Xylose-Assisted Glycolic Acid Overproduction in Engineered Issatchenkia orientalis

Glycolic acid (GA) is an important platform chemical with applications in biodegradable polymers, cosmetics, and pharmaceutical synthesis; however, traditional petrochemical routes raise environmental concerns, and current microbial production platforms remain constrained by acid toxicity. In this study, systematic metabolic engineering combined with fermentation optimization was implemented to construct an efficient GA biosynthetic pathway in the acid-tolerant non-conventional yeast Issatchenkia orientalis. To redirect the metabolic flux of the glyoxylate cycle, the Arabidopsis thaliana glyoxylate reductase (AtGLYR), with a catalytic efficiency approximately 74-fold higher than that of the native yeast enzyme, was heterologously expressed, coupled with the overexpression of endogenous isocitrate lyase (ICL) and the knockout of the competing malate synthase (MLS) gene. Furthermore, a heterologous xylose assimilation pathway was integrated into the engineered strain to rescue the growth deficiency caused by mls deletion. Subsequent fermentation optimization via an ethanol-xylose co-feeding strategy and calcium carbonate supplementation effectively further improved GA accumulation. Ultimately, the optimized engineered strain yielded a maximum GA titer of 712.42 mg/L in shake-flask fermentation. This work validates the potential of I. orientalis as a robust chassis microbe for sustainable GA biosynthesis and broadens the product portfolio of metabolic engineering applications for this yeast strain.