Integrated Base Editing and Microfluidics Boost Microbial Lipid Production from Lignin

Lignin, a recalcitrant aromatic biopolymer, represents a promising feedstock for sustainable biorefining. In this study, we characterized the transcriptional response of the fungus Curvularia clavata J1 to alkali lignin (AL) and developed a Cu2+-inducible MCM5–AID base editing system for genome-wide C-to-T and G-to-A mutagenesis. Transcriptomic analysis revealed that AL exposure triggered upregulation of genes involved in electron transport, lipid catabolism, and iron homeostasis. Using droplet microfluidics, we conducted ultrahigh-throughput screening and identified a mutant strain, M6, exhibiting superior phenotypic traits. When cultivated in lignosulfonate medium, M6 showed a 33–36% increase in lipid production and a 75–87% enhancement in laccase activity. Transcriptional profiling further indicated reinforced metabolic pathways related to fatty acid, steroid, and glycerolipid biosynthesis, redirecting carbon flux toward the formation of oil-enriched single-cell protein. This study establishes an integrated platform for lignin valorization and underscores the synergy between synthetic biology and microfluidics for precision engineering of fungal hosts. For comparative transcriptomic analysis of the wild-type (WT) strain under alkaline lignin (AL) conditions, mycelia were inoculated in 100 mL of minimal medium (MM) containing either 1% (w/v) AL (WT_L) or 1% glucose (WT_G) as the carbon source, and cultured at 30°C with shaking at 200 rpm for four days. To compare the transcriptional profiles of the M6 mutant and WT grown on lignosulfonate, both strains were cultivated in MM supplemented with 0.3% (w/v) glucose and 1% (w/v) lignosulfonate under the same conditions. After incubation, mycelia were harvested, washed two times with sterilized water, and processed for RNA extraction. Biological triplicates were prepared for each experimental condition.