Adaptive laboratory evolution and metabolic regulation of genetic Escherichia coli W3110 toward low-carbon footprint production of 5-aminolevulinic acid

Background: The 5-aminolevulinic acid (5-ALA) is a prodrug that has been approved by FDA for photodynamic therapy of cancer. 5-ALA is intracellularly metabolized to protoporphyrin IX, which upon illumination with red light results in photooxidative damage to cells. However, microbial 5-ALA production is challenged by strain tolerance and limited metabolic engineering, while carbon capture is rarely conducted. In this study, E. coli W3110 was equipped with recombinant ALAS production and concomitant CO₂ assimilation capacity for low-carbon footprint 5-ALA production. Methods: A stable E. coli strain expressing recombinant ALAS was further improved by adaptive laboratory evolution (ALE). Glycine distribution between biomass and 5-ALA was deciphered using C¹³ isotope analysis based on C¹³–2-glycine. Metabolic flux was enforced by overexpressing phosphoenolpyruvate carboxylase (ppc) and knockout of phosphate acetyltransferase (pta). The ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK) were added to confer CO₂ assimilation ability in RSSL strain. Significant findings: The adaptive RL8 strain demonstrated 129% and 205% increase in biomass and 5-ALA, respectively. C¹³ isotope analysis revealed that RSSL assimilated CO₂ into biomass and generated glycine from glucose for maximal 5-ALA production. Overexpression of ppc and knockout of pta led to energy conservation, reduced acetate production, thus increasing 5-ALA accumulation (9.53 g/L). Carbon uptake via RuBisCO/PRK significantly reduced CO₂ emission to -2.32 g-CO₂/g-DCW.