Study On The Regulation Mechanism Of Pigment Synthesis In High-Yield Lycopene Mutanta Based On Blakeslea Trispora

As a kind of lipid-soluble vitamin with important functions,lycopene distributes widely in nature and possesses functions such as cancer resistance,oxidation resistance and cardiac-cerebral vascular disease resistance.As a result,lycopene is widely utilized in foods,feeds,health foods and cosmetics etc.The requirement of lycopene increases with the increase of the population,while extraction of lycopene from plants cannot meet the requirement for lycopene of people.Therefore,it is of critical importance to exploit microorganisms with high produce capacity of lycopene for the health foods development and the pharmaceutical production.Blakeslea trispora is the only strain to produce lycopene in practice.A lot of investigations have been done to improve the production of lycopene,however,the production as well as the quality of lycopene still cannot meet the requirement of this product from people,which mainly ascribe to the uncertainty of the regulation mechanism of lycopene synthesis,and further hindering the improvement of lycopene production strain by genetic engineering manipulation.Therefore,it is imperative to study the regulation mechanism of lycopene synthesis in Blakeslea trispora,and it can provide important theoretical support for genetic modification and regulation of fermentation conditions of the strain.In the present study,we gained high lycopene production strain by ARTP mutation and established the high-throughput screening methods.Besides,we investigated the molecular mechanisms of the high lycopene production strain through transcriptome analysis,and improved the lycopene production performance of Blakeslea trispora by oxidative stress,which provides an important foundation for further genetic transformation of this strain.We gained the following innovative results:1)B.trispora?-?serves as the initial strains,after mutation by ARTP,we constructed a 24-well plate high throughput screening strategy,and the highly lycopene productive strain WY239 was screened from 500 alive mutated strains.After 120h shake bottle fermentation culture of the WY239 strain,the concentration of lycopene was 892.46 mg/L,which was improve by 45%compared to the initial strain.We found this mutated strain WY239 possessed high hereditary stability according to the results of five generations of culture fermentation.2)The results of transcriptome analysis on the initial and mutant strains showed that total 2257 differential genes were gained,and 1445 genes were up-regulated expression,812 genes were down-regulated expression.Most of the genes whose expression levels have changed significantly are related to metabolic pathways such as glycolysis,fatty acid metabolism,amino acid metabolism,nucleotide metabolism,carotenoid synthesis,and biosynthesis of sterols.Sugar metabolism,amino acid metabolism,and fatty acid synthesis were significantly downregulated,and metabolic pathways favoring the increase of acetyl-CoA,a precursor of lycopene,appeared to be significantly upregulated,such as fatty acids,valine,leucine,and isoleucine.In the pathway of carotenoid synthesis,the main metabolic pathway for the ynthesis of lycopene was significantly enhanced,and metabolic branch-related genes appeared significantly down-regulated,such as sterols,squalene and other synthetic pathways.3)The effect of oxidative stress on the yield of lycopene was studied.Both the original strain and utagenesis was subjected to hydrogen peroxide stress.The results showed that,when 40?L of 30%H₂O₂was added to 25 mL fermentation broth at 60 h of fermentation,the highest yield was 980.03±0.03 mg/L,with an incense of 63.3%compared to the control.Compared with the control,the supplementation of H₂O₂improves the enzyme activity of CAT and SOD.The highest pigment production of WY239 strain was 120.69 mg/L after H₂O₂ treatment,and the production is 25.56%before the H₂O₂ addition and was83.91%of the initial starting strain.4)The segment produced by fungi is important and natural,and we gained a fungi strain that could produce soluble red pigment.The IT1-IT4 sequence of this fungal ribosomal RNA gene is 99%similar to Talaromyces purpurogenum and clustered on the phylogenetic tree constructed by eighbor-Joining.The strain produced the highest red pigment production at 120 h,and the pigment was an extracellular secondary metabolite.Spectroscopy and high performance liquid chromatography analysis showed that the pigment has a maximum absorption peak at 495 nm and consists of more than four single pigment components,which is different from the current common lycopene and metharubin,and the igment has strong stability to temperature,light and pH,but is easily degraded by oxidants and reducing agents.The pigment is an extracellular water-soluble pigment.Compared with the intracellular fat-soluble pigment of Trichothecium sp,it has obvious advantages in pigment extraction and has great development value.