Mechanism Research On Biosynthetic Hypocrellin By Shiraia Bambusicola

As a significantly medical fungus in China,Shiraia bambusicola can produce hypocrellin.The photo-activated hypocrellin can generate reactive oxygen species?ROS?.Abundant ROS can induce cellular oxidative stress,which damages cellular macromolecules and induces cell apoptosis.Therefore,hypocrellin can be used as a photosensitizer and applied in areas of food,agriculture,and medicine.Based on the essential applications and theoretical values,the researches on optimum,purification,and application of hypocrellin have been particularly reported in these decades.However,deficiency of suitable molecular tools limits the researches on hypocrellin biosynthesis.S.bambusicola also presents the extraordinary ability to defense oxidative stresses,including hypocrellin and other environments.This ability ensures to maintain normal activities.To date,reports on the antioxidant network have not yet mentioned.In this project,Shiraia sp.SUPER-H168 was used as the original strain.We constructed a stable platform for analysis of gene relative expressions,established effective systems for gene expression and gene knockout,exploited the essential genes for hypocrellin biosynthesis,and decoded the antioxidant system of S.bambusicola by resisting different oxidative stresses from the environment or toxic metabolites.The results were described as the following points.?1?Construct a stable technology of the relative-expression-analysis in S.bambusicolaUnder two different culture conditions?with or without hyprocrellin productions?,we tested stabilities and expression abundances of the nine candidate reference genes,including actin?Act?,citrate synthase A?CsA?,citrate synthase B?CsB?,cytochrome oxidase,?CyO?,phosphoglycerate kinase?PhoK?,pyruvate decarboxylase?PyrD?,tubulin?Tub?,18S ribosomal RNA?18S rRNA?and glyceraldehyde-3-phosphate dehydrogenase?GAPDH?.As the most unstable genes,Tub and Act were not feasible for relative expression analysis based on GeNorm results.CyO and GAPDH were the more stable reference genes.These results were similar to those of NormFinder algorithm.In order to determine the optimal combination of reference genes,we further used GeNorm to analyze V_n/_n+1 values of candidate genes.Therein,the value of V₂/₃ was 0.119,lower than the threshold value?0.15?.Based on their most stable characteristics,CyO and GAPDH were chosen as the gene combination to normalize relative expression level of other genes.The normalized gene relative expression of polyketide synthase indicated that this method was reliable and can be used to normalize the relative expression of other genes.?2?Construct an efficient expression system of S.bambusicola.Sorbitol was used as a stabilizing agent.We can obtain high-quality protoplast by degrading the cell walls using the enzymes,including lysing enzyme,driselase,?-glucuronidase,and D-glucanase.The expression plasmid was transformed into protoplast by a method of polyethylene-glycol and calcium dichloride.Positive strains were screened on regeneration medium with Hygromycin B and presented a green fluorescent signal,while no fluorescent signal was detected for the wild-type strain.We used different lengths of GAPDH promoter to activate green fluorescent protein and screened the suitable length promoter based on the green fluorescent signal.Therein,GAPDH promoter?about 2000 bp?presented higher fluorescent signal and can be utilized for gene expression of S.bambusicola.?3?Establish an effective CRISPR/Cas9 system.Growth rates and hypocrellin yields of those strains,including S.bambusicola with Cas9plasmid,S.bambusicola with sgRNA plasmid,wild-type S.bambusicola,presented no differences.These results suggested that CRISPR elements are nontoxic to S.bambusicola and can be used for genome editing.As a global regulator,the transcriptional factor was used as a target gene.In order to improve the genome-editing efficiency,we prepared a homologous donor.This donor and CRISPR plasmid were co-transformed into protoplast.As a result,the genome-editing efficiency was up to 23.07%.Then we optimized exogenous U6 promoters for sgRNA transcription.Therein,the efficiency was slightly improved when we used U6-1 and U6-3 to activate sgRNA.The value was 27.27%and 33.33%,respectively.In contract,the value was 20%when we used U6-2 promoter for sgRNA transcription.?4?Exploit the essential genes in hypocrellin pathway.We have screened corresponding genes for hypocrellin biosynthesis by antiSMASH analysis.Based on the relative expression platform,we determined that relative expressions of several genes,including polyketide synthase,monooxygenase,and transcription factor,were relative to hypocrellin biosynthesis.CRISPR system was used to knock out genes,including polyketide synthase,monooxygenase,and transcriptional factor.These corresponding mutants don't produce hypocrellin.In contrast to wild S.bambusicola,the relative expressions of the genes for hypocrellin biosynthesis were clearly down-regulated.These results suggested that these essential roles of these two genes in hypocrellin biosynthesis.In addition,the overexpression of muticopper oxidase in hypocrellin pathway enhanced its relative expression with 5-fold.The relative expressions of genes,such as polyketide synthase and monooxygenase,were also improved by above 7-fold.Meanwhile,the relative expression of the transcriptional factor was increased by 9.69-fold.These enhancements of gene transcriptional levels and relevant synergistic effect ensure the overexpression strain produce 3.18 g·L^-11 hypocrellin,increasing 5-fold yield than that of the wild S.bambusicola.?5?Decode the antioxidant system of S.bambusicola.Oxidative stresses stimulated superoxide dismutase,which ensures abundant superoxide radical transform to non-toxic H₂O.Meanwhile,catalase?CAT?yields were also enhanced.The CAT production still remained high levels after 72 h treatment,which guaranteed that abundant H₂O₂ was catalyzed to H₂O.In contrast,glutathione peroxidase?GPx?production kept low level after H₂O₂ treatment.GPx activity was improved after 48 h,which also guaranteed that abundant H₂O₂ was catalyzed to H₂O.Contents of reduced glutathione?GSH?contents in the treated S.bambusicola were also up-regulated.The contents still maintained high quantities after 96 h treatment.Based on the synergistic effect of GPx,the increasing GSH quickly conversed H₂O₂ to H₂O.This up-regulated antioxidant system ensures S.bambusicola resist oxidative stress.Based on gene knockout and compliment experiment,the major facilitator superfamily?MSF?transporter in hypocrellin pathway was determined to efflux hypocrellin from cells.In summary,oxidative stresses from hypocrellin induced the antioxidant system,ensured normal cellular redox level,and remained continuous biosynthesis of hypocrellin.