Production of High Levels of 3<i>S</i>,3′<i>S</i>-Astaxanthin in <i>Yarrowia lipolytica</i> via Iterative Metabolic Engineering

Abstract

Astaxanthin is a highly value-added keto-carotenoid compound. The astaxanthin 3<i>S</i>,3'<i>S</i>-isomer is more desirable for food additives, cosmetics, and pharmaceuticals due to health concerns about chemically synthesized counterparts with a mixture of three isomers. Biosynthesis of 3<i>S</i>,3'<i>S</i>-astaxanthin suffers from limited content and productivity. We engineered <i>Yarrowia lipolytica</i> to produce high levels of 3<i>S</i>,3'<i>S</i>-astaxanthin. We first assessed various β-carotene ketolases (CrtW) and β-carotene hydroxylases (CrtZ) from two algae and a plant. HpCrtW and HpCrtZ from <i>Haematococcus pluvialis</i> exhibited the strongest activity in converting β-carotene into astaxanthin in <i>Y. lipolytica</i>. We then fine-tuned the HpCrtW and HpCrtZ transcriptional expression by increasing the rounds of gene integration into the genome and applied a modular enzyme assembly of HpCrtW and HpCrtZ simultaneously. Next, we rescued leucine biosynthesis in the engineered <i>Y. lipolytica</i>, leading to a five-fold increase in biomass. The astaxanthin production achieved from these strategies was 3.3 g/L or 41.3 mg/g dry cell weight under fed-batch conditions, which is the highest level reported in microbial chassis to date. This study provides the potential for industrial production of 3<i>S</i>,3'<i>S</i>-astaxanthin, and this strategy empowers us to build a sustainable biorefinery platform for generating other value-added carotenoids in the future.