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.

Abstract The properties of three mutants of the industrial-scale L-lysine–producing strain Corynebacterium glutamicum VKPM B-11969 were studied. These mutants had one of the anaplerotic pathways for synthesizing lysine precursors, oxaloacetate, blocked due to inactivation of the ppc (encodes phosphoenolpyruvate carboxylase) or pyc (encodes pyruvate carboxylase) genes, or one of the pyruvate synthesis pathways was disrupted due to inactivation of the pyk gene (encodes pyruvate kinase). Inactivation of pyruvate carboxylase, which catalyzes the synthesis of oxaloacetate from pyruvate and CO 2 , did not affect L-lysine production. Inactivation of pyruvate kinase, which catalyzes the synthesis of pyruvate from phosphoenolpyruvate, resulted in a decrease in the rate of L-lysine synthesis (by 20–25%). The synthesis of L-lysine decreased to the greatest extent with the inactivation of phosphoenolpyruvate carboxylase, by 30–50% depending on the growing conditions. It was concluded that, in the B-11969 strain, under conditions of active synthesis of L‑lysine, the formation of oxaloacetate occurs predominantly with the participation of phosphoenolpyruvate carboxylase. However, the increase in the expression of the ppc gene in the producer strain led to an increase in the activity of phosphoenolpyruvate carboxylase, but not in the production of L-lysine, which allowed us to assume that in the B-11969 strain the stage of oxaloacetate synthesis is not limiting for the production of lysine.