An efficient Agrobacterium-mediated system based on the pyrG auxotrophic marker for recombinant expression in the filamentous fungus Penicillium rubens.

OBJECTIVES: This work aimed to construct a versatile, effective, and food-grade Agrobacterium tumefaciens-mediated transformation (ATMT) system for recombinant expression in the filamentous fungus Penicillium rubens (also known as Pencillium chrysogenum). RESULTS: In this study, the wild-type P. chrysogenum VTCC 31172 strain was re-classified as P. rubens by a multilocus sequencing analysis. Further, the pyrG gene required for uridine/uracil biosynthesis was successfully deleted in the VTCC 31172 strain by homologous recombination to generate a stable uridine/uracil auxotrophic mutant (ΔpyrG). The growth of the P. rubens ΔpyrG strain could be restored by uridine/uracil supplementation, and a new ATMT system based on the uridine/uracil auxotrophic mechanism was established for this strain. The optimal ATMT efficiency could reach 1750 transformants for 106 spores (equivalent to 0.18%). In addition, supplementation of uridine/uracil at the concentrations of 0.005-0.02% during the co-cultivation process significantly promoted transformation efficiency. Especially, we demonstrated that the pyrG marker and the amyB promoter from the koji mold Aspergillus oryzae were fully functional in P. rubens ΔpyrG. Expression of the DsRed reporter gene under the regulation of the A. oryzae amyB promoter lighted up the mycelium of P. rubens with a robust red signal under fluorescence microscopy. Furthermore, genomic integration of multiple copies of the Aspergillus fumigatus phyA gene under the control of the amyB promoter significantly enhanced phytase activity in P. rubens. CONCLUSIONS: The ATMT system developed in our work provides a safe genetic platform for producing recombinant products in P. rubens without using drug resistance markers.

The Distribution of Autoantibodies by Age Group in Children with Type 1 Diabetes versus Type 2 Diabetes in Southern Vietnam.

Asian children are increasingly being diagnosed with type 1 diabetes (T1D) or type 2 diabetes (T2D), and the presence of coexisting islet autoimmune antibodies complicate diagnosis. Here, we aimed to determine the prevalence of islet cell autoantibodies (ICAs) and glutamic acid decarboxylase 65 autoantibodies (GADAs) in children with T1D versus T2D living in Vietnam. This cross-sectional study included 145 pediatric patients aged 10.3 ± 3.6 years, with 53.1% and 46.9% having T1D and T2D, respectively. ICAs were reported in only 3.9% of pediatric T1Ds, which was not significantly different from the 1.5% of those with T2D. Older children with T1D were positive for either ICAs, or ICAs and GADAs (5-9 and 10-15 years), whereas only a small proportion of children aged 0-4 years were positive for GADAs (18%). Notably, 27.9% of children with T2D aged 10-15 were positive for GADAs, and all were classified as overweight (n = 9) or obese (n = 10). GADAs were more commonly observed in T1D patients younger than four years than ICAs, which were more prevalent in older children (5-15 years). Even though few children with T2D carried ICAs and GADAs, finding a better biomarker or an appropriate time to confirm diabetes type may require further investigation.