Psilocybin Biosynthesis Based On Optimization Of Fungal Chassis

Psilocybin is a naturally occurring tryptamine derivative in the genus Psilocybe,which acts on the human 5-hydroxytryptaminereceptor and has the potential to treat a variety of mental disorders.At present,there have been many clinical studies on the use of psilocybin for the treatment of depression,obsessive-compulsive disorder,posttraumatic stress disorder,tobacco addiction,alcoholism,advanced cancer anxiety and other psychiatric diseases.Psilocybin for the treatment of treatment-resistant depression and major depression has been granted Breakthrough Therapy status by the FDA.The chemical synthesis method of psilocybin uses high cost of raw materials and there is a risk of environmental pollution in the phosphorylation step.Heterologous synthesis of psilocybin by applying biosynthesis methods allow simple production processes from cheap starting substrates such as glucose,which can effectively reduce production costs,satisfy the high yield and purity required for pharmaceutical applications,and avoid the use of hazardous chemicals and environmental pollution.Furthermore,microbial cell factories can rapidly produce various valuable tryptamine derivatives,thereby obtain psilocybin-related derivatives,enriching the types of natural products.In this study,with the purpose of effective psilocybin synthesis,the biosynthesis of psilocybin and its derivatives was studied by CRISPR-Cas9 multi-site gene editing technologyand metabolic pathway optimization method,the result is as follows:1.Heterologous biosynthesis of psilocybin and its halogenated derivatives in Saccharomyces cerevisiae.Saccharomyces cerevisiae is an important industrial microorganism with a variety of eukaryotic cell organelles and membrane structures in its cells,which can provide different environments and compartments for the synthesis of natural products.In particular,it has great potential for producing aromatic compounds.In this study,CRISPR-Cas9 multi-site gene editing technology was used to integrate the psilocybin biosynthesis pathway genes Tdc、Psi H、Psi K、Psi M into the genome of Saccharomyces cerevisiae,in the commonly used laboratory strains of Saccharomyces cerevisiae BY4741,CEN.PK2-1D,CEN.PK113-5D.The CEN.PK113-5D strain with faster growth and less by-products was selected as the chassis cell for further optimization.Meanwhile,In the psilocybin biosynthesis process,we found that the hydroxylation capacity of P450 oxidase Psi H was insufficient,thereby affecting the synthetic yield of psilocybin.Subsequently,the biosynthesis pathway of psilocybin was optimized by increasing the copy number of P450 oxidase,and the yield of psilocybin in the shake flask was increased from 15 mg/L to 114 mg/L,which was 7.6 times that of the starting strain.In addition,according to the principle of precursor-directed biosynthesis,and the promiscuity of the enzyme,we exogenously added the analogs of precursor tryptophan to the culture,and successfully obtained 4 novel non-natural psilocybin halogenated derivatives with enhanced lipid solubility.2.Heterologous biosynthesis of psilocybin in Acremonium chrysogenum.This study also tried to develop a new industrial microorganism Acremonium chrysogenum as a chassis cell for heterologous synthesis of psilocybin.The secondary metabolite cephalosporin C synthesized by Acremonium chrysogenum requires serine and methionine as precursors.Thionine can enhance intracellular S-adenosylmethionine,and serine is the main synthetic precursor of tryptophan.While the biosynthesis of psilocybin requires tryptophan as a precursor,and S-adenosylmethionine acts as a methyl donor to promote the N-methylation of tryptamine,so there is a sufficient amount of precursors required for psilocybin synthesis in Acremonium chrysogenum,which can be used as a chassis cell for psilocybin biosynthesis studies.In this study,an element expressing psilocybin biosynthesis genes Tdc、Psi H、Psi K、Psi M was constructed by combining the 2A self-cleaving peptide with the TEV protease recognition site,in fungi cells.The results showed that psilocybin was heterologously expressed in the chassis of the industrial microorganism Acremonium chrysogenum successfully.