Host analysis-guided selection and targeted engineering (HASTE) of Lipomyces tetrasporus for the conversion of CO2-derived feedstocks.

Cost-competitive bioproduction calls for utilizing cellulosic sugars or acetate from electro-reduction of CO2. Here, we identified a novel Lipomyces tetrasporus strain (i.e., NRRL Y-11562 from 24 Lipomyces species) capable of effectively assimilating diverse substrates (acetate, glucose, and xylose). We characterized this new chassis by integrative 13C-metabolic flux analysis, metabolomic analysis, and RNA sequencing with large language model-based data processing. During acetate metabolism, L. tetrasporus demonstrated a highly active TCA cycle, rapid citrate and malate accumulation, strong NADH/NADPH production, and acetyl-CoA synthase activity. Moreover, this crab-tree negative oleaginous yeast showed strong metabolic flexibility for co-utilization of sugars with acetate or acetate-rich electrocatalytic solutions under stressed conditions (e.g., O2 limitation and inhibitors) along with over 70% of flux through the oxidation pentose phosphate pathway during glucose and acetate co-conversion, higher than model yeasts such as Saccharomyces cerevisiae and Yarrowia lipolytica. Using the metabolic insights as a guide, we engineered L. tetrasporus for malate production, achieving 7.5 g/L (0.25 g/g yield) in shake flasks with glucose-acetate media and 28.8 g/L malic acid (0.20 g/g yield) in fed-batch with corn-stover hydrolysate.