| With the concerns of increasing energy demands,consuming away of fossil fuels, and environment pollution,the search for sustainable supplies of energy and renewable materials are becoming more and more important in the coming years. About 35%-50%dry weight of plant are cellulose,which are the most abundant renewable resource in the world.Available cellulosic feedstock from agriculture and other sources can be transformed to the fermentable syrup composed of polysaccharides and monosaccharides which can be fermented to the clean liquid biofuels and other value-added products alternative to fossil fuels,becoming the research focus of many countries.Generally,the biorefinery from lignocellulose to bioethanol is envisioned to comprise four major sections:pretreatment of feedstock, production of cellulase,enzymatic hydrolysis,and sugar fermentation to ethanol by Saccharomyces cerevisiae,and recover of ethanol.As a key problem for production on commercial scale,conversion of cellulosic biomass requires using large amounts of cellulases,which makes the process costly.Penicillium decumbens JU-A10,a catabolic-repression resistant mutant from the wild strain P.decumbens 114-2 suffered several rounds of mutagenesis,could produce abundant cellulase and hemicellulase,which were enzyme complexes hydrolyzing lignocelluloses.But the productions and activities of cellulase were not sufficient for industrial demands,and the strain needed to be further improved.As the genetic background of P.decumbens is not clear yet and there is no suitable genetic tool,we still have difficulties in designing rational methods for its cellulase improvement.Genome shuffling has been demonstrated as an effective method for the rapid improvement of cellular phenotypes in recent years.In this paper,genome shuffling was used to improve cellulase activities of P.decumbens JU-A10,and the main results were as follows:1.Establishment of an effective two-layer cellulose screening plate The ingredients in the two-layer cellulose screening plate were optimized.To detect halos of cellulose hydrolysis and improve efficiency of each screening plate, the upper layer was supplemented with sodium deoxycholate,reaching a final concentration of 0.2%to restrict colonies extension.The ball-milled microcrystalline cellulose and glucose were used as selection pressure to choose the mutants out,and the volume of each upper medium is 7 ml.The cellulase activity was mainly positive related to the size of cellulose hydrolysis halo in the plate.JU-A10 could only form cellulose hydrolysis halo in the two-layer plate containing 2%ball-milled microcrystalline cellulose and less than 1%glucose.The mutants with better cellulase production or resistance to catabolite repression could be selected when the concentration of ball-milled microcrystalline cellulose and glucose gradually increased during screening.2.The protocols of protoplast preparation,protoplasts regeneration and protoplasts fusion of double parents inactivatedProtoplasts were prepared from the mycelia harvested after 24-h culture in mycelia culture medium.Protoplasts were isolated using an enzyme combinations composed of 4 mg ml-1 Snailase,4 mg ml-1 cellulase,and 2 mg ml-1 Lywallzyme in pH 6.5 citric acid buffer,supplemented with 0.6 M NaCl as an osmotic stabilizer.A maximum number of 106/ml protoplasts were obtained when treated at 35℃for 1 h. The final population of protoplasts in total cells was 95%,and a high regeneration ratio of 90%was occurred on the regeneration medium.The parent protoplasts purified could lose their ability to regenerate after irradiation of UV for 30 minutes or incubation at 50℃for 50 minutes.The protoplasts were gathered together after the previous two kinds of inactivation,and fused in the system composed of 40%PEG;35℃,10 mM Ca2+.Large numbers of multi-cellular aggregates were observed in the fusion system described above under phase contrast microscope.Although the parent cells might be nonviable after inactivation treatment, they could form viable recombinants when fused with the protoplasts treated by another method.3.Improvement of the ability to produce cellulase after two rounds of genome shufflingFour mutants were selected after primary two-layer screening and secondary fermentation:UE-5 and UE-6 were obtained after UV and EMS mutagenesis;NII-5 and NII-8 were chosen after 30 key N+ ion implantation.The initial parents' pool was composed of four developed mutants with different advantages respectively.Two successive rounds of protoplast fusion were carried out,and after each round,the concentration of ball-milled cellulose or glucose in the plates used for selection was increased.The four mutants were used as parents of the first round for genome shuffling,then the colonies quickly regenerating and forming cellulose hydrolysis halos on screening plate SM2(2%ball-milled cellulose,2%glucose) were chosen for further fermentation assay.The 6 strains with further improvements were selected as the parents for the second round of shuffling.After screening on plates SM3(5% ball-milled cellulose,2%glucose) and secondary fermentation assay,three fusants: GS2-15,GS2-21,GS2-22 with significant improvement were acquired.They could produce cellulase as much as 190%of the parent strain JU-A10.4.The reasons why the fusants GS2-15,GS2-21,GS2-22 produce more cellulaseFirstly,some morphological characteristics of the fusants changed during the process of genome shuffling.The width of mycelia and the colony diameter of the fusants were smaller than that of JU-A10,while the volume of spores became larger. The mycelia of the fusants were more easily fragmented in the late fermentation stage (90 h) comparing with JU-A10.There may be somewhat relationship between the improved cellulase production and morphological changes,and it is probably that the easy fragmentation of mycelia is helpful to the release of cellulase,and the work is underway in our lab now.Secondly,the randomly amplified polymorphic DNA(RAPD) system in P. decumbens was established after detailed optimization of reaction parameters.Most of RAPD bands from the three fusants were the same with JU-A10,but some of them were distinct.The genetic similarity varied from 0.842 to 0.905 indicating variability among the three fusants and JU-A10.The dendrogram showed three distinct clades by the genetic similarity 0.87,the first containing JU-A10 and GS2-22,the second and the third containing GS2-15 and GS2-21,respectively.The present work shows the usefulness of RAPD molecular markers for genetic characterization to establish phylogenetic relations,and RAPD would therefore be a suitable tool to discriminate the diversity of the fusants and parent strain.Thirdly,the extracellular and intracellular cellulase,protein concentration,and biomass when they were cultured with the corncob residue as carbon sources were studied.The fusants had robust growth,higher protein concentration and cellulase activities compared with JU-A10.Interestingly,the fusants synthesized sharply intracellular cellulase in very early fermentation stage.The improvements of the fusants were possibly due to their enhanced growth rates,earlier cellulase synthesis and higher secretion of extracellular proteins.Fourthly,the fusants had better performances on the liquid fermentation culture containing 2%glucose as sole carbon source.They consumed the glucose more quickly,and the biomass and cellulase activities were also higher,comparing to JU-A10.The SDS-PAGE and CMCase activity staining showed that the fusants had their own new special protein bands,although in despite of most of the protein profiles were nearly the same as the JU-A10.Some protein bands showing CMCase activity expressed stronger.These differential protein patterns suggested that extracellular protein of the fusants had changed.5.Cellulases production of the fusants using lignocellulosics as carbon source and in 5-L fermentorThe cellulase production of the fusants and JU-A10 were investigated with corn stover,wheat straw,and bagasse as carbon sources.The fusants could synthesize more enzymes earlier,and both cellulases and xylanases were observably higher than that of JU-A10.The pH value is an important factor effecting cellulase production and is very important to keep the enzyme stability during the fermentation.In 5-L fermentor assay, the fusants produced cellulase more quickly,and the cellulase activities were further improved compared to that in 500-ml flask.However,the pH value in late fermentation stage rose up nearly to 7,resulting in losing much of enzymatic activities, which is not beneficial to industrial production.The performances of several kinds of buffering agent were tested in 500-ml flask by the fusant GS2-15,and the performance with addition of 0.08%CaCO3 was the best one.The pH value and enzymatic activities were very stable in late fermentation stage in 5-L fermentor by GS2-15,and the highest FPase activity reached 15.04 FPU ml-1,the concentration of extracellular reached 5.81 mg ml-1,the CMCase activity reached 133.12 IU ml-1,β-glucosidase reached 5.18 IU ml-1,and xylanase activity reached 411.34 IU ml-1.6.Cloning of aβ-glueosidase gene from P.decumbens Peni-1,and purification and characterization of theβ- glucosidaseAβ-glucosidase gene(bgl) in Peni-1 was cloned by PCR and RT-PCR.DNA sequencing results showed that bgl had an open reading frame of 2586 bp with 5 introns and encoded a polypeptide of 862 amino acids.There were three amino acids different from the fusant GS2-15:Lys→Arg(the second site),Gly→Ser(the 482 site), Val→Ile(the 489 site).The protein ofβ-glucosidase was purified by gel filtration chromatography,and enzymatic characteristics were studied.The thermal stability of the enzyme purified was determined,and about 80%of enzyme activity remained after incubation at 60℃for 12 h.The optimum reaction temperature is 70℃.The pH optima with salicin as substrates were determined as 5.0.The Km ofβ-glucosidase toward salicin was 2.6 mmol L-1,and was 0.2 mmol L-1 toward pNPG.There were some differences between the extracellular protein profiles of Peni-1 and GS2-15.The regulation mechanism ofβ-glucosidase synthesis in Peni-1 might be different,which make it producing moreβ-glucosidase.7.The determination of best ratio of FPA andβ-glucosidaseEnzymatic broth from Peni-1 and GS2-15 were mixed in order to make the final ratio of FPA andβ-glucosidase at 1:1,1:2,1:3,1:4,1:5 and 1:6,and enzymatic activities before or after mixture were tested.The best ratio of FPA andβ-glucosidase was found to be 1:3 in P.decumbens cellulase system.Consequently,the cellulase system of P.decumbens IU-A10 or GS2-15 was still not optimal enough.This offered theoretical evidence for further strain improvement.
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