Low-Carbon-Footprint Production of High-End 5-Aminolevulinic Acid via Integrative Strain Engineering and RuBisCo-Equipped <i>Escherichia coli</i>
Ying-Chen Yi, Chengfeng Xue, I‐Son Ng
Abstract
5-Aminolevulinic acid (ALA) is an imperative compound that has been widely applied in the agricultural and medical fields. The ALA synthesis in Escherichia coli via the C4 pathway depends on ALA synthase (ALAS) and releases CO2 in a single-step reaction. However, enhancement of the ALA production by increasing the protein’s quality and quantity with simultaneous CO2 reduction has never been reported. In this study, the expression and activity of ALAS were improved by employing chaperone and rare tRNAs. Moreover, the pdxY gene was introduced for pyridoxal phosphate (PLP) regeneration, a cofactor for ALAS, and significantly increased ALA production to 14.3 g/L through stepwise strain engineering with an optimal feeding strategy. The leading excess CO2 emission was further reused as a second carbon source for cell growth and ALA synthesis by cloning the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) and phosphoribulokinase (Prk) in E. coli. As a result, the co-application of RuBisCo, Prk, and ALAS reduced CO2 emission by 53.8% in combination with pdxY and pRARE. Finally, the crude ALA was demonstrated and applied in the antibacterial photodynamic therapy (aPDT) against three different pathogens. The robust strain, RSSA-BY with a lower carbon footprint, achieved the highest cell growth and efficient ALA production for aPDT for the first time, providing a vital and green bioprocess toward an advanced and sustainable future.