Selection And Construction Of High Malonyl-CoA Flux Saccharomyces Cerevisiae Using SCRaMbLE And High-throughput Screening

Malonyl-coenzyme A is a key precursor of various biofuels and biochemicals compounds,such as fatty acid derived products.lit is also a very important precursor for the synthesis of pharmaceutical products,such as flavonoids,and polyketides.Malonyl-CoA derived products have important applications in many fields such as medicine,chemical industry and biofuels.Therefore,the construction of high-yield malonyl-CoA platform strains ha.s great potential for industrial production.Saccharomyces cerevisiae has clear genetic background,advanced gene manipulation tools and good robustness.Thus,it is a potential microbial cell factory to synthesize malonyl-CoA derived products.Currently,the study to improve malonyl-CoA synthesis mainly focuses on rational engineering,including strengthening the upstream pathway of malonyl-CoA synthesis to increase its supply,and weakening downstream metabolic pathways such as phospholipids pathwayto reduce malonyl-CoA consumption.However,the malonyl-CoA flux is still relatively low to sati'sfy the industrial production demand.Thus,it is necessary to identify new targets by irrational engineering to enhance its synthesis.Synthetic yeast genome project(Sc2.0 Project)aims to construct a highly modified Saccharomyces cerevisiae genome.A large amount of loxPsym(gene recombination site)are inserted downstream of the stop condon of non-essential genes to facilitate genetic manipulation and molecular research.It can achieve rapid evolution through the genome rearrangement system and obtains evolved strains with important application potential in agriculture,industry and other fields.In this study,based on malonyl-CoA sensor we constructed in previous work,we used in vitro synthetic chromosome S.cerevisiae strain(SynV)to quickly construct mutation library through SCRaMbLE,and obtained high malonyl-CoA synthesis strains using high-throughput screening.First,the malonyl-CoA sensor was introduced into SynV strain,and the genome rearrangement was induced by adding inducer ?-estradiol.The genome mutation library was screened by fluorescence-activated cell sorting(FACS)and then was subject to next round of selection by microplate reader.Then the 3-hydroxypropionic acid synthetic pathway was introduced into the evolved strain to confirm the malonyl-CoA improvement.After several rounds of FACS screening,about 20,000 cells were screened out of more than one million cells.After re-selection in the microplate reader from the 20,000 mutants,a high-yield mutantion was finally selected.the fluorescence intensity of the mutation strain was measured,and the fluorescence value increased by 283%compared to the wild-type.3-hydroxypropionic acid production increased by 116.4%,reached 101.5mg/L in the evolved strain.Besides irrational selection new target to enhance malonyl-CoA synthesis,we also improve its synthetic through introduction of citrate lyase pathway.The accumulation of cytoplasmic citric acid was improved and the flux of tricarboxylic acid cycle(TCA)was weaken.The results showed that the deletion of ICL1 gene,or the deletion of both ICL1 and IDH1 genes,deletion EAF7 gene increased the fluorescence by 5 19.4%,359.4%and 244.3%compared with the wild-type strain,respectively,which showed the highest improvement among the optimization strategies.In this study,SCRaMbLE coupled with biosensor based selecttion was used for high-throughput screening of high malonyl-CoA yield mutation strain.Genomic sequencing of high-yield mutantion will be performed to find new key targets for malonyl-CoA biosynthesisto further improve the production of important downstream products through rational optimization strategies.