Mechanism Of Rhodotorula Mucilaginosa On Promoting Laccase Production Of Pleurotus Eryngii Var. Ferulae In Co-culture

Laccase（EC1.10.3.2） is a copper-containing polyphenol oxidase,which has been widely paid attention to and applied because of its unique enzymatic properties.The applications of laccase include lignin degradation,wastewater treatment,dye decolorization,biosensor and so on.Compared with other methods of increasing laccase production,microbial co-culture has been developed as an efficient,convenient and sustainable fermentation strategy to obtain large amounts of laccase.In the previous study,our group found that co-culture of Rhodotorula mucilaginosa（non-laccase-producing strain）and Pleurotus eryngii var.ferulae（laccase-producing strain）could significantly increase laccase production,but the molecular and transcriptional regulatory mechanisms are still unclear.In this study,the co-culture strains were taken as the research object to explore the regulatory mechanism of high yield laccase in fungal co-culture.By comparing transcriptomic global analysis of differential genes,functional clustering,laccase isoenzymes and putative transcription factors,the study objects were determined.To establish an efficient genetic transformation system of P.eryngii var.ferulae,and explore multifunctional endogenous transcription factors,so as to provide reliable molecular biological means for the study of P.eryngii var.ferulae mechanism.The enzyme and biological function of ssPOXA3a/b were analyzed.Mining and verification of transcription factors in response to co-culture laccase production;To analyze the transcriptional regulation mechanism of putative transcription factors and seek their universality in other co-culture systems.The main research results are as follows:（1）Global analysis of P.eryngii var.ferulae differential transcripts in co-culture.RNA-seq analysis showed that genes concerning xenobiotic biodegradation and expenditure of energy were upregulated.However,genes related to oxidative stress were downregulated.In addition,the transcription levels of laccase isoenzymes were not consistent in the co-culture system:3 laccase genes（lacc1,lacc2,lacc12）were upregulated,and 3 laccase genes（lacc4,lacc6,lacc9）were downregulated.The enhancement in laccase activity can be due to upregulation of a laccase heterodimer encoded by the genes poxa3 and sspoxa3a（or sspoxa3b）,whose expression levels were increased by 459%and 769%（or 585%for ssPOXA3b）compared with those of a control,respectively.β-Carotene produced by R.mucilaginosa upregulated the transcription of poxa3 only.Combining these results with an analysis of cis-acting responsive elements indicated that four transcription factors（TFs）had potential regulatory effects on the transcription of laccase genes.It was supposed that TFa regulated lacc transcription by binding with methyl jasmonate and heat shock response elements.The expression of TFb,TFc,and TFd was regulated byβ-carotene.However,β-carotene had no effect on TFa expression.（2）The homologous resistance marker carboxin（cbx）was used to establish a polyethylene glycol-mediated transformation（PMT）system in P.eryngii var.ferulae.Optimization of the transformation process greatly improved the number of positive transformants.In particular,we optimized:（i）protoplast preparation and regeneration;（ii）screening methods;and（iii）transformation-promoting factors.The optimized transformation efficiency reached 72.7CFU/μg,which is higher than the average level of Pleurotus sp.（10–40 CFU/μg）.Moreover,three endogenous promoters(P_pfgpd1,P_pfgpd2,and P_pfsar1)were screened and evaluated for different transcription initiation characteristics.A controllable overexpression system was established using these three promoters that satisfied various heterologous gene expression requirements,such as strong or weak,varied,or stable expression levels.（3）The present study revealed that neither ssPOXA3a nor ssPOXA3b per se has a catalytic ability,whereas their combination with POXA3（and especially ssPOXA3b）enhances the activity,thermostability,and p H stability of POXA3.In P.eryngii var.ferulae,there was no regulatory relationship between ssPOXA3a/b and POXA3 at the transcriptional level.However,ssPOXA3a/b overexpression had a negative feedback effect on lacc6 transcripts.By contrast,poxa3 transcripts had no effect on any other laccase isoenzyme.Overexpression of sspoxa3a/b resulted in small fungal pellets,thin mycelial walls,and facilitated laccase secretion.However,poxa3 overexpression had no influence on pellet morphology.（4）Mining and validation of laccase transcription factors in response to co-culture.We found that TFa,TFb and TFc could not change the expression level of laccase in P.eryngii var.ferulae by overexpressing the four putative transcription factors predicted by transcriptome respectively.Overexpression of TFd inhibited the expression of laccase.Through the point-to-point test of yeast single hybrid,it was found that TFd could not directly bind to the poxa3promoter,which was an indirect regulation.In order to explore transcription factors that directly bind to the laccase promoter and respond to the co-culture process,a yeast single-hybrid screening system using poxa3 promoter as bait and P.eryngii var.ferulae c DNA as screening library was established.Combined with transcriptomics and bioinformatics analysis,Ptf6 was confirmed as an important GATA transcription factor.（5）To analyze the response mechanism of Ptf6 in co-culture and the regulation mechanism of laccase gene.We found an essential GATA transcription factor Ptf6 by yeast one-hybrid assay using poxa3 promoter as bait and P.eryngii var.ferulae c DNA as the gene library.Ptf6 responds to the fungal co-culture process in two forms,which are the enhancement of its own transcripts and the increase of alternatively spliced isoform Ptf6-αproportion.Ptf6-αwith regulatory activity can bind to four kinds of GATA-motifs on lacc promoter with different affinities.Ptf6-αcan positively activate multiple laccase subunit genes and some other lignin degrading enzymes（manganese peroxidase and versatile peroxidase）by overexpression and RNA interference of ptf6.The Ptf6-regulatory mechanism was also validated in the co-culture process of three other white-rot fungi and R.mucilaginosa.