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Kluyveromyces marxianus  is a yeast that could be identified from kefir and can use a broad range of substrates, such as glucose and lactate, as carbon sources. The lactate produced in kefir culture can be a substrate for  K. marxianus . However, the complexity of the kefir microbiota makes the traits of  K. marxianus  difficult to study. In this research, we focused on  K. marxianus  cultured with lactate as the sole carbon source. The optimal growth and released protein in lactate culture were determined under different pH conditions, and the LC–MS/MS-identified proteins were associated with the tricarboxylic acid cycle, glycolysis pathway, and cellular stress responses in cells, indicating that autolysis of  K. marxianus  had occurred under the culture conditions. The abundant glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) was cocrystallized with other proteins in the cell-free fraction, and the low transcription level of the  GAP1  gene indicated that the protein abundance under autolysis conditions was dependent on protein stability. These results suggest that lactate induces the growth and autolysis of  K. marxianus , releasing proteins and peptides. These findings can be fundamental for  K. marxianus  probiotic and kefir studies in the future.

Growth and autolysis of the kefir yeast Kluyveromyces marxianus in lactate culture