Targeting pathway expression to subcellular organelles improves astaxanthin synthesis in Yarrowia lipolytica

Metabolic engineering approaches for the production of high-value chemicals in microorganisms mostly use the cytosol as general reaction vessel. However, sequestration of enzymes and substrates, and metabolic cross-talk frequently prevent efficient synthesis of target compounds in the cytosol. Organelle compartmentalization in eukaryotic cells suggests ways for overcoming these challenges. Here we have explored this strategy by expressing the astaxanthin biosynthesis pathway in sub-organelles of the oleaginous yeast Yarrowia lipolytica. We first showed that fusion of the two enzymes converting β-carotene to astaxanthin, β-carotene ketolase and hydroxylase, performs better than the expression of individual enzymes. We next evaluated the pathway when expressed in compartments of lipid body, endoplasmic reticulum or peroxisome, individually and in combination. Targeting the astaxanthin pathway to subcellular organelles not only accelerated the conversion of β-carotene to astaxanthin, but also significantly decreased accumulation of the ketocarotenoid intermediates. Anchoring enzymes simultaneously to all three organelles yielded the largest increase of astaxanthin synthesis, and ultimately produced 858 mg/L of astaxanthin in fed-batch fermentation (a 141-fold improvement over the initial strain). Our study is expected to help unlock the full potential of subcellular compartments and advance LB-based compartmentalized isoprenoid biosynthesis in Y. lipolytica.