Gene manipulation is essential for metabolic engineering and synthetic biology, but the current general gene manipulation methods are not applicable to the non-model strain Corynebacterium glutamicum (C. glutamicum) ATCC14067, which is used for amino acid production. Here, we report an effective and sequential deletion method for C. glutamicum ATCC14067 using the exonuclease-recombinase pair RecE + RecT (RecET) for recombineering via a designed self-excisable linear double-strand DNA (dsDNA) cassette, which contains the Cre/loxP system, to accomplish markerless deletion. To the best of our knowledge, this is the first effective and simple strategy for recombination with markerless deletion in C. glutamicum ATCC14067. This strategy provides a simple markerless deletion strategy for C. glutamicum and builds a solid basis for producer construction.

The 4S pathway of biodesulfurization, which can specifically desulfurize aromatic S-heterocyclic compounds without destroying their combustion value, is a low-cost and environmentally friendly technology that is complementary to hydrodesulfurization. The four Dsz enzymes convert the model compound dibenzothiophene (DBT) into the sulfur-free compound 2-hydroxybiphenyl (HBP). Of these four enzymes, DszC, the first enzyme in the 4S pathway, is the most severely affected by the feedback inhibition caused by HBP. This study is the first attempt to directly modify DszC to decrease its inhibition by HBP, with the results showing that the modified protein is insensitive to HBP. On the basis of the principle that the final HBP product could show a blue color with Gibbs reagent, a high-throughput screening method for its rapid detection was established. The screening method and the combinatorial mutagenesis generated the mutant AKWC (A101K/W327C) of DszC. After the IC 50 was calculated, the feedback inhibition of the AKWC mutant was observed to have been substantially reduced. Interestingly, the substrate inhibition of DszC had also been reduced as a result of directed evolution. Finally, the recombinant BL21(DE3)/ BADC*+C* (C* represents AKWC) strain exhibited a specific conversion rate of 214.84 μmol HBP /g DCW /h, which was 13.8-fold greater than that of the wild-type strain. Desensitization engineering and the overexpression of the desensitized DszC protein resulted in the elimination of the feedback inhibition bottleneck in the 4S pathway, which is practical and effective progress toward the production of sulfur-free fuel oil. The results of this study demonstrate the utility of desensitization of feedback inhibition regulation in metabolic pathways by protein engineering.

AZ31 alloys were extruded by direct extrusion and bending-shear deformation (DEBS). The microstructure characteristic and texture evolution of DEBSed AZ31 sheets were investigated by electron backscattered diffraction (EBSD). It is found that DEBS technique could effectively refine grains and weaken texture. Besides, we also investigate how twinning affects dynamic recrystallization during hot extrusion. {10-12} extension twins can offer nucleation sites and enough energy to trigger dynamic recrystallization. Moreover, the character of direct extrusion and bending-shear die can lead to the activation of non-basal slip system and further dramatically weaken the basal texture of the microstructure with many preactivated basal slip systems.

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