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Secretion of a low and high molecular weight ?-glycosidase by Yarrowia lipolytica.

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 ?-glycosidases in a comparative cultivation approach. RESULTS:A low sized ?-glucosidase from Pyrococcus furiosus (CelB; 55 kDa) and a large sized ?-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.

SUBMITTER: Swietalski P

PROVIDER: S-EPMC7216700 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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

Swietalski Paul P Hetzel Frank F Seitl Ines I Fischer Lutz L

Microbial cell factories 20200511 1

<h4>Background</h4>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 ...[more]

PMID: 32393258

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Project description:β-Carotene is a provitamin A precursor and an important antioxidant that is used widely in the aquaculture, food, cosmetic, and pharmaceutical industries. Oleaginous Yarrowia lipolytica has been demonstrated as a competitive producer microorganism for the production of hydrophobic β-carotene through rational engineering strategies. However, the limited understanding of the complexity of the metabolic network between carotenoid biosynthesis and other cellular processes has hampered further advancement. Genome-scale mutagenesis and high-throughput screening of mutagenesis libraries have been extensively employed in gene mining or in the identification of key targets associated with particular phenotypes. In this study, we developed a fluorescence-activated cell-sorting approach for the effective high-throughput screening of high-β-carotene-producing strains. Using this approach, millions of mutants were screened rapidly, and new gene targets involved in lipid metabolism, sterol metabolism, signal transduction, and stress response were identified. The disruption of the genes affecting fatty acid oxidation, lipid composition, and sterol transcriptional regulation (4CL-8, GCS, and YIsterTF) increased β-carotene significantly. By engineering these targets in a high-β-carotene production, a strain that produced 9.4 g/L β-carotene was constructed. Here, we used a flow cytometry approach to improve screening efficiency and eliminate the interference of intermediate metabolites. The targets obtained in this study can be used in studies focusing on metabolic engineering in the future for improving carotenoid production. IMPORTANCE β-Carotene is a high-value-added product that is widely used in the aquaculture, food, cosmetic, and pharmaceutical industries. In our previous study, Yarrowia lipolytica has been engineered extensively to produce β-carotene. To further improve its production, high-throughput screening and the identification of new beneficial gene targets are required. Herein, we developed a fluorescence-activated cell-sorting approach for the effective high-throughput screening of high-β-carotene-producing strains. Using this approach, millions of mutants were screened rapidly, and new gene targets involved in lipid metabolism, sterol metabolism, signal transduction, and stress response were identified. The disruption of the genes affecting fatty acid oxidation, lipid composition, and sterol transcriptional regulation (4CL-8, GCS, and YIsterTF) increased β-carotene significantly. By engineering these targets in a high-β-carotene production, a strain that produced 9.4 g/L β-carotene was constructed.

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Dataset Information

Secretion of a low and high molecular weight ?-glycosidase by Yarrowia lipolytica.

Publications

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

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