Effects Of Ultrasound And Lights On Hypocrellin Biosynthesis In Shiraia Bambusicola

Shiraia bambusicola is a kind of pathogenic fungi of Brachystachyum Keng and other species of bamboos whose fruiting bodies have been used as traditional Chinese medicine to treat cough and phlegm,promote blood circulation and relieve pain.Hypocrellins,the major active ingredient of S.bambusicola,are a naturally occurring fungal perylenequinones.Among the class of hypocrellins,hypocrellin A(HA)as a new type of nonporphyrin photosensitizer,has drawn much attention because of its high photodynamic therapy(PDT)effects on antibacterial,antitumor and anticancer.Due to the limitation of the wild sources of the fruiting body of S.bambusicola,solid and liquid cultures of mycelium are becoming a promising alternative for HA production.However,the lower HA production in mycelial culture has become the bottleneck of the large-scale production.In this paper,two kinds of abiotic elicitation,including ultrasound and lights,were studied on the growth and HA biosynthesis of S.bambusicola.Futhermore,we optimized the cultural conditions,revealed the mechanism of ultrasound and lights on the fungus,and provided a basis and reference for the development of the photosensitive drug.First,we studied the effect of ultrasound on the growth and HA biosynthesis of S.bambusicola.A low intensity ultrasound(US,0.28 W/cm2 at 40 k Hz)was conducted thrice of repeated US exposure(5-min)with an interval of 12 h to stimulate HA production of S.bambusicola after 72 h of the initial submerged cultures.US not only increased the content of HA in mycelia,but also stimulated the release of HA into the medium with the highest total production of HA(247.67 mg/ L),which is 3-fold of control.US resulted in the decreased pellet diameter,fluffier pellets,the enhanced membrane permeability,the alternation of fatty acid compounds.The reactive oxygen species(ROS)generation and up-regulated expression of the genes about HA biosynthesis and release(including PKS,Mono,FAD,O-mef and MFS)have been observed.These results indicated that the enhancement of HA production was mainly due to both the stimulated cellular biosynthesis and the enhanced fungal exudation of HA.Meanwhile,we investigated on the effect of light/dark shift on S.bambusicola.Our results showed a light/dark shift(24: 24 h)at 200 lux increased HA content in mycelia and total production of HA(181.67 mg/L),about 73% increase over the dark control.Moreover,light/dark shifting induced the formation of smaller and more compact fungal pellets,beneficial to large scale of HA production.Futhermore,RT-q PCR analysis showed that the light/dark shift up-regulated the expression levels of two ROS related genes including superoxide-generating NADPH oxidase(Nox)and cytochrome c peroxidase(CCP)and induced the generation of ROS.With the treatment of vitamin C,we found that ROS was involved in the up-regulated expression of key biosynthetical genes for hypocrellins and improved HA production induced by the light/dark shift.It indicated preliminarily that there was a close relationship between ROS and HA biosynthesis under light/dark shift.Moreover,to further explore the effect of light on HA biosynthesis,transcriptomes of S.bambusicola under darkness and red light cultivation were sequenced.Finally,a total of 24,804 unigenes were assembled.We used the five databases of NR,Swiss-Port,KOG,GO and KEGG to annotate the obtained unigenes,and calculated the expression of unigenes in the darkness group(control)and red light treatment group by RPKM method.In the red light group,a total of 310 differentially expressed unigenes(DEGs)were identified,among which 155 unigenes were up-regulated while 155 unigenes were down-regulated.GO classification of DEGs revealed that ‘transmembrane transport'(GO:0055085)and ‘integral component of membrane'(GO:0016021)were all significantly affected by red light,suggested that red light could influence the cell membrane and transmembrane transport.In addition,the enhancement of HA biosynthesis and release under red light was mainly related to the up-regulated expression levels of HA synthesis and transport genes.This study explored the impacts of ultrasound and lights on growth and HA biosynthesis of S.bambusicola,revealed the mechanism of HA synthesis through the changes of morphology,redox and genes expression levels.And provided a foundation for theoretical study and industial application for the subsequent large-scale fermentation of Shiraia cultures.