초록
<P><B>Abstract</B></P> <P>Cellular metabolic networks should be carefully balanced using metabolic engineering to produce the desired products at the industrial scale. As the precursor for the biosynthesis of the neurotransmitter serotonin, 5-hydroxytryptophan (5-HTP) is effective in treating a variety of diseases, such as depression, fibromyalgia, obesity, and cerebellar ataxia. Due to the lack of an efficient synthetic method, commercial production of 5-HTP is only achieved by extracting from the seeds of <I>Griffonia Smplicifolia</I>. This study reports efficient microbial production of 5-HTP via metabolically engineered <I>Escherichia coli.</I> Firstly, human tryptophan hydroxylase I (<I>TPH1</I>) gene was functionally expressed. For endogenous supply of the cofactor tetrahydrobiopterin (BH4), human BH4 biosynthesis and regeneration pathway was reconstituted. Whole-cell bioconversion resulted in high-level production of 5-HTP (~1.2 g/L) from 2 g/L <SMALL>L</SMALL>-tryptophan in shake flasks. Further metabolic engineering efforts were employed to achieve 5-HTP biosynthesis from simple carbon sources. The whole biosynthetic pathway was divided into three functional modules, <SMALL>L</SMALL>-tryptophan module, the hydroxylation module, and the BH4 module. By reducing the copy number of <SMALL>L</SMALL>-tryptophan module, replacing TPH1 with a more stable mutant form, and promoter regulation of the BH4 module, 5-HTP was produced at a final titer of 1.3 g/L in the shake flask and 5.1 g/L in a fed-batch fermenter with glycerol as the carbon source, both of which were the highest ever reported for microbial production of 5-HTP.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The cofactor BH4 biosynthesis and regeneration pathway was reconstituted in <I>Escherichia coli</I>. </LI> <LI> <I>De novo</I> production of 5-hydroxytryptophan (5-HTP) from glycerol/glucose was achieved. </LI> <LI> Highest production of 5-HTP was achieved through a combined metabolic pathway engineering strategy. </LI> </UL> </P>