Bacterial microcompartment-directed polyphosphate kinase promotes stable polyphosphate accumulation in E. coli.

Area of Science:

• Microbiology
• Biotechnology
• Environmental Science

Background:

• Biological phosphate removal from wastewater often relies on transient bacterial polyphosphate accumulation.
• Polyphosphate levels in E. coli are regulated by polyphosphate kinase (PPK1) and exopolyphosphatases (PPX, GPPA).
• Current methods using enzyme overexpression yield only temporary increases in polyphosphate.

Purpose of the Study:

• To investigate if partitioning PPK1 from cytoplasmic exopolyphosphatases can increase and stabilize E. coli polyphosphate levels.
• To enhance biological phosphate removal efficiency through improved polyphosphate storage.
• To explore the role of subcellular compartmentalization in metabolic product sequestration.

Main Methods:

• Co-expression of E. coli PPK1 fused with a microcompartment-targeting sequence.
• Co-expression of an artificial operon of Citrobacter freundii bacterial microcompartment genes.
• Utilizing bacterial microcompartments to encapsulate targeted PPK1 and sequester polyphosphate.

Main Results:

• Encapsulation of targeted PPK1 led to persistent phosphate uptake and stable increases in cellular polyphosphate throughout cell growth.
• Targeted PPK1 increased polyphosphate within microcompartments 8-fold compared to non-targeted PPK1.
• Compartmentalization prevented PPX polyphosphatase from reducing elevated polyphosphate levels, unlike with non-targeted PPK1.

Conclusions:

• Subcellular compartmentalization of PPK1 effectively sequesters polyphosphate, preventing its degradation by cytoplasmic enzymes.
• This strategy significantly enhances and stabilizes cellular polyphosphate levels, offering a promising approach for biological phosphate removal.
• Preventing enzyme access to metabolic products via compartmentalization is a viable strategy for metabolic engineering.