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Heterologous expression of a gene for thermostable xylanase from Chaetomium thermophilum in Pichia pastoris GS115

Heterologous expression of a gene for thermostable xylanase from Chaetomium thermophilum in Pichia pastoris GS115. BY Dr. Abdul Ghaffar Assistant Professor Department of Chemistry and Biochemistry University of Agriculture, Faisalabad. Xylanase .

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Heterologous expression of a gene for thermostable xylanase from Chaetomium thermophilum in Pichia pastoris GS115

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  1. Heterologous expression of a gene for thermostable xylanase from Chaetomium thermophilum in Pichia pastoris GS115 BY Dr. Abdul Ghaffar Assistant Professor Department of Chemistry and Biochemistry University of Agriculture, Faisalabad

  2. Xylanase • Hydrolyze the , 1, 4-D-xylosidic linkage in xylan or arabino-xylans • Used for production of hydrolysates from agro-industrial wastes of crops such as wheat straw, rice straw, rice husk, banana stem and corncob • Increase animal feed digestibility • Bleaching in Pulp & Paper industry • Nutritional improvement of foods (e.g. clarification of juices; making bread fluffier) • Production of ethanol, methane and other products • Improving the cleaning ability of detergents, • Improving the extraction of oil from oil rich plant material

  3. THERMOPHILICFUNGAL XYLANASES • Produce thermostable enzymes • Grow up to 60 C or more (Optimum 45 C) • Produce inducible enzymes • C. thermophile first isolated in 1949 • C. thermophile-NIBGE1isolated from rhizospere of wheat • Produces multiple xylanases & cellulases • Optimum growth temperature of 45-50 °C and pH 5-8

  4. Chaetomium thermophile NIBGE 1 Chaetomium thermophile on Ball Milled Cellulose Agar C. thermophile NIBGE 1 observed under microscope. The mature fruiting bodies (perithecia) range from 100-200 m in diameter and are oval shaped C. thermophile grows & sporulates profusely on wheat straw

  5. Objectives • Isolation of xylanase gene from C. thermophile • Characterization of xylanase gene • Cloning of xylanase gene in pPIC3.5K and pPIC9K and its expression in P. pastoris • Evaluation of the efficiency of native and yeast secretion signals for xylanase production • Assessment of the efficiency of bacterial and yeast expression systems • Characterization the recombinant xylanase

  6. Plan of work • Extraction of fungal chromosomal DNA • Xylanase gene isolation by PCR amplification • Cloning of xyn 11A gene in pTZ57R/T • Sequencing of amplified gene • Expression of xylanase gene in P. pastoris GS115 • Expression of xylanase gene with native secretion signal using pPIC3.5K • Expression of xylanase gene with α-MF secretion signal using pPIC9K • Screening of recombinant P. pastoris through His- medium, geneticin resistance, PCR and enzyme assay • Xylanase activity assay • Characterization of recombinant xylanase • Native and SDS PAGE • Activity staining • Optimum temperature and pH • Thermostability

  7. Isolation of genomic DNA from Chaetomium thermophile Fungal genomic DNA isolation method with few modifications Spermidine-SDS buffer 1 2 Genomic DNA Agarose gel electrophoresis of genomic DNA of C. thermophile after its isolation from fresh mycelia. The isolated genomic DNA was electrophoresed on 0.8(%) agarose gel and its integrity was checked after ethidium bromide staining.

  8. PCR amplification of xylanase gene Designing of primers from C. thermophile xylanase 11 A gene Accession No. AJ 508931 P1 Xyn-11 A- (34 mer) 5´- G GCG ATA GCT AGC ATG GTC AAC TTC TCA ACT CTC - 3´ P2 Xyn-11 A-R (29 mer) 5´- GGA AGG GCC CGC ACT GCA TGC TTG TTA GC - 3´ PCR profile: Process Temperature Time Denaturation 95 C 5 min Denaturation 95 C 1 min Annealing temperature 55 C 1 min Extension 72 C 1 min Number of cycles 35 Final Extension 72 C 10 min 876 bp xylanase gene M 1 2 3 4 10000 8000 6000 5000 4000 3500 3000 2500 2000 1500 1000 750 500 250 pTZ57R/T cloning vector for cloning of PCR product (Mead et al., 1986). Agarose gel electrophoresis of PCR products. The expected size of xyn11A gene was 876 bp.

  9. Confirmation of recombinant pTZ57R/T Contin…. Determination of directional cloning through restriction analysis 2886 bp M 1 2 10000 4000 3000 2000 1500 1000 750 500 250 678 bp 198 bp Agarose gel electrophoresis of pTZ57R/T with xyn11A gene after restriction with Kpn1. Xylanase gene in required orientation produced a fragment of 678 bp (lane 1) while other orientation produced a fragment of 198 bp (lane 2).

  10. Sequence of xyn11A gene of C. thermophile NIBGE 1 Nucleotide and deduced amino acid sequence of the xyn11A gene from C. thermophile NIBGE 1. The full length xylanase gene consists of 863 bp with an intron of 35 nucleotides (uppercase, bold and underlined). Amino acids with italic, bold and underlined represent glycosyl hydrolases of family 11 active site. ACCESSION No. AY366479

  11. Sequence of xyn11A gene of C. thermophile NIBGE 1 Contin… Alignment of homologous xylanase sequences of C. thermophile NIBGE 1 with other NCBI documented C. thermophile and C. gracile. The gene structure was almost similar with other Chaetomium species in coded DNA sequences for amino acid but was quite different in the intron region.

  12. Pichia pastoris • Eukaryotic expression system • Protein processing, folding, post-translational modifications • Easy to manipulate • Faster, easier, less expensive • Generally gives higher expression levels 10-100 folds

  13. Pichia pastoris cont…. • Methylotropic yeast • Methanol to formaldehyde by alcohol oxidase • AOX has poor affinity towards O2, large amount of enzyme is needed • Strong AOX promoter • Expression regulated by methanol

  14. Pichia pastoris cont….. • Three vectors: • pPIC3.5K (for intracellular expression) • pPIC9K (for extracellular expression) • pAO815 (creation of multicopy genes and intracellular expression) • Pichia strains available • GS115 • KM71

  15. Expression vector pPIC3.5K • Intracellular expression of gene • Five unique restriction site for inframe cloning of gene • Requires ATG for proper translation initiation • Insertion at His4 after linearization with Sac1

  16. Expression vector pPIC9K • Secretes the recombinant protein to medium • Uses alpha factor secretion signals • Four unique restriction sites for inframe cloning of gene • Insertion at His4 after linearization with Sac1

  17. Strategies for cloning and expression of xyn11A gene with native and α-mating factor secretion signal. Schematic expression of xylanase in P. pastoris

  18. PART 1.1 Expression of xylanase with native secretion signal

  19. Cloning of xyn810 into pPIC3.5K • pET810 and pPIC3.5K digestion with EcoR1 & Not1 M 1 2 3 10000 2000 1500 1000 750 500 250 9,000 bp 5900 bp 891 bp Agarose gel electrophoresis of pET810 and pPIC3.5K. After digestion with EcoRI and NotI.

  20. Cloning of xyn810 in pPIC3.5K Contin…... M 1 2 3 4 M 1 2 10000 8000 3000 1500 1000 750 500 250 9,000 bp 9891 bp 10000 2000 1500 1000 750 500 250 891 bp Agarose gel electrophoresis of pPIC3.5K810. The plasmid was digested with EcoRI and NotI to confirm the cloning of xylanase gene in it. Agarose gel electrophoresis after linearization of pPIC3.5K810 with SacI. Lane M: 1 kb DNA marker. Lane 1: pPIC3.5K. Lane 2: pPIC3.5K810.

  21. Expression of xyn810 in P. pastoris • Optimization of geneticin resistance for P. pastoris • Transformation (10 μg of pPIC3.5K810 & pPIC3.5K as –ve control separately into electro-competent cells of P. pastoris • Selection of transformants over Minimal methanol MM (His deficient) medium • Selection over geneticin • Expression of xylanase in BMMY medium • Induction with Methanol • Sampling • Xylanase activity assay with DNS • zymolyase treatment • Xylanase activity of cell lysate 2.96 U/mL Time course of recombinant xylanase production by P. pastoris GS115 in BMMY medium

  22. PART 1.2 Expression of xylanase with α-MF secretion signal

  23. Expression of xylanase gene with α-MF secretion signal • The xylanase gene without native secretion signal but with intron (xyn698) • Xylanase gene without native secretion signal and without intron (xyn669) • Extracellular expression in P. pastoris GS115 using pPIC9K.

  24. Cloning of xyn11A gene into pTZ57R/T M 1 2 3 4 • Removal of native secretion signal through PCR amplification with P6 and P7 • Cloning in pTZ57R/T • Transformation in E. coli TOP10F` • White blue selection over amp and tet. • The clone was designated as pAG698 (xylanase gene without native secretion signal, with intron) 10000 2000 1000 750 500 250 698 bp Agarose gel electrophoresis after PCR amplification of xylanase gene.

  25. Cloning of xyn698 into pPIC9K • Plasmid isolation (pAG698) • Restriction analysis with EcoR1 and Not1 • Ligation into pPIC9K • Restriction analysis of pPIC9K698 with EcoR1 and Not1 1 2 3 4 5 6 M 1 2 3 M 9300 bp 2886 bp 10000 8000 3000 1500 1000 750 500 250 10000 2000 1500 1000 750 500 250 698 bp 698 bp Agarose gel electrophoresis of pPIC9K698 after restriction with EcoRI and NotI. Lanes 1-3: pPIC9K698. Agarose gel electrophoresis after restriction of plasmid DNA with EcoRI and NotI. Lanes 1 and 2: pAG698. Lanes 3-6: pPIC9K Lane M: 1 kb DNA marker.

  26. Cloning of xyn698 into pPIC9K contin….. (B) Cloning of xylanase gene of C. thermophile in pPIC9K. (A) 5′ region of xyn11A/pPIC9K construct showing last 22 amino acids of α-MF secretion signal, the tetrapeptide (bold characters) of pPIC9K plasmid and a dipeptide (bold italic) placed ahead of the first two amino acids of the mature xyn11A sequence. (B) 3′ region of xyn11A/pPIC9K construct, showing last five amino acids of xyn11A sequence and a stop codon, 15 nucleotides of the pPIC9K and NotI site used for sub-cloning into the pPIC9K plasmid.

  27. Expression of xyn698 in P. pastoris • Linearization of pPIC9K698 and the parent vector pPIC9K (as control) with SacI • Electroporation into P. pastoris GS115. • Selection on Minimal Methanol (histidine-deficient) medium

  28. Expression of xyn698 in P. patorisconti... • Confirmation of transformation • Selection of P. pastoris GS115 different concentrations of geneticin (0.75, 1.25, 1.75, 2.25 and 3 mg/mL) B A Selection of recombinant P. pastoris on YPD plates. (A): Colonies of P. pastoris with xyn669 (upper three) and P. pastoris with xyn698 at geneticin concentration 0.75 mg/mL. (B) P. pastoris with xyn698 at geneticin concentration 3 mg/mL.

  29. Expression of xyn698 in P. patorisconti... M 1 2 • Confirmation of xyn698 integration into host genome • PCR amplification with xylanse specific primers for confirmation of gene insertion 10000 6000 3000 2000 1500 1000 750 500 250 698 bp Agarose gel electrophoresis of PCR amplified xyn698.

  30. Expression of xyn698 in P. pastoris conti... • Methanol utilization fast (Mut+) colonies were selected in minimal methanol medium • Culture in 5 mL of MGY medium in a 50 mL centrifuge tube • At log phase (16-18 h) resuspended cells in buffered methanol complex medium BMMY (OD600=0.8) • Induction with CH3OH after every 24 h • Sampling at 0, 12, 24, 36, 48, 72, 96 h • Xylanase activity assay with DNS

  31. Expression of xyn698 in P. pastoris conti... Xylanase activity of recombinant P. pastoris with xyn698 at different time intervals Time course of recombinant xylanase (xyn698) production by two colonies of P. pastoris GS115in BMMY medium.

  32. Characterization of recombinant xylanase • Polyacrylamide gel electrophoresis of recombinant xylanase • Activity staining with Congo red for xylanase • Optimum temperature • Optimum pH • Thermostability

  33. Polyacrylamide gel electrophoresis of recombinant xylanase 1 2 M 1 2 3 Xyn11A Xyn11A SDS-PAGE of secretary proteins of P. pastoris. Lane M: protein marker (Fermentas). Lanes 1 and 2: xylanase of P. pastoris Lane3: xylanase of C. thermophile Native polyacrylamide gel electrophoresis of secretory proteins of P. pastoris. Lane 1: P. pastoris having pPICK9K698. Lane 2: positive control from xylanase of C. thermophile.

  34. Activity staining for xylanase 1 2 B A Xylanase activity from the supernatant of a P. pastoris clone expressing xylanase. The native gel was overlaid on xylan agarose gel at 45 °C for 20 min. The xylan hydrolysis bands were stained with Congo red and developed with NaCl solution and acetic acid. Lane 1: xylanases from C. thermophile. Lane 2: recombinant xylanase from P. pastoris. Xylanase activity from the supernatant of a P. pastoris clone expressing xylanase. Twenty microlitres of supernatant were loaded into the xylan agarose plate (pH 6) and incubated at 45 °C for 20 min. The transparent zones after staining with Congo red indicated xylanase activity. (a) Samples of different time intervals from P. pastoris Rec26 and C. thermophile (lower right). (b) Samples from different colonies of P. pastoris and C. thermophile (topmost) after 96h of induction.

  35. Optimum temperature • Xylanase activity assay with DNS at temperature ranges from 30 to 70 °C • Optimum temperature for the activity of enzyme was found to be 45 °C Effect of temperature on optimum activity of recombinant xylanase. Xylanase from P. pastoris was analyzed for substrate hydrolysis at different temperature.

  36. Optimum pH • Xylanase activity assay with DNS at range of pH 3.0-10.5 • Optimum pH for the activity of enzyme was found to be 6.0 Effect of pH on activity of recombinant xylanase. The xylanase activity was determined through DNS method at 45 °C in citrate phosphate buffer of different pH.

  37. Thermostability • Pre-incubation of enzyme in the absence of substrate at 10, 20, 30, 40, 50, 60, 70 and 80 °C for 10, 20 and 30 min, respectively. • 91(%) of maximal activity after incubation at 50 °C for 10 min • 68(%) of maximal activity after 30 min incubation at 50 °C • 38(%) of maximal activity after incubation at 70 °C for 30 min Thermostability of recombinant xylanase (xyn698). The enzyme was pre-incubated at different temperatures in the absence of xylan for 10, 20 and 30 min.

  38. Future Prospects • Efforts will be carried out to enhance the secretion of xyn enzyme in yeast • The recombinant xyn gene in yeast will be studied for its growth and thermodynamics. • The other characteristics such as protein sequencing, folding, glycosylation, three dimensional structure will be studied. • These facts will be compared with the wild type gene.

  39. Acknowledgements Dr. Farooq Latif Mr Zahid Mukhtar Mr Sher Afzal Khan All lab fellows Director NIBGE

  40. Thanks…...

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