Determination Of The Biosynthetic Pathway And Gene Cluster Of Sporormielones From Sporormiella Minima(No.40-1-4-1)

Sporormielone A is a new type of 2,3,3b,4-tetramethylcyclopenta[a]indene-1,6,8(3a H,3b H,8a H)-trione with a novel skeleton isolated from the rice fermented extract of the endophytic fungus S.minima(No.40-1-4-1)from the lichen Cladonia subulata(L.)Wigg.in our preliminary works.Up to now,only 3 compounds with similar structure have been reported,they all have a three-ring system which contains a dimethylcyclopentenone unit.It is speculated that these compounds may be polyketones derived from orsellinic acid derivatives by gene knockout or chemical communication methods,but the details of the biosynthetic pathway have not been clarified.In addition,a sesquiterpene alkaloid(S)-6-hydroxy-3,4a,5-trimethyl-4,4adihydro-1H-benzo[f]indole-2,7-dione whose structure is similar to sporormielone A was obtained from the rice fermented extract of S.minima in our preliminary works at the same time with sporormielone A.Therefore,it is not clear whether sporormielone A derives from polyketide or terpenoid pathway.In order to clarify the biosynthesis of these compounds,[1-13C]acetate and [2-13C]acetate were used as isotope substrate for feeding experiments and the labeled compounds were obtained.The biosynthetic pathway of sporormielone A was identified as polyketide pathway according to the rules of labeled carbon atoms in target compounds.Then,the candidate polyketide synthase genes related to the biosynthesis of sporormielone A was identified by whole genome sequence analysis and differential transcriptome analysis,and the polyketide synthase gene related to the biosynthesis of sporormielone A was identified as g7393 by knockout experiments with the use of CRISPR/Cas9 system.Then,combined with the results of differential transcriptome analysis,we deduced the biosynthetic gene cluster of sporormielone A.Ultimately,the possible biosynthetic pathway of sporormielone A was deduced based on the results of isotope labeling experiments and functional analysis of the genes in the biosynthetic gene cluster of sporormielone A,which laid a foundation for the elucidation of the biosynthetic mechanism of these compounds.