PERTANIKA JOURNAL OF TROPICAL AGRICULTURAL SCIENCE

Meyerozyma guilliermondii strain SO, a newly isolated yeast species from spoilt orange, has been used as a host to express the recombinant proteins using methylotrophic yeast promoters. However, as a novel yeast expression system, the vacuolar proteases of this yeast have not been determined, which may have contributed to the low level of heterologous protein secretions. Thus, this study aimed to determine intra- and extracellular proteolytic activity and identify the putative vacuolar proteases using bioinformatics techniques. A clear zone was observed from the nutrient agar skimmed milk screening plate. Proteolytic activity of 117.30 U/ml and 75 U/ml were obtained after 72 h of cultivation for both extracellular and intracellular proteins, respectively. Next, the Hidden Markov model (HMM) was used to detect the presence of the vacuolar proteases (PEP4 and PRB1) from the strain SO proteome. Aspartyl protease (PEP4) with 97.55% identity to Meyerozyma sp. JA9 and a serine protease (PRB1) with 70.91% identity to Candida albicans were revealed. The homology with other yeast vacuolar proteases was confirmed via evolutionary analysis. PROSPER tool prediction of cleavage sites postulated that PEP4 and PRB1 might have caused proteolysis of heterologous proteins in strain SO. In conclusion, two putative vacuolar proteases (PEP4 and PRB1) were successfully identified in strain SO. Further characterization can be done to understand their specific properties, and their effects on heterologous protein expression can be conducted via genome editing.

• Abu, M. L., Nooh, H. M., Oslan, S. N., & Salleh, A. B. (2017). Optimization of physical conditions for the production of thermostable T1 lipase in Pichia guilliermondii strain SO using response surface methodology. BMC Biotechnology, 17, Article 78. https://doi.org/10.1186/s12896-017-0397-7

• Ahmad, M., Hirz, M., Pichler, H., & Schwab, H. (2014). Protein expression in Pichia pastoris; Recent achievements and perspectives for heterologous protein production. Applied Microbiology and Biotechnology, 98, 5301-5317. https://doi.org/10.1007/s00253-014-5732-5

• Armenteros, J. J. A., Tsirigos, K. D., Sønderby, C. K., Petersen, T. N., Winther, O., & Brunak, S. (2019). SignalP 5.0 improves signal peptide predictions using deep neural networks. Nature Biotechnology, 37, 420-423. https://doi.org/10.1038/s41587-019-0036-z

• Bailey, T. L., & Noble, W. S. (2003). Searching for statistically significant regulatory modules. Bioinformatics, 19(suppl.2), 1116-1125. https://doi.org/10.1093/bioinformatics/btg1054

• Barrett, A. J., Rawlings, N. D., & Woessner, J. F. (1998). Handbook of proteolytic enzymes. Academic Press.

• Boer, E., Steinborn, G., Kunze, G., & Gellissen, G. (2007). Yeast expression platforms. Applied Microbiology and Biotechnology, 77, 513-523. https://doi.org/10.1007/s00253-007-1209-0

• Bourbonnais, Y., Larouche, C., & Tremblay, G. M. (2000). Production of full-length human pre-elafin, an elastase specific inhibitor, from yeast requires the absence of a functional yapsin 1(Yps1p) endoprotease. Protein Expression and Purification, 20, 485-491. https://doi.org/10.1006/prep.2000.1338

• Chung, B. H., & Park, K. S. (1998). Simple approach to reducing proteolysis during the secretory production of human parathyroid hormone in Saccharomyces cerevisiae. Biotechnology and Bioengineering, 57, 245-249. https://doi.org/10.1002/(SICI)1097-0290(19980120)57:2

• Clare, J. J., Rayment, F. B., Ballantine, S. P., Sreekrishna, K., & Romanos, M. A. (1991). High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem intergrations of the gene. Biotechnology Journal, 9, 455-60. https://doi.org/10.1038/nbt0591-455

• Copley, K. S., Alm, S. M., Schooley, D. A., & Courchesne, W. E. (1998). Expression, processing and secretion of a proteolytically sensitive insect diuretic hormone by Saccharomyces cerevisiae requires the use of a yeast strain lacking genes encoding the Yap3 and Mkc7 endoproteases found in the secretory pathway. Biochemical Journal, 330, 1333-1340. https://doi.org/10.1042/bj3301333

• Crowley, E. M., Roeder, K., & Bina, M. (1997). A statistical model for locating regulatory regions in genomic DNA. Journal of Molecular Biology, 268(1), 8-14. https://doi.org/10.1006/jmbi.1997.0965

• Delic, M., Valli, M., Graf, A. B., Pfeffer, M., Mattanovich, D., & Gasser, B. (2013). The secretory pathway: Exploring yeast diversity. FEMS Microbiology Reviews, 37(6), 872-914. https://doi.org/10.1111/1574-6976.12020

• Egel-Mitani, M., Andersen, A. S., Diers, I. I., Hach, M., Thim, L., Hastrup, S., & Vad, K. (2000). Yield improvement of heterologous peptides expressed in yps1-disrupted Saccharomyces cerevisiae strains. Enzyme and Microbial Technology, 26, 671-677. https://doi.org/10.1016/s0141-0229 (00)00158-7

• Ernst, J., & Kellis, M. (2012). ChromHMM: Automating chromatin-state discovery and characterization. Nature Methods, 9, 215-216. https://doi.org/10.1038/nmeth.1906.

• Falush, D., Matthew, S., & Jonathan, K. P. (2003). Inference of population structure using multilocus genotype data: Linked loci and correlated allele frequencies. Genetics, 164, 1567-1587.

• Fariselli, P., Finocchiaro, G., & Casadio, R. (2003). SPEPlip: The detection of signal peptide and lipoprotein cleavage sites. Journal of Bioinformatics, 19, 2498-2499. https://doi.org/10.1093/bioinformatics/btg360

• Feyder, S., De Craene, J. O., Bär, S., Bertazzi, D. L., & Friant, S. (2015). Membrane trafficking in the yeast Saccharomyces cerevisiae model. International Journal of Molecular Sciences, 16(1), 1509-1525. https://doi.org/10.3390/ijms16011509

• Forgac, M. (2000). Structure, mechanism and regulation of the clathrincoated vesicle and yeast vacuolar H(+)-ATPase. Journal of Experimental Biology, 203, 71-80.

• Frith, M. C., Hansen, U., & Weng, Z. (2001). Detection of cis-element clusters in higher eukaryotic DNA. Journal of Bioinformatics, 17, 878-889.

• Gales, M., & Young, S. (2007). The application of hidden Markov models in speech recognition. Foundation and Trends in Signal Processing, 1(3), 195-304. https://doi.org/10.1561/2000000004

• Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D., & Bairoch A. (2005). Protein identification and analysis tools on the ExPASy server. In J. M. Walker (Ed.), The proteomics protocols handbook (pp 571-607). Humana Press.

• Gellissen, G., Kunze, G., Gaillardin, C., Cregg, J. M., Berardi, E., Veenhuis, M., & Van Der Klei, I. (2005). New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica–a comparison. FEMS Yeast Research, 5(11), 1079-1096. http://doi.org/10.1016/j.femsyr.2005.06.004

• Gleeson M. A. G., White C. E., Meininger D. P., & Komives E. A. (1998). Generation of protease-deficient strains and their use in heterologous protein expression. In D. R. Higgins & J. M. Cregg (Eds.), Pichia protocols (pp. 81-94). Humana Press. https://doi.org/10.1385/0-89603-421-6:81

• Gomez-Lopera, J. F., Matinez-Aroza, J., Roman-Roldan, R., Roman-Galvez, R., & Blanco-Navarro, D. (2017). The evaluation problem in discrete semi-hidden Markov models. Mathematics and Computers in Simulation, 137, 350-365. https://doi.org/10.1016/j.matcom.2016.12.002

• Gonzalez-Lopez, C. I., Szabo, R., Blanchin-Roland, S., & Gaillardin, C. (2002). Genetic control of extracellular protease synthesisin the yeast Yarrowia lipolytica. Genetics, 160, 417-427.

• Haussler, D., Krogh, A., Mian, I. S., & Sjolander, K. (1993). Protein modeling using hidden Markov models: Analysis of globins. In 1993 Proceedings of the Twenty-sixth Hawaii International Conference on System Sciences (Vol. 1, pp. 792-802). IEEE Publishing. https://doi.org/10.1109/HICSS.1993.270611

• Hensing, M. C., Rouwenhorst, M., Heijnen, R. J., Van, J. J., Dijken, J. P., & Pronk, J. T. (1995). Physiological and technological aspects of large-scale heterologous-protein production with yeasts. Antonie Van Leeuwenhoek, 67(3), 261-279. https://doi.org/10.1007/BF00873690

• Hughey, R., & Krogh, A. (1996). Hidden Markov models for sequence analysis: Extension and analysis of the basic method. Bioinformatics, 12(2), 95-107. https://doi.org/10.1093/bioinformatics/12.2.95

• Idiris, A., Tohda, H., Kumagai, H., & Takegawa, K. (2010). Engineering of protein secretion in yeast: Strategies and impact on protein production. Applied Microbiology and Biotechnology, 86, 403-417. https://doi.org/10.1007/s00253-010-2447-0

• Imai, K., & Nakai, K. (2020) Tools for the recognition of sorting signals and the prediction of subcellular localization of proteins from their amino acid sequences. Frontiers in Genetics, 11, Article 607812. https://doi.org/10.3389/fgene.2020.607812

• Johanna, M., Smeekens, H. X., & Wu, R. (2017). Global analysis of secreted protein and glycoproteins in Saccharomyces cerevisiae. Journal of Proteome Research, 16(2), 1039-1049. https://doi.org/10.1021/acs.jproteome.6b00953

• Jones, D. T., Taylor, W. R., & Thornton, J. M. (1992). The rapid generation of mutation data matrices from protein sequences. Computer Applications in the Biosciences, 8, 275-282. https://doi.org/10.1093/bioinformatics/8.3.275

• Jones, E. W. (1991). Tackling the proteases problem in Saccharomyces cerevisiae. Methods in Enzymology, 194, 428-453. https://doi.org/10.1016/0076-6879(91)94034-a

• Jonson, L., & Rehfeld, J. F. (2004). Enhanced peptide secretion by gene disruption of CYM1, a novel protease in Saccharomyces cerevisiae. European Journal of Biochemistry, 271(23-24), 4788-4797. https://doi.org/10.1111/j.1432-1033.2004.04443.x

• Kang, H. A., Kim, S. J., Choi, E. S., Rhee, S. K., & Chung, B. H. (1998). Efficient production of intact human parathyroid hormone in a Saccharomyces cerevisiae mutant deficient in yeast aspartic protease 3 (YAP3). Applied Microbiology and Biotechnology, 50,187-192. https://doi.org/10.1007/s002530051275

• Kerry-Williams, S. M., Gilbert, S. C., Evans, L. R., & Balance, D. J. (1998). Disruption of the Saccharomyces cerevisiae YAP3 gene reduces the proteolytic degradation of secreted recombinant human albumin. Yeast, 14, 161-169. https://doi.org/10.1002/(SICI)1097-0061(19980130)14:2

• Kim, H., Yoo, S. J., & Kang, H. A. (2014). Yeast synthetic biology for the production of recombinant therapeutic proteins. FEMS Yeast Research, 15, 1-16. https://doi.org/10.1111/1567-1364.12195

• Kirkwood, B., & Sterne, J. (2003). Essential medical statistics (2nd Ed.). Wiley-Blackwell.

• Kobayashi, K., Kuwae, S., Ohya, T., & Ohda, T. (2000). High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation. Journal of Bioscience and Bioengineering, 89, 55-61. https://doi.org/10.1016/s1389-1723(00)88050-0

• Krogh, A., Brown, M., Mian, I. S., Sjölander, K., & Haussler, D. (1994). Hidden Markov models in computational biology: Applications to protein modeling. Journal of Molecular Biology, 235(5), 1501-1531. https://doi.org/10.1006/jmbi.1994.1104

• Krogh, A., Larsson, B., von Heijne, G., & Sonnhammer, E. L. (2001). Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. Journal of Molecular Biology, 305(3), 567-580. https://doi.org/10.1006/jmbi.2000.4315

• Krogh, A., Sonnhammer, E. L. L., & Ka, L. (2007). Advantages of combined transmembrane topology and signal peptide prediction - The Phobius web server. Nucleic Acids Research, 35, W429-W432. https://doi.org/10.1093/nar/gkm256

• Küberl, A., Schneider, J., Thallinger, G. G., Anderl, I., Wibberg, D., Hajek, T., Jaenicke, S., Brinkrolf, K., Goesmann, A., Szczepanowski, R., Pühler, A., Schwab, H., Glieder, A., & Pichler, H. (2011). High-quality genome sequence of Pichia pastoris CBS7435. Journal of Biotechnology, 154(4), 312-320. https://doi.org/10.1016/j.jbiotec.2011.04.014

• Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33(7) 1870-1874. https://doi.org/10.1093/molbev/msw054

• Leow, T. C., Rahman, R. N. Z. R. A., Basri, M., & Salleh, A. B. (2004). High level expression of thermostable lipase from Geobacillus specie strain T1. Bioscience, Biotechnology, and Biochemistry, 68(1), 96-103. https://doi.org/10.1271/bbb.68.96

• Li, H., & Durbin, R. (2011). Inference of human population history from individual whole-genome sequences. Nature, 475(7357), 493-496. https://doi.org/10.1038/nature10231

• Li, S. C., & Kane, P. M. (2009). The yeast lysosome-like vacuole: Endpoint and crossroads. Biochimica et Biophysica Acta, 1793, 650-663. https://doi.org/10.1016/j.bbamcr.2008.08.003

• Low, K. O., Mahadi, N. M., & IIias, R. M. (2013). Optimization of signal peptide for recombinant protein secretion in bacterial hosts. Applied Microbiology and Biotechnology, 97(9), 3811-3826. https://doi.org/10.1007/s00253-013-4831-z

• Madzak, C., Gaillardin, C., & Beckerich, J. M. (2004). Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: A review. Journal of Biotechnology, 109, 63-81. https://doi.org/10.1016/j.jbiotec.2003.10.027

• Mahyon, N. I. (2017). Structural investigation of alcohol oxidase from Meyerozyma Guilliermondii and the use of its promoter for recombinant protein expression (Master thesis). Universiti Putra Malaysia, Malaysia.

• Martínez, J. L., Liu, L., Petranovic, D., & Nielsen, J. (2012). Pharmaceutical protein production by yeast: Towards production of human blood proteins by microbial fermentation. Current Opinion in Biotechnology, 23, 965-971. https://doi.org/10.1016/j.copbio.2012.03.011

• Mattanovich, D., Branduardi, P., Dato, L., Gasser, B., Sauer, M., & Porro, D. (2012). Recombinant protein production in yeasts. Methods in Molecular Biology, 824, 329-358. https://doi.org/10.1007/978-1-61779-433-9_17

• Mergulhao, F., Summers, D. K., & Monteri, G. A. (2005). Recombinant protein secretion in Escherichia coli. Biotechnology Advances, 23(3), 177-202. https://doi.org/10.1016/j.biotechadv.2004.11.003

• Mousavi, P., Mostafavi, Z., & Morowvat, M. H. (2017). In silico analysis of several signal peptides for the excretory production of reteplase in Escherichia coli. Current Proteomics, 14(4), 326-335. https://doi.org/10.2174/1570164614666170809144446

• Nasir, N. M., Leow, C., Oslan, S., Salleh, A., & Oslan, S. (2020). Molecular expression of a recombinant thermostable bacterial amylase from Geobacillus stearothermophilus SR74 using methanol-free Meyerozyma guilliermondii strain SO yeast system. BioResource, 15(2), 3161-3172

• Negahdaripour, M., Nezafat, N., & Hajighahramani, N. (2017). In silico study of different signal peptides for secretory production of interleukin- 11 in Escherichia coli. Current Protein and Peptide Science, 14(2), 112-121. https://doi.org/10.2174/1570164614666170106110848

• Nevoigt, E. (2008). Progress in metabolic engineering of Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews, 72, 379-412. https:/doi.org/10.1128/MMBR.00025-07

• Ohmuro-Matsuyama, Y., & Yamaji, H. (2017). Modification of a signal sequence for antibody secretion from insect cells. Journal of Cytotechnology, 70(3), 891-898. https://doi.org/10.1007/s10616-017-0109-0

• Oslan, S. N., Salleh, A. B., Rahman, R. A., Basri, M., & Chor, A. L. (2012). Locally isolated yeasts from Malaysia: Identification, phylogenetic study and characterization. Acta Biochimica Polonica, 59 (2), 225-229.

• Oslan, S. N., Salleh, A. B., Rahman, R. A., Zaliha, R. N., Leow, T. C., Sukamat, H., & Basri, M. (2015). A newly isolated yeast as an expression host for recombinant lipase. Cellular and Molecular Biology Letters, 20(2), 279-293. https://doi.org/10.1515/cmble-2015-0015

• Oslan, S. N., Salleh, A. B., Rahman, R. A., Zaliha, R. N., Leow, A. T. C., & Basri, M. (2014). Pichia pastoris as a host to overexpress the thermostable T1 lipase from Geobacillus zalihae. Journal of Biosciences, 3(1), 7-17. https://doi.org/10.5176/2251-3140_3.1.45

• Petersen, T. N., Brunak, S., von Heijne, G., & Nielsen, H. (2011). SignalP 4.0: Discriminating signal peptides from transmembrane regions. Nature Methods, 8, 785-786. https://doi.org/10.1038/nmeth.1701

• Rahman, R. N. Z. A., Razak, C. N., Ampon, K., Basri, M., Yunus, W. M. Z. W., & Salleh, A. B. (1994). Purification and characterization of a heat stable alkaline protease from Bacillus stearothermophilus F1. Applied Microbiology and Biotechnology, 40, 822-827. https://doi.org/10.1007/BF00173982

• Rajewsky, N., Vergassola, M., Gaul, U., & Siggia, E. D. (2002). Computational detection of genomic cis-regulatory modules applied to body patterning in the early Drosophila embryo. BMC Bioinformatics, 3, Article 30. https://doi.org/10.1186/1471-2105-3-30

• Rawlings, N. D., Barrett, A. J., Thomas, P. D., Huang, X., Bateman, A., & Finn, R. D. (2018). The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Research, 46, D624-D632. https://doi.org/10.1093/nar/gkx1134

• Savojardo, C., Martelli, P. L., Fariselli, P., & Casadio, R. (2018). DeepSig: Deep learning improves signal peptide detection in proteins. Journal of Bioinformatics, 34, 1690-1696. https://doi.org/10.1093/bioinformatics/btx818

• Silva, C. I., Teles, H., Moers, A. P., & Eggink, G. (2011). Secreted production of collagen-inspired gel-forming polymers with high thermal stability in Pichia pastoris. Biotechnology and Bioengineering, 108, 2517-525. https://doi.org/10.1002/bit.23228

• Singh, B., Kapur, N., & Kaur, P. (2012). Speech recognition with Hidden Markov Model: A review. International Journal of Advanced Research in Computer Science and Software Engineering, 2(3), 401-403.

• Sinha, J., Plantz, B. A., Inan, M., & Meagher, M. M. (2005). Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: Case study with recombinant ovine interferon-tau. Biotechnology and Bioengineering, 89,102-112. https://doi.org/10.1002/bit.20318

• Sinha, S., van Nimwegen, E., & Sigga, E. D. (2003). A probabilistic method to detect regulatory modules. Journal of Bioinformatics, 19(Suppl. 1), i292-i301. https://doi.org/10.1093/bioinformatics/btg1040

• Song, J., Tan, H., Perry, A. J., Akutsu, T., Webb, G. I., & Whisstock, J. C. (2012). PROSPER: An integrated feature-based tool for predicting protease substrate cleavage sites. PLoS ONE, 7(11), Article e50300. https://doi.org/10.1371/journal.pone.0050300

• Suryawanshi, H. K., & Pandya, N. D. (2017). Screening, identification of alkaline proteases producing fungi from soil of different habitats of Amalner Tahsil [Maharashtra] and their applications. International Journal of Applied Sciences and Biotechnology, 5(3), 397-402. https://doi.org/10.3126/ijasbt.v5i3.18304

• Tang, H., Coram, M., Wang, P., Zhu, X., & Risch, N. (2006). Reconstructing genetic ancestry blocks in admixed individuals. American Journal of Human Genetics, 79, 1-12. https://doi.org/10.1086/504302

• Valli, M., Tatto, N. E., Peymann, A., Gruber, C., Landes, N., Ekker, H., Thallinger, G. G., Mattanovich, D., Gasser, B., & Graf, A. B. (2016). Curation of the genome annotation of Pichia pastoris (Komagataella phaffii) CBS7435 from gene level to protein function. FEMS Yeast Research, 16(6), 1-12. https://doi.org/10.1093/femsyr/fow051

• Van Den Hazel, H. B., Morten, C. K. B., & Jakob, R. W. (1996). Biosynthesis and function of yeast vacuolar proteases: Review. Yeast, 12, 1-16. https://doi.org/10.1002/(SICI)1097-0061(199601)12:1<1

• Van Ooyen, A. J., Dekker, P., Huang, M., Olsthoorn, M. M., Jacobs, D. I., Colussi, P. A., & Taron, C. H. (2006). Heterologous protein production in the yeast Kluyveromyces lactis. FEMS Yeast Research, 6, 381-392. https://doi.org /10.1111/j.1567-1364.2006.00049.x

• Wang, K., Li, M., Hadley, D., Liu, R., Glessner, J., Grant, S. F. A., & Bucan, M. (2007). PennCNV: An integrated hidden Markov model designed for high-resolution copy number variation detection in whole-genome SNP genotyping data. Genome Research, 17(11), 1665-1674. https://doi.org/10.1101/gr.6861907

• Werten, M. W., & de Wolf, F. A. (2005). Reduced proteolysis of secreted gelatin and Yps1-mediated alpha-factor leader processing in a Pichia pastoris kex2 disruptant. Applied and Environmental Microbiology, 71, 2310-2317. https://doi.org/10.1128/AEM.71.5.2310-2317.2005

• Wu, M., Shen, Q. M., Yang, Y., & Zhang, S. (2013). Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HAS/PTH in Pichia pastoris GS115. Journal of Industrial Microbiology and Biotechnology, 40, 589-599. https:doi.org/10.1007/s10295-013-1264-8

• Yao, X. Q., Zhao, H. L., Xue, C., Zhang, W., Xiong, X. H., Wang, Z. W., Li, X. Y., & Liu, Z. M. (2009). Degradation of HSA-AX15 (R13K) when expressed in Pichia pastoris can be reduced via the disruption of YPS1 gene in this yeast. Journal of Biotechnology, 139, 131-136. https://doi.org/10.1016/j.jbiotec.2008.09.006

• Yoon, B. (2009). Hidden Markov Models and their applications in biological sequence analysis. Current Genomics, 10(6), 402-415. https://doi.org/10.2174/138920209789177575

• Zamani, M., Nezafat, N., & Negahdaripour, M. (2015). In silico evaluation of different signal peptides for the secretory production of human growth hormone in E. coli. International Journal of Peptide Research and Therapeutics, 21(3), 261-268. https://doi.org/10.1007/s10989-015-9454-z