초록
<P>5-Hydroxytryptophan (5-HTP) is a drug that is clinically effective against depression, insomnia, obesity, chronic headaches, etc. It is only commercially produced by the extraction from the seeds of <I>Griffonia simplicifolia</I> because of a lack of synthetic methods. Here, we report the efficient microbial production of 5-HTP via combinatorial protein and metabolic engineering approaches. First, we reconstituted and screened prokaryotic phenylalanine 4-hydroxylase activity in <I>Escherichia coli</I>. Then, sequence- and structure-based protein engineering dramatically shifted its substrate preference, allowing for efficient conversion of tryptophan to 5-HTP. Importantly, <I>E. coli</I> endogenous tetrahydromonapterin (MH4) could be utilized as the coenzyme, when a foreign MH4 recycling mechanism was introduced. Whole-cell bioconversion allowed the high-level production of 5-HTP (1.1–1.2 g/L) from tryptophan in shake flasks. On this basis, metabolic engineering efforts were further made to achieve the <I>de novo</I> 5-HTP biosynthesis from glucose. This work not only holds great scale-up potential but also demonstrates a strategy for expanding the native metabolism of microorganisms.</P><P><B>Graphic Abstract</B><BR><IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/asbcd6/2014/asbcd6.2014.3.issue-7/sb5002505/production/images/medium/sb-2014-002505_0005.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/sb5002505'>ACS Electronic Supporting Info</A></P>