Cloning and expression of Aspergillus niger phytase gene in yeast platform and Target gene alteration to improve enzyme characterization

Improvement of phytase Thermostability and Catalytic Efficiency by Site-Directed Mutagenesis for Industrial application

ByArdeshir HesampourIslamic Azad UniversityJuly 2015

1IntroductionPhosphatases are a diverse class of enzymes and can act on a variety of phosphate esters

Phytases ( myo -inositol hexaphosphate phosphohydrolases) are a subgroup of acid phosphatases which hydrolyze phytic acid to myo -Inositol and inorganic phosphates.

2Phytase producing organismFungi, yeasts and bacteriaFungi especially Aspergillus niger is main producer of commercial Phosphatase

Aspergillus niger phytase(histidine phosphatase, 3-phytases) has been well characterized.

Gene encoding Phytase (phyA) has been cloned and sequenced several time.

A.niger phytase, high catalytic efficiency in compare with other phytase producers. But low expression

3Expression of Phytase by YeastsAdvantages of phyA expression in Yeast hosts:

Correct Glycosylation profile of Phytase protein(10 N- glycosylation residue).Active expression of phytase in different yeast hosts are done.High phytate affinityHigh expression productionNon pyrogen and pathogenUtilize broad range of carbon sourcesCost effective bulk recombinant protein production

Saccharomyces serviciaPichia pastorisHansenula polymorphaArxula adenivora

4Phytase Industrial applicationsSoil Amendment:50% of soil appears to be in the form of phytate and its derivatives that require a especial class of phosphatase i.e. phytases for hydrolysisApplications in Feed (Feed additive) Inhibit environmental pollutionPotential in AquaculturePreparation of Myo-Inositol Phosphates

*Industrial Application criteria : High Temperature (feed pelleting) / Low pH Activity

*Protein engineering methods to adopt Phytase for Industrial applications

5GoalRecombinant expression of Aspergillus niger phytase (phyA) in methylotrophic yeast P. pastoris

Protein engineering (site directed mutagenesis) of WT recombinant phytase to improve biochemical properties

Comparison ofbiochemical propertiesof recombinant enzymes containing the mutationsandwildenzyme

Characterization of recombinant phytase

Stage IStage II6

7 Native Aspergillus niger PhyA gene (Gene Bank p34752)MGVSAVLLPLYLLSGVTSGLAVPASRNQSSCDTVDQGYQCFSETSHLWGQYAPFFSLANESVISPEVPAGCRVTFAQVLSRHGARYPTDSKGKKYSALIEEIQQNATTFDGKYAFLKTYNYSLGADDLTPFGEQELVNSGIKFYQRYESLTRNIVPFIRSSGSSRVIASGKKFIEGFQSTKLKDPRAQPGQSSPKIDVVISEASSSNNTLDPGTCTVFEDSELADTVEANFTATFVPSIRQRLENDLSGVTLTDTEVTYLMDMCSFDTISTSTVDTKLSPFCDLFTHDEWINYDYLQSLKKYYGHGAGNPLGPTQGVGYANELIARLTHSPVHDDTSSNHTLDSSPATFPLNSTLYADFSHDNGIISILFALGLYNGTKPLSTTTVENITQTDGFSSAWTVPFASRLYVEMMQCQAEQEPLVRVLVNDRVVPLHGCPVDALGRCTRDSFVRGLSFARSGGDWAECFA

The amino acid sequence of native protein and its N-terminal native signal peptide(Underlined)Phytase Protein sequence selection and sequence OptimizationStage IExpression of phyA in P.pastoris8pPink-HC expression vector

AOX1 Promoter-mating factor signalMultiple Cloning SiteCYC1 TerminatorADE2 GeneTRP2 Sequence

9Cloning and expression of phyA gene in Pichia PastorisDigestion of pTG19T-phyA with 5Xho I/ 3Kpn IDigestion of pPink-HC with Xho I/ Kpn I Ligation and transformation in E. coli DH5Plasmid purification

pPinkHC-phyA (Linearization) by Afl II

Transformation of pPinkHC-phyA to P.Pastoris

Screening of phyA integrant clones by specific primers

Sbu Forward 5'-CTCGAGGCTTCTAGAAACCAATCTTCTT-3'Sbu Reverse 5'-GGTACCCTACTAAGATCTAGCGAAACAT-3'

Expression and characterization of recombinant phytase

10Phytase EngineeringStage II11Phytase Engineering parameters

Thermostability

Animal feed commonly pelleted, a commercially attractive phytase should be able to withstand the temperature that are reach temporarily during the pelleting process (60-90 C).

All components of animal feed, including enzymes, are exposed to average temperature of 80 C

Kinetic efficiency12Mutant designing predictions13Candidate point selection Approaches

Comparison of the amino acid sequence of the phyA phytase with that of a thermostable and highly homologous counterpart

Amino acids accessibility and interactions investigation

Introduction of possibly stabilizing amino acids based on the 3D structure of protein

14Structural studies

PhyA structural comparison with homologue thermostable phytases

PDB files analysis by YASARA

15

Candidate points accessibility and interactions analysis

DSSP

Secondary structure assignments

Solvent exposure of proteins

Atomic coordinatesWhat IF :Work in water environmentMultiple Alignment with thermostable homologue phytases16Determination of substituted points

A.aculeatus RCEF 4894 , withstand up to 90C for 10 min

17

Amino acids substitution and modeling

Homology Modelling

The 3D structure of the native A.niger PhyA phytase (Access No. 3K4P) was used as the template.Substituted amino acids (Single and combined) were modeled by Swiss model Modeller , a protein structure homology-modelling server. (Swiss Pdb-Viewer)

18Model validation

PROCHECK/ Model Validation

DSSP / Secondary structureGeometrical featuresSolvent exposure of modeled proteins

19Structural Refinement by Molecular Dynamics (MD)MD simulations can provide great details regarding the motion of individual particles as a function of time in realistic environmentsResults can be used to study the relation between structure and function of biomacromolecules Structural refinement was performed using Gromacs 4.5.3 A pair of simulations was carried out, one in 300 K and the other in 353 K for 4ns and pressure of 1 bar was set under isotropic condition

WT and Mutants heat treatment simulation20Analysis of MD resultsTotal H bond determinationTotal salt Bridge determinationMeasure the number of strong hydrogen bonds formed between residues i and i+10 in the primary sequenceH bond energy calculation21Experimental mutant substitution22Candidate points substitution strategies

De novo design

Rational designSite Directed Mutagenesis (SDM)

Directed evolution

23Quick Change Site Directed Mutagenesis (SDM)

24Designing of substitutions primer

Sequences in italics and bold represent the designated mutant for the target amino acid residue25Single and combined substituted phytase mutants

For successful thermostability engineering, many mutations have to generated and analysed individually, followed by combination of the few stabilizing amino acid exchanges26

27

Synthetic Aspergillus niger phyA in P.pastoris, Gene Bank Accession No. JN 193562.1MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKRASRNQSSCDTVDQGYQCFSETSHLWGQYAPFFSLANESVISPEVPAGCRVTFAQVLSRHGARYPTDSKGKKYSALIEEIQQNATTFDGKYAFLKTYNYSLGADDLTPFGEQELVNSGIKFYQRYESLTRNIVPFIRSSGSSRVIASGKKFIEGFQSTKLKDPRAQPGQSSPKIDVVISEASSSNNTLDPGTCTVFEDSELADTVEANFTATFVPSIRQRLENDLSGVTLTDTEVTYLMDMCSFDTISTSTVDTKLSPFCDLFTHDEWINYDYLQSLKKYYGHGAGNPLGPTQGVGYANELIARLTHSPVHDDTSSNHTLDSSPATFPLNSTLYADFSHDNGIISILFALGLYNGTKPLSTTTVENITQTDGFSSAWTVPFASRLYVEMMQCQAEQEPLVRVLVNDRVVPLHGCPVDALGRCTRDSFVRGLSFARSGGDWAECFARS

Amino acid sequence of phytase. The amino acid sequence of synthetic phytase protein of P.pastoris (the alpha mating factor signal peptide of S.cerevisiae is underlined).

Stage I Results28Cloning and expression of synthetic phyA gene in pPink-HC

M: ladder1 : pTG19T-phyA plasmid2: Double digested pTG19T-phyADigestion of pTG19T-phyA with Xho I/ Kpn I

Digestion of pPink-HC with Xho I/ Kpn I 1: ladder2: Double digested pPink-HC 3: pPink-HC plasmidConfirmation by Colony PCRM: ladder1-4: phyA gene(1350bp)5: negative control

bp

3000

1500

12001000

500

400

300

200

10029Transformation and screening of pPinkHC-phyAPichia Pastoris transformation by Electroporation method

Screening of transformants on PAD media

30Genome extraction and Positive colony screening

1: Integrated phyA gene , PCR product with phyA specific primer2: Integrated phyA gene ,1705bp PCR product with -mating factor primer3: negative control M: ladder

1-3 : transformant P.pastorisM : ladder31Expression of phytase in P. pastorisGrowth of recombinant P.pastoris in BMGY media at 30 C

Methanol induction and expression of recombinant phytase in BMMY media at 30 C

32Phytase expression analysis Phytase activity assay SDS-PAGE AnalysisSpecific Activity (U/mg)= Enzyme Activity (U/ml) Total Protein (mg/ml)Phytase activity: 179 U/mlTotal protein: 2.71 mg/mlSpecific activity: 66.05 U/mg

Recombinant phytase50-70 kDaEnzyme + sodium phytateIncubation for 30 minStopping buffer (Sulphuric acid, Ammonium molybdate, Acetone)

Measurement of released Pi at OD380 nm33Purification of recombinant phytase

Smear of 50-65KDaFPLC Method / Size exclusion by Sephadex G100

34Purification of recombinant 2 steps

Two purification steps analysis

Purification stepTotal activity (U)Total protein (mg)Specific activity (U/mg)Recovery(%)Purification(fold)Culture supernatant1792.7166.051001Amicon ultrafiltration1681.8789.8393.851.36Sephadex G100840.69121.7346.921.84Enzyme reactions (n=3) ,Values are mean. 35Glycosylation analysis

Due to heavy yeast glycosylation, the expressed phytase revealed as a smear band on SDS-PAGE with molecular size ranging from 50 to 65 KDa.

Recombinant phytase Smear of 50-65KDa49 KDaAfter Endo H treatment

Endo H protein36Michaelis Menten Kinetics

Kinetic parameters of the purified Recombinant phytase of P.pastoris Km(M)Vmax(mol min-1mg-1)Kcat (s-1)(Kinetic efficiency) (s-1mol-1)P.Pastoris recombinant WT phytase148 1135 0.9168.75 2.11.14106Values are mean SE (n=3).

Graph pad prism 537Phytase characterization

38Phytase thermostability

39Results ofPhytase Engineering(Stage II)40Candidate points determination

S205N & S206A T314S Q315RV62NT150A 41Three-dimensional conformation model

42Quick change site directed mutagenesis(substituted PCR fragment Products)

M : ladder

1-6 and 8-9:substituted phyA gene+pTG19

7: Negative control43Confirmation of mutations

Confirmation of mutations by sequencing

Confirmation of mutations by Colony PCR

M :ladder

1-4: Positive clones with 1350 bp phyA gene

5: negative control44Cloning and expression of mutant phytases in Pichia Pastoris

Substitution of designed points in phyA gene + pTG19T vector by Quick change PCR methodDpn I digestion of PCR productsE.Coli transformation and nick repairDigestion of pTG19T-phyA with Xho I/ Kpn IDigestion of pPink-HC with Xho I/ Kpn I Ligation and transformation in E. coli DH5Plasmid purificationpPinkHC-phyA(mutant) plasmids Linearization by Afl II

Transformation of pPinkHC-phyA(mutants) to P.Pastoris

Screening of phyA integrant clones by specific primers'

Expression and analysis of mutant phytases

45Mutant phytasesThermostability analysis46

Mutant phytases(single substitutions) thermostabilityWT Vs phyA P3 and phyA P4

Among the mutants, the P3(V62N )and P4(T151A) substitutions exhibited higher residual activity at 70C with no statistical significance47Mutant phytases (Double substitutions)thermostabilityWT Vs phyA P7, phyA P8 and phyA P10

P7 (S205N S206A), P8 (T314S Q315R), and P10 (T151A V62N)

Showed improvement in residual activity with a greater thermal retention (P