Enhancing Astaxanthin Biosynthesis in Synthetic Yeast Through Combinatorial Metabolic Engineering and Genome-Scale Evolution

Astaxanthin, a high-value keto-carotenoid with exceptional antioxidant capacity, has significant commercial potential for industrial applications. Microbial biosynthesis via engineered synthetic yeast presents an environmentally sustainable production platform. In this study, we developed a multistrategy optimization framework to enhance astaxanthin biosynthesis in synthetic yeast. Our systematic approach initiated with the construction of a de novo astaxanthin pathway in synthetic yeast strain 2369R, achieving a baseline production of 0.11 mg/L. Through rigorous screening of heterologous enzymes, we identified optimal variants of β-carotene hydroxylase (CrtZ) and ketolase (CrtW) that increased the titer to 0.65 mg/L. Subsequently, the combined enhancement of MVA pathway flux (via tHMG1 overexpression) and lipid metabolism regulation (through DGK1 overexpression) synergistically boosted astaxanthin production to 2.59 mg/L. Through combinatorial implementation of genome-scale diversification using the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system coupled with an absorption-based semi-high-throughput screening platform (A₄₅₀/A₆₀₀), we successfully isolated an elite mutant strain, YgM97, that achieved 6.85 mg/L astaxanthin production in shake-flask culture. This represents a remarkable 61.27-fold enhancement compared with the parental strain. Transcriptomic and genomic analyses subsequently revealed the potential molecular mechanisms underlying this significant yield improvement. Collectively, this study demonstrates the powerful synergy between rational metabolic engineering and randomized genome evolution, providing a novel paradigm for high-value compound biosynthesis in a microbial chassis.