Characterization and engineering of peroxisome targeting sequences for compartmentalization engineering in Pichia pastoris.
Pichia pastoris
compartmentalization engineering
geraniol
high‐throughput screening
peroxisome targeting signal type 1 (PTS1)
Journal
Biotechnology and bioengineering
ISSN: 1097-0290
Titre abrégé: Biotechnol Bioeng
Pays: United States
ID NLM: 7502021
Informations de publication
Date de publication:
03 Apr 2024
03 Apr 2024
Historique:
revised:
03
03
2024
received:
27
12
2023
accepted:
19
03
2024
medline:
3
4
2024
pubmed:
3
4
2024
entrez:
3
4
2024
Statut:
aheadofprint
Résumé
Peroxisomal compartmentalization has emerged as a highly promising strategy for reconstituting intricate metabolic pathways. In recent years, significant progress has been made in the peroxisomes through harnessing precursor pools, circumventing metabolic crosstalk, and minimizing the cytotoxicity of exogenous pathways. However, it is important to note that in methylotrophic yeasts (e.g. Pichia pastoris), the abundance and protein composition of peroxisomes are highly variable, particularly when peroxisome proliferation is induced by specific carbon sources. The intricate subcellular localization of native proteins, the variability of peroxisomal metabolic pathways, and the lack of systematic characterization of peroxisome targeting signals have limited the applications of peroxisomal compartmentalization in P. pastoris. Accordingly, this study established a high-throughput screening method based on β-carotene biosynthetic pathway to evaluate the targeting efficiency of PTS1s (Peroxisome Targeting Signal Type 1) in P. pastoris. First, 25 putative endogenous PTS1s were characterized and 3 PTS1s with high targeting efficiency were identified. Then, directed evolution of PTS1s was performed by constructing two PTS1 mutant libraries, and a total of 51 PTS1s (29 classical and 22 noncanonical PTS1s) with presumably higher peroxisomal targeting efficiency were identified, part of which were further characterized via confocal microscope. Finally, the newly identified PTS1s were employed for peroxisomal compartmentalization of the geraniol biosynthetic pathway, resulting in more than 30% increase in the titer of monoterpene compared with when the pathway was localized to the cytosol. The present study expands the synthetic biology toolkit and lays a solid foundation for peroxisomal compartmentalization in P. pastoris.
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : National Key Research and Development Program of China
ID : 2021YFC2103200
Organisme : National Natural Science Foundation of China
ID : 22278361
Organisme : National Natural Science Foundation of China
ID : 32200052
Organisme : National Natural Science Foundation of China
ID : 32300053
Organisme : China Postdoctoral Science Foundation
ID : 2023M733096
Organisme : Fundamental Research Funds for the Central Universities
ID : 226-2023-00015
Organisme : Fundamental Research Funds for the Central Universities
ID : 226-2022-00214
Organisme : Fundamental Research Funds for the Central Universities
ID : 226-2023-00085
Informations de copyright
© 2024 Wiley Periodicals LLC.
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