Recombinant expression vector for salty peptide expression and production method using the same

Salt substitute technology using repetitive expression of salty peptides in the GRAS yeast Kluyveromyces lactis

This technology involves a recombinant expression vector for yeast transformation that repeatedly links the alpha-mating signal sequence of the yeast Kluyveromyces lactis with genes encoding salty peptides, the yeast transformed with this vector, and a method for producing salty peptides using it. It also includes newly designed salty peptides based on their binding affinity to salt taste receptor candidates, as well as salt substitutes containing them.

Excessive sodium intake is a risk factor for hypertension and cardiovascular disease, but reducing salt in food by more than 10% significantly degrades taste, making low-sodium efforts difficult. Existing substitutes or soy-derived salty proteins have limitations in taste or manufacturing processes. Even when attempting to produce peptides using microorganisms, Pichia pastoris is not a GRAS-certified strain and requires the use of explosive methanol, while Saccharomyces cerevisiae has low production yields, hindering commercialization.

This technology uses food-safe certified yeast as a host, secretes and expresses the peptide gene in a long-chain form with multiple repeats, and then cleaves it with endopeptidase to obtain unit peptides, thereby increasing production efficiency. It utilizes a pKLAC2-based vector with the LAC4 operator. Applicable to low-sodium processed food seasonings, low-salt diets for hypertensive patients, and savory seasonings for convenience foods and snacks, it enables the production of salty peptides through safe yeast fermentation without methanol, reducing sodium intake without sacrificing taste.

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Key Features:

• A recombinant expression vector for yeast transformation comprising the Kluyveromyces lactis alpha-mating signal sequence and a polynucleotide encoding a salty peptide selected from SEQ ID NOs: 1 to 6
• A recombinant expression vector in which the polynucleotide encoding the salty peptide is repeated 10 to 100 times and further includes the LAC4 operator
• A step of culturing the transformed yeast and isolating and purifying the salty peptide from the yeast or culture medium through endopeptidase treatment
• A salt substitute containing as an active ingredient a salty peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 6

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This invention was developed with support from the Ministry of Education for research on the interaction between human GPCR sweet taste receptors and sweet substances, and for the development of new sweet materials.

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