Construction And Gene Mining Of Genomic Large Fragment Editing System In Candida Glycerinogenes

Genomic large fragment editing is an important technical method on microbial genome reduction by deleting non-essential genes,optimizing biochemical metabolic networks and gene research.Industrial strain Candida glycerinogenes is a diploid yeast with the advantages of multiple resistance to stress and high temperature concentrated mash fermentation.It has the potential to become the synthetic biological industrial chassis cell.And its unique stress resistance makes large number of unknown genes need to be excavated.However,up to now,there was no relevant research on its genomic large fragment editing.In this study,the technical method of deleting large DNA fragments was explored,and new functional genes were obtained by characterizing and analyzing the tolerance and fermentation characteristics of mutant strains,which provided a technical basis for the gene mining of C.glycerinogenes in the future.The main research results were as follows:(1)Based on bioinformatics analysis,a number of large genomic fragments without essential genes were identified as regions to be deleted.By optimizing the gene editing parameters of paired sg RNA-mediated CRISPR/Cas9,several large DNA fragments with different lengths were deleted,achieving 25 kb and 50 kb long fragment knockout with 4.5%and 3.0%-knockout efficiency,respectively.It indicated the availability of CRISPR/Cas9 for large fragment deletion in C.glycerinogenes genome reduction,and preliminarily established a method for editing genomic large fragments of C.glycerinogenes.(2)In order to investigate the function of knocking out unknown genes in large DNA fragments,the phenotype of the mutant strains was analyzed.It showed that the biomass ofΔ7.8 kb strain,deleted the 7.8 kb-unannotated-information DNA,in conventional culture was lower than others;the biomass of Δ7.8 kb and Δ25 kb strain,deleted one of the repeat region,decreased by 17.3% and 21.4% under high temperature,respectively.The Δ44kb strain could rapidly synthesize ethanol,while the Δ25 kb strain have weak ethanol synthesis ability.It was found that the fermentation differences of Δ44 kb strain may be the result of the comprehensive regulation of multiple genes in the pathways of glucose consumption,by-product generation,tolerance and other pathways affected by the deletion of large DNA fragments in the genome.The glycerol accumulation ability of Δ50kb strain and Δ25 kb strain was weak.The Δ50 kb strain and Δ25 kb strain lacking repeat sequences had extremely weak glucose utilization performance during glycerol fermentation.It is speculated that the deletion of repeat sequences may affect the energy metabolism of strain.Quantitative PCR results showed that the reducing power-related genes G6 PD and 6PGDH of Δ7.8 kb strain with the highest glycerol production per unit cell were significantly up-regulated,indicating that the deletion of this fragment may affect pentose phosphate pathway flux,thereby affecting glycerol synthesis.Δ7.8 kb strain become weaker under various pressures such as high temperature,high temperature and high sugar,hydrogen peroxide,lactic acid,2-phenylethanol(2-PE),acetic acid,and furfural.(3)Bioinformatics analysis and mining of unknown genes found that the deletion fragment of Δ7.8 kb strain contained several suspected reverse transcriptase(RT)encoding genes CgP2,CgP3 and CgP4.The strains that inhibited CgP2 expression were weak under furfural,lactic acid,acetic acid,2-PE,and high temperature pressure.The growth of CgP4-inhibiting strains became weaker under high temperature and acetic acid.CgP2 and CgP4 were co-expressed as expression cassette Cg RT.Cg RT-overexpressing recombinant strain showed obvious growth advantages under hydrogen peroxide,acetic acid,lactic acid,phenethyl alcohol,and high temperature;the inhibition of Cg RT significantly weakened the tolerance performance under various stresses.The recombinant Saccharomyces cerevisiae expressing Cg RT had obvious growth advantages under the conditions of 2-PE,furfural,high glucose and acetic acid.Transcriptional level showed that genes involved in DNA damage repair were significantly up-regulated in overexpression strains.When a 2-PE-tolerant strain overexpressing Cg RT was used for 2-PE synthesis,both the sugar consumption rate and the2-PE production were improved.The results showed that the Cg RT overexpression strain with improved tolerance could better tolerate 2-PE in the synthesis of phenethyl alcohol,indicating that the genes obtained by mining were significantly related to stress resistance.