Multi-omics dissect the molecular mechanisms driving high-lipid production in a laboratory-evolved Chlamydomonas mutant

Abstract

Enhancing lipid accumulation in microalgae is critical for commercial viability but often compromises growth. We previously generated through UV mutagenesis and iterative selection a Chlamydomonas reinhardtii mutant (H5) that retains parental growth while producing 3.2-fold more lipids (Sharma et al., 2015; Abdrabu et al., n.d.). Here, we present multi-omic analyses elucidating the molecular basis of this phenotype. Whole-genome sequencing revealed over 3000 mutations including a frameshift in the regulatory domain of 6-phosphofructokinase (PFK1). Six independent CLiP mutants in affected genes also showed elevated lipids, including a PFK1 mutant, validating functional relevance. Transcriptomics revealed upregulation of glycolytic genes and nutrient acquisition pathways under nutrient-replete conditions. Metabolomics identified an 8.31-fold malonate increase (p = 8.5 × 10⁻⁴), linking glycolysis to lipid synthesis. Lipidomics showed increased TAG diversity and lack of betaine lipids. Epigenomics revealed genome-wide hypermethylation, potentially stabilizing the phenotype. Together, these data suggest PFK1 deregulation drives metabolic reprogramming enabling lipid accumulation without growth penalty, demonstrating how evolutionary selection generates sophisticated metabolic solutions for engineering industrial microalgal strains.