| As the crisis of depletion of air pollution energy increases,the search for new and renewable energy-free energy sources is imminent,and bio-produced fatty acids,as a technology that can be converted into biodiesel,are receiving more and more attention.The traditional transformation of microorganisms is a way of using genetic recombination,which is time consuming and laborious.As a new generation of gene editing and regulation technology,CRISPR/Cas9 and dCas9 systems have been widely used in a variety of species to rapidly modify the expression of genes and regulatory genes.The CRISPR system uses gRNA to direct Cas9 or dCas9 proteins to target DNA sequences,but how to select effective gRNAs is a key issue.In this study,CRISPR-dCas9 orthogonal technology was used to regulate two key nodes in the regulation of two metabolic pathways in high-yield fatty acid Saccharomyces cerevisiae,and 16 different expression suppression combinations were used to find optimal inhibition:by inhibiting PGI1 gene Increasing the pentose phosphate pathway increases NADPH in cells,inhibits the IDH2 gene to increase the amount of acetyl-CoA,and increases the precursors of fatty acid synthesis,ultimately increasing 22%fatty acid production.Fatty acids are involved in the synthesis of lipid droplets and membrane structures in cells.How to increase the proportion of fatty acids in Saccharomyces cerevisiae has not been studied.The study also uses CRISPR-Cas9 technology to block multiple pathways involved in fatty acids,delete genes in the entire pathway,and direct carbon to fatty acid synthesis and accumulation,thereby increasing fatty acid production.We found that blocking fatty acid degradation pathways,phospholipid degradation pathways,and DAG synthesis pathways facilitated the synthesis and storage of fatty acids.Our research can promote the production of fatty acids in yeast,and verify that CRISPR technology can accelerate the metabolic engineering of yeast species and increase yield and traits. |
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