Single-base Mutation Evolution And Genome Rearrangement Of Astaxanthin-produced Saccharomyces Cerevisiae

Astaxanthin is a a red,fat-soluble carotenoid pigment with tremendous antioxidant activity and great commercial value.It is widely used in the field of medical cosmetics.Microbial production of astaxanthin via metabolic engineering has become a promising alternative.This study aimed to use synthetic biology to increase the production of astaxanthin in Saccharomyces cerevisiae and to releval the MVA pathway target.The whole genome of strains SyBE_Sc2110M1,SyBE_Sc2110M3 and SyBE_Sc307001 was re-sequenced.Through bioinformatics analysis,non-synonymous mutations and frameshift genes were found in the coding region(CDS)of the two mutant strains.It can be divided into four parts,which are transcriptional regulation,transposons,cell wall function and unknown function proteins.Based on the results of comparative genomic analysis,the high frequency genes CSS1,DAN4,FLO9 and the mutant strain SyBE_Sc2110M3 specific genes YBR012W-B and YLR410W-B were knocked out individually in the control strain SyBE_Sc307001.After knocking out YBR012W-B,CSS1,and DAN4,astaxanthin production increased.Nevertheless,individually deletion of CSS1 or DAN4 enhanced astaxanthin yield by 59.6% and 36.3%,respectively.Fed batch fermentation was conducted in 5-L bioreactor amplifying the astaxanthin-producing S.cerevisiae strain SyBE_Sc2110M3.Eventually,a titer of 217.9 mg/L astaxanthin was obtained after 140 h cultivation,which is the highest reported titer in microbial cells as known.At that point,the astaxanthin ratio reached 89.4%.In order to achieve large-scale rearrangement of the genome and further increase the yield of astaxanthin.S.cerevisiae containing the SynV chromosome was introduced,combined with the existing sitespecific recombination system Cre-LoxPSym system and cell disassembly technology,a strain was screened out.The high-producing astaxanthin mutant strain SyBE_Sc307030 had an astaxanthin yield 1.5 times that of SyBE_Sc2110M3.