Genetic Transformation And Cultural Condition Optimization For A Cellulase Hyper-producing Mutant EMM

Talaromyces stipitatus EMM is a cellulase hyper-producing mutant that originated from T.stipitatus OPC4.It shows a good prospect of industrialization in the production of cellulase production for its high protein production and stability of enzyme activity.However,its low ?-xylosidase seriously affects the synthesis ability of lignocellulosic.In this paper,we modify it by means of genetic engineering.The key of the CaCl2-PEG transformation is how to prepare protoplasts and improve regeneration of protoplasts.Conditions for Talaromyces stipitatus EMM protoplasts preparation and regeneration were optimized by the preparation of materials,the age of fungus,the osmotic pressure stabilizer,the type of enzyme,the concentration of the enzyme,the time of enzymatic hydrolysis and regeneration.The optimal conditions were obtained as follows:the mycelia was cultured for 48 h,then treated by enzyme mixture containing 50 mg/m L of lysing enzymes?from Trichoderma harzianum?in the osmotic pressure stabilizer of 1.2MMgSO₄ at 30? for 3h;obtained protoplasts were regenerated in liquid medium containing 1M sorbital over night and then in solid medium in this conditions,the protoplast yields was 8.73±0.8×108 /m L and regeneration rate was 17.7±1.9%.As a cellulase production strain,?-xylosidase activity of Trichoderma reesei is high.Clone the genes of ?-xylosidase from T.reesei Rut-C30 and Talaromyces stipitatus EMM and replace the original ?-xylosidase gene of Talaromyces stipitatus EMM with the ?-xylosidase gene of T.reesei Rut-C30 added with CBH promoter and terminator to improve ?-xylosidase activity of EMM.After tansfered 3 times in PDB medium containing hygromycin B,we got 37 transformants on the plates containing hygromycin B.PCR analyses of the transformants revealed that there were 10 positive transformants containing hph gene and there were 2 HR transformants and 8 NHEJ transformants.The fermentation results revealed FPA activity of the transformants was improved by 60% but ?-xylosidase activity was not improved compared with the original EMM.Because of the selection intensity and stability of the hygromycin B marker is not high,it is needed to pass through the liquid culture medium containing B to screen the positive transformation,and the efficiency is not high.A uracil auxotoph?strain EMU6?was isolated by resistance to 5-fluoroorotic acid after UV mutagenesis from EMM.We cloned a wild-type pyrF gene encoding orotate phosphoribosyl tansferase?OPRTase,EC 2.4.2.10?from OPC4.Based on the sequence analysis,mutated pyrF gene of EMU6 has one nucleotide deletion at 1160 bp.To confirm the pyrF gene could be used as a selection marker,we transformed the strain EMU6 with the pyrF gene carried by plasmid pGEF2,using the protopiast polyethyleneglucol?PEG?method.PCR analyses of the transformants revealed that the pyrF gene was integrated into the genome of EMU6 and made it growing on the uracil-free medium.From these results,we can come to the conclusion that we have developed the transformation system with the pyrF gene as a selection marker successfully.To construct the recycling pyrF mark gene,we addad a direct repeat sequences of 490 bp to the side of the pyrF.After the transformants were slected,the recycling pyrF mark gene could be deleted by homologous recombination by resistance to 5-fluoroorotic.The development of he recycling pyrF mark gene makes using Talaromyces stipitatus as the cell factories to express heterologous protein possible in the future.