High production of glycolic acid by Klebsiella pneumoniae: Engineering metabolic pathways and optimizing culture conditions

Growing concerns regarding environmental pollution caused by non-biodegradable plastic waste have intensified the demand for sustainable biodegradable bioplastics. Glycolic acid (GA), the monomer of polyglycolic acid (PGA), is a key precursor in the production of biodegradable plastics. In this study, a systematic metabolic engineering strategy was employed to enhance GA production in Klebsiella pneumoniae via the xylose oxidation pathway. As a first step, wild-type K. pneumoniae CU (a non-GA-producing strain) was transformed with the pETM-XFEA plasmid harboring four genes involved in the xylose oxidation pathway, resulting in KPGA0, which produced 4.0 ± 0.3 g/L of GA. To further increase metabolic flux, the xylA gene was deleted in KPGA0, yielding the KPGA1 strain, which produced 5.3 ± 0.4 g/L of GA—a 1.32-fold increase. Due to the accumulation of acetic acid and ethylene glycol as major byproducts in KPGA1, genes responsible for their production were subsequently deleted to construct KPGA11. Under optimized culture conditions (pH, agitation speed, and aeration), KPGA11 achieved 15.8 ± 0.6 g/L of GA, a 2.98-fold increase compared to KPGA1. Furthermore, fed-batch cultivation of KPGA11 resulted in a GA titer of 70.1 ± 3.1 g/L, a productivity of 0.730 ± 0.033 g/L/h, and a conversion yield of 0.283 ± 0.003 g/g. To the best of our knowledge, this study is the first to demonstrate high-titer GA production through upregulation of the xylose oxidation pathway in K. pneumoniae, and also reports the highest GA production achieved to date in a microbial system utilizing a carbon source.