Gradient radiation breeding and culture domestication of menaquinone producing strains.

By comparing the survival rate and positive mutation rate of the primary mutagenic strain and progeny mutagenic strain under different radiation doses, the results showed that the tolerance of the mutagenic strain to radiation dose increased with the increase of the mutagenic generations. We adopted an improved gradient radiation breeding strategy to improve the breeding efficiency. The strains were treated with radiation in four stages. The first stage was low energy N+ ion implantation (ion energy 15 keV, dose 80 × 2.6 × 1013 cm-2). In the second stage, the energy and dose of N+ ion reached to 20 keV, 90 × 2.6 × 1013 cm-2. In the third stage, 60Co-γ radiation (dose of 1.56 kGy) was used. In the fourth stage, the radiation dose of 60Co-γ increased to 1.82 kGy. After each stage of radiation, the MK (Menaquinone) precursor 1, 4-dihydroxy-2-naphthalate (DHNA) was used as the stress factor to domesticate the mutant strains. By gradually increasing the concentration of DHNA in the culture medium, the substrate tolerance of Flavobacterium sp. was effectively improved. By measuring SOD (superoxide dismutase) activity and malondialdehyde, it showed that the cell damage caused by radiation mutagenesis to the offspring mutant was less than that of the primary mutant. Changes in membrane permeability and membrane potential of the mutant strains were reflected in changes in fluorescence intensity of luciferin diacetate and rhodamine 123, which could explain the enhanced substrate tolerance of strain F-2. After gradient radiation breeding and culture acclimation, the biomass of mutant Strain F-2 was 6.59 g/L, and the MK yield was 9.59 mg/L.

Impact of metformin on survival outcome of esophageal squamous cell carcinomas patients undergoing surgical resection: a multicenter retrospective study.

BACKGROUND: Diabetes mellitus is a recognized risk factor for esophageal squamous cell carcinomas (ESCC), and metformin is a recognized protective factor for some gastrointestinal tumors. But knowledge is limited regarding the effect of metformin on survival outcome of ESCC patients with type 2 diabetes mellitus (T2DM). We assessed the impact of post-diagnosis metformin use on overall survival (OS) and disease-free survival (DFS) in ESCC with T2DM undergoing surgical resection. METHODS: A retrospective analysis was performed on 3,523 patients with ESCC who met the study conditions after surgical resection. Log-rank and Cox regression models were used to evaluate the relationship between metformin and T2DM and ESCC survival rate, and adjusted according to age, gender, BMI, smoking, drinking and staging, et al. RESULTS: Among included ESCC patients, 619 were associated with type 2 diabetes, while the remaining 2,904 were not associated with type 2 diabetes. The 5-year OS (28.43%) of patients with T2DM was significantly lower than that of patients without T2DM (32.75%), P=0.037. DFS in 5 years were 27.30% (with T2DM) and 31.75% (without T2DM) (P=0.030), respectively. Compared with patients without T2DM, patients with T2DM presented worse OS [adjusted risk ratio (HRadj) =1.19] and DFS (HRadj =1.17; P<0.001). Among the 619 patients with type 2 diabetes, 485 were treated with metformin and 134 were not treated with metformin. Patients treated with metformin had significantly improved OS [adjusted risk ratio (HRadj) =0.89; P=0.031) and DFS (HRadj =0.90; P=0.013). CONCLUSIONS: T2DM was again associated with poorer survival in ESCC patients, and metformin may improve the prognosis of these patients.

Morphological regulation of Aspergillus niger to improve citric acid production by chsC gene silencing.

The mycelial morphology of Aspergillus niger, a major filamentous fungus used for citric acid production, is important for citric acid synthesis during submerged fermentation. To investigate the involvement of the chitin synthase gene, chsC, in morphogenesis and citric acid production in A. niger, an RNAi system was constructed to silence chsC and the morphological mutants were screened after transformation. The compactness of the mycelial pellets was obviously reduced in the morphological mutants, with lower proportion of dispersed mycelia. These morphological changes have caused a decrease in viscosity and subsequent improvement in oxygen and mass transfer efficiency, which may be conducive for citric acid accumulation. All the transformants exhibited improvements in citric acid production; in particular, chsC-3 showed 42.6% higher production than the original strain in the shake flask. Moreover, the high-yield strain chsC-3 exhibited excellent citric acid production potential in the scale-up process.The citric acid yield and the conversion rate of glucose of chsC-3 were both improved by 3.6%, when compared with that of the original strain in the stirred tank bioreactor.

Biotransformation of menadione to its prenylated derivative MK-3 using recombinant Pichia pastoris.

Prenylated quinones, especially menaquinones, have significant physiological activities, but are arduous to synthesize efficiently. Due to the relaxed aromatic substrate specificity and prenylation regiospecificity at the ortho- site of the phenolic hydroxyl group, the aromatic prenyltransferase NovQ from Streptomyces may be useful in menaquinone synthesis from menadione. In this study, NovQ was overexpressed in Pichia pastoris. After fermentation optimization, NovQ production increased by 1617%. Then the different effects of metal ions, detergents and pH on the activity of purified NovQ were investigated to optimize the prenylation reaction. Finally, purified NovQ and cells containing NovQ were used for menadione prenylation in vitro and in vivo, respectively. Menaquinone-1 (MK-1) was detected as the only product in vitro with γ,γ-dimethylallyl pyrophosphate and menadione hydroquinol substrates. MK-3 at a concentration of 90.53 mg/L was detected as the major product of whole cell catalysis with 3-methyl-2-buten-1-ol and menadione hydroquinol substrates. This study realized whole cell catalysis converting menadione to menaquinones.