β-carotene is a precursor of vitamin A and has multiple physiological functions. Producing β-carotene by microbial fermentation has attracted much attention to consumers’ preference for natural products. This study focused on improving β-carotene production by constructing codon-adapted genes and minimizing intermediate accumulation. The codon-adapted CarRA and CarB genes from the industrial strain of Blakeslea trispora were integrated into the genome of the Yarrowia lipolytica to construct YL-C0, the baseline strain for producing β-carotene. Thereafter, the β-carotene biosynthetic pathway’s metabolic balance was accurately regulated to reduce the intermediates’ accumulation. Notably, the β-carotene content increased by 21 times to reach 12.5 dry cell weight (DCW) mg/g when minimizing HMG-CoA and FPP accumulation. Further, we improved the expression levels of the CarRA and CarB genes to minimize the accumulation of phytoene and lycopene. Total production of β-carotene of 1.7 g/L and 21.6 mg/g DCW was achieved. These results reveal that the rate-limiting enzymes CarRA and CarB of B. trispora exhibited higher catalytic activity than the same enzymes from other microorganisms. Promoting metabolic balance by minimizing the accumulation of intermediates is a very effective strategy for increasing β-carotene. The β-carotene-producing strain constructed in this study has established the foundation for its potential use in industrial production. These successful engineering strategies also provide a foundation for large-scale production of other terpenoids.

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Microbial oil triacylglycerol (TAG) from the renewable feedstock attract much attention. The oleaginous yeast Yarrowia lipolytica has become the most studied for lipid biosynthesis. Fatty acid desaturases catalyze the introduction of a double bond into fatty-acid hydrocarbon chains to produce unsaturated fatty acids. Desaturases are known to enhance lipid accumulation. In this study, we have achieved a significant increase in lipid production and increase the unsaturated fatty acids content in Y. lipolytica. By comparing the expression of the native genes of-9 stearoyl-CoA desaturase (SCD) and 12 desaturase (12D), and an exogenous 15 desaturase (15D) from flax in the strain with deleted peroxisomal biogenesis factor 10 (PEX10) and overexpressed diacylglyceride acyl-transferase (DGA1), we found that the strain with overexpressed 15 desaturase accumulated 30.7% lipid. Simultaneously, we explored the effect of two copies of desaturase genes (12D-SCD, 15D-SCD, 12D-15D) on lipid production, and found co-expression of 12D and 15D accumulated 42.6% lipid. The lipid content was further increased by 56.3% through the deletion of the multifunctional enzyme (MFE1) and the overexpression of acetyl-CoA carboxylase (ACC1). Finally, the lipid productivity of 50 g/L and maximal lipid content of 77.8% DCW are obtained using a 5-L stirred-tank bioreactor during the stationary phase in the engineered YL-10. Our result demonstrated that the 12 and 15 desaturases play an important role in lipid production in Y. lipolytica and provides an effective strategy for biodiesel development.