Study On The Metabolic Pathway Of Saccharomyces Cerevisiae To Synthesize Longifolene

Longifolene is a tricyclic sesquiterpene composed of three isoprene units,which are often used in perfume and spice industries.Longifolene has the characteristics of the anti-termite activity and high volume combustion heat,so it has good application prospects.Biosynthesis of longifolene is green,energy-saving,sustainable,and has important research significance.As a chassis cell,S.cerevisiae is a mature eukaryotic expression system with an endogenous Mevalonate(MVA)pathway,suitable for heterologous enzyme expression and has great potential for the synthesis of terpenoids.In this study,Saccharomyces cerevisiae CENPK113-5D was used as the expression strain to screen the longifolene synthase genes lgfs,psTPS,and TPS5 from three plant sources of Norway spruce,Pinus sylvestris,Taiwan cedar.The biosynthesis pathway of longifolene in Saccharomyces cerevisiae was established.The gene psTPS from Pinus sylvestris was finally determined as the most suitable enzyme gene.First,the endogenous MVA pathway of Saccharomyces cerevisiae was metabolically regulated by over-expression of four key rate-limiting enzyme genes of the MVA pathway,namely the acetyl-CoA acetyltransferase gene(atoB)derived from Escherichia coli,Farnesyl pyrophosphate synthase gene(erg20),IPP isomerase gene(idil)and truncated 3-hydroxy-3-methylglutaryl-CoA reductase(thmgl).The four genes were integrated into Saccharomyces cerevisiae chromosomes.Then,the Saccharomyces cerevisiae chassis strain was modified by knocking out the genes encoding phosphatidic acid phosphatase(dppl and lppl)and the negative transcription factor(roxl).The expression level of the key enzyme gene(erg9)for squalene synthesis was down-regulated.The Saccharomyces cerevisiae which the endogenous competition pathway was inhibited were obtained.Next,by screening the endogenous molecular chaperones,the disulfide isomerase gene(PDI1)was selected,whose overexpression could optimize the expression of longifolene synthase in Saccharomyces cerevisiae.Finally,by regulating the synthesis pathway of intracellular acetyl-CoA to promote the production of longifolene,the main methods included the knockout of the alcohol dehydrogenase gene ADH1,the overexpression of the alcohol dehydrogenase gene ADH2,acetaldehyde dehydrogenase gene(ALD6)and the acetyl-CoA synthase gene mutant(ACSL641P)derived from Salmonella enterica.Based on the above strategies,a genetically engineered strain of Saccharomyces cerevisiae with high-yield longifolene was constructed.The yield of longifolene reached 27.30 mg/L after fermentation at the shake flask level,and the total yield of longifolene reached 165.96 mg/L after fed-batch fermentation at the 5 L fermentor level.It is currently the highest yield of biosynthesis of longifolene by using Saccharomyces cerevisiae as the chassis strain.