A strain of the yeast Komagataella kurtzmanii, a producer of recombinant peptidase SerP38 from the yellow mealworm Tenebrio molitor, has been obtained. The level of proenzyme secretion was 20-50 mg/L. It was shown that the target His 6 -tagged protein was produced in two forms during secretion in yeast. One of them was a monomer that was efficiently purified via Ni-NTA chromatography and then activated with trypsin. Another form accumulated in the culture medium as oligomers prone to aggregation in the presence of Ni 2+ ions and was not activated by trypsin treatment. Aggregation is likely the result of the polypeptide-chain misfolding.

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The yeast strain Komagataella kurtzmanii VKPM Y‐727 shows a significant defect in sorbitol utilization compared to closely related yeast K. phaffii (including strains formerly identified as Pichia pastoris). Our aim was to investigate the factors that determine the phenotype of the wild‐type strain and to obtain a K. kurtzmanii strain with an improved ability to utilize sorbitol. We sequenced and annotated the genome of K. kurtzmanii VKPM Y‐727 and compared it with that of K. phaffii GS115. Five K. phaffii GS115 genes that might be involved in sorbitol metabolism were selected and transferred into K. kurtzmanii Y‐727. The transfer of the modified SOR1 gene resulted in an increased growth rate of K. kurtzmanii in sorbitol, despite the fact that Y‐727 already contains its own SOR1 gene without any apparent mutations. The enzymes encoded by the SOR1 genes were analyzed in vitro and found to have similar properties. Differences in promoter activity were assessed using lacZ as a reporter gene, and the PSDH727 (promoter of SOR1 (SDH727) from K. kurtzmanii Y‐727) promoter was shown to be 1.5–2.0 times weaker than PSDH115 (promoter of SOR1 (SDH115) from K. phaffii GS115). Moreover, both promoters were less active in K. kurtzmanii than in K. phaffii when evaluated in cells grown in synthetic complete media with glucose or sorbitol. Thus, SOR1 gene expression was identified as a bottleneck in sorbitol metabolism in K. kurtzmanii. Also, the positive effect of additional modified SOR1 gene copies was observed in both yeasts, as K. kurtzmanii and K. phaffii could grow on synthetic complete media with sorbitol three times faster than the original K. phaffii GS115 strain.

A strain of the Komagataella kurtzmanii yeast, a producer of recombinant peptidase SerP38 from the yellow mealworm Tenebrio molitor, has been obtained. The level of the pro-enzyme secretion was 20-50 mg/L. It was shown that, during secretion in yeast, the target His6-tagged protein was produced in two forms. One of them was a monomer that was efficiently purified by Ni-NTA chromatography and then activated with trypsin. Another form accumulated in the culture medium as oligomers prone to aggregation in the presence of Ni2+ ions and was not activated by trypsin treatment. Aggregation is likely the result of incorrect folding of the polypeptide chain. Tenebrio molitor, S1 family serine peptidase, SerP38, yeast, Komagataella kurtzmanii, ion-dependent aggregation