Synthetic biology-based cell engineeringof the yeast Pichia pastoris

Background.

Yeasts are important hosts for production of industrial enzymes and biopharmaceutical proteins (Zhu et al. 2019; De et al. 2021). Zero-growth conditions have been identified as favorable for recombinant protein production in yeasts, as they limit the production of the byproduct biomass, which poses severe technical constraints to production processes (Looser et al. 2015, Zavec et al. 2020). Synthetic biology accelerates the development of superior chassis strains, by applying engineering principles and standardized orthologous parts and tools (e.g. switches) to biology and cell engineering (Cao et al. 2018; Gao et al. 2021; Patra et al. 2021; Thak et al. 2020).

Aims and methods.

The aim of this project is to develop and design molecular switches leading to higher protein production in the methylotrophic yeast Pichia pastoris at slow growth.

The first step of this thesis will be the design and selection of suitable tools and parts necessary for regulating the metabolism of slow-growing P. pastoris (using the comprehensive omics dataset of P. pastoris cultivated in retentostats generated in the CD-Laboratory and literature survey). Examples for design of tools and switches for P. pastoris are given by e.g. Ito et al. 2020 or Ergün et al. 2020; these will be used for artificially deregulating metabolic engineering target genes to decouple them from growth rate. The impact of selected molecular switches will then be experimentally verified by implementing them in P. pastoris chassis strains, using state-of-the-art high throughput methods such as Golden Gate Cloning and CRISPR/Cas9 (all established in our laboratory for P. pastoris (Prielhofer et al., 2017, Gassler et al., 2018).

P. pastoris strains containing the most promising modification(s) will then be characterized for their impact on growth and production of recombinant model proteins in small scale and bioreactor cultivations (as described e.g. in Prielhofer et al. 2018). 

The PhD project is part of the CD Laboratory for growth decoupled protein production in yeast, and will be carried out at the Institute of Microbiology and Microbial Biotechnology at the Department of Biotechnology.

Cao J, de la Fuente-Nunez C, Ou RW, Torres MT, Pande SG, Sinskey AJ, Lu TK. 2018. Yeast-Based Synthetic Biology Platform for Antimicrobial Peptide Production. ACS Synth Biol. 7(3):896-902. doi: 10.1021/acssynbio.7b00396.
De S, Mattanovich D, Ferrer P, Gasser B. 2021. Established tools and emerging trends for the production of recombinant proteins and metabolites in Pichia pastoris. Essays Biochem. EBC20200138. doi: 10.1042/EBC20200138.
Ergün BG, Demir İ, Özdamar TH, Gasser B, Mattanovich D, Çalık P. 2020. Engineered Deregulation of Expression in Yeast with Designed Hybrid-Promoter Architectures in Coordination with Discovered Master Regulator Transcription Factor. Adv Biosyst. 4(4):e1900172. doi: 10.1002/adbi.201900172.
Gao J, Jiang L, Lian J. 2021. Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products. Synth Syst Biotechnol. 6(2):110-119. doi: 10.1016/j.synbio.2021.04.005.
Gassler T, Heistinger L, Mattanovich D, Gasser B, Prielhofer R. 2019. CRISPR/Cas9-Mediated Homology-Directed Genome Editing in Pichia pastoris. Methods Mol Biol. 1923:211-225. doi: 10.1007/978-1-4939-9024-5_9.
Ito Y, Terai G, Ishigami M, Hashiba N, Nakamura Y, Bamba T, Kumokita R, Hasunuma T, Asai K, Ishii J, Kondo A. 2020. Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression. Nucleic Acids Res. 48(22):13000-13012. doi: 10.1093/nar/gkaa1066.
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(6 Pt 2):1177-93. doi: 10.1016/j.biotechadv.2015.05.008.
Patra P, Das M, Kundu P, Ghosh A. 2021. Recent advances in systems and synthetic biology approaches for developing novel cell-factories in non-conventional yeasts. Biotechnol Adv. 47:107695. doi: 10.1016/j.biotechadv.2021.107695.
Prielhofer R, Barrero JJ, 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(1):123. doi: 10.1186/s12918-017-0492-3.
Prielhofer R, Reichinger M, Wagner N, Claes K, Kiziak C, Gasser B, Mattanovich D. 2018. Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose-regulated GTH1 promoter of Pichia pastoris. Biotechnol Bioeng. 115(10):2479-2488. doi: 10.1002/bit.26800.
Thak EJ, Yoo SJ, Moon HY, Kang HA. 2020. Yeast synthetic biology for designed cell factories producing secretory recombinant proteins. FEMS Yeast Res. 20(2):foaa009. doi: 10.1093/femsyr/foaa009.
Zhu T, Sun H, Wang M, Li Y. 2019. Pichia pastoris as a Versatile Cell Factory for the Production of Industrial Enzymes and Chemicals: Current Status and Future Perspectives. Biotechnol J. 14(6):e1800694. doi: 10.1002/biot.201800694.
Zavec D, Gasser B, Mattanovich D. 2020. Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications. Biotechnol Bioeng. 117(5):1394-1405. doi: 10.1002/bit.27303.