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Amino acid biosynthesis is essential for cell growth, protein synthesis, and metabolic regulation. Cells generate essential and non-essential amino acids from metabolic intermediates to sustain vital biological functions. These intermediates originate from key metabolic pathways: glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway. Important precursors include α-ketoglutarate, pyruvate, oxaloacetate, phosphoenolpyruvate, and erythrose-4-phosphate, which...
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Engineering Tryptophan Synthase via In Vivo Directed Evolution for High-Level l-Cysteine Production.

Xingyu Zhu1,2, Hengwei Zhang1,3, Di Zhang1

  • 1Key Laboratory of Industrial Biotechnology of the Ministry of Education, School of Biotechnology, Laboratory of Applied Microorganisms and Metabolic Engineering, Jiangnan University, Wuxi 214122, China.

Journal of Agricultural and Food Chemistry
|March 15, 2026
PubMed
Summary
This summary is machine-generated.

Researchers improved the industrial production of l-cysteine using tryptophan synthase (TrpS). Directed evolution yielded a superior TrpS mutant, achieving high l-cysteine titers and efficient whole-cell immobilization for enhanced biosynthesis.

Keywords:
directed evolutionimmobilizationl-cysteinesensortryptophan synthase

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Area of Science:

  • Biocatalysis
  • Enzyme Engineering
  • Metabolic Engineering

Background:

  • Direct synthesis of l-cysteine from l-serine and sodium hydrosulfide is catalyzed by tryptophan synthase (TrpS).
  • Current TrpS efficiency is limited under high substrate concentrations, hindering industrial application.
  • Enhancing TrpS catalytic efficiency is crucial for cost-effective l-cysteine production.

Purpose of the Study:

  • To develop a directed evolution platform for improving tryptophan synthase (TrpS) for l-cysteine biosynthesis.
  • To identify TrpS variants with enhanced catalytic activity and stability.
  • To establish an industrially viable process for l-cysteine production.

Main Methods:

  • A directed evolution platform was established, incorporating in vivo continuous mutagenesis.
  • A highly sensitive l-cysteine biosensor and fluorescence-activated cell sorting (FACS) were used for high-throughput screening.
  • Whole-cell immobilization strategy was employed to enhance biocatalyst stability and reusability.

Main Results:

  • Multiple TrpS variants were successfully generated, with the V139M/A302P mutant showing significantly improved activity and stability.
  • The engineered TrpS mutant achieved an l-cysteine titer of 116.4 g/L under optimized conditions.
  • Immobilized cells produced a maximum l-cysteine titer of 113.05 g/L with a 93.3% conversion rate in a single batch.

Conclusions:

  • The developed directed evolution platform is efficient for screening improved biocatalysts.
  • The engineered TrpS mutant and immobilization strategy offer a promising route for industrial l-cysteine biosynthesis.
  • This study provides a robust and scalable method for producing l-cysteine.