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.