Break

In our previous work, one combined biosynthetic pathway was engineered to produce 5-hydroxy-L-tryptophan (5-HTP) with glycerol as carbon source in E. coli. In the present work, the low specific bioactivity of tryptophan hydroxylase and low stability of two co-enzymes (pterin-4α-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) ) were identified to be two factors which seriously hinders further enhancement of 5-HTP production. A gene-circuit based high-throughput screening method was developed to evolve the above proteins. One mutant of TPH-8 was screened out to have 1.5-fold bioactivity improvement, and the mutants of PCD 94 or DHPR 109 were also obtained to have obviously improved stabilities. By combining the evolution of all the three proteins, the titer of 5-HTP was improved 3.7 fold compared with those with the original strain in the shake flask. The present work would pave one new road for the bioproduction of 5-hydroxy-L-tryptophan in industry.