Molecular Cloning Of Gene Encoding A Laccase From Lenzites Gibbosa And Heterologous Expression Of This Gene In Pichia Pastoris

Laccases (EC1.10.3.2) belong to multicopper phenol oxidases that showing the wide distribution in plants, bacteria, insects, and fungi(particularly in white rot fungi). Laccases may be essential for lignin decomposition. Laccases catalyze the oxidation of a variety of chemical compounds (such as phenolic and their derived compounds, aromatic carboxylic acid and so on). For biotechnological and industrial purposes, laccases providing larger-scale applications such as paper manufacture, environmental conservation, textile industry, conversation of toxic compounds, and biobleaching.The white-rot fungus Lenzites gibbosa (Pers.) Hemmi belonging to the family of Polyporaceae, isolated from rotted wood in Changbai Mountain. Recently, The cultural characteristics, extracellular oxidative systems from L. gibbosa, and its decolorization ability has been studied. A novel MnP-encoding genes was described, and the heterologous expression system in Pichia pastoris was established successfully. This study present cloning and characterization of two laccase encoding genes of L. gibbosa, named Lg-lacl and Lg-lac2. The expression of two genes were monitored by real-time PCR analysis. Further more, the heterologous expression system in Pichia pastoris of two L. gibbosa laccases were also established successfully. The research conclusions of the present study are listed as follows:1. L. gibbosa was pre-grown on PDA plates at28℃for6days as inoculum sources. Then the fungus was transferred into LNAS medium supplemented with2g ussuri poplar for9days to harvest the mycelial mats.Using liquid nitrogen frozen preservation at-80℃, used for subsequent experiment genomic DNA and the extraction of total RNA.2. Using degenerate PCR, RT-PCR, RACE, and Genome walking-PCR, the full-length cDNAs and genes of two laccases from L. gibbosa were obtained then named Lg-lacl and Lg-Lac2, respectively. The translational start codon are ATG and stop codon are all TAG. Both genes encoding for two precursor polypeptide of520amino-acid residues. The predicted open reading frames (ORFs) are all1,563-bp long. The full-length cDNAs of Lg-lacl and Lg-lac2consist of1744,1873nucleotides excluding the poly(A) tail which contain a59and73nucleotides of5' untranslating region, and a122and237nucleotides of3'untranslating region, respectively.The predicted two preprotein products have80%identity, whereas both mature proteins are82%identical at the amino acid level. To examine the respective structure of the regulatory regions of both genes further,5'upstream and3'downstream non-coding flanking regions of two genes were also amplified using genome walking-PCR, respectively. The cDNA and genomic sequences have separately been submitted to the NCBI GenBank under accession numbers JF817354and JF906786(Lg-lacl), and JF817353and JF906787(Lg-lac2), respectively.At nucleotide level of cDNA, Lg-lacl shows the highest degree of identity with lcc2from T. versicolor which is up to81%homology, whereas Lg-lac2shares the highest similarity to lac2from T. velutina which is up to82%homology. Both cDNAs are also highly homologous to the laccase cDNAs from T. versicolor, T. sp.I-62, T. sp.48424, Coriolus hirsutus, P. sanguineus, Polyporus brumalis, and Ganoderma lucidum with identities ranging from75%to81%.The full-length genes of Lg-lacl and Lg-lac2consist of3934,3887nucleotides, respectively. From the translational start codon to the stop codon, Lg-lacl have2106bp and Lg-lac2consist2165bp. Both genes contain10introns and share the same length of exons, Both genes show highly homologous to the laccase genes from Trametes sp. AH28-2, Pycnoporus sanguineus, Pycnoporusc innabarinus, Trametes villosa, Trametes versicolor with identities ranging from73%to75%.3. The G+C content of Lg-Lacl and Lg-Lac2were58.80%and60.27%at the same time.The G+C content of Lg-Lac1'and Lg-Lac2'were55.75%and58.19%. both G+C content very high of cDNA and DNA.Account for L. gibbosa that the genetic structure is more complicated. Amino acid composition, Lg-Lacl and Lg-Lac2alanine in the highest content, homocysteine, lysine, methionine content at least.Respectively, The molecular mass of Lg-Lacl and Lg-Lac2were53.7KD and53.9KD, the pI were4.69and5.65. There were46and42amino acid residues with negative charge (Asp+Glu)19and24amino acid residues with positive charge (Arg+Lys). The putative protein contained5813and5864atoms with a molecular formula of C1960H2819N464O656S13and C1984H2850N472O668S18. The protein was an unstable protein with a instability index of54.25and51.16. The aliphatic index were78.22and83.25. both the total average hydrophobicity is0.008.4. The most probable splice site of signal peptide of Lg-lacl was predicted to be located between the21st and22nd amino acid, which was recognized as ANG-AA. The most probable splice site of signal peptide of Lg-lac2was predicted to be located between the21st and22nd amino acid, which was recognized as VVG-AI. The protein contained two hydrophobic regions which were located between the67th-95th,392th-352th,378th-401th amino acid; The protein contained two hydrophobic regions which were located between the67th-95th,392th-353th,263th-279th amino acid.5.We get Lg-Lac1'and Lg-Lac2'promoter sequences, and5'upstream include the eukaryotic promoters transcription regulation of the basic components, a TATA-box located in the initiator codon.respectively, located in-93bp and-107bp,respectively, The core promoter Sequence of Laccase gene promoter region are located in-55～-104bp and-65～-115bp. Lg-Lac1'and Lg-Lac2'of L. gibbosa promoter sequence still exists many potential exogenous induced with things response components, CAAT box, respectively, Lg-Lac1'and Lg-Lac2'are situated in-359,-445 bp and-702,-776bp; AP2components, respectively, Lg-Lacl'and Lg-Lac2'are situated in-501bp and-119,173,348,--365,-731bp; Heat strike components (CreA)(SYGGRG), respectively, Lg-Lacl'and Lg-Lac2'are situated in-501bp and-697bp; NIT2components, respectively, Lg-Lacl'and Lg-Lac2'are situated in-121bp and-154.6. Quantitative Real-time PCR experiments was using to compare the relative expression levels of Lg-lacl and Lg-lac2. Transcript of Lg-lacl peaked on day5in the low-copper concentration medium then went down, whereas Lg-lac2level peaked on day5in the high-copper concentration medium then went down. During day5to day13, transcript levels for Lg-lacl and Lg-lac2were slightly influenced by different copper concentrations. Lg-lac2transcripts were expressed approximately the same levels with Lg-lacl under any copper-free medium. In the low-Cu concentration medium, transcripts of both isoforms peaked on day5then decreased. In the high-Cu concentration medium, transcripts of Lg-lacl peaked on day9then decreased, but transcripts of Lg-lac2peaked on day5which was recorded about3-fold higher than Lg-lacl transcript.7. The full-length cDNAs of Lg-lacl and Lg-lac2from RT-PCR were used for heterologous expression in Pichia pastoris. The cDNAs were digested by restriction enzymes and ligated with the expression plasmid pPICZB. The recombinant plasmids pPICZB/Lg-Lacl and pPICZB/Lg-Lac2were all transformed into P. pastoris by electroporation. The recombinant Pichia colonies were screened using the PCR method. The result showed that the recombinant pPICZB/Lg-Lacl and pPICZB/Lg-Lac2were all successfully integrated into the genome of P. Pastoris.The two laccase-positive transformants as well as the laccase-negative control transformant SMD1168H (pPICZB) were fermented with BMMY liquid medium at28℃and induced by adding0.5%(v/v) methanol daily. Under the induction of methanol, extracellular laccase activities could be detected in culture supernatants of the two laccase-positive transformants and were measured every interval of12h, indicating that laccases were secreted from two transformmed yeast cells in an active form. The two laccase-positive transformants both appeard laccase activity at24h, and the highest yields of laccases were reached following a72-hours growth. The highest laccase activity were0.172and0.175μkat·1-1, respectively. However, no extracellular laccase activity was detected in culture supernatants of the negative control-SMD1168H(pPICZB).