High-Level Productivity of Lacto-N-neotetraose in Escherichia coli by Systematic Metabolic Engineering.

Lacto-N-neotetraose (LNnT) is a critical component of human milk oligosaccharides. This study introduces a systems metabolic engineering method to produce LNnT in Escherichia coli. First, 12 target genes contributing to LNnT production were identified using a double-plasmid system. Subsequently, combinatorial optimization of the copy number was performed to tune the target gene expression strength. Next, the CRISPR/Cas9 system was used to block the UDP-Gal and UDP-GlcNAc competitive pathways, and the titer of LNnT reached 1.16 g/L (E27). Moreover, the lactoylglutathione lyase (GloA) was deleted to block the competing metabolite pathway from glycerol to lactate, and the titer of LNnT (1.46 g/L) was 26% higher than that of strain E27. Finally, the LNnT productivity was increased to 0.34 g/L/h in a 3 L bioreactor, which was 36% higher than the recently reported LNnT productivity. This research work opens an innovative framework for the effective production of LNnT.

Module-Guided Metabolic Rewiring for Fucosyllactose Biosynthesis in Engineered Escherichia coli with Lactose De Novo Pathway.

Fucosyllactose (FL) has garnered considerable attention for its benefits on infant health. In this study, we report an efficient E. coli cell factory to produce 2'/3-fucosyllactose (2'/3-FL) with lactose de novo pathway through metabolic network remodeling, including (1) modification of the PTSGlc system to enhance glucose internalization efficiency; (2) screening for ß-1,4-galactosyltransferase (ß-1,4-GalT) and introduction of lactose synthesis pathway; (3) eliminating inhibition of byproduct pathways; (4) constructing antibiotic-free and inducer-free FL strains; and (5) up-regulating the expression of genes in the GDP-l-fucose module. The final engineered strains BP10-3 and BP11-3 produced 4.36 g/L for 2'-FL and 3.23 g/L for 3-FL in shake flasks. In 3 L bioreactors, fed-batch cultivations of the two strains produced 40.44 g/L for 2'-FL and 30.42 g/L for 3-FL, yielding 0.63 and 0.69 g/g glucose, respectively. The strategy described in this work will help to engineer E. coli as a safe chassis for other lactose-independent HMOs production.

Multi-level metabolic engineering of Escherichia coli for high-titer biosynthesis of 2'-fucosyllactose and 3-fucosyllactose.

Fucosyllactoses (FL), including 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL), have garnered considerable interest for their value in newborn formula and pharmaceuticals. In this study, an engineered Escherichia coli was developed for high-titer FL biosynthesis by introducing multi-level metabolic engineering strategies, including (1) individual construction of the 2'/3-FL-producing strains through gene combination optimization of the GDP-L-fucose module; (2) screening of rate-limiting enzymes (α-1,2-fucosyltransferase and α-1,3-fucosyltransferase); (3) analysis of critical intermediates and inactivation of competing pathways to redirect carbon fluxes to FL biosynthesis; (4) enhancement of the catalytic performance of rate-limiting enzymes by the RBS screening, fusion peptides and multi-copy gene cloning. The final strains EC49 and EM47 produced 9.36 g/L for 2'-FL and 6.28 g/L for 3-FL in shake flasks with a modified-M9CA medium. Fed-batch cultivations of the two strains generated 64.62 g/L of 2'-FL and 40.68 g/L of 3-FL in the 3-L bioreactors, with yields of 0.65 mol 2'-FL/mol lactose and 0.67 mol 3-FL/mol lactose, respectively. This research provides a viable platform for other high-value-added compounds production in microbial cell factories.

Permeabilization and immobilization of whole-cell Pseudomonas nitroreducens SP.001 to improve its l-glutaminase performance.

BACKGROUND: L-Glutaminase is considered to be an important industrial enzyme in both the pharmaceutical and food industries, especially for producing functional glutamyl compounds, such as l-theanine. Pseudomonas nitroreducens SP.001 with intracellular l-glutaminase activity has been screened previously. In the present study, three physical permeabilization methods were used to improve l-glutaminase activity. Then, the whole-cell immobilization conditions of permeabilized cells using sodium alginate as an embedding agent were optimized to enhance the enzyme's stability and reusability. The characteristics of the immobilized cells were investigated in comparison with those of permeabilized cells. RESULTS: The results obtained showed that cell permeabilization using osmotic shock with 155 g L-1 sucrose markedly improved enzyme activity. Then, an effective procedure for immobilization of permeabilized P. nitroreducens cells was established. The optimum conditions for cell immobilization were: sodium alginate 40 g L-1 , calcium chloride 30 g L-1 , cell mass 100 g L-1 and a curing time of 3 h. After successful immobilization, characterization studies revealed that the thermostability and pH resistance of l-glutaminase from immobilized cells were enhanced compared to those from permeabilized cells. Moreover, the immobilized biocatalyst could be reused up to 10 times and retained 80% of its activity. CONCLUSION: The stability and reusability of the permeabilized cells were improved through the immobilization. These findings indicated that immobilized whole-cell l-glutaminase from P. nitroreducens SP.001 possesses more potential for various industrial biotechnological applications than free cells. © 2020 Society of Chemical Industry.

Correlation between SOX2 and Survivin clinical features in patients with salivary adenoid cystic carcinoma.

OBJECTIVE: In this study, expression of cancer stem cells (CSCs)-related factor-Sex-determining region of Y chromosome-related high-mobility-group box 2 (SOX2) and anti-apoptotic specific factor- Survivin in salivary adenoid cystic carcinoma (SACC) was detected to provide important clues for effective SACC prevention and treatment by combining clinical pathological parameters analysis. METHODS: Paraffin and fresh specimens were collected from SACC patients who underwent surgery at the Oral and Maxillofacial Surgery Department of Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital. The experimental group was designed as SACC tissue, and the control group normal paracancerous normal gland tissue. (1) SOX2 and Survivin expression were detected using immunohistochemistry and analyzed by comnining clinical pathological parameter analysis. (2) mRNA and protein expression levels of SOX2 and Survivin were detected using RT-PCR, Western Blot. RESULTS: 1. Immunohistochemistry: (1) SOX2 was mainly expressed on the nucleus. The SOX2 positive rate was 28.57% in clinical stage I-II, and 76.92% in stage III-IV. (2) Survivin was mainly expressed in the cytoplasm. The Survivin positive rate was 61.90% in clinical stage I-II, and 76.92% in stage III-IV. (3) There was a clear correlation between SOX2 and Survivin. 2. RT-PCR and Western Blot: The mRNA and protein expression levels of SOX2 and Survivin were significantly higher in the experimental group than in the control group (P < 0.01). CONCLUSION: (1) The mRNA and protein expression level of SOX2 and Survivin was significantly higher in SACC tissues than in paracancerous normal salivary gland tissues, indicating that both of the two are tissue-specific and may become SACC oncogenes. (2) SOX2 and Survivin are significantly correlated in expression, which may coorinatively participate in SACC incidence and development.

A research on quality of life score (QOLS) of patients with trigeminal neuralgia (TN).

BACKGROUND: To evaluate the clinic effect of treatment of trigeminal neuralgia (TN). METHODS: The current study aims to develop a multiple-scale characteristics quality of life (QOL) for patients with TN. After interview, the individual questionnaire was acquired. indicating the QOL has a good responsiveness and surveying effects of the dynamic state of health on TN patients. CONCLUSION: It is feasible to form multiple-scale characteristics QOL for patients with TN.

[Curative effect of warm gutta-percha filling technique combined with different sealers in root canal filling].

PURPOSE: To evaluate the clinical curative effect of warm gutta-percha filling technique combined with different sealers in root canal filling. METHODS: Ninety-five patients with pulpitis who underwent root canal filling from June 2015 to June 2017 were selected. They were randomly divided into group A (n=30), group B (n=33) and group C(n=32). Group A was treated with warm gutta-percha filling technique + iRoot SP, group B was treated with warm gutta-percha filling technique + AH plus, and group C was treated with warm gutta-percha filling technique + Vitapex. The quality of root canal filling at 7 days after surgery was compared among the three groups, and the pain degree was assessed. Statistical analysis was performed using SPSS 19.0 software package. RESULTS: There was no significant difference in the rate of suitable filling among the three groups (P>0.05). The rates of level 1 pain in group A and group B (6.7% and 6.1%) were significantly lower than that in group C (28.1%) (P<0.05). CONCLUSIONS: The root canal sealing effects of warm gutta-percha filling technique combined with iRoot SP or AH plus or Vitapex are good. However, compared with Vitapex, iRoot SP and AH plus can relieve symptoms of pain after root canal treatment more effectively. They are suggested as the preferred sealers in root canal filling.