Random Mutagenesis Of Zygosaccharomyces Rouxii Producing D-Arabitol And Metabolic Engineering Modificationn

D-arabitol is an important functional sugar alcohol with a wide range of applications and significant development potential and market value in the food,pharmaceutical and chemical industries.At present,most of the studies on the production of D-arabitol by biological methods are focused on the screening of natural strains and the optimization of fermentation conditions,but less research has been conducted on metabolic engineering modifications.Zygosaccharomyces rouxii is a natural D-arabitol producing strain,which is commonly used in soy sauce brewing and is a safe strain for food production.In this study,a D-arabitol high-producing strain was obtained by ARTP mutagenesis using Z.rouxii as the starting strain;subsequently,the fermentation conditions for D-arabitol production by the mutant strain were optimized.Finally,the genes and metabolic pathways associated with D-arabitol production were explored in conjunction with transcriptomic analysis results to elucidate the molecular mechanisms underlying the enhanced D-arabitol production in the mutant strains and to further enhance D-arabitol production by metabolic engineering of Z.rouxii accordingly.The main findings were as follows:(1)The lethality rate curve of the original strain Z.rouxii ST 109 was determined using different ARTP treatment times,followed by mutagenesis screening to obtain the mutant strain Z.rouxii M 075,by which D-arabitol production reached 42.01 g/L,an increase of 49.6% compared to the original strain.(2)Optimization of fermentation conditions,including medium composition and culture conditions,was carried out for the mutant strain Z.rouxii M 075.The optimal medium composition for D-arabitol fermentation was obtained as follows: glucose concentration 300 g/L,tryptone 12 g/L,yeast extract 12 g/L,ammonium sulfate 1 g/L,fumaric acid 10 g/L,Triton X-100 1.5%(v/v).The optimal culture conditions were:5%(v/v)inoculum,96 h fermentation time,and 30℃.Under the optimal conditions,the production of D-arabitol was 58.19 g/L,which increased by 38.9%.Subsequently,a variable temperature fermentation strategy was adopted to further improve the D-arabitol production according to the strain growth and different optimal fermentation temperatures.And the production can be reached to 64.22 g/L with a10.36% increase compared to that of 30℃ constant temperature fermentation..(3)Transcriptomic techniques were used to analyze the differences between the mutant strain Z.rouxii M 075 and the original strain,and differently expressed genes were obtained.The analysis of GO enrichment and KEGG metabolic pathway of differential genes were also carried out for comparison,and the role of key genes and proteins on D-arabitol production was preliminary analyzed from a functional perspective.(4)Targeted metabolic modification of the pentose phosphate pathway of Z.rouxii M 075 was carried out based on the results of transcriptomic analysis.Two key genes upstream of the metabolic pathway were overexpressed,and two recombinant plasmids p GAP-gene were successfully constructed using seamless cloning technology.The two genes were transformed separately and integrated into the genome of Z.rouxii successfully.The results showed that the overexpression of gnd or zwf could help enhancing the production of D-arabitol.