Aims:  This study attempted to fully explore the expression potentials of Pichia pastoris for producing porcine insulin precursor (PIP) through PIP copy number optimization. Methods and Results:  Multi‐copy strains were screened employing a highly efficient improved in vivo method and their copy numbers were quantified by real‐time qPCR. A range of Mut+P. pastoris strains harbouring 0, 1, 3, 6, 12, 18, 29, 52 copies of PIP were obtained. After 96 h methanol induction, a bell‐shaped correlation curve was observed between gene dosage and protein yield, and the maximum PIP expression level of 181 mg l−1 was achieved by a 12‐copy strain. Specific growth rate and methanol utilization capacity were found to decrease remarkably for high copy strains (>12 copies). Transcriptional analysis of KAR2 suggested higher copy strains were suffering more from ER stress. Conclusions:  A copy number around 12 is optimal for secretory expression of PIP in P. pastoris. Excess PIP gene dosage (>12 copies) significantly impaired the growth of P. pastoris hosts. Significance and Impact of the Study:  The methods developed and the discoveries made by this systematical investigation will be helpful to the application and understanding of Pichia pastoris expression system for heterologous overexpression.

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Aims:  Subcellullar localizations and cross‐immunities of GAPDHs from six common pathogenic bacteria in aquaculture were investigated. Methods and Results:  Subcellullar localizations of GAPDHs of Edwardsiella tarda EIB202, Edwardsiella ictaluri ATCC33202, Aeromonas hydrophila LSA34, Vibrio anguillarum MVM425, Vibrio alginolyticus EPGS020401 and Vibrio harveyi VIB647 were analysed with Western blotting, indirect immunofluorescence and flow cytometry examinations. Immunoprotections of different recombinant GAPDHs against these pathogens were investigated with zebrafish model. Western blotting of subcellular extractions showed that all GAPDHs were secreted into extracellular medium and periplasmic space. In addition, GAPDHs were demonstrated to distribute in the outer membranes except MVM425 and VIB647. And, GAPDHs were confirmed to be present on the surface of these bacteria with indirect immunofluorescence and flow cytometry examinations. The remarkable cross‐protective immunities of these recombinant GAPDHs were induced in zebrafish, and the relative protective survivals were almost over 60%. Conclusions:  Localizations of GAPDHs from these pathogenic bacteria were similar to many other causative agents. And, GAPDHs could be important protective antigens and give remarkable cross‐immunity against different pathogens. Significance and Impact of the Study:  Recombinant GAPDH could be designed as a broad spectrum vaccine candidate against multiple microbial infections in aquaculture.

Carbon nanofibers were in-situ grown on graphite-felt, and then Ru was supported to synthesize Ru/CNFs/GF structured catalyst. The comparisons of pressure gradient and reaction results clearly indicated that the Ru/CNFs/GF structured catalyst was more proper than powdered Ru/CNFs catalyst for sorbitol hydrogenolysis in a trickle bed reactor. For the structured catalyst, increasing the linear velocities would increase the sorbitol conversion, but only slightly change the glycols selectivity, which was due to the intensified mass transfer. Nevertheless, increasing the thickness of the carbon nanofibers layer would significantly decrease the glycols selectivity, which was due to the increased diffusion distance for hydrogen and glycols in the mesopores of the carbon nanofibers layer.