Novel extracellular lipases from Trichosporon asahii MSR54: Role in biotechnology and physiology

1. Novel extracellular lipases from TrichosporonasahiiMSR54: Role in biotechnology and physiology Prof. Rani Gupta Department of Microbiology University of Delhi, South Campus New Delhi – 110 021

2. Lipases DRUG INDUSTRIES • Lipases (E.C. Number 3.1.1.3) are triacylglycerol hydrolases that catalyze hydrolysis of long-chain triacylglycerides. • Under micro-aqueous conditions, lipases possess the unique ability to carry out the reverse reaction leading to esterification. FOOD INDUSTRY COSMETICS Application of Lipases OLEO CHEMICAL AGR0- CHEMICALS LEATHER INDUSTRY DRAINAGE CLEANING DETERGENT

3. Trichosporon asahii MSR54 • Previously isolated from petroleum sludge; submitted at MTCC with accession number 9549. • Genome wide survey revealed three extracellular lipases: • TALipA (27 kDa) • TALipB (33 kDa) • TALipC(54 kDa) • Even though yeast lipases are potential catalysts, still they have not been exploited to a great extent: • Most of them are pathogenic • Multiple isozymes are expressed under similar culture conditions that have overlapping substrate specificities Rectangular cells; budding with Cream to tan membranous colony Zone of hydrolysis on oil plate pseudohyphae

4. Phylogenetic analysis by nr-BLASTP TALipB TALipA TALipC • TALipA and TALipB shared 63% homology among themselves and have close affinity with bacterial lipases. • TALipC is totally distinct and has close affinity with fungal lipases

5. Sequence analysis with the fungal lipases retrieved from LED • TALipA and TALipB were present in Clade I and closer to Yarrowia lipases • TALipC was present on second branch of clade II and close to Malassezia and Candida antarctica lipases

6. In silico analysis of lipase TALipA • TALipA has highest homology with Bacillus subtilis lipase (32%); PDB ID: 1I6WA Multiple sequence alignment of TALipA with Bacillus subtilis lipases N-terminal is similar to B. subtilis lipase Penta peptide “AHSMG” (common in bacteria) oxyanion hole “GX” type L48 is Ist and M116 is IInd Predicted catalytic residues: S177, D205 and H237

7. In silico analysis of lipase TALipB • TALipB has highest homology with Candida antartica CALB (22%); PDB ID: 3O0D Multiple sequence alignment of TALipB with CALB oxyanion hole “GX” type Conserved penta peptide “GHSMG” (common in yeasts) Predicted catalytic residues: S154, D228 and H261

8. In silico analysis of lipase TALipC • TALipC has highest homology with Yarrowia lipolytica YlLip2 (32%); PDB ID: 3O0D Multiple sequence alignment of TALipC with YlLip2 N-terminal is from parvovirus oxyanion hole “GX” type P187 is 1st and L164 is 2nd Conserved penta peptide “GHSLG” (common in yeasts) Core region showed affinity with YlLIP2 C-terminal is very heterogeneous and asprich domain Predicted catalytic residues: S279, D334 and H360

9. Strategy used for gene isolation of lipases Genome survey of T. asahii available on http://www.nuccore/406870529 Protein to Gene Purified protein were subjected to N-terminal and LC-MS/MS Three extracellular lipases genes were identified which corresponds to 27kDa, 33kDaand 54kDa proteins Peptide matching with the lipase sequences retrieved from genome mining of T. asahii TALipC (54 kDa) TALipA (27 kDa) Intron analysis by vector NTI software Lip27 and Lip33 were intron free, While Lip54 has two introns. Primers were designed using genome database

10. Cloning and sequencing of TALipC (54 kDa) Intron 2 Intron 1 Exon 1 Exon 2 Exon 3 Lip 54 kDa has two introns, therefore it was amplified by overlapping PCR. 54R1 Lip54F 54F1 54R2 34 bp fragment Synthesized by Company Amplification of Exone 1 = 567 bp Amplification of Exone 2 = 854 bp Taking these fragments together as target DNA they were further amplified using primer Lip54F & 54R2 for 10 cycles 1.4 kb M 1 2 3 4 Colony PCR Taking these fragments together as target DNA they were further amplified using primer Lip54F & Lip54R for 10 cycles Lip54 The amplified product was cloned in PCR4 vector and sequenced.

11. Expression of lipases in E. coli BL21, pET 22b system TALipA, B and C were expressed in functional form in Pichiapastoris X33 as well.

12. Purification of lipases - Ni-NTA affinity chromatography TALipA TALipB TALipC Singh R, Gupta N, Goswami VK and Gupta R (2006) A simple activity staining protocol for lipases and esterases. Applied Microbiology and Biotechnology 70(6): 679-82

13. Comparative biochemical characterization

14. Enantio-selectivity • Enatio-selectiove lipases have a big market in the pharma sector • 56% drugs are chiral and 88% are marketed as racemates as they are difficult to separate by chemical processes Enantio-selectivity studied using esterification of racemic phenyl ethanol with lauric acid in different solvent Solvent 1 Solvent 2 R-chiral selectivity S-chiral selectivity Solvent-based enantio-inversion

15. Enantio-inversion - TAlipA Iso-propanol Hexane R-selectivity S-selectivity 1,4-dioxane

16. Summary – Part 1 • All the three lipases are unique to the database • TALipA and TALipB have close affinity with bacterial lipases, while TALipC has affinity with yeast and fungal lipases • TALipA and TALipB have closer homology to each other while TALipC is more distant • All the three enzymes are thermostable, solvent stable and enantio- selective • TAlipA and TALipC exhibit solvent-based enantio-inversion • Of all, TALipC is most unique with very interesting biochemical properties

17. Role of lipases in cellular physiology • Construction of lipase knockdown • Developed a strategy for A. tumefaciens mediated transformation in T. asahii MSR54 for performing vector based RNA interference • Comparative analysis of wild T. asahii and lipase knockdown strains • Effect on cellular growth and morphology • Effect on strains in biofilm formation

18. Construction of lipase knockdown Lipase knockdown was performed by vector based RNA interference The prerequisite for RNAi is the presence of DICER (A key enzyme for RNAi) Genome mining of T. asahii Proteins A1Q1_05439: Dicer of Rhizoctoniasolani (86% homology) T. asahii dicer homologue The RNAi vector used in the study was modified pCAMBIA1300 vector having MCS from pFGC5942 Bilichak A, Yao Y and Kovalchuk I (2014) Transient down-regulation of the RNA silencing machinery increases efficiency of Agrobacterium-mediated transformation of Arabidopsis. Plant Biotechnology Journal 12(5):590-600

19. Strategy for lipase knockdown Vector pCAMBIA1300 modification Step I Identification of unique target sequence (UTS) (200 bp) Step II Cloning of UTS in modified pCAMBIA1300 E. coli DH5α (200 bp) Step III Plasmid isolation and transformation in Agrobacterium tumefaciens Step IV A. tumefaciens mediated transformation in T. asahiiMSR54 Step V

20. Strategy for lipase knockdown Step I: Vector modification TEF1 (yeast promoter) in place of CaMV35S promoter (plant promoter) BstXI AatII Lane A: Ladder, Lane B: Linearized vector pCAMBIA 1300 (with BstXI) Lane C: Double digested vector pCAMBIA 1300 (with and AatII) lower band corresponds to CaMV 35S promoter, Lane D: Amplified TEF1 promoter. Step II: Identification of Unique target sequence for silencing These sequence should be as short as 200 bp and unique to the targeted protein

21. Strategy for lipase knockdown STEP III: Molecular Sub-cloning of the silencing fragments into binary vector pCAMBIA1300-TEF1 The sense and antisense repeat of unique target sequences of TALipA, TALipB and TALipC were assembled in the binary vector using the introduced restriction enzyme sites. Transformed in E. coli DH5α Confirmed by colony PCR

22. Strategy for lipase knockdown Step IV: Agrobacterium mediated transformation in T. asahiiMSR54 Place two concentric plates one inside other pour the YPD inside and YEB in outer plate after solidification of the first Transformed Agrobacterium primary culture 250 rpm, 28 °C for 48 h Inoculate the inner plate with fungi Agrobacterium grown in YEB media + Acetosyringone Grow it at 28°C till fungal growth reached to periphery of the first plate Inoculate the outer plate with Agrobacterium contained requisite plasmids Co-cultivation till 48 h at 28 ºC Co-cultivation method

23. Agrobacterium - yeast attachment by light microscopy Agrobacterium - yeast attachment by scanning electron microscopy

24. Morphological changes in lipase knockdown strains in stationary phase Yeast Medium control ΔTALipA ΔTALipB ΔTALipC ΔTALipA and ΔTALipC predominantly retained yeast form in stationary phase and lost the property of yeast to fungal transition Lipase Production Medium control ΔTALipA ΔTALipB ΔTALipC Lower growth was observed in LPM

25. Role of lipases in biofilm formation • XTT assay • SEM analysis • Antibiotic susceptibility Comparative analysis of biofilm production by T. asahii MSR54 in YPD and LPM till 96 h Higher biofilm in lipase production medium Biofilm analysis of knockdown strains by XTT assay • All the knockdown strain showed reduction in biofilm • Highest reduction was observed in ΔTALipC (0.6 after 96h)

26. Biofilm analysis by SEM T. asahii MSR54 ∆TALipA Massive biofilm with profuse extracellular matrix having all fungal morphology Intermediate biofilm reduction ∆TALipB ∆TALipC large reduction in the biofilm with numerous blastospores showing no hyphal stage Intermediate biofilm reduction

27. Change in antibiotic susceptibility due to less biofilm production T. asahiiMSR54 lipase knockdown strains with amphoterecinB T. asahii has SMIC (sessile minimum inhibitory concentration) of 16 μg/ml and 2 μg/ml for amphotericin B and fluconazole, respectively. lipase knockdown strains with fluconazole • Drastic decrease in the SMIC for lipase knockdown strains. • Among all the maximum decrease in SMIC was found in ∆TALipC where it reduced to 0.02 μg/ml for both the drugs which was same as that of lipase inhibited condition (in presence of orlistat).

28. Summary - Part 2 • All the knockdown strains predominantly retained yeast stage and lost the property of yeast to fungal transition • There was a decrease in biofilm formation in all the knockdown strains with drastic decrease in ΔTALipC strain • Antibiotic susceptibility of knockdown strain was reducedfor all the knockdown strains with maximum decrease in ∆TALipC

29. Future paths for Trichosporon lipases

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