High-efficient Transmembrane Transport Of Hexose In Candida Glycerinogenes

As the first step in the process of biological fermentation,transmembrane transport of sugar is accomplished by a series of sugar transporters in yeast.However,low efficient sugar transport under the condition of high gravity fermentation has always been the rate-limiting step of industrial fermentation.Therefore,it is very important to explore the efficient sugar transporters from the excellent industrial strains and to study the mechanism of efficient sugar transport.Candida glycerinogenes is an excellent industrial strain with efficient sugar consumption,rapid growth,high sugar conversion rate and able to high gravity fermentation.However,sugar transporters from C.glycerinogenes are still poorly understood,especially under the condition of high sugar concentration.In this study,C.glycerinogenes was used as the research material and found that it has outstanding ability of fermented sugars utilization and fermentation properties.Based on the hidden Markov model of sugar transporter,14 candidate sugar transporter genes were screened from C.glycerinogenes genomic information and predicted that their encoded proteins have 11 or 12 transmembrane structures.Ultimately,five candidate proteins,CgHxt1-2 and CgHxt4-6,were identified as sugar transporters in the hxt-null Saccharomyces cerevisiae.CgHxt4 is an efficient sugar transporter found to maintain high efficiency of glucose transport both at low and high glucose concentration.Meanwhile,the key sugar transporter genes were knockout in C.glycerinogenes.It is found that different glucose transporters play a major role in different glucose concentration,especially knockout of CgHxt4 seriously inhibits the cell growth at high glucose concentration.The relative transcription level of sugar transporter genes in C.glycerinogenes was analyzed by qRT-PCR.It was found that the transcriptions of glucose transporter genes were regulated in varying degrees by glucose concentrations and osmotic pressure conditions.The transcription of CgHXT4 was induced by high glucose concentration and osmotic pressure,and the induced expression of CgHXT4 required the transcription regulator Hog1.In addition,by observing subcellular localization of fluorescent fusion proteins,it was found that the endocytosis of glucose transporters,which mediated by ubiquitin at post-translational modification level,was also related to glucose concentration.It is worth noting that the endocytosis of CgHxt4 is weak,and it is not sensitive to the change of sugar concentration.Further,compared with the efficient sugar transporters in S.cerevisiae,CgHxt4 has more prominent ability of sugar transport at high glucose concentration.CgHxt4 is able to bind to the cell membrane more stably during the fermentation process with high glucose concentration,which supports for sustained sugar transport and also is an important factor for the efficient glucose transport of CgHxt4 at high glucose concentrations.Meanwhile,the specific site N329 in the YYX(T/P)conserved motif of CgHxt4 was found by amino acid sequence alignment.By mean of site-directed mutation,it was found that the specific site N329 was a key site contributing to the efficient glucose transport of CgHxt4.The three-dimensional structure analysis of mutant proteins revealed that the slightly longer hydrophilic side chain of asparagine at N329,which located at the entrance of the channel,might bind to the sugar molecules better and induced sugar into the solvent-accessible channel of sugar transporter.At last,in view of the outstanding sugar transport efficiency of CgHxt4,CgHXT4 was overexpressed in S.cerevisiae model strain,and the cell growths,glucose consumptions and ethanol productions of recombinant strains were enhanced at high glucose concentration.In this study,transport efficiency,expression regulation and structure-function relationship of hexose transporters have been studied,which discussed the mechanism of efficient transmembrane transport of hexose by C.glycerinogenes and was to provide technical support and theoretical guidance for biological fermentation production.