Engineering microalgae for robust glycolate biosynthesis: Targeted knockout of hydroxypyruvate reductase 1 combined with optimized culture conditions enhance glycolate production in Chlamydomonas reinhardtii.

Microalgae-based glycolate production through the photorespiratory pathway is considered an environmentally friendly approach. However, the potential for glycolate production is limited by photoautotrophic cultivation with low cell density and existing strains. In this study, a targeted knockout approach was used to disrupt the key photorespiration enzyme, Chlamydomonas reinhardtii hydroxypyruvate reductase 1 (CrHPR1), leading to a significant increase in glycolate production of 280.1 mg/L/OD. The highest potency yield reached 2.1 g/L under optimized mixotrophic conditions, demonstrating the possibility of synchronizing cell growth with glycolate biosynthesis in microalgae. Furthermore, the hypothesis that the cell wall-deficient mutant facilitates glycolate excretion was proposed and validated by comparing the glycolate accumulation trends of various Chlamydomonas reinhardtii strains. This study will facilitate the development of microalgae-based biotechnology and shed lights on the continuous advancement of green biomanufacturing for industrial application.