Genome Mining And Heterologous Efficient Synthesis Of Celangulin In Saccharomyces Cerevisiae

Celangulin is a natural botanical insecticide with high efficiency and low toxicity and derived from the insecticidal plant Celastrus angulatus.We take celangulin as the research object to carry out the synthetic element mining and heterologous efficient synthesis,because of the low content of botanical pesticides in plants and extraction process is time-consuming.In this study,we obtain a chromosome-scale reference genome of C.angulatus.Combined with transcriptomic analyses,we mine the biosynthetic elements related to celangulin biosynthesis and preliminarily shed light on the intermediateα-agarofuran biosynthetic pathway.Specifically,we constructed a high-efficient Saccharomyces cerevisiae cell factories to produceα-agarofuran according to the synthetic biology and metabolic network design and reconstruction technology including the biological element mining,chassis cell design and construction,and the adaptation between biological modules.An acid catalyzed synthesis method was used to produceβ-dihydroagarofuran,which is the parent ring of celangulin V,and the mechanism of chiral inversion in the synthesis process was speculated by density functional theory（DFT）calculations.The genome size of C.angulatus is 322.07 MB,including 23 chromosomes,and the N50 value is 5.29 MB.Genome combined with transcriptome analysis revealed sixteen sesquiterpene synthases（STSs）,the function of STSs was verified in vitro by recombinant protein.Among them,CaTPS8 and CaTPS18 are involved in celangulin biosynthesis pathway,which can catalyze the formation ofγ-eudsemol;we obtained nine P450s that may involve in celangulin biosynthesis pathway,their functions were verified in vivo of S.cerevisiae and microsomes of recombinant yeast in vitro,among them,CaCYP21,CaCYP22,CaCYP23 and CaCYP28 can further catalyze oxidation ofγ-eudsemol toα-agarofuran.In order to realize the heterologous and efficient synthesis of key intermediates,we constructed a high-efficient S.cerevisiae cell factories to produceα-agarofuran.We introduced CaCYP18 into HO loci in strain Sc027,knocked out the Gal80,overexpressed the upc2-1,and P_ERG9 was replaced with glucose concentration sensitive P_HXT1,and constructed a S.cerevisiae cell factory to enable high-level production ofγ-eudsemol.Glucose was the sole carbon source to sequentially control gene expression between the competitive squalene andγ-eudsemol pathways.Finally,theγ-eudsemol production of recombinant strain LWG003-CaTPS18 was 342.32 mg/L,which was 4.2times higher than that of the original strain,and the yield of fed fermentation in 5 L bioreactor was 2.56 g/L.CaCYP23 were further expressed,the yield ofα-agarofuran reached 1.86 mg/L.Then,we studied the adaptation between CaCYP23 and CaCPRs and constructed the fusion protein CaCYP23-CaCPRs.When CaCYP23 and CaCPR3were co-expressed,the efficiency of CaCYP23 was the highest,which could convert 3.2%γ-eudsemol toα-agarofuran,and was 2.67 times that of the original strain.In order to realize the efficient synthesis of celangulin,we carried out the acid catalyzed synthesis research on the key intermediatesβ-dihydroagarofuran at the same time.We heterologously expressed 7-epi-α-eudsemol synthases in yeast chassis cell,and the yield was 208.3 mg/L,which ensured the high-efficient production of 7-epi-α-eudsemol.By treating different eudesmane-type sesquiterpene with different concentrations of acid,we obtained the optimum synthetic method ofβ-dihydroagarofuran.When treated 7-epi-α-eudsemol with 4M HCl,the highest yield ofβ-dihydroagarofuran was 17%,which was 1.67 times that of chemical synthesis.At the same time,we calculated and speculated the chiral inversion mechanism of 7-epi-α-eudsemol converted toβ-dihydroagarofuran by density functional theory（DFT）.Under acidic conditions,7-epi-α-eudsemol is first converted toγ-eudsemol,then spiro sesquiterpenes were formed,and finally hydroxyl attacked the carbocation intermediate of spiro sesquiterpenes to form chiral inversion β-dihydroagarofuran.This chiral inversion mechanism was first reported in the formation of sesquiterpenes.The study will provide new ideas for the analysis of synthetic pathway and heterologous and efficient production of botanical pesticides,and accelerate the research process of synthetic biology of natural products.