The Construction Of An Expression Vector Using18S RDNA As Integration Site For The Host Of Candida Glycerinogenes WL2002-5

Candida glycerinogenes WL2002-5is an osmotolerant yeast strain with extremely highosmotic pressure, which has been used for the commercial production of glycerol for decades.Compared with many other yeast strains, C. glycerinogenes has high growth rate, high glycerolyield, and a high tolerance to glucose. In addtion, C. glycerinogenes can be also applied as apotential excellent cell factory for the biosynthesis of glycerol derivatives, such as1,3-propanediol,3-hydroxypropionic acid, and dihydroxyacetone. However, the lack of geneticstrategy for enzyme expression hinders these potential applications. These potentialapplications require efficient genetic engineering strategy to increase the production capabilityof target compounds in C. glycerinogenes. In this study, we do the following work in the targetof developing a conversion system used for C. glycerinogenes:1. We have constructed an integrative expression vector pUSGZ used for C.glycerinogenes. The vector constructed possessed of the backbone of plasmid pUC19,18SrDNA sequence of integration site of C. glycerinogenes, phleomycin-resistant gene (ble) ofselectable marker, multiple clone sites (MCS). An osmolarity induced promoter of Cggpd(PCggpd) from C. glycerinogenes was employed to develop the integrative vector to expressheterogeneous genes. The linearized plasmid pUSGZ was integrated into the18S rDNA siteof C. glycerinogenes by electroporation transformation.2. Green fluorescent protein was used as a reporter to verify the characterization ofpUSGZ in C. glycerinogenes. The reporter gene gfp was fused to the downstream of PCggpdto construct the vector pUSGZG. After linearization, plasmid pUSGZG was integrated into C.glycerinogenes by electroporation transformation. The expression of GFP was inspected byfluorescence microscopy, and gfp was expressed in C. glycerinogenes successfully.3. The expression of GFP under different osmotolerant medium was inspected toinvestigate the performance of the system. The recombinant cells were cultivated in YEPDcontaining additional osmotic pressure stabilizers (glucose, NaCl, KCl or sorbitol). Theexpression levels of GFP were accordingly strengthened with the increased concentration ofthese compounds. Among the tested inducements, high concentration glucose and NaCl caninduce the higher expression of a report gene gfp. It’s because that PCggpd activity is variedunder different extracellular osmotic pressure.It supplied an excellent strategy for foreigngenes expression in C. glycerinogenes as an available genetic strategy for metabolicengineering in future.4. To quantify the transcription level of gfp driven by PCggpd, the gfp mRNA contentsof recombinant C. glycerinogenes under different conditions were investigated. Similar to thefluorescent intensities, the mRNA levels of gfp varied from different osmotic pressurestabilizer concentrations. The high induction efficiency of glucose and NaCl enabled theregulation of recombinant metabolism enzymes in further genetic engineering by therecombinant PCggpd.