Fragment containing the whole riboflavin(rib)operons of B.cereus ATCC14579 was detected from GenBank and annotated.The rib operon of ATCC14579 was cloned with Pn,its native promoter,or with P43,the vegetative growth promoter,into the plasmid.Expression analysis showed that heterologous rib operon was operative in B.subtilis.Integrative plasmid with P43-rib fragment was integrated into the chromosome of B.subtilis RH33,yielding transformant B.subtilis PY.With optimized medium components,4.3 g·L -1 of riboflavin was achieved in batch culture of B.subtilis PY,which was 27%enhancement compared to the host strain.Real-time reverse transcription polymerase chain reaction(RT-PCR)analysis indicated that the transcriptional level of ribA maintained 2.8-fold higher with the expression of herterologous rib operon.Furthermore,the stability of B.subtilis PY was increased form 45%to 87%.The high transcriptional level of rib gene and higher stability of B.subtilis PY could explain the increased riboflavin production.
The present study is focused on protein degradation during astaxanthin synthesis in Haematococcus plu- vialis under high irradiance and nitrogen deficient conditions. It was found that with the onset of astaxanthin synthesis in the cultures of high light and nitrogen-free （HF）, high light and nitrogen-repletion （HR）, and low light and nitrogen-free （LF）, （1） endopeptidase （EP） activities increased along with decrease in protein content, （2） asparagine in HF and HR rose significantly before the first 4 and 5 day, but fell after that time. While, it increased slowly and continuously in LF, （3） ammonium increased continuously in HF and HR, whereas in LF, it was detected on the sixth day, and increased slowly on the following days. By contrast, in low light and nitrogen-repletion culture, （LR）, the contents of protein and asparagine as well as EP activity were maintained relatively constant, no astaxanthin and ammonium were detected. Furthermore, when HF was sealed and bubbled with CO2-free gas （02 and N2）, astaxan- thin content increased as the protein level decreased. These results strongly suggest that （1） the degraded protein served as a substitutive carbon source, to some extent, for the biosynthesis of astaxanthin, （2） endopeptidase was involved in the degradative process, （3） for detoxification, part of the ammonium generated by protein degradation was transiently stored in asparagine, whereas the rest of it was expelled into the culture broth.
