NDLI: Secretion of a low and high molecular weight β-glycosidase by Yarrowia lipolytica

Content Provider	Springer Nature : BioMed Central
Author	Swietalski, Paul Hetzel, Frank Seitl, Ines Fischer, Lutz
Abstract	Background The secretory production of recombinant proteins in yeast simplifies isolation and purification but also faces possible complications due to the complexity of the secretory pathway. Therefore, correct folding, maturation and intracellular transport of the recombinant proteins are important processing steps with a higher effort needed for complex and large proteins. The aim of this study was to elucidate the secretion potential of Yarrowia lipolytica for low and high molecular weight β-glycosidases in a comparative cultivation approach. Results A low sized β-glucosidase from Pyrococcus furiosus (CelB; 55 kDa) and a large sized β-galactosidase isolated from the metagenome (M1; 120 kDa) were integrated into the acid extracellular protease locus using the CRISPR–Cas9 system to investigate the size dependent secretion of heterologous proteins in Y. lipolytica PO1f. The recombinant strains were cultivated in the bioreactor for 78 h and the extra- and intracellular enzyme activities were determined. The secretion of CelB resulted in an extracellular volumetric activity of 187.5 µkatoNPGal/Lmedium, while a volumetric activity of 2.98 µkatoNPGal/Lmedium was measured during the M1 production. However, when the amount of functional intra- and extracellular enzyme was investigated, the high molecular weight M1 (85%) was secreted more efficiently than CelB (27%). Real-time PCR experiments showed a linear correlation between the transcript level and extracellular activity for CelB, while a disproportional high mRNA level was observed regarding M1. Interestingly, mass spectrometry data revealed the unexpected secretion of two endogenous intracellular glycolytic enzymes, which is reported for the first time for Y. lipolytica. Conclusion The results of this study provide deeper insights into the secretion potential of Y. lipolytica. A secretion limitation for the low-size CelB was observed, while the large size M1 enzyme was produced in lower amounts but was secreted efficiently. It was shown for the first time that Y. lipolytica is a promising host for the secretion of heterologous high molecular weight proteins (> 100 kDa), although the total secreted amount has to be increased further.
Related Links	https://microbialcellfactories.biomedcentral.com/counter/pdf/10.1186/s12934-020-01358-5.pdf
Ending Page	13
Page Count	13
Starting Page	1
File Format	HTM / HTML
ISSN	14752859
DOI	10.1186/s12934-020-01358-5
Journal	Microbial Cell Factories
Issue Number	1
Volume Number	19
Language	English
Publisher	BioMed Central
Publisher Date	2020-05-11
Access Restriction	Open
Subject Keyword	Applied Microbiology Biotechnology Microbiology Microbial Genetics and Genomics Enzymology Genetic Engineering Yarrowia lipolytica PO1f CRISPR Cas9 Protein secretion Recombinant enzyme production Yeast expression system Unconventional protein secretion CelB M1
Content Type	Text
Resource Type	Article
Subject	Applied Microbiology and Biotechnology Bioengineering Biotechnology
Journal Impact Factor	4.3/2023
5-Year Journal Impact Factor	5.5/2023

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