Efficient Corn Grits Utilization For Hypocrellin Production By Shiraia Bambusicola Under Solid State Fermentation

Shiraia bambusicola is a Chinese medicinal fungus,it is used for the treatment of rheumatoid arthritis,sciatica,lumbar muscle strain,injury and cold stomach pain,and other diseases in the folk.As the main active component of Shiraia bambusicola,hypocrellin is a good photosensitizer which can react with oxygen to generated reactive oxygen species?ROS?under illumine condition.These ROS can damage biomacromolecule and cause cell death.Hypocrellin showed good pharmacological activities in antibacterial,antivirus and antitumor,and has great potential application value in the fields of food,feed,environmental protection and medical treatment.So far,hypocrellin were mainly obtained from natural extraction,while the production was too low to meet its current medical demand and scientific research use.Shiraia bambusicola in nature has many defects such as regional limitation,short growing season and low yield,so it is the main trend to obtain hypocrellin by biological fermentation.Compared with liquid state fermentation?LSF?,solid state fermentation?SSF?has advantages of low energy consumption,low production cost,no waste water discharge,high volume productivity and no contamination of miscellaneous bacteria,and some application can be directly used without extraction and purification,so the production of hypocrellin under SSF has become the focus of this study.In our previous work,we isolated a high-yield hypocrellin producing strain,Shiraia sp.SUPER-H168,and fermented substrate and cultural conditions by Shiraia sp.SUPER-H168 under SSF were studied.In this paper,following studies were carried out.Influences of light on growth,reproduction and hypocrellin production by Shiraia bambusicola were studied.Growth of Shiraia bambusicola was analyzed under SSF.Optimizations of hypocrellin production by Shiraia bambusicola under SSF?including inoculum type,agitation,and amylase addition?were studied.The purification,characterization and gene analysis of a new?-glucosidase from Shiraia bambusicola under SSF were studied.Amylases in Shiraia bambusicola were predicted and analyzed.Enhanced hypocrellin production by overexpression of amylase gene and hemoglobin gene in Shiraia bambusicola were studied.The main results were shown below.?1?Influences of light on Shiraia bambusicola.Pigment production reached the highest level 13.73 mg per dish under dark condition.Under blue light condition,pigment production decreased to the lowest level 2.83 mg per dish.All incubations under different light conditions had stimulating effects on aerial hyphae and suppressing effects on hypocrellin biosynthesis compared with dark condition.Under blue light condition,the colony with profuse growth of aerial mycelium was formed.Light promoted sexual development and inhibited asexual reproduction,especially blue light strongly inhibited asexual development.Under dark condition,there was only asexual development,and asexual spores reached the highest level.Sexual spore was short rod-like,and asexual spore was spherical.Four types of hyphae,namely surface hyphae,aerial hyphae,biofilm hyphae and penetrative hyphae,were observed by light microscope and scanning electron microscope?SEM?.It was found that hypocrellin was only produced by biofilm hyphae,and thickness of biofilm hyphae was about 300?m under dark condition.?2?Optimization of hypocrellin production by Shiraia bambusicola under SSF.When inoculated with fungal seed culture,the period of SSF was 15 d.The first agitation was conducted on the third day,intermittent agitation was conducted every 36 h.bacterial mesophilic?-amylase(2.85 U·gds^-1)was added at the start time and glucoamylase(29.78U·gds^-1)was added on the third day.After optimization,pigment production reached 60.74mg·gds^-1 within 13 d,and the residual content of starch in solid substrate was 27.42%.?3?A new?-glucosidase AMY33 from Shiraia bambusicola under SSF was purified by alcohol precipitation,anion-exchange and gel filtration chromatography.Molecular mass of it was 108.2 kDa.The optimum pH and temperature of the purified?-glucosidase were 4.5 and60°C,respectively,using p-nitrophenyl-?-glucopyranoside??-pNPG?as a substrate.The K_m,V_max,and k_cat/K_m of the?-glucosidase were 0.52 mmol·L^-1,3.76 U·mg^-1,and 1.3×10⁴L·s^-1·mol^-1,respectively.K_m with maltose was 0.62 mmol·L^-1,and K_m with starch was 2.38mg·mL^-1.Transglycosylation activities were observed with maltose and sucrose as substrates,while no transglycosylation with trehalose.AMY33 had an activity of?-1,2-glycoside of sucrose and a weak activity of?-1,1-glycoside of trehalose.DNA and its corresponding full-length cDNA were cloned and analyzed.The?-glucosidase coding region consisted of a2997-bp open reading frame encoding a 998-amino acid protein with a 22-amino acid signal peptide;one 48-bp intron was located.Predicted isoelectric point of AMY33 was 5.08.In total,twenty-four amylase genes in Shiraia sp.SUPER-H168 were predicted and analyzed,including ten genes of?-glucosidase,three genes of glucoamylase,eight genes of?-amylase and three genes of starch debranching enzyme.?4?Relative expression levels of twenty-four amylase genes in Shiraia sp.SUPER-H168were investigated by real-time quantitative PCR when various carbohydrates,including glucose,sucrose,maltose,amylose,amylopectin and corn flour,were used as carbon source.Relative expression levels of amy33,amy365-1 and amy130 reached the highest level when maltose,amylose and amylopectin were used as carbon source,respectively.Overexpression of amylase was studied,and five overexpression strains were constructed.Under LSF,biomasses and pigment productions were both gradually increased and residual sugar was decreased in five overexpression strains than those of wild type strain.Under SSF,pigment production of amy365-1 and amy130 coexpression strain reached 71.85 mg·gds^-1,which was2.83 fold than that of wild type strain.When amy365-1 or amy130 was overexpressed,expression of amy33 was also increased.After SSF,the residual content of starch in solid substrate of amy365-1 and amy130 coexpression strain was 19.56%.?5?Overexpression of vgb was studied,and two overexpression strains were constructed.Under LSF,when amy365-1 and vgb were coexpressed,pigment production reached the highest level 3681 mg?L^-1 at 96 h,which was 5.24 fold than that(703 mg?L^-1)of wild type strain.Under SSF,pigment production of amy365-1 and vgb coexpression strain reached the highest level in this study 75.89 mg·gds^-1.When vgb was overexpressed,relative expression level of amy365-1 was increased,and amylase activity under SSF was increased.Furthermore,period of SSF was reduced from 13 d to 11 d.After SSF,the residual content of starch in solid substrate of amy365-1 and vgb coexpression strain was 16.87%.