Mechanism Of Sugar Transport In Candida Glycerinogenes At High Concentrations And Its Application

High concentration sugar（high gravity）fermentation is one of the advanced fermentation technologies with high production intensity and energy saving and environmental protection.Currently,research related to this technology is focused on process,the tolerance and metabolism of the strain.As the research progresses,most of the production strains in the fermentation industry are found to have poor sugar uptake performance in high concentration sugar matrices,which is one of the bottlenecks limiting the further development of high gravity fermentation.Moreover,efficient transport mechanisms at high sugar concentrations associated with this technology have not been reported.Candida glycerinogenes is an industrial yeast strain with excellent resistance performance,which grows well and performs rapid sugar metabolism at ultra-high glucose concentrations.In this dissertation,we took this yeast strain as the research object,and mined its novel efficient hexose transporters,and investigated their mechanisms of transcriptional regulation,transport mode and ubiquitination modification under high sugar concentration and their modification applications,in order to provide a basis and new genetic resources for the engineering modification of high gravity fermentation strains.In this dissertation,we used this yeast strain as a research object to mine its novel and efficient hexose transporters,and investigated their mechanisms of transcriptional regulation,transport patterns and ubiquitination modifications at high sugar concentrations and their modification applications.These studies will provide the basis and new genetic resources for the engineering transformation of high gravity fermentation strains,and provide a basis for the study of sugar transport mechanism in non-traditional yeast and the later application development of this yeast.The main contents and results of this dissertation are as follows.（1）High-concentration sugar fermentation studies showed that C.glycerinogenes exhibited better sugar consumption than the industrial ethanol yeast S.cerevisiae ZWA46 and Angel yeast at high glucose concentrations.Based on the whole genome sequencing results,five hexose transporters（Cg Hxt1-2 and Cg Hxt4-6）with glucose transport function were mined from C.glycerinogenes.Further analysis of their transport properties showed that Cg Hxt4 had the best transport performance at high sugar concentration and was significantly better than S.cerevisiae hexose transporters（Sc Hxt1,Sc Hxt2,Sc Hxt3 or Sc Hxt7）.Transcriptomic analysis showed that Cg HXT4 and Cg HXT6 exhibited significant transcriptional up-regulation at high and low glucose concentrations,respectively.Then,knockout strains of these two transporters were constructed,and it was found that the knockout of Cg HXT4 led to a severe decrease in the fermentation performance of C.glycerinogenes at high glucose concentrations,which further confirmed that Cg Hxt4 is a key efficient hexose transporter mediating the rapid fermentation of high glucose concentrations.（2）To examine whether the hexose transporter Cg HXT4 is regulated by the sugar signaling and high osmotic glycerol（HOG）pathways,deletion strains Cg RGT1Δand Cg HOG1Δ,key genes of these two pathways,were constructed.Transcriptome and q RT-PCR results showed that the transcript levels of Cg HXT4 were severely reduced in these two deletion strains compared to that in wild-type Cg WT strains at high glucose concentrations.Transcriptome analysis showed that the transcriptional regulation of downstream target genes,including Cg HXT4,in C.glycerinogenes at high glucose concentrations was mainly dependent on the Sln1 branch pathway of HOG pathway and the Snf1-Mig1 and c AMP-PKA branch pathways of glucose signaling pathway.Promoter function analysis showed that potential binding sites for the HOG pathway transcription factor Sko1 and the glucose signaling pathway transcription factor Rgt1 were present on the promoter of Cg HXT4,P_{Cg HXT4},and were much more numerous than those on P_{Sc HXT1}.Further studies on P_{Cg HXT4}showed that the region between-1000 bp and the start codon"ATG"was mainly regulated by Rgt1,and the region between-2400 bp and-1001 bp was mainly regulated by Sko1.In addition,the transcript levels of Cg HXT4 in Cg WT,Cg HOG1Δ,Cg SKO1Δand Cg RGT1Δunder different conditions indicated that high sugar or/and high osmolality induced the transcription of Cg HXT4.The above results suggest that efficient transcriptional regulation of Cg HXT4 by sugar signaling and the HOG pathway at high glucose concentration is one of the mechanisms of high sugar transport in C.glycerinogenes.（3）A homology model of Cg Hxt4 was established based on the structure of Arabidopsis thaliana Stp10,and Cg Hxt4 was found to be an active proton-coupled homotransporter.Alanine scan mutations were performed on 16 conserved residues potentially involved in glucose coordination,and the results showed that N322A,F325A or G426A all resulted in a significant decrease in the transport performance of Cg Hxt4 at high glucose concentrations.Ten transmembrane segment（TMs）chimeras of Cg Hxt5 with Cg Hxt4 were constructed（i.e.,the TMs of Cg Hxt5 were replaced with those of Cg Hxt4 one by one）,and it was found that the transport performance of Cg Hxt5 containing Cg Hxt4 TM7 was significantly enhanced at high glucose concentrations.The 21 residues located in the TMs of Cg Hxt4 were mutated one by one to the corresponding residues in Cg Hxt5,and it was found that mutation of both residues（N321 and P427）resulted in a significant decrease in the transport performance of Cg Hxt4 at high concentrations of glucose.The saturation mutants of these two residues were constructed and their roles were examined using various analytical methods,and it was found that N321 and P427 are likely to play a role in glucose coordination and regulation of transport conformational flexibility,respectively.Based on the above results,an efficient working mode of Cg Hxt4 was obtained.（4）Subcellular localization analysis showed that the MLS of Cg Hxt4 was better than that of Sc Hxt1 during high concentration glucose fermentation.The results of alanine mutation of two ubiquitinated residues（K9 and K538）with high confidence showed that the MLS of mutant Cg Hxt4.2A was significantly improved during the fermentation of high glucose concentration.Based on these results,recombinant strains with Cg Hxt4 or its mutant Cg Hxt4.2A overexpressed were constructed and their effects on improving the ethanol productivity of C.glycerinogenes were examined.It was found that both of them not only enhanced the sugar consumption and growth performance of C.glycerinogenes,but also significantly increased the ethanol productivity,with Cg Hxt4.2A achieving the best results.The Cg TT-Cghxt.2A strain overexpressing Cg Hxt4.2A was used for the ethanol fermentation of non-detoxified sugarcane bagasse hydrolysate,and the results showed that the ethanol productivity of this strain was 18%-25%higher than that of the control strain Cg TT,respectively,and the fermentation time was shortened by 6 h.The above results suggest that the higher MLS of Cg Hxt4 is one of the mechanisms of high sugar transport in C.glycerinogenes,and increasing the expression or/and MLS of this transporter can effectively enhance the sugar consumption and lignocellulosic ethanol fermentation performance of the strain.（5）In order to solve the current problem of lack of co-fermentation（i.e.,simultaneous consumption of glucose and xylose）as the main obstacle to efficient lignocellulose utilization,studies on the xylose transport properties of the hexose transporters of C.glycerinogenes and the modified application of Cg Hxt4 were carried out.Five hexose transporters（Cg Hxt1-2 and Cg Hxt4-6）with xylose transport function were identified in C.glycerinogenes,among which Cg Hxt4 was found to have higher background xylose transport performance than the natural xylose utilization yeast-derived xylose transporters Ss Xut1,Ci Gxs1 and Dh Xyl HP.Based on the homology model of Cg Hxt4 and its molecular docking results with D-xylose or D-glucose,it was found that N351 residue may be involved in the coordination of both xylose and glucose.The result of point mutation in this residue showed that N351S significantly enhanced the xylose transport performance and slightly decreased the glucose transport performance of Cg Hxt4.To enhance the stability of membrane localization of Cg Hxt4 in the late stage of mixed sugar fermentation,the mutant Cg Hxt4.2A.S(Cg Hxt4^{K9A,N351S,K538A})was constructed.The results of mixed sugar fermentation showed that this mutant promoted the co-utilization of glucose and xylose by the recombinant strain.Finally,overexpression of this mutant in C.glycerinogenes achieved efficient utilization of undetoxified bagasse hydrolysate and elevated the D-xylonate titer to 44.5±1.7 g/L,which was 49.3%higher than the control strain.The above study showed that Cg Hxt4 has a good xylose transport capacity,can transport both glucose and xylose after being modified,and can confer the strain the ability to utilize lignocellulosic hydrolysate efficiently.