Expression Strategy And Application Of Exogenous Genes In Candida Glycerinogenes

Candida glycerinogenes is a highly efficient industrialized production strain of glycerol.It has strong resistance to high osmotic pressure,high temperature,high sugar,and organic acids,which is a potential biotechnology host strain.There are still many gaps in genetic and molecular biology of the strain,which hinders the development of its industrial application potential.Yeast is one of the most important eukaryotic expression systems for exogenous gene expression.There are many factors affecting the expression of exogenous gene.In this study,codon preference,promoter and secondary structure of RNA were used to study the expression strategy of exogenous genes in the strain,aiming at providing a basis for the transformation of industrial anti-reverse yeast and the production of high value-added products.The research of this thesis is as follows:In order to optimize the expression of foreign genes from codon preference,Codon-W was used to analyze the codon usage of C.glycerinogenes.The results showed that the average GC content of C.glycerinogenes was 40.3%,the average GC3s content was 37.2%,and the optimal codon had 25 and ended mostly in A/U.By expressing exogenous genes from different sources,such as GFP,ldhA,xylB,xylD,kivD,it was found that the codon preference of GFP and ldhA were similar to that of the host and easy to express successfully.However,the GC content of xylB,xylD and kivD were all more than 65%.The codon preference ended with G/C,the CAI values were about 0.2,and the ENc values were all above 40.The genes were only transcribed but not expressed until codon optimization.The results showed that codon synonymous mutation could optimize exogenous genes,reduce GC content and ENc value,increase CAI value,and regulate exogenous genes expression efficiency in C.glycerinogenes.Based on the promoter expression strategy,the effects of different promoters on the expression of GFP,xylB and ldhA were further studied.Specifically,five constitutive promoters PGAP,PPDC,PPGK1,PCS1 and PLSC2 were selected from C.glycerinogenes to express GFP,and the starting intensity of PGAP was the strongest,followed by PPDC and PPGK1.Low pH inducible promoters PGMT1,PGUK1 and constitutive promoters PGAP,PPDC,PPGK1 were used to express xylB and ldhA.The results showed that the constitutive promoter regulated expression genes did not change significantly at pH 5.0 and 4.0,but decreased significantly at pH 3.0.The transcriptional levels of xylB and ldhA expressed by low-pH inducible promoter PGMT1 increased 14.3 times and 23.3 times,respectively,compared with pH 5.5,resulting in2.6 times and 3.2 times higher xylonate and lactic acid yields,respectively.Studies showed that C.glycerinogenes could be used as host can use different promoter expression strategies for different foreign genes,and can control the expression of foreign genes.Taking GFP and codon optimized xylB as the research objects,the expression strategies of minimum free energy change of secondary structure near translation initiation region and copy number on expression in C.glycerinogenes were investigated.It was found that the fluorescence intensity of GFP decreased by 49.6%when the secondary structure in the translation initiation region?G₀=-3.3 Kcal·mol^-1 decreased to?G₁₂=-12.0 Kcal·mol^-1,while the base pairing probability of?G₄=-6.3 Kcal·mol^-1 compared with?G₀ decreased,and the fluorescence intensity increased by 31.1%.When the secondary structure of xylB translation initiation region?G₀=-6.2 Kcal·mol^-1 was raised to?G₁=-2.4 Kcal·mol^-1,the stability of mRNA secondary structure was decreased,the probability of base pairing was reduced,and the activity of expressed enzyme was increased by 40.4%.Increasing the number of foreign gene copies resulted in the fluorescence intensity of the recombinant C.g-6GFP being 3.9 times higher than that of the single copy,the activity of C.g-4xylB01 and the transcription level of xylB being 1.5 and 2.8 times higher than that of the single copy,respectively,and the yield of xylic acid reached 24.5 g·L^-1.While the enzymatic activity and transcription level of 6 xylB copy numbers decreased by 35.7%and 36.5%compared with 4.The results showed that the expression of exogenous genes in C.glycerinogenes could be controlled to some extent by changing the minimum free energy of the secondary structure of the translation initiation region and optimizing the number of copies.