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<p>5-Hydroxytryptophan (5-HTP) is widely used as a natural remedy for sleep disorders. In terms of biosafety, bio-derived 5-HTP is preferred over chemically synthesized 5-HTP. However, the low titer of 5-HTP in the reported microbiological methods (&lt; 10&#xa0;g/L) limits the industrialization of 5-HTP biosynthesis. In the present study, a Trp-accumulating <i>E. coli</i> strain TRP1 was constructed by blocking the degradation path (Δ<i>tnaA</i>), branching paths (Δ<i>pheA</i>, Δ<i>tyrA</i>) and repression system (Δ<i>trpR</i>, Δ<i>trpL</i>). Next, the hydroxylation module employing a phenylalanine hydroxylase mutant XcPAH<sup>W179F</sup> (XC2) coupled with an MH4 regenerating system (CvPCD-EcFolM system) was screened to convert L-Trp into 5-HTP. Protein engineering was performed on hydroxylase XC2 based on the molecular dynamics simulation of the enzyme-substrate complex, and the strain TRP1-XC4 harboring the triple-mutant XcPAH<sup>L98I/A129K/W179F</sup> (XC4) was able to produce 319.4&#xa0;mg/L 5-HTP. Genome editing was carried out focused on accelerating product efflux (strengthening YddG) and increasing MH4 supply (strengthening FolM, FolE and FolX), resulting in a strain TRP5-XC4 to produce 13.9&#xa0;g/L 5-HTP in 5&#xa0;L fed-batch fermentation with a space-time yield of 0.29&#xa0;g/L/h, which is the highest production and productivity record for 5-HTP biosynthesis. This study successfully provided an engineered strain and an efficient green method for the industrial synthesis of 5-HTP.</p> Graphical Abstract <p></p>

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Engineered phenylalanine hydroxylase coupled with an effective cofactor synthesis and regeneration system for high-yield production of 5-hydroxytryptophan

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年3月6日

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  • Yulin Ai,
  • Yusong Huang,
  • Hongru Zhao,
  • Bingmei Su,
  • Juan Lin

摘要

5-Hydroxytryptophan (5-HTP) is widely used as a natural remedy for sleep disorders. In terms of biosafety, bio-derived 5-HTP is preferred over chemically synthesized 5-HTP. However, the low titer of 5-HTP in the reported microbiological methods (< 10 g/L) limits the industrialization of 5-HTP biosynthesis. In the present study, a Trp-accumulating E. coli strain TRP1 was constructed by blocking the degradation path (ΔtnaA), branching paths (ΔpheA, ΔtyrA) and repression system (ΔtrpR, ΔtrpL). Next, the hydroxylation module employing a phenylalanine hydroxylase mutant XcPAHW179F (XC2) coupled with an MH4 regenerating system (CvPCD-EcFolM system) was screened to convert L-Trp into 5-HTP. Protein engineering was performed on hydroxylase XC2 based on the molecular dynamics simulation of the enzyme-substrate complex, and the strain TRP1-XC4 harboring the triple-mutant XcPAHL98I/A129K/W179F (XC4) was able to produce 319.4 mg/L 5-HTP. Genome editing was carried out focused on accelerating product efflux (strengthening YddG) and increasing MH4 supply (strengthening FolM, FolE and FolX), resulting in a strain TRP5-XC4 to produce 13.9 g/L 5-HTP in 5 L fed-batch fermentation with a space-time yield of 0.29 g/L/h, which is the highest production and productivity record for 5-HTP biosynthesis. This study successfully provided an engineered strain and an efficient green method for the industrial synthesis of 5-HTP.

Graphical Abstract