Mutational impact of classical strain improvement on Penicillium chrysogenum

We have examined the impact of mutations introduced into P. chrysogenum during CSI in amino acid metabolism, and the results
shows that many of the mutations served to inactive competing biosynthesis
pathway to optimize the precursor flux towards cysteine biosynthesis. In
addition, the work shows that other mutations in metabolism in a subtler manner
contributed to optimized penicillin production, whereas some mutations had no
effect, likely reflecting side effects of the rather harsh way the mutations were
induced during the CSI programme. Here in this thesis, a number of genes were
studied and their functions were identified. However, many other genes obtained
mutations and their functions still remain unknown. Along with the genome sequencing
techniques, the advent of novel genome editing system, namely CRISPR/
Cas9 technology provides a powerful tool for unravelling the exact gene and
pathway functions of interest. This can be approached by deleting certain genes
and observing the changes and effects on phenotypes. Of interest would be the
removal of the direct sulfhydrylation pathway by deleting the Pc22g16570 gene
encoding for the enzyme catalyzing the first-step reaction. This will contribute
to a better understanding of the role of the two pathways for cysteine biosynthesis
in P. chrysogenum. This should be combined with techniques of monitoring the
levels of intracellular and extracellular metabolites. By use of these technologies,
the underlying mechanism of classical strain improvement on P. chrysogenum
strains for enhanced penicillin production will be revealed at an unprecedented
detail and also will give guidance for increasing the titers of valuable secondary
metabolites in cells in a rational manner.