Simple and efficient expression of Agaricus meleagris pyranose dehydrogenase in Pichia pastoris

Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression hosts more suitable for genetic engineering. Expression in Escherichia coli resulted in no soluble or active PDH. Heterologous expression in the methylotrophic yeast Pichia pastoris was investigated using two different signal sequences as well as a codon-optimized sequence. A 96-well plate activity screening for transformants of all constructs was established and the best expressing clone was used for large-scale production in 50-L scale, which gave a volumetric yield of 223 mg L−1 PDH or 1,330 U L−1 d−1 in space–time yield. Purification yielded 13.4 g of pure enzyme representing 95.8% of the initial activity. The hyperglycosylated recombinant enzyme had a 20% lower specific activity than the native enzyme; however, the kinetic properties were essentially identical. This study demonstrates the successful expression of PDH in the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, the feasibility of large-scale production of the enzyme with this expression system together with a simplified purification scheme for easy high-yield purification is shown

Effect of Different Water Salinities on the Larvae of the Blue Bream <i>Ballerus ballerus</i> (Linnaeus, 1758) during Rearing

The influence of water salinities of 3, 5, and 7 ppt on the growth and survival of Ballerus ballerus (L.) larvae was studied. The control group was fish reared in freshwater (0 ppt). The larvae showed high tolerance to water salinities of 5–7 ppt. The mean final weight of the larvae ranged from 48.6 to 64.1 mg, with corresponding mean total lengths from 18.9 to 22.6 mm, depending on the water salinity level. The best larval length increments were recorded in water with salinity of 3 ppt. They were only slightly lower in 0 ppt water, and there were no statistically significant differences between the breeding rates calculated for larvae reared in 3 ppt water. Depending on the salinity level of the water, the final survival rate of the blue bream larvae ranged from 83.5 to 98.6%. The blue bream larvae reared in water with salinity levels of 5 and 7 ppt were statistically smaller than the others, but the results obtained were equally satisfactory

Effect of Salinity on the Gut Microbiome of Pike Fry (Esox lucius)

The increasing popularity of pike in angling and fish farming has created a need to increase pike production. However, intensive pike farming is subject to limitations due to diseases and pathogens. Sodium chloride (NaCl) could be a good alternative to chemotherapeutics, especially for protecting the fish against pathogens and parasites at early life stages. However, the impact of high salinity on the symbiotic bacteria inhabiting freshwater fish is still unclear. Therefore, our objective was to analyze the gut microbiome to find possible changes caused by salinity. In this study, the influence of 3&permil; and 7&permil; salinity on pike fry was investigated. High-throughput 16S rRNA gene amplicon sequencing was used to profile the gut microbiome of the fish. It was found that salinity had a statistically significant influence on pike fry mortality. Mortality was highest in the 7&permil; salinity group and lowest in the 3&permil; group. Microbiological analysis indicated that Proteobacteria and Actinobacteria predominated in the pike gut microbiome in all examined groups, followed by lower percentages of Bacteroidetes and Firmicutes. There were no statistically significant differences in the percent abundance of bacterial taxa between the control group and groups with a higher salinity. Our results suggest that salinity influences the gut microbiome structure in pike fry, and that 3&permil; salinity may be a good solution for culturing pike at this stage in their development