Synthetic plug-in fatty acid pathway for stress-adaptive lipid accumulation in Chlamydomonas reinhardtii

Lipid accumulation in microalgae is typically induced by stress but often comes at the expense of growth and energy stability. Here, we introduced the plsXYC module from Cyanobacterium aponinum into Chlamydomonas reinhardtii, aiming to enhance stress-responsive lipid production by a plug-in prokaryotic acyltransferase route. The initial wild-type strain produced lipids at 135.1 mg/g-dry cell, while the engineered CrXYC sustained the highest lipid yield of 385 mg/g-DCW across light and dark cycles. Transcriptomics revealed that light-dark shifts drove 65% of expression variance and elevated ATP levels. During heterotrophic culture under nitrogen starvation, CrXYC preserved ATP up to 1.6-fold higher than the parental background. Carbon repartition was proved using ¹³C-isotopes, and redox reinforcement showed coordinated upregulation of lipid assembly pathways and repression of starch biosynthesis. This shift coincided with enhanced superoxide scavenging activity, while broad antioxidant capacity remained unchanged. Together, we demonstrate that introducing an orthogonal acyl-acyl carrier protein entry route sustains lipid accumulation under stress while preserving growth and energy balance.