[3S05Am] 20. Plant Biotechnology

11:00 AM - 11:20 AM JST(2:00 AM - 2:20 AM UTC)

[3Plant-04] Rewiring lipid metabolism via synthetic plug-in pathway in Chlamydomonas reinhardtii

○Priskila A. Diankristanti¹, I-Son Ng¹ (1. National Cheng Kung University (Taiwan))

Keywords:

Microalgae,Lipid accumulation,Cyanobacteria,Synthetic pathway

Lipid production in microalgae usually comes at the cost of growth, particularly under elevated salinity, nitrogen starvation, and dark conditions. Cyanobacteria, however, exhibit natural resilience to such environments while maintaining efficient carbon flux. A key feature is the acyl-ACP recycling route involving phosphate acyltransferase (plsX), glycerol-3-phosphate acyltransferase (plsY), and 1-acyl-sn-glycerol-3-phosphatase (plsC), which provides a shortcut into glycerolipid assembly and bypasses canonical acetyl-CoA carboxylation. In this study, we integrated the plsX, plsY, and plsC genes from halotolerant Cyanobacterium aponinum PCC 10605 into model green microalgae Chlamydomonas reinhardtii CC400, to enhance stress-adaptive lipid accumulation. This gene cluster acts as a “plug-in” orthogonal acyl-entry module which complements native eukaryotic metabolism without inferring with upstream carbon fixation.Strain performance was evaluated under high salinity, nitrogen starvation, and transitions between mixotrophic and heterotrophic culture. Interestingly, principal component analysis of transcriptome profiles revealed that metabolic mode (mixo- vs heterotrophy) was the dominant source of variance, accounting for 65.7% of total expression differences (PC1), whereas strain identity contributed more modestly to separation (PC2, 19.8%). The highest lipid content of 385 mg/g-dry cell weight was observed in CrXYC strain. Moreover, CrXYC preserved ATP up to 1.6-fold higher than the parental background during heterotrophic nitrogen starvation. The association between elevated ATP and continued lipid accumulation highlights how orthogonal entry pathways may help buffer energy decline during stress. Carbon repartition was proofed using ¹³C-isotope analysis, showing more acetate-derived carbon being assimilated into cellular biomass with capacity scaling with cassette complexity. Finally, transcriptional genes associated with starch synthesis (STA, PGM, GLPT) declined across both cultivation modes, while lipid assembly genes (DGAT, GPAT, LPAAT) showed sustained induction. Importantly, this rerouting did not collapse growth but instead created a productive bias in allocation. Our study demonstrates that a compact orthogonal pathways can provide a scalable framework for building strains that remain productive when conditions change, a prerequisite for sustainable carbon conversion at industrial scale.