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The effects of wheat-gluten hydrolysates (WGH) and their ultrafiltration fractions on multiple-stress tolerance and ethanol production in yeast during very-high-gravity (VHG) fermentation were examined. The results showed that WGH and WHG-ultrafiltration-fraction supplementations could significantly enhance the growth and viability of yeast and further improve the tolerance of yeast to osmotic stress and ethanol stress. The addition of MW < 1 kDa fractions led to 51.08 and 21.70% enhancements in cell-membrane integrity, 30.74 and 10.43% decreases in intracellular ROS accumulation, and 34.18 and 26.16% increases in mitochondrial membrane potential (ΔΨ) in yeast under osmotic stress and ethanol stress, respectively. Moreover, WGH and WHG-ultrafiltration-fraction supplementations also improved the growth and ethanol production of yeast during VHG fermentation, and supplementation with the <1 kDa fraction resulted in a maximum biomass of 16.47 g/L dry cell and an ethanol content of 18.50% (v/v) after VHG fermentation.

Considerable attention has been given to the development of robust fermentation processes, but microbial contamination and phage infection remain deadly threats that need to be addressed. In this study, a robust Escherichia coli BL21(DE3) strain was successfully constructed by simultaneously introducing a nitrogen and phosphorus (N&P) system in combination with a CRISPR/Cas9 system. The N&P metabolic pathways were able to express formamidase and phosphite dehydrogenase in the host cell, thus enabled cell growth in auxotrophic 3-(N-morpholino) propanesulfonic acid medium with formamide and phosphite as nitrogen and phosphorus sources, respectively. N&P metabolic pathways also allowed efficient expression of heterologous proteins, such as green fluorescent protein (GFP) and chitinase, while contaminating bacteria or yeast species could hardly survive in this medium. The host strain was further engineered by exploiting the CRISPR/Cas9 system to enhance the resistance against phage attack. The resultant strain was able to grow in the presence of T7 phage at a concentration of up to 2 × 10 7 plaqueforming units/ml and produce GFP with a yield of up to 30 μg/10 9 colony-forming units, exhibiting significant advantages over conventional engineered E. coli. This newly engineered, robust E. coli BL21(DE3) strain therefore shows great potential for future applications in industrial fermentation. K E Y W O R D Santimicrobial, anti-phage, CRISPR/Cas9 system, Escherichia coli, N&P pathway

Two novel polysaccharides DOP1-DES and DOP2-DES with molecular weight of 297,911 Da and 30,400 Da were extracted by using DESs from Dendrobium officinale for the first time, and purified with macroporous resin AB-8, Sephadex G75 and Sephadex G200 chromatography. Besides, the effect of extraction temperature, molar ratio of DES, reaction time and compound enzymes concentration on the extraction yield of polysaccharides were investigated. Monosaccharide composition analysis showed DOP1-DES and DOP2-DES were composed of D-glucose and D-mannose with the ratio of 2.2:1 and 3.7:1, respectively. Structural features of DOP1-DES and DOP2-DES were investigated by a combination of FT-IR, methylation analysis and NMR, which indicated that both polysaccharides had a similarly backbone consisting of (1, 4)-linked β-D-Manp and (1, 4)-linked β-D-Glcp with different ratio. Simultaneously, the two fractions possessed considerable antioxidant activity.

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