The <i>Pichia pastoris</i> enzyme production platform: From combinatorial library screening to bench-top fermentation on residual cyanobacterial biomass

Korbinian Sinzinger; Ulrike Obst; Samed Güner; Manuel Döring; Magdalena Haslbeck; Doris Schieder; Volker Sieber

doi:10.1016/j.jobab.2023.12.005

Volume 9 Issue 1

Feb. 2024

Turn off MathJax

Article Contents

Article Navigation > Journal of Bioresources and Bioproducts > 2024 > 9(1): 43-57

Korbinian Sinzinger, Ulrike Obst, Samed Güner, Manuel Döring, Magdalena Haslbeck, Doris Schieder, Volker Sieber. The Pichia pastoris enzyme production platform: From combinatorial library screening to bench-top fermentation on residual cyanobacterial biomass[J]. Journal of Bioresources and Bioproducts, 2024, 9(1): 43-57. doi: 10.1016/j.jobab.2023.12.005

Citation:

Korbinian Sinzinger, Ulrike Obst, Samed Güner, Manuel Döring, Magdalena Haslbeck, Doris Schieder, Volker Sieber. The Pichia pastoris enzyme production platform: From combinatorial library screening to bench-top fermentation on residual cyanobacterial biomass[J]. Journal of Bioresources and Bioproducts, 2024, 9(1): 43-57. doi: 10.1016/j.jobab.2023.12.005

Citation:

PDF( 3137 KB)

The Pichia pastoris enzyme production platform: From combinatorial library screening to bench-top fermentation on residual cyanobacterial biomass

doi: 10.1016/j.jobab.2023.12.005

a.
Chair of Chemistry of Biogenic Resources, Campus Straubing for Biotechnology and Sustainability, Technical University of Munich, Straubing 94315, Germany
b.
Catalysis Research Center, Technical University of Munich, Garching 85748, Germany
c.
SynBiofoundry@TUM, Technical University of Munich, Straubing 94315, Germany
d.
School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia 4072, Australia

More Information

Corresponding author: E-mail address: doris.schieder@tum.de (D. Schieder)
Available Online: 2023-12-26
Publish Date: 2024-02-01

Abstract

Abstract

The demand for industrial enzymes is continually rising, fueled by the growing need to shift towards more sustainable industrial processes. However, making efficient enzyme production strains and identifying optimal enzyme expression conditions remains a challenge. Moreover, the production of the enzymes themselves comes with unavoidable impacts, e.g., the need to utilize secondary feedstocks. Here, we take a more holistic view of bioprocess development and report an integrative approach that allows us to rapidly identify improved enzyme expression and secretion conditions and make use of cyanobacterial waste biomass as feed for supporting Pichia pastoris fermentation. We demonstrate these capabilities by producing a phytase secreted by P. pastoris that is grown on cyanobacterium hydrolysate and buffered glycerol-complex (BMGY) medium, with genetic expression conditions identified by high-throughput screening of a randomized secretion library. When our best-performing strain is grown in a fed-batch fermentation on BMGY, we reach over 7000 U/mL in three days.
- Protein secretion,
- Phytase,
- Cyanobacterial biomass,
- Fed-batch fermentation,
- Pichia pastoris

Declaration of Competing Interest
There are no conflicts to declare.
Supplementary materials
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jobab.2023.12.005.
¹ Korbinian Sinzinger and Ulrike Obst contributed equally to this work and should be considered co-first authors.

FullText(HTML)

References(47)

References

Akbarzadeh, A., Dehnavi, E., Aghaeepoor, M., Amani, J., 2015. Optimization of recombinant expression of synthetic bacterial phytase in Pichia pastoris using response surface methodology. Jundishapur J. Microbiol. 8, e27553.

Bae, H.D., Yanke, L.J., Cheng, K.J., Selinger, L.B., 1999. A novel staining method for detecting phytase activity. J. Microbiol. Methods 39, 17–22. doi: 10.1016/S0167-7012(99)00096-2

Bai, Y.G., Yang, P.L., Wang, Y.R., Shi, P.J., Luo, H.Y., Meng, K., Wu, B., Yao, B., 2009. Phytase production by fermentation of recombinant Pichia pastoris in monosodium glutamate wastewater. World J. Microbiol. Biotechnol. 25, 1643–1649. doi: 10.1007/s11274-009-0058-7

Briones-Nagata, M.P., Martinez-Goss, M.R., Hori, K., 2007. A comparison of the morpho-cytology and chemical composition of the two forms of the cyanobacterium, Nostoc commune Vauch., from the Philippines and Japan. J. Appl. Phycol. 19, 675–683. doi: 10.1007/s10811-007-9240-1

Canton, B., Labno, A., Endy, D., 2008. Refinement and standardization of synthetic biological parts and devices. Nat. Biotechnol. 26, 787–793. doi: 10.1038/nbt1413

Celińska, E., Borkowska, M., Białas, W., Korpys, P., Nicaud, J.M., 2018. Robust signal peptides for protein secretion in Yarrowia lipolytica: identification and characterization of novel secretory tags. Appl. Microbiol. Biotechnol. 102, 5221–5233. doi: 10.1007/s00253-018-8966-9

Chandra, R., Iqbal, H.M.N., Vishal, G., Lee, H.S., Nagra, S., 2019. Algal biorefinery: a sustainable approach to valorize algal-based biomass towards multiple product recovery. Bioresour. Technol. 278, 346–359. doi: 10.1016/j.biortech.2019.01.104

Chen, C.C., Cheng, K.J., Ko, T.P., Guo, R.T., 2015. Current progresses in phytase research: three-dimensional structure and protein engineering. ChemBioEng Rev. 2, 76–86. doi: 10.1002/cben.201400026

Chen, C.C., Wu, P.H., Huang, C.T., Cheng, K.J., 2004. A Pichia pastoris fermentation strategy for enhancing the heterologous expression of an Escherichia coli phytase. Enzyme Microb. Technol. 35, 315–320. doi: 10.1016/j.enzmictec.2004.05.007

Choi, S.P., Nguyen, M.T., Sim, S.J., 2010. Enzymatic pretreatment of Chlamydomonas reinhardtii biomass for ethanol production. Bioresour. Technol. 101, 5330–5336. doi: 10.1016/j.biortech.2010.02.026

Curran, K.A., Karim, A.S., Gupta, A., Alper, H.S., 2013. Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications. Metab. Eng. 19, 88–97. doi: 10.1016/j.ymben.2013.07.001

David G., 2018. Chapter 26—economics of food and feed enzymes: status and prospectives. In: Carlos S. N., Vikas K. (Eds. ). Enzymes in Human and Animal Nutrition. USA: Academic Press, 487–514.

de Farias Silva, C.E., Barbera, E., Bertucco, A., 2019. Biorefinery as a promising approach to promote ethanol industry from microalgae and Cyanobacteria. Bioethanol Production from Food Crops. Amsterdam: Elsevier, 343–359.

Delic, M., Valli, M., Graf, A.B., Pfeffer, M., Mattanovich, D., Gasser, B., 2013. The secretory pathway: exploring yeast diversity. FEMS Microbiol. Rev. 37, 872–914. doi: 10.1111/1574-6976.12020

Engler, C., Gruetzner, R., Kandzia, R., Marillonnet, S., 2009. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4, e5553. doi: 10.1371/journal.pone.0005553

Fasahati, P., Wu, W.Z., Maravelias, C.T., 2019. Process synthesis and economic analysis of cyanobacteria biorefineries: a superstructure-based approach. Appl. Energy 253, 113625. doi: 10.1016/j.apenergy.2019.113625

Han, Y.M., Lei, X.G., 1999. Role of glycosylation in the functional expression of anAspergillus nigerPhytase (phyA) inPichia pastoris. Arch. Biochem. Biophys. 364, 83–90. doi: 10.1006/abbi.1999.1115

Helian, Y.K., Gai, Y.M., Fang, H., Sun, Y.M., Zhang, D.W., 2020. A multistrategy approach for improving the expression of E. coli phytase in Pichia pastoris. J. Ind. Microbiol. Biotechnol. 47, 1161–1172. doi: 10.1007/s10295-020-02311-6

Herrmann, K.R., Ruff, A.J., Infanzón, B., Schwaneberg, U., 2019. Engineered phytases for emerging biotechnological applications beyond animal feeding. Appl. Microbiol. Biotechnol. 103, 6435–6448. doi: 10.1007/s00253-019-09962-1

Hesampour, A., Siadat, S.E.R., Malboobi, M.A., Mohandesi, N., Arab, S.S., Ghahremanpour, M.M., 2015. Enhancement of thermostability and kinetic efficiency of Aspergillus niger PhyA phytase by site-directed mutagenesis. Appl. Biochem. Biotechnol. 175, 2528–2541. doi: 10.1007/s12010-014-1440-y

Karbalaei, M., Rezaee, S.A., Farsiani, H., 2020. Pichia pastoris: a highly successful expression system for optimal synthesis of heterologous proteins. J. Cell. Physiol. 235, 5867–5881. doi: 10.1002/jcp.29583

Lee, M.E., DeLoache, W.C., Cervantes, B., Dueber, J.E., 2015. A highly characterized yeast toolkit for modular, multipart assembly. ACS Synth. Biol. 4, 975–986. doi: 10.1021/sb500366v

Liang, S.L., Li, C., Ye, Y.R., Lin, Y., 2013. Endogenous signal peptides efficiently mediate the secretion of recombinant proteins in Pichia pastoris. Biotechnol. Lett. 35, 97–105. doi: 10.1007/s10529-012-1055-8

Liu, W.C., Inwood, S., Gong, T., Sharma, A., Yu, L.Y., Zhu, P., 2019. Fed-batch high-cell-density fermentation strategies for Pichia pastoris growth and production. Crit. Rev. Biotechnol. 39, 258–271. doi: 10.1080/07388551.2018.1554620

Looser, V., Bruhlmann, B., Bumbak, F., Stenger, C., Costa, M., Camattari, A., Fotiadis, D., Kovar, K., 2015. Cultivation strategies to enhance productivity of Pichia pastoris: a review. Biotechnol. Adv. 33, 1177–1193. doi: 10.1016/j.biotechadv.2015.05.008

Madden, K., Tolstorukov, I., Cregg, J., 2015. Electroporation of Pichia pastoris. Genetic Transformation Systems in Fungi, Volume 1. Cham: Springer, 2015: 87–91. doi: 10.1007/978-3-319-10142-2_8

Massahi, A., Çalık, P., 2015. In-silico determination of Pichia pastoris signal peptides for extracellular recombinant protein production. J. Theor. Biol. 364, 179–188. doi: 10.1016/j.jtbi.2014.08.048

Mitra, M., Mishra, S., 2019. Multiproduct biorefinery from Arthrospira spp. towards zero waste: current status and future trends. Bioresour. Technol. 291, 121928. doi: 10.1016/j.biortech.2019.121928

Möllers, K.B., Cannella, D., Jørgensen, H., Frigaard, N.U., 2014. Cyanobacterial biomass as carbohydrate and nutrient feedstock for bioethanol production by yeast fermentation. Biotechnol. Biofuels 7, 64. doi: 10.1186/1754-6834-7-64

Morse, N.J., Gopal, M.R., Wagner, J.M., Alper, H.S., 2017. Yeast Terminator function can be modulated and designed on the basis of predictions of nucleosome occupancy. ACS Synth. Biol. 6, 2086–2095. doi: 10.1021/acssynbio.7b00138

Navone, L., Vogl, T., Luangthongkam, P., Blinco, J.A., Luna-Flores, C., Chen, X.J., von Hellens, J., Speight, R., 2021. Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris. Microb. Cell Fact. 20, 8. doi: 10.1186/s12934-020-01499-7

Obst, U., Lu, T.K., Sieber, V., 2017. A modular toolkit for generating Pichia pastoris secretion libraries. ACS Synth. Biol. 6, 1016–1025. doi: 10.1021/acssynbio.6b00337

Perez-Pinera, P., Han, N.R., Cleto, S., Cao, J.C., Purcell, O., Shah, K.A., Lee, K., Ram, R., Lu, T.K., 2016. Synthetic biology and microbioreactor platforms for programmable production of biologics at the point-of-care. Nat. Commun. 7, 12211. doi: 10.1038/ncomms12211

Prielhofer, R., Barrero, J.J., Steuer, S., Gassler, T., Zahrl, R., Baumann, K., Sauer, M., Mattanovich, D., Gasser, B., Marx, H., 2017. GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris. BMC Syst. Biol. 11, 123. doi: 10.1186/s12918-017-0492-3

Prielhofer, R., Maurer, M., Klein, J., Wenger, J., Kiziak, C., Gasser, B., Mattanovich, D., 2013. Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris. Microb. Cell Fact. 12, 5. doi: 10.1186/1475-2859-12-5

Qin, X., Qian, J., Xiao, C., Zhuang, Y., Zhang, S., Chu, J., 2011. Reliable high-throughput approach for screening of engineered constitutive promoters in the yeast Pichia pastoris. Lett. Appl. Microbiol. 52, 634–641. doi: 10.1111/j.1472-765X.2011.03051.x

Rajkumar, A.S., Varela, J.A., Juergens, H., Daran, J. MG., Morrissey, J.P., 2019. Biological parts for Kluyveromyces marxianus synthetic biology. Front. Bioeng. Biotechnol. 7, 97. doi: 10.3389/fbioe.2019.00097

Ranjan, B., Satyanarayana, T., 2016. Recombinant HAP phytase of the thermophilic mold Sporotrichum thermophile: expression of the Codon-optimized phytase gene in Pichia pastoris and applications. Mol. Biotechnol. 58, 137–147. doi: 10.1007/s12033-015-9909-7

Shen, W., Xue, Y., Liu, Y.Q., Kong, C.X., Wang, X.L., Huang, M.M., Cai, M.H., Zhou, X.S., Zhang, Y.X., Zhou, M., 2016. A novel methanol-free Pichia pastoris system for recombinant protein expression. Microb. Cell Fact. 15, 178. doi: 10.1002/asjc.1200

Sinzinger, K., Schieder, D., Rühmann, B., Sieber, V., 2022. Towards a cyanobacterial biorefinery: carbohydrate fingerprint, biocomposition and enzymatic hydrolysis of Nostoc biomass. Algal Res. 65, 102744. doi: 10.1016/j.algal.2022.102744

Stadlmayr, G., Mecklenbräuker, A., Rothmüller, M., Maurer, M., Sauer, M., Mattanovich, D., Gasser, B., 2010. Identification and characterisation of novel Pichia pastoris promoters for heterologous protein production. J. Biotechnol. 150, 519–529.

Tai, H.M., Yin, L.J., Chen, W.C., Jiang, S.T., 2013. Overexpression of Escherichia coli phytase in Pichia pastoris and its biochemical properties. J. Agric. Food Chem. 61, 6007–6015. doi: 10.1021/jf401853b

Vogl, T., Sturmberger, L., Kickenweiz, T., Wasmayer, R., Schmid, C., Hatzl, A.M., Gerstmann, M.A., Pitzer, J., Wagner, M., Thallinger, G.G., Geier, M., Glieder, A., 2016. A toolbox of diverse promoters related to methanol utilization: functionally verified parts for heterologous pathway expression in Pichia pastoris. ACS Synth. Biol. 5, 172–186. doi: 10.1021/acssynbio.5b00199

Weis, R., Luiten, R., Skranc, W., Schwab, H., Wubbolts, M., Glieder, A., 2004. Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena. FEMS Yeast Res. 5, 179–189. doi: 10.1016/j.femsyr.2004.06.016

Xiong, A.S., Yao, Q.H., Peng, R.H., Han, P.L., Cheng, Z.M., Li, Y., 2005. High level expression of a recombinant acid phytase gene in Pichia pastoris. J. Appl. Microbiol. 98, 418–428. doi: 10.1111/j.1365-2672.2004.02476.x

Yarimizu, T., Nakamura, M., Hoshida, H., Akada, R., 2015. Synthetic signal sequences that enable efficient secretory protein production in the yeast Kluyveromyces marxianus. Microb. Cell Fact. 14, 20. doi: 10.1186/s12934-015-0203-y

Zang, J.K., Zhu, Y.F., Zhou, Y., Gu, C.J., Yi, Y.F., Wang, S.X., Xu, S.Q., Hu, G.W., Du, S.J., Yin, Y.N., Wang, Y.L., Yang, Y., Zhang, X.Y., Wang, H.K., Yin, F.F., Zhang, C., Deng, Q., Xie, Y.H., Huang, Z., 2021. Yeast-produced RBD-based recombinant protein vaccines elicit broadly neutralizing antibodies and durable protective immunity against SARS-CoV-2 infection. Cell Discov. 7, 71.

Relative Articles

Supplements(0)

Cited By

Proportional views