Cloning And Functional Characterization Of Sugar Transporter Genes From Candida Tropicalis And Using Saccharomyces Cerevisiae As A Host

Energy is the basic material for human survival and develop, and fossil oil, as a liquid energy, has been considered as the blood of modern civilization. However, the fossil oil resources on are not renewable but limited, and showing an accelerating depletion trend. In order to seek alternative resources, people have focused their attention on bio-ethanol as the fuel. The most concern in the word is the fermentation of lignocellulosic hydrolysate to ethanol. The hydrolysate from lignocellulose mainly include hexoses ?such as glucose, mannose, and galactose? and pentoses ?such as xylose and arabinose?. However, Saccharomyces cerevisiae, the most excellent ethanologenic yeast, cannot utilize all the monosaccharides mentioned above in the lignocellulosic hydrolysate. To resolve this question, modification of S. cerevisiae is particularly important for large-scale production of bio-ethanol by genetic engineering. Transmembrane transport of monosaccharides is the first and most critical step to decide whether sugar can be metabolized by S. cerevisiae. Therefore, it is very important to explore the efficient sugar transporters and study their functional properties.This experiment use Candida tropicalis strain SHC-03 possessing good sugar metabolic ability as a research material. First, we tested its ability for utilization of different monosaccharides. Second, according to the prediction and annotation results in KEGG ?Kyoto Encyclopedia of Genes and Genomes? database, we screened 15 genes which probably have the function for sugar transport out from all 6254 genes of C. tropicalis, and heterologously expressed them in engineered S. cerevisiae and tested their sugar transport function by functional complementation assays. Third, transcriptional changes of the 15 candidate sugar transporter genes in response to xylose, glucose and mixed xylose and glucose were measured in C. tropicalis by RT-qPCR. Last, molecular structures of the monosaccharide transporter proteins identified out of 15 candidate genes were further predicted and their phylogenies were analyzed. The main results obtained in this study were listed as follows:?1? Growth performances of C. tropicalis strain SHC-03 in different sugar sources indicated that C. tropicalis has a wide range of sugar utilization ability. It can not only efficiently utilize hexoses of glucose, fructose, mannose, and galactose, but also utilize pentose of xylose and disaccharides of sucrose and cellobiose. Fifteen target genes were screened out from 6254 functional genes of C. tropicalis, and cloned and transformed into S. cerevisiae YXI. in which all the sugar transporter genes are knocked out but the xylose metabolic pathway is introduced, via recombinant expression plasmid.Functional complementation assays of 15 candidate genes found that CTRG₀0554, CTRG₀1164. CTRG 01165, CTRG₀1648, and CTRG₀4615 exhibited sugar transport function, therefore were identified as new sugar transporter genes. Though growth rate analyses of the recombinant strains, we found that different sugar transporters have transport preference for different monosaccharides. All of the 5 sugar transporters can transport glucose, xylose, and fructose. Among which, CTRG₀1648p showed the strongest ability for glucose and xylose transport; and CTRG₀1164p showed the strongest ability for fructose transport. CTRG₀0554p, CTRG₀1648p, and CTRG₀4615p can transport all of the monosaccharides except for ribose tested in this study. CTRG₀0554p showed better utilization ability for xylose and mannose than the rest monosaccharides tested in this study. CTRG₀1648p and CTRG₀4615p showed strong ability to transport glucose, mannose, fructose and xylose, therefore they were considered as efficient sugar transporters. Moreover, the transport ability of CTRG₀1648p for the 4 monosaccharides mentioned above are significantly better than the control of ScHXT7p. No transporter that is specific for xylose was found in this study.?2? Under three conditions of xylose, glucose, and mixed xylose and glucose used as carbon source, expression levels of the 15 candidate genes were studied in this study. Among the 15 candidate sugar transporter genes,8 genes?CTRG 00554, CTRG₀1165, CTRG₀1322,CTRG₀1648, CTRG₀2338, CTRG₀3572, CTRG₀4303, and CTRG₀6250? showed induced expression under all of the three conditions, and the expression levels of CTRG₀0554 and CTRG₀4303 under xylose condition were 15 times higher than that under glucose condition, exhibiting significant induced expression by xylose. Although CTRG₀1164p and CTRG₀4615p showed the ability for glucose and xylose transport, expression levels of these two genes showed no significant increase under three conditions of xylose, glucose, and mixed xylose and glucose. In this study, we found that five genes, including CTRG 01322, CTRG 02338, CTRG₀3572, CTRG₀4303, and CTRG₀6250, with no sugar transport function also showed induced expression under the three sugar conditions mentioned above. Their specific functions need to be further studied.?3? Prediction of protein structures of the 15 candidate sugar transporters indicated that their structures meet the characteristics of Major facilitator superfamily ?MFS?.A phylogenetic tree was created based on the multiple protein sequence alignment of these 15 candidate sugar transporters and other reported 27 sugar transporters by Maximum Likelihood method. Clustering results showed that the five new identified sugar transporters of the fifteen candidate proteins were divided into three groups. Combined with the previous reports, we found that these three sugar transporter groups of A, B, and C have their own characteristics on the structure and function.In conclusion, using C. tropicalis strain SHC-03 as the research object, we successfully identified 5 new sugar transporter genes from 15 candidate genes of C. tropicalis through gene cloning, expression and functional complementation assays in S. cerevisiae. Different transporter genes showed different transport ability and transcriptional response to monosaccharides tested in this study. A few genes identified in this study have strong abilities to transport several monosaccharides. The achievements obtained in this study can not only enrich the resource library for genetic modification of S. cerevisiae, but also help us to better understand the characteristics of yeast sugar transporters. This study has important significance to improve the production efficiency of fuel ethanol from lignocellulosic materials.