# Engineering the TCA cycle regulator GarA to increase erythromycin production in Saccharopolyspora erythraea

## TL;DR

Scientists modified a protein called GarA in a bacteria to boost antibiotic production, which could help in making more medicines.

## Contribution

The study shows that engineering GarA can enhance erythromycin production in Saccharopolyspora erythraea.

## Key findings

- Phosphorylation of GarA occurs at the N-terminal ETTS motif in S. erythraea.
- Engineered S. erythraea with phosphoablative GarA produced twice as much erythromycin.
- Altered amino acid metabolism suggests GarA influences precursor supply for antibiotic biosynthesis.

## Abstract

Actinobacteria are important for industrial production of antibiotics, fine chemicals and food and a source of new compounds for drug discovery. Their central metabolism is regulated by a conserved protein GarA that is unique to the Actinobacteria and has been studied in Mycobacterium tuberculosis and Corynebacterium glutamicum. GarA regulates the TCA cycle and glutamate metabolism by direct binding to enzymes to modulate their activity on glutamate and alpha-ketoglutarate. Given the importance of the TCA cycle in the synthesis of acyl-CoA precursors for antibiotic biosynthesis, and increasing evidence for the role of nitrogen regulators in control of secondary metabolism, we hypothesized that engineering GarA could be used to enhance production of valuable metabolites. His6-tagged GarA was introduced into Saccharopolyspora erythraea, an overproducer of the polyketide antibiotic erythromycin. Phosphorylation of GarA was detected at the N-terminal ETTS motif, suggesting that it is regulated by protein kinases like in M. tuberculosis. GarA expression was observed at all growth stages, and a truncated form lacking the phosphorylation site accumulated during late fermentation. Engineered S. erythraea expressing phosphoablative GarA produced twofold more erythromycin, both in standard fermentation broth and in minimal medium. To investigate the mechanism for the increased titre, the engineered strain was characterized for transcription of erythromycin biosynthetic genes, as well as its ability to metabolize glutamate and its intracellular and extracellular aa content. The observed alterations in aa metabolism are consistent with the role of GarA as a TCA cycle regulator that may influence precursor supply for polyketide biosynthesis.