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Microbial production of isoprene from renewable feedstock is a promising alternative to traditional petroleum-based processes. Currently, efforts to improve isoprenoid production in Saccharomyces cerevisiae mainly focus on cytoplasmic engineering, whereas comprehensive engineering of multiple subcellular compartments is rarely reported. Here, we propose dual metabolic engineering of cytoplasmic and mitochondrial acetyl-CoA utilization to boost isoprene synthesis in S. cerevisiae. This strategy increases isoprene production by 2.1-fold and 1.6-fold relative to the recombinant strains with solely mitochondrial or cytoplasmic engineering, respectively. By combining a modified reiterative recombination system for rapid pathway assembly, a two-phase culture process for dynamic metabolic regulation, and aerobic fed-batch fermentation for sufficient supply of acetyl-coA and carbon, we achieve 2527, mg l−1 of isoprene, which is the highest ever reported in engineered eukaryotes. We propose this strategy as an efficient approach to enhancing isoprene production in yeast, which might open new possibilities for bioproduction of other value-added chemicals.

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Astaxanthin is a highly valued carotenoid with strong antioxidant activity and has wide applications in aquaculture, food, cosmetic, and pharmaceutical industries. The market demand for natural astaxanthin promotes research in metabolic engineering of heterologous hosts for astaxanthin production. In this study, an astaxanthin-producing Saccharomyces cerevisiae strain was created by successively introducing the Haematococcus pluvialis β-carotenoid hydroxylase (crtZ) and ketolase (bkt) genes into a previously constructed β-carotene hyperproducer. Further integration of strategies including codon optimization, gene copy number adjustment, and iron cofactor supplementation led to significant increase in the astaxanthin production, reaching up to 4.7 mg/g DCW in the shake-flask cultures which is the highest astaxanthin content in S. cerevisiae reported to date. Besides, the substrate specificity of H. pluvialis CrtZ and BKT and the probable formation route of astaxanthin from β-carotene in S. cerevisiae were figured out by expressing the genes separately and in combination. The yeast strains engineered in this work provide a basis for further improving biotechnological production of astaxanthin and might offer a useful general approach to the construction of heterologous biosynthetic pathways for other natural products.

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Because it is an outstanding antioxidant with wide applications, biotechnological production of astaxanthin has attracted increasing research interest. However, the astaxanthin titer achieved to date is still rather low, attributed to the poor efficiency of β-carotene ketolation and hydroxylation, as well as the adverse effect of astaxanthin accumulation on cell growth.To address these problems, we constructed an efficient astaxanthin-producing Saccharomyces cerevisiae strain by combining protein engineering and dynamic metabolic regulation. First, superior mutants of β-carotene ketolase and β-carotene hydroxylase were obtained by directed coevolution to accelerate the conversion of β-carotene to astaxanthin. Subsequently, the Gal4M9-based temperature-responsive regulation system was introduced to separate astaxanthin production from cell growth. Finally, 235 mg/L of (3S,3′S)-astaxanthin was produced by two-stage, high-density fermentation. This study demonstrates the power of combining directed coevolution and temperature-responsive regulation in astaxanthin biosynthesis and may provide methodological reference for biotechnological production of other value-added chemicals.

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