Cloning And Expressing The Fae Gene Of Penicillium Chrysogenum In Pichia Pastoris

Feruloyl esterases (FAEs) act synergistically with xylanases to hydrolyzeester-linked ferulic (FA) and diferulic (diFA) acid from cell wall material and thereforeplay a major role in the degradation of plant biomass. Microbial sources producingferuloyl esterase are very wide. The feruloyl esterase activities of wild strains are verylow, thus limiting the industrialization and applications of FAEs in all fields.Penicillium chrysogenum HA4089was used as research materials, and over-productionstrains were constructed to improve the yield of FAE by means of molecular biolog andgene engineering.Using PCR technique， the feruloyl esterase A (faeA) geonomic gene wassuccessfully cloned from Penicillium chrysogenum HA4089. Sequence analysissuggested that the length of faeA from strain HA4089was933bp, containing two exonsand one introns. Using overlapping fusion PCR protocol, the faeA cDNA gene wascloned. The expression vector pPIC9K-PcfaeA was constructed by ligation of faeAdigested by EcoRI and AvrII with pPIC9k digested with the same enzymes. Sequencinganalysis showed that the length of faeA cDNA gene sequence length was783bp,encoding260amino acids. The identity of faeA cDNA from strain HA4089with faeAgene reported in NCBI Genbank was97.69%, indicated that the clonded cDNA geneencoded feruloyl esterase A. Comparision of cloned faeA cDNA gene with faeA geneof GenBank indicatd that6differences were found,12K (lysine),30th R (arginine),49Y (tyr),170L (leucine),211V (valine),245E (glutamic acid) respectively.Using electrotransformation method, P. pastoris GS115strains were transformedwith linearized expression vector pPIC9K-PcfaeA by SacI. Through observation oftransformants on plate containing ethyl ferulate,5strains with clear transparent circlewere screened and then extracted to prepare chromosomal DNA respectively, as atemplate of PCR amplification. After PCR identification,4transformants with highFAE activity were determined, and the highest enzyme activity of transformant3#reached20.67U/mL. SDS-PAGE analysis showed that there was a clear protein band in32kDa, suggesting the achievement of expression of faeA in P. pastoris GS115. Crudeenzyme properties of recombinant FAEA were studied. The optimal temperature andpH of recombinant FAEA was50℃and4.5repectively. Afer incubation in50℃for 20min, the enzymatic activity remains more than90%. The recombinant enzyme wasstable in pH4.0-6.0, and remains more than90%of the enzyme activity.The faeC geonomic gene was successfully cloned from P. chrysogenum HA4089by PCR amplification. Sequence analysis revealed that the length of faeC from strainHA4089was750bp, containing no intron. The gene could be directly used inheterologous expression. Sequencing analysis showed that the length of faeC gene is762bp, encoding249amino acids. Compared with FAEC in GenBank database, therewas only one amino acid sequence difference in24th of histidine, which was replacedby the Y (Tyr). The difference in amino acids does not affect the subsequent expression.The faeC double-digested by EcoRI and AvrII was ligated with pPIC9K digested bysame restricted endonuclease, resulting the expression vector pPIC9K-PcfaeC. Thevectpr pPIC9K-PcfaeC was linearizated by SacI and electrotransformed into P.pastorisGS115strain. Through observation of transformants on plate containing ethyl ferulate,6transformants with clear transparent circle were screened and then extracted to preparechromosomal DNA respectively, as a template of PCR amplification. After PCRidentification,5transformants with high FAE activity were determined, and the highestenzyme activity of transformant4#reached20.14U/mL. SDS-PAGE analysis showedthat there is a band in32kDa, suggesting the achievement of expression of faeC in P.pastoris. Crude enzyme properties of recombinant FAEC were investigated. Theoptimal temperature and pH of recombinant FAEC was45℃and6.0repectively. Aferincubation in55℃for1h, the enzymatic activity remains more than45%. Therecombinant enzyme was stable in pH5.0-7.0, and remains more than85%of theenzyme activity.