Recombinant Corynebacterium glutamicum Adenylosuccinate synthetase (purA)

Basic Research Questions

What is the functional role of adenylosuccinate synthetase (PurA) in C. glutamicum metabolism?

PurA catalyzes the conversion of inosine monophosphate (IMP) to adenylosuccinate in the de novo purine biosynthesis pathway, a critical step for AMP synthesis. Its activity directly influences intracellular AMP/GMP ratios, which regulate nucleotide pools and downstream cellular processes like transcription and energy metabolism . Experimental validation involves:

• Gene deletion studies: ΔpurA strains exhibit adenine auxotrophy and reduced AMP levels (Table 1) .

• Metabolite profiling: Quantifying intermediates like IMP, AMP, and adenosine in mutants (e.g., ΔpurA vs. wild type) using HPLC or LC-MS .

Table 1: Intracellular purine intermediate concentrations in C. glutamicum strains (μmol/gCDW)

Why is C. glutamicum a preferred host for recombinant PurA studies compared to E. coli?

C. glutamicum offers:

• Low protease activity: Reduces recombinant protein degradation .

• Secretion efficiency: Properly folded proteins can be secreted extracellularly via signal peptides (e.g., cspB) .

• Safety: GRAS (Generally Recognized As Safe) status and absence of endotoxins .
  Methodologically, constitutive promoters (e.g., PsodA*) and auto-inducible systems (e.g., P₄−N₁₄) are optimized for stable expression .

Advanced Research Questions

How can metabolic flux analysis resolve contradictions in purine intermediate accumulation in recombinant C. glutamicum strains?

Discrepancies in IMP/AMP levels between ΔpurA and ΔguaB2 mutants (Table 1) arise from:

• Feedback regulation: AMP inhibits PurF (glutamine phosphoribosylpyrophosphate amidotransferase) .

• Salvage pathway activation: ΔpurA strains compensate via adenine phosphoribosyltransferase (APT) .
  To address this:

• Use ¹³C metabolic flux analysis to quantify de novo vs. salvage pathway contributions.

• Engineer dual mutants (e.g., ΔpurA ΔguaB2) to block competing pathways .

What CRISPR-Cas9 strategies improve PurA expression in C. glutamicum?

Advanced strain engineering involves:

• Promoter optimization: Replace native purA promoter with strong constitutive/inducible variants (e.g., tac) .

• Ribosome binding site (RBS) engineering: Modulate translation initiation efficiency via RBS libraries .

• Multiplex deletions: Combine ΔpurA with Δpgi (phosphoglucose isomerase) to redirect carbon flux toward nucleotide precursors .

How do transcriptomic and proteomic datasets explain low recombinant PurA yields in C. glutamicum?

Key bottlenecks identified via RNA-seq and proteomics:

• Resource competition: Recombinant PurA expression downregulates ribosomal proteins and ATP synthases, impairing growth .

• Misfolding stress: Overexpression triggers chaperone upregulation (e.g., dnaK) .
  Mitigation strategies:

• Use fed-batch bioreactors to decouple growth and production phases .

• Co-express folding chaperones (e.g., GroEL/ES) .

What experimental controls are critical when analyzing PurA activity in engineered strains?

To ensure data validity:

• Internal standards: Spike stable isotope-labeled AMP/IMP during metabolite extraction .

• Enzyme kinetics: Compare Kₘ and Vₘₐₓ of recombinant vs. native PurA using spectrophotometric assays .

• Genetic controls: Include complemented strains (e.g., ΔpurA + plasmid-borne purA) to confirm phenotype reversibility .