Example 3
Effectiveness of Newly Evolved TpH Background Strain Using Schistosoma mansoni TpH
One of the 7 evolved high 5HTP-producers was selected to further evaluate if the mutations identified were only specifically beneficial to hsTpH2 or could be widely applicable to others. The chosen evolved strain was first cured to lose the evolution plasmid (e.g. the hsTpH gene) and this was immediately followed by re-introducing the E. coli tyrA gene. Upon restoration of the strain's tyrosine auxotrophy, the resulting strain was transformed with pHM2, which is identical to pHM1 used in the earlier evolution study except that the hsTpH gene was replaced with a Schistosoma mansoni TpH gene (SEQ ID NO:9). The 5HTP production of the resulting strain was compared to a wild-type strain carrying pHM2 in the presence of 100 mg/l tryptophan. Results showed the wild-type transformants could only produce ˜0.05 mg/l 5HTP while the newly evolved background strain transformants accumulated >20 mg/l. These production results demonstrated that the mutations acquired in the evolved background strain were not only beneficial to hsTpH but also to other TpHs; possibly applicable also to other aromatic amino acid hydroxylases (e.g. tyrosine hydroxylase).
