RESUMO
This paper describes the development of a straightforward method for site-directed gene mutagenesis in Streptococcus zooepidemicus, inspired by the mechanism of natural competence regulated by ComX in other streptococci. An alternative sigma factor comX gene was overexpressed from a plasmid in S. zooepidemicus and electrocompetent cells were prepared. As proof of concept, a DNA cassette with two targeting regions flanking a kanamycin resistance gene was spliced in an overlap extension PCR and electroporated. The cassette was then integrated in the genomic DNA by homologous recombination. Next, the gene SeseC_00180 (fibrinogen- and Ig-binding protein precursor) was selected as target for markerless gene deletion and the impact of its loss on the resulting hyaluronan production was determined. The new method of site-directed mutagenesis is significant because it is not necessary to clone the DNA cassette in an auxiliary vector, electroporating it in S. zooepidemicus cells is enough, which allows to bypass the problems with hard to clone DNA sequences and speeds up the whole process of mutation generation in S. zooepidemicus.
Assuntos
Streptococcus equi , Sequência de Bases , Deleção de Genes , Mutagênese Sítio-Dirigida , Plasmídeos/genética , Streptococcus equi/genéticaRESUMO
Monascus purpureus was grown in submerged liquid culture using ammonium sulfate, sodium nitrate, and peptone as nitrogen sources while initial medium pH was adjusted to 2.5, 5.5, 6.5, or 8.0. The combined effect of culture pH and nitrogen source on the biosynthesis of yellow (ankaflavin and monascin) and orange (rubropunctatin and monascorubrin) pigments, plus the mycotoxin citrinin, was evaluated chromatographically. Optimum cultivation conditions, that is, initial pH 2.5 and 8.8 g/L peptone as a nitrogen source, resulted in high levels of production of yellow and orange pigments (sum of pigment concentration 1,138 mg/L) and negligible citrinin concentration (2 mg/L).
