# Proteomics Analysis of _Zygosaccharomyces mellis_ in Response to Sugar Stress
## 1 Introduction  
Honey is a natural and stable sweetener that is primarily composed of sugars (70-85%), including fructose, sucrose, glucose, maltose, raffinose and others [1]. Due to high osmotic stress, most microbes cannot survive in honey, except osmotolerant and halotolerant microorganisms, such as yeast _Zygosaccharomyces_ spp., which can cause food spoilage. These yeasts are found in a wide range of sugary foods, including fruit juices, wine and honey [2], leading to economic losses in food manufacture and health hazard. Among these species, _Z. roxtii_ has been extensively studied for its osmotic tolerance in food fermentation under high sugar or high salt stress. Another yeast isolated from honey, _Z. mellis_, also has remarkably high sugar tolerance [3]. Exploring the molecular mechanisms of resistance under high sugar stress is helpful for developing strategies for food production. Althoughprevious studies have reported the isolation and identification of _Z. mellis_ and the effect of a high-glucose environment on the transcription expression profile, there is \(a\) lack of in-depth studies on its hypertonic resistance [4; 5; 6]. iTRAQ-based proteomic technology could screen differentially expressed proteins (DEPs) that are involved in cell biological processes and help us to better understand the hypertonic mechanism of _Z. mellis_.  
The metabolic pathways and response mechanisms of yeasts, especially _Z. rouxii_ and _Saccharomyces cerevisiae_, under high osmotic conditions have been widely reported [7; 8; 9]. The content of major metabolites in the stress response in yeast, including glycerin, trehalose, D-arachnoid and erythroid, is increased under the stress of high concentrations of sugar, NaCl and ethanol [7; 10]. At the same time, cells can adapt to stress by regulating the activity of superoxide dismutase (SOD), intracellular peroxidase, adenosine triphosphatase (ATPase) and mitochondrial ATPase [11]. In addition, the high osmolarity glycerol mitogen-activated protein kinase (HOG- MAPK) pathway, which plays a crucial role in signal transduction in yeast, can promote glycerin accumulation and regulate other physiological processes [12; 13].  
With the development of genomics, proteomics and other molecular technologies, the tolerance mechanisms of yeast can also be studied from a comprehensive perspective at the protein level. For example, in _Z. rouxii_, the proteins involved in carbohydrate and energy metabolism, amino acid metabolism, response to stimulus, etc., were shown to be regulated in response to high sugar concentrations [14]. Unlike _Z. rouxii_, under stationary-phase stress conditions, the cell replication, oxidative stress responses and proteolytic activity of _S. cerevisiae_ were decreased, whereas cell-wall renewal, the regulation of solute transport across the cell membrane and de novo arginine synthesis were enhanced [15]. The metabolic processes of _Z. rouxii_ and _S. cerevisiae_ have been well reported; however, there was few reports on the mechanism of osmo-tolerance in _Z. mellis_. Due to the different and extremely complex metabolic processes, the mechanisms of other yeasts adapted to high-sugar environments need to be further studied.  
DNA metabarcoding is a high-throughput molecular identification approach for identifying mixed samples from plants, animals or fungi [16; 17]. In this study, the yeasts isolated from honey were identified by DNA barcoding and metabarcoding. Furthermore, the mechanism associated with the sugar tolerance of _Z. mellis_ was investigated by proteomic techniques. The findings contribute to a better understanding of the high sugar tolerance features of _Z. mellis_.