoter and the hybrid signal peptide PROSCW10-α. Meanwhile, the co-expression of transcription factors, translation factors, and molecular chaperones resulted in a 2.94-fold enhancement in extracellular hOPN production compared with that in XPSA01, resulting in a titer of 23.6 mg/L. Finally, the above strategies were combined to obtain the recombinant strain XPSA01-CP, which achieved titers of 35.6 and 128.5 mg/L hOPN in shake-flask fermentation and 3-L bioreactor, respectively. To the best of our knowledge, this is the highest extracellular yield of hOPN ever reported.
Display omitted
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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
.
PDF
