Recombinant Escherichia coli O8 L-threonine 3-dehydrogenase (tdh)

Introduction to Recombinant Escherichia coli O8 L-Threonine 3-Dehydrogenase (Tdh)

Recombinant Escherichia coli O8 L-threonine 3-dehydrogenase (Tdh) is an engineered enzyme critical in the oxidative degradation of L-threonine. It catalyzes the NAD(P)+-dependent oxidation of L-threonine to 2-amino-3-oxobutyrate, a key intermediate in microbial threonine catabolism . The recombinant form is produced via heterologous expression in E. coli systems, enabling scalable production for industrial and research applications .

2.1. Molecular Properties

• Gene: Encoded by the tdh gene (UniProt ID: A1AHF3) .

• Domain: Full-length protein (1–341 amino acids) with catalytic residues conserved in the medium-chain alcohol dehydrogenase superfamily .

• Cofactors: Requires NAD+ and zinc for activity, distinguishing it from other short-chain dehydrogenases .

Table 1: Sequence Features of Recombinant Tdh

2.2. Catalytic Mechanism

Tdh oxidizes L-threonine to 2-amino-3-oxobutyrate, which nonenzymatically decarboxylates to aminoacetone and CO₂. Subsequent CoA-dependent cleavage by 2-amino-3-oxobutyrate lyase yields glycine and acetyl-CoA .

3.1. L-Threonine and L-Isoleucine Production

Inactivation of tdh in E. coli redirects carbon flux toward L-threonine accumulation, enhancing precursor availability for L-isoleucine biosynthesis:

• Key Studies:

  • Deletion of tdh in E. coli NXU102 increased L-isoleucine production by 72.3% (4.34 → 7.48 g·L⁻¹) compared to the wild-type strain .

  • Transcriptomic analysis revealed upregulation of aspartate pathway genes and oxidative phosphorylation, improving metabolic efficiency .

Table 2: Impact of tdh Knockout in E. coli Strains

3.2. Industrial Relevance

• Strain Optimization: tdh deletion in E. coli MDS-205 reduced threonine degradation, increasing production by 83% .

• CRISPR/Cas9 Editing: Simultaneous knockout of tdh, ltaE, and yiaY enhanced carbon flux toward L-isoleucine while improving bacterial growth .

4.1. Recombinant Expression

• Expression Systems: E. coli, yeast, baculovirus, or mammalian cells .

• Purity: >85% via SDS-PAGE, confirmed by N-terminal sequencing .

5.2. Biotechnological Innovations

• Plasmid Engineering: Strains like E. coli BKIIM B-3996 use recombinant plasmids (e.g., pVIC40) to overexpress feedback-resistant threonine operons while deleting tdh .

Challenges and Future Directions

• Byproduct Accumulation: Acetic acid buildup in tdh-deficient strains requires balancing glycolytic and TCA cycle fluxes .

• Transcriptomic Profiling: Further studies on global gene regulation could optimize carbon allocation in industrial strains .