K. marxianus produces different secreted enzymes when different carbon sources are used. For example, pectinase is produced when glucose or pectin is used as a carbon source 15, and inulinase is produced when inulin or sucrose is used as a carbon source 16. Therefore, it would be interesting to identify the proteins produced by K. marxianus in kefir culture. Lactate is the primary acid that is produced in kefir culture, and its level can range from 0.6 to 1% (w/v) 17,18. On the other hand, application of the lactate assimilation trait of K. marxianus was reported to reduce the L-lactate feedback inhibition of Lactococcus lactis for nisin production 7. This indicates that K. marxianus could play the same role during kefir production and the potential of lactate usage for biotechnological applications. In this study, to study microbiota interactions in kefir, we focused on using lactate as the sole carbon source to culture K. marxianus. The proteins released from K. marxianus under lactate culture conditions were identified.
DL-lactate was used as the sole carbon substrate for batch cultivation of K. marxianus at different pH values. Even under the optimal growth conditions at pH 4.18, as shown in the results (Table 1), the released protein concentrations and the SDS-PAGE analysis of released protein indicated that some of the yeast cells were still autolyzed (Supplementary Fig. S1). The mechanism of autolysis in K. marxianus is based on the biosynthesis of lytic enzymes, which is similar to the mechanism in S. cerevisiae 19,20, and optimum autolysis was observed at pH 4.5 in culture 20. In this study, the optimum culture pH for autolysis was observed to be pH 5.2 (Table 1). However, the culture conditions were different from those in this study 20, since this work lacked fermentable carbon sources in the culture medium. In the presence of 0.2 M MgCl2, the final cell concentrations and the released protein concentrations in the culture medium increased and decreased, respectively (Fig. 1c, d). The presence of excess Mg2+ ions facilitates protein secretion in K. marxianus (0.2 M MgSO4) 10 and decreases autolysis in marine bacteria (0.5 M MgCl2) and S. cerevisiae (0.1% MgCl2) 11,21. Although the mechanism remains unclear, it was proposed that Mg2+ ions might help organize cell membranes in both eukaryotes and prokaryotes 10. The GAP1 and HSP12 proteins found in the culture filtrate were also related to the maintenance of cell membrane integrity and stability 22,23. These results suggest that K. marxianus may bear the stress that could decompose membranes. Nonetheless, the inhibition of autolysis suggested that the presence of 0.2 M MgCl2 helped K. marxianus resist the stress occurring under the lactate culture conditions (Fig. 1c, d).
GAP1 is a moonlighting (multifunctional) protein that is involved in glycolysis and constitutively expressed when fermentable or nonfermentable carbon sources are used 24. Some glyceraldehyde 3-phosphate dehydrogenases are related to aggregation and apoptosis 25,26. However, GAP1 was reported to be related to flocculation and contributes to cell wall integrity in K. marxianus 22,27. The expression of GAP1 from K. marxianus in S. cerevisiae induces flocculation 27. In this study, even though the yeast was separated from the proteins, stable GAP1 crystallized in the liquid (Fig. 2a). These results indicate that GAP1 plays an important role in K. marxianus flocculation.
LC-MS/MS analysis of the total protein content revealed the identities of the proteins released after autolysis, other than the crystallized GAP1, and it also provided a glimpse of the metabolic pathways in K. marxianus. Enzymes in central metabolic pathways, such as the tricarboxylic acid cycle, glycolysis/gluconeogenesis, and pentose phosphate pathway, were identified (Supplementary Table S2). Although DL-lactate was the only carbon source that was supplied to the culture medium, proteins related to galactose and inulin metabolism were also expressed (Supplementary Table S2). These results suggest that K. marxianus has the potential to assimilate galactose and inulin under these culture conditions. Two lactate metabolism-related proteins, namely, D-lactate dehydrogenase [cytochrome] 2 and cytochrome b2, were identified (Supplementary Table S2). D-lactate dehydrogenase [cytochrome] 2 catalyzes the conversion of D-lactate to pyruvate with the reduction of two molecules of cytochrome. The association between these identified lactate assimilation proteins and the expression of the central metabolism enzymes indicates the lactate assimilation pathway in K. marxianus. It was reported that the PGU1 promoter is activated in the presence of lactate in S. cerevisiae 6. In the lactate culture of K. marxianus, the transcription levels of PGU1 were higher than those of GAP1 and INU1 (Fig. 4). However, none of the analyzed peptides matched the PGU1 protein in our results. Among the 15 most abundant proteins, the average emPAI% of HSP12 was 18.7%, and that of the second abundant protein, GAP1, was 3.51% (Table 2). There was a 15.19% difference between these two proteins, and the third most abundant protein differed from GAP1 by only 1.21% (Table 2). These results suggest that most of the proteins were digested to unidentified peptides under the autolysis conditions. Therefore, PGU1 might be digested to unidentified peptides. However, none of the peptides matched PGU1, even though no enzyme digest fingerprints were considered (data not shown), which needs to be further investigated.
GAP1 was crystallized in the cell-free fraction and was found to be the second most abundant protein in the results (Table 2). However, the transcription level of GAP1 was lower than that of ACT (Fig. 4). These results suggest that the protein abundance in the autolysis condition was dependent on the stability of the protein, not on the gene transcription level. The most abundant protein, HSP12, was reported to increase membrane stability under different stress conditions in S. cerevisiae, such as heat shock and oxidative and osmotic stresses 23. The expression of HSP12 in S. cerevisiae may also contribute to lifespan extension by protecting the membrane from desiccation 28,29. The relatively high expression of HSP12 (Fig. 4) under the autolysis conditions suggests that HSP12 might play a similar role in protecting K. marxianus cells. Additionally, Marchal, et al. 30 demonstrated the contribution of peptides from HSP12 of S. cerevisiae to sweet perception after yeast autolysis in wine. HSP12 from K. marxianus autolysis might also contribute additional flavor in kefir culture.
Protein abundance was related to protein stability and to K. marxianus abundance under the autolysis conditions. However, one cannot exclude the possibility that proteins might also be secreted into the medium rather than released from dead yeast cells. For example, GAP1, enolase and pyruvate decarboxylase are secreted into the extracellular space in Candida albicans without N-terminal signal peptides 31. These nonconventional protein secretion pathways were also found in S. cerevisiae 32 and are related to nonclassical export (NCE) genes 33 such as NCE102, which is also encoded in the K. marxianus genome 34. Although 513 proteins were identified, only 15 proteins had an abundance ratio higher than 0.5% (Table 2). Additionally, the 11 out of 15 most abundant proteins (Table 2) were reported as moonlighting proteins that do not have N-terminal signal peptides and might be secreted by nonconventional protein secretion pathways 35–42. The alternative functions of these moonlighting proteins were reported to bind plasminogen, extracellular matrix protein (fibronectin, vitronectin and laminin) and kininogen and inhibit transcription 35–39,41,42. Most of these studies focused on the extracellular matrix protein binding activities of these moonlighting proteins on the cell surface of the opportunistic pathogenic yeasts C. albicans, Candida parapsilosis and Candida tropicalis. Considering that the moonlighting proteins in this study were identified from the cell-free fraction, the alternative functions of these proteins might be different from those in previous reports. However, this could imply that the moonlighting proteins released from K. marxianus possibly competitively bind to extracellular matrix proteins and reduce the ability of those pathogenic yeasts to adhere to the cell surface. On the other hand, the secreted proteins might also be digested into peptides as the rest of the proteins are released by autolysis and become substrates for other symbiotic-related proteins in kefir. The well-known symbiotic interaction between Lactobacillus bulgaricus and Streptococcus thermophilus in yogurt is that S. thermophilus produces formate to stimulate L. bulgaricus, and L. bulgaricus liberates free amino acids and peptides from milk proteins to stimulate S. thermophilus 43. In this study, lactate was used as a substrate for K. marxianus to grow and induce autolysis of K. marxianus to release proteins and peptides into the cell-free fraction. This suggests that the lactate produced by lactic acid bacteria would stimulate K. marxianus to release proteins and peptides. However, the functions of those proteins and peptides remain to be discovered. The proposed interaction model of lactic acid bacteria and K. marxianus is presented in Fig. 5. Nonetheless, the potential of K. marxianus lactate culture for preventing pathogenic yeast invasion and the roles of the released proteins and peptides in kefir culture need further investigation in the future.
K. marxianus has received recognition for its potential industrial applications in recent years due to its thermotolerance, rapid growth rates, and broad substrate spectrum 4. Although knowledge of its biochemistry and genetics is limited compared to that for S. cerevisiae, many studies based on different biochemical principles, such as nonhomologous end joining 44, homologous recombination 45,46, and the CRISPR/Cas9 mechanism 47, have been applied to develop genetic engineering tools. These studies used fermentable substrates for culture, such as glucose and galactose. Therefore, knowledge on culturing nonfermentable substrates is limited. In this study, the optimal lactate culture conditions, the released proteins and the transcription levels of several genes were determined in K. marxianus. Autolysis occurred in all of the experimental lactate culture conditions, and the LC-MS/MS results that present various heat shock proteins and moonlighting proteins can be the foundation of K. marxianus probiotic and kefir research.