Construction And Application Of A Flow Cytometry Based Plating-free System For Fungal Transformant Screening Technology

Recent progress in genomic editing and synthetic biology tools has successfully resolved the problem of low gene targeting frequency and led to improvements in strains for the production of enzymes and desirable chemicals in filamentous fungi.The improvement of strains regarding their industrially important traits is mainly performed through manipulation by genetic engineering.However,such manipulation is typically complicated and laborious.It includes plating of transformed protoplasts,picking the candidate transformants from selection plates,culturing the colonies for sporulation,harvesting the conidia of the transformants for PCR verification,and performing flask cultures for phenotypic analysis.These labor-intensive procedures delay the potential industrial exploitation of filamentous fungi.There is thus a need to develop a plating-free system to overcome these limitations.Myceliophthora thermophila is a cellulolytic thermophilic filamentous fungus that has exceptional potential to produce enzymes,chemicals,and biofuels directly from renewable biomass.This study developed a flow cytometry-based plating-free system to directly screen and isolate the transformed protoplasts in industrial fungus Myceliophthora thermophila.This system combines genetic engineering via the foot-and-mouth disease virus(FMDV)2A peptide and the CRISPR–Cas9 system,strain screening by flow cytometry,and direct sorting of colonies for deep-well-plate incubation and phenotypic analysis,while avoiding culturing transformed protoplasts in plates,colony picking,conidiation and cultivation.This system was able to efficiently and conveniently obtain the desired mutant strains without plating the transformed protoplasts through conventional transformation and screening,which usually involves laborious and time-consuming procedures.As a proof of concept,this study chose glucoamylase as the target protein for production via strain engineering to test this system.This study applied F2 A peptide to co-express the target gene Tegla A and the reporter gene gfp.To improve glucoamylase production,five genes involved in protein expression and secretion,including Mtres-1,Mtalp-1,Mtspr-14,Mtcre-1 and Mtexo-1 genes were selected for gene editing by CRISPR–Cas9 system.Notably,the protein secretion level and enzyme activities in Mt YM6 were 17.3-and 25.1-fold higher than in the wild-type strain Mt WT.The expression of the key amylase regulator amy R and two major amylase genes(Mycth＿72393and Mycth＿2300079)were monitored by q RT-PCR in the Mt YM6 and Mt WT strains under4 h of induction with 2% starch.The q RT-PCR experiments showed that the expression levels of amy R,Mycth＿72393,and Mycth＿2300079 were significantly promoted in the Mt YM6 mutant.Markedly,the Mycth＿72393 and Mycth＿230007 genes showed 51-fold and 37-fold higher expression in the Mt YM6 mutant than in Mt WT.Collectively,these results demonstrate that the hyperproducing M.thermophila strains can rapidly,conveniently,and efficiently generate the target protein using the FCPF system.In summary,this study developed a versatile plating-free manipulation system based on flow cytometry for directly sorting the transformed protoplasts generated by genetic engineering.This study suggest that the flow cytometry-based plating-free system can be a convenient and efficient tool for strain engineering in fungal biotechnology for industrial applications.