Metabolic Engineering Of Escherichia Coli For The Production Of 5-Hydroxytryptophan

5-Hydroxytryptophan(5-HTP)is the biosynthetic precursor of the neurotransmitter serotonin and the amine hormone melatonin.It has been shown to be effective in the treatment of a variety of diseases,including depression,insomnia and chronic headaches.Currently,extraction from the seeds of Griffonia Simplifolia is the main commercial way for 5-HTP production,due to the lack of an efficient synthetic method.However,the material supply is seasonally and regionally dependent,resulting in limited production and high market price of 5-HTP.This study reports efficient microbial production of 5-HTP via metabolically engineered Escherichia coliHuman tryptophan hydroxylase(TPH)isoforms,TPH1 and TPH2 were firstly introduced into E.coli strain BL21(DE3)and their expression patterns and activities were studied.Cells expressing TPH were able to catalyze L-tryptophan into 5-HTP with externally added tetrahydrobiopterin(BH4)as the co-factor.For endogenous supply of BH4,human BH4 biosynthesis and regeneration pathway was reconstituted using a two-plasmid expression system.Whole-cell bioconversion resulted in high-level production of 5-HTP(1.24 g/L)from 2 g/L L-tryptophan in shake flasks.Further introduction of L-tryptophan biosynthetic pathway enabled 5-HTP biosynthesis from simple carbon sources.Recombinant strain HTP4-1 produced 22.4 mg/L 5-HTP in M9Y medium.We improved this titer to 314.8 mg/L,a 13-fold improvement,through the optimation of culture medium and fermentation conditions in shake flasks.However,the final concentration of L-tryptophan was found to be as high as 3971.8 mg/LCellular metabolic networks should be carefully balanced using metabolic engineering to produce the desired products at the industrial scale.For this purpose,the whole 5-HTP biosynthetic pathway was divided into four functional modules,L-tryptophan module,the BH4 synthesis module,the BH4 regeneration module,and the hydroxylation module.Accumulation of L-tryptophan was lowered by reducing the copy number of L-tryptophan module;Replacing TPH1 with a more stable mutant form enhanced the hydroxylation activity;and promoter regulation of the BH4 biosynthesis and regeneration modules reduced the metabolic burden caused by high-level protein expression.5-HTP was produced at a final titer of 1.29 g/L using the modified strain HTP101-LMT in the shake flasks.In a fed-batch fermentation using glycerol as the carbon source where the cell density reached an OD600 of 94.6,5-HTP production was further improved to 5.1 g/L,along with 8.5 g/L of L-tryptophan production.Next,we lowered L-tryptophan accumulation and increased the stability of tryptophan hydroxylation pathway through the integration of tryptophan synthetic pathway into E.coli genome.Regulation of the copy number of tryptophan hydroxylating plasmid,together with promoter substitution of aroHfbr gene helped to elevate 5-HTP production.Recombinant strain TRPmut/pSCHTP-LMT was able to produce 1.61 g/L 5-HTP in shake flasks,which presented a 24.8%improvement compared to HTP101-LMT.Whereas,L-tryptophan concentration was further decreased to 0.25 g/L,resulting an increase of the concentration ratio of 5-HTP to L-tryptophan from 0.78:1 for HTP101-LMT to 6.44:1 for TRP/pSCHTP-LMT,which would reduce the complexity for downstream 5-HTP isolation and purification.The role of aroHfbr gene in L-tryptophan and 5-HTP biosynthesis was explored in a sequential deletion of aroHfbr gene and its isoenzymes aroF and aroG.Deletion of aroHfbr impaired cell growth as well as L-tryptophan and 5-HTP synthesis,whereas deletion of aroF and aroG showed no visible influence with only a small decrease of L-tryptophan accumulation for aroG deletion.Double deletion of both aroHfbr and aroG showed a severe inhibition for cell growth,while no 5-HTP or L-tryptophan synthesis was detected,which suggested an important role of both aroHfbr and aroG in aromatic amino acids biosynthesis.