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<b>Abstract</b>— <p>In the metabolic engineering of producer strains, strong constitutive promoters are an attractive alternative to inducible promoters, since they do not require the addition of expensive and sometimes toxic inducing substances to the fermentation medium. In this work, we have compared the <i>Yarrowia lipolytica</i> constitutive promoters: pEXP1, pRPS2, pRPL22 (native) and hybRPS2_499, hybRPL22 (synthetic) with the inducible promoter pEYK1-3AB. The synthetic promoters hybRPS2 and hybRPL22 are modified versions of the ribosomal promoters pRPS2 and pRPL22, respectively. Initially, promoter strength was assessed by the expression level of the reporter gene encoding the green fluorescent protein hrGFP. It was shown that the strength of the promoters increased in the following order: pRPS2, pRPL22, pEXP1, hybRPL22, hybRPS2_499, while the level of pEYK1-3AB activity with 1.5% erythritol induction was comparable to the level of unmodified pRPS2 and pRPL22. During heterologous expression of the <i>CarRP-GGPPSs7</i> genes under the control of the studied promoters, the highest β-carotene production was observed in the recombinant <i>Y. lipolytica</i> strain with the pEXP1 promoter, while the hybRPS2_499 promoter showed the result comparable to the pEYK1-3AB promoter induced by the addition of 1.5% erythritol. Thus, we have shown that in constructing heterologous biosynthetic pathways in <i>Y. lipolytica,</i> a strong constitutive promoter can successfully compete with an inducible variant. However, in our case, the use of the strongest promoter did not lead to the highest yield of the target product. Therefore, rapid identification of the best producer strain requires testing a wide range of promoters of varying strengths.</p>

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Comparison of Efficiency of Different Promoters by β-Carotene Production in the Yeast Yarrowia lipolytica

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  • 发表日期：

    2026年2月24日

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  • A. A. Cherenkova,
  • M. O. Taratynova,
  • O. E. Melkina

摘要

Abstract—

In the metabolic engineering of producer strains, strong constitutive promoters are an attractive alternative to inducible promoters, since they do not require the addition of expensive and sometimes toxic inducing substances to the fermentation medium. In this work, we have compared the Yarrowia lipolytica constitutive promoters: pEXP1, pRPS2, pRPL22 (native) and hybRPS2_499, hybRPL22 (synthetic) with the inducible promoter pEYK1-3AB. The synthetic promoters hybRPS2 and hybRPL22 are modified versions of the ribosomal promoters pRPS2 and pRPL22, respectively. Initially, promoter strength was assessed by the expression level of the reporter gene encoding the green fluorescent protein hrGFP. It was shown that the strength of the promoters increased in the following order: pRPS2, pRPL22, pEXP1, hybRPL22, hybRPS2_499, while the level of pEYK1-3AB activity with 1.5% erythritol induction was comparable to the level of unmodified pRPS2 and pRPL22. During heterologous expression of the CarRP-GGPPSs7 genes under the control of the studied promoters, the highest β-carotene production was observed in the recombinant Y. lipolytica strain with the pEXP1 promoter, while the hybRPS2_499 promoter showed the result comparable to the pEYK1-3AB promoter induced by the addition of 1.5% erythritol. Thus, we have shown that in constructing heterologous biosynthetic pathways in Y. lipolytica, a strong constitutive promoter can successfully compete with an inducible variant. However, in our case, the use of the strongest promoter did not lead to the highest yield of the target product. Therefore, rapid identification of the best producer strain requires testing a wide range of promoters of varying strengths.