Sustainable β-carotene production by engineered S. cerevisiae using sucrose and agricultural by-products

β-carotene, a carotenoid precursor to vitamin A, is widely employed in the food, pharmaceutical, and nutraceutical sectors. In this study, we present an economically sustainable strategy for β-carotene biosynthesis in Saccharomyces cerevisiae by engineering the yeast to utilize sucrose and agricultural by-products as alternative carbon and nitrogen sources. Specifically, the deletion of the GAL80 gene facilitated effective β-carotene synthesis directly from sucrose, circumventing the costly requirement for galactose induction. Using this engineered yeast strain, we achieved β-carotene titers of up to 23.30 ± 4.22 mg/L and content levels of 2.29 ± 0.16 mg/g dry cell weight (DCW). To further improve the economic viability and environmental sustainability, we evaluated the use of agricultural by-products—molasses as a carbon source and fish meal as a nitrogen source—in a fed-batch fermentation process, highlighting the potential of these substrates to replace refined feedstocks while achieving competitive β-carotene production levels. This approach yielded substantial β-carotene titers of 17.02 ± 0.40 mg/L and content levels of 2.90 ± 0.21 mg/g DCW. It also significantly reduced medium costs by up to 73% compared to conventional yeast extract and peptone-based media, demonstrating the practical potential of these low-cost, sustainable substrates for industrial applications. This study uniquely highlights the successful application of unrefined agricultural by-products, addressing key challenges in cost and sustainability. These findings represent an important advancement toward developing economically competitive and environmentally responsible microbial platforms for the production of β-carotene and other high-value biochemicals.