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Microbial factories lacking the ability of dynamically regulating the pathway enzymes overexpression, according to in situ metabolite concentrations, are suboptimal, especially when the metabolic intermediates are competed by growth and chemical production. The production of higher alcohols (HAs), which hijacks the amino acids (AAs) from protein biosynthesis, minimizes the intracellular concentration of AAs and thus inhibits the host growth. To balance the resource allocation and maintain stable AA flux, this work utilizes AA-responsive transcriptional attenuator ivbL and HA-responsive transcriptional activator BmoR to establish a concentration recognition-based auto-dynamic regulation system (CRUISE). This system ultimately maintains the intracellular homeostasis of AA and maximizes the production of HA. It is demonstrated that ivbL-driven enzymes overexpression can dynamically regulate the AA-to-HA conversion while BmoR-driven enzymes overexpression can accelerate the AA biosynthesis during the HA production in a feedback activation mode. The AA flux in biosynthesis and conversion pathways is balanced via the intracellular AA concentration, which is vice versa stabilized by the competition between AA biosynthesis and conversion. The CRUISE, further aided by scaffold-based self-assembly, enables 40.4 g L-1 of isobutanol production in a bioreactor. Taken together, CRUISE realizes robust HA production and sheds new light on the dynamic flux control during the process of chemical production.

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2-O-α-D-glucopyranosyl-sn-glycerol (2-αGG) is a high value product with wide applications. Here, an efficient, safe and sustainable bioprocesses for 2-αGG production was designed. A novel sucrose phosphorylase (SPase) was firstly identified from Leuconostoc mesenteroides ATCC 8293. Subsequently, SPase mutations were processed with computer-aided engineering, of which the activity of SPaseK138C was 160% higher than that of the wild-type. Structural analysis revealed that K138C was a key functional residue moderating substrate binding pocket and thus influences catalytic activity. Furthermore, Corynebacterium glutamicum was employed to construct microbial cell factories along with ribosome binding site (RBS) fine-tuning and a two-stage substrate feeding control strategy. The maximum production of 2-αGG by these combined strategies reached 351.8 g·L-1 with 98% conversion rate from 1.4 M sucrose and 3.5 M glycerol in a 5-L bioreactor. This was one of the best performance reported in single-cell biosynthesis of 2-αGG, which paved effective ways for industrial-scale preparation of 2-αGG.

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The biological treatment of wastewater with high concentrations of ammonia nitrogen has become a hot research issue, but there are limited reports on the mechanism of ammonia nitrogen utilization by microorganisms. In this paper, a transcriptomic approach was used to investigate the differences in gene expression at 500.0 mg/L (Amo 500) and 100.0 mg/L (Amo 100) ammonium concentrations to reveal the mechanism of ammonia nitrogen removal from water by Pseudomonas stutzeri F2. The transcriptome data showed 1015 (459 up-regulated and 556 down-regulated) differentially expressed genes with functional gene annotation related to nitrogen source metabolism, glycolysis, tricarboxylic acid cycle, extracellular polysaccharide synthesis, energy conversion and transmembrane transport, revealing the metabolic process of ammonium nitrogen conversion to biological nitrogen in P. stutzeri F2 through assimilation. To verify the effect of ammonium transporter protein (AmtB) of cell membrane on assimilation, a P. stutzeri F2-ΔamtB mutant strain was obtained by constructing a knockout plasmid (pK18mobsacB-ΔamtB), and it was found that the growth characteristics and ammonium removal rate of the mutant strain were significantly reduced at high ammonium concentration. The carbon source components and dissolved oxygen conditions were optimized after analyzing the transcriptome data, and the ammonium removal rate was increased from 41.23% to 94.92% with 500.0 mg/L ammonium concentration. The study of P. stutzeri F2 transcript level reveals the mechanism of ammonia nitrogen influence on microbial assimilation process and improvement strategy, which provides a new strategy for the treatment of ammonia nitrogen wastewater.

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2-O-α-D-glu-copyranosyl-sn-glycerol is a high value-added product with prospective application in food, cosmetics, health products and pharmaceutical industries. However, industrial scale of 2-O-α-D-glu-copyranosyl-sn-glycerol has not yet been applied in China, and there are few related reports on 2-O-α-D-glu-copyranosyl-sn-glycerol synthesis. The purpose of this experiment is to develop a method for catalyzing the synthesis of food-grade 2-O-α-D-glu-copyranosyl-sn-glycerol using whole cells of "Generally Recognized as Safe" (GRAS) recombinant Bacillus subtilis. In our work, a recombinant B. subtilis 168/pMA5-gtfA that heterologously expressing Leuconostoc mesenteroides sucrose phosphorylase was constructed and used as a whole-cell catalyst to synthesize 2-O-α-D-glu-copyranosyl-sn-glycerol. Optimizing the culture temperature, time and whole cell transformation conditions has increased the yield of 2-O-α-D-glu-copyranosyl-sn-glycerol. The results showed that 1.43 U/mL of sucrose phosphorylase was achieved in B. subtilis 168/pMA5-gtfA after culturing for 20 h at 30 °C in fermentation medium. The highest conversion rate reached 75.1%, and the yield of 2-O-α-D-glu-copyranosyl-sn-glycerol was 189.3 g/L with an average transformation rate of 15.6 mmol/(L·h) after 48 hours whole-cell transformation with the sucrose concentration of 1 mol/L and the glycerol concentration of 2.5 mol/L at 30 °C, OD600 40 and pH 7.0. This is the highest yield of 2-O-α-D-glu-copyranosyl-sn-glycerol synthesized catalytically by recombinant B. subtilis that was ever reported, and this study provides the theoretical and experimental basis for the industrial production and application of 2-O-α-D-glucopyranosyl-sn-glycerol.

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OBJECTIVE: To investigate the mRNA expression of galectin-3 and its clinical significance in acute myeloid leukemia (AML) patients carrying AML1/ETOfusion gene. METHODS: RQ-PCR method was used to detect the expression of galectin-3 mRNA in bone marrow mononuclear cells of 53 AML patients with AML1/ETO+, ELISA was used to detect the expression of galectin-3 protein in peripheral blood, and the correlations of galectin-3 expression with clinical and laboratory features and outcomes were analyzed. RESULTS: The mRNA and protein levels of galectin-3 were significantly higher in newly diagnosed AML1/ETO+ AML patients compared with the control ( P<0.001). Galectin-3 mRNA and protein expressions were positively correlated (r=0.732, P<0.001). Galectin-3 protein was significantly decreased during the period of complete remission (CR)( P<0.001). The mRNA expression of galectin-3 was negatively correlated with the count of white blood cells ( P=0.014), and positively correlated with CD34 expression and additional cytogenetic aberrations (ACA) ( P=0.001, P=0.026). There was no significant difference in CR, partial remission (PR), induction death (early mortality) between galectin-3 high-expression group and low-expression group ( P>0.05), but there was significant difference in recurrence rate between the two groups ( P=0.029). The median overall survival (OS) rate and disease-free survival (DFS) rate were shortened in the high-expression group ( P=0.007, P=0.015) and the cumulative incidence of relapse was increased ( P=0.045), but there was no significant difference in the cumulative incidence of CM(155mm]mortality ( P>0.05). Cox regression analysis suggested galectin-3 mRNA level an independent indicator of OS and DFS in AML1/ETO+ AML patients. CONCLUSION: Bone marrow galectin-3 mRNA level may be an important reference index for evaluating the prognosis and guiding the treatment of AML1/ETO+ AML patients.

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OBJECTIVE: To study the effect of safflower injection on the proliferation and apoptosis of human leukemia cell line HEL and the relevant molecular mechanisms. METHODS: HEL cells were treated with different concentrations of safflower injection. HEL cells without safflower injection treatment were used as the control group. MTT method was used to detect the inhibitory rate of the HEL cells at 24, 48 and 72 hours after various concentrations of safflower injection treatment (10, 20, 30, 40 and 50 mg/mL). The cell cycle and apoptosis were detected using flow cytometry and the HOXB3-mRNA expression was measured by RT-PCR at 48 hours after safflower injection treatment (10, 20 and 30 mg/mL). RESULTS: Compared with the control group, various concentrations of safflower injection inhibited HEL cell proliferation in a dose-dependent manner (P<0.05). At 48 hours after various concentrations of safflower injection treatment, the number of treated cells in the G2/M phase increased, but that in the S phase decreased, and the apoptosis rate was significantly higher than that in the control group, with a dose-dependent manner (P<0.05). The expression of HOXB3-mRNA in safflower injection-treated cells decreased in a dose-dependent manner compared with the control group (P<0.05). CONCLUSIONS: Safflower injection can inhibit proliferation and induce apoptosis of HEL cells in vitro, and its underlying mechanisms may involve down-regulation of the HOXB3-mRNA expression.

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