Enhancing Longifolene Synthesis In Engineered Saccharomyces Cerevisiae Using Multiple Strategies

Terpene compounds are a diverse class of natural compounds with isoprene as their structural unit.Among them,longifolene belongs to the tricyclic sesquiterpenoids and is the main component of heavy turpentine oil.It is characterized by a unique woody and ambergris fragrance and is used in the perfume and insect repellent industries.Longifolene also exhibits antibacterial activity,high density,and high combustion heat,making it widely applicable in various fields.Microbial synthesis of longifolene offers environmental friendliness,high renewability,and low production costs,which hold significant implications for national carbon neutrality and peak carbon policies.Saccharomyces cerevisiae possesses a natural mevalonate pathway(MVA)that lays a solid foundation for sesquiterpenoid production.Moreover,various gene manipulation tools and methods in Saccharomyces cerevisiae have matured,indicating a promising future for the biosynthesis of sesquiterpenoids using this organism.In this study,the brewer’s yeast strain CEN.PK113-5D was used as the chassis host.We employed computer-aided prediction to remove the N-terminal mitochondrial targeting peptide of longifolene synthase and fused it with longifolene synthase from Pinus sylvestris(PSTPS)and farnesyl pyrophosphate synthase(FPS)to improve the catalytic efficiency of longifolene synthase.Acetyl-Co A in the cytoplasm is mainly generated by decarboxylation of pyruvate through the pyruvate dehydrogenase bypass(PDH).Therefore,this study screened and overexpressed key enzyme genes in the PDH bypass to increase the supply of cytoplasmic acetyl-Co A through a push-pull metabolic strategy.This included overexpression of pyruvate decarboxylase 5(PDC5),alcohol dehydrogenase 2(ADH2),aldehyde dehydrogenase 6(ALD6),and acetyl-Co A synthetase 1(ACS1).The study also investigated the effect of exogenous acetate addition as a carbon source on intracellular acetyl-Co A levels.Subsequently,the study targeted the tricarboxylic acid cycle(TCA)by introducing an ATP-citrate lyase(ACL)pathway to increase the supply of cytoplasmic acetyl-Co A.This involved four enzymes: ACL,malic enzymes(MEs),endogenous citrate transporter(CTP1),and malate dehydrogenase(MDH3),creating a citrate cleavage enzyme cycle to enhance the concentration of acetyl-Co A in the cytoplasm.Furthermore,considering the substantial energy supply required for product synthesis,the study screened formate dehydrogenases(FDH)from different sources to boost the supply of intracellular cofactors NAD(P)H.Ultimately,through combinatorial optimization,a recombinant yeast strain with high longifolene production was constructed.The highest longifolene yield in shake flasks reached 78.637 mg/L.When this strain was subjected to 5 L fed-batch fermentation,the final longifolene yield reached2063.9 mg/L,which is the highest yield reported to date.