Directed Evolution of Brain-Derived Neurotrophic Factor for Improved Folding and Expression in Saccharomyces cerevisiae

ABSTRACTBrain-derived neurotrophic factor (BDNF) plays an important role in nervous system function and has therapeutic potential. Microbial production of BDNF has resulted in a low-fidelity protein product, often in the form of large, insoluble aggregates incapable of binding to cognate TrkB or p75 receptors. In this study, employingSaccharomyces cerevisiaedisplay and secretion systems, it was found that BDNF was poorly expressed and partially inactive on the yeast surface and that BDNF was secreted at low levels in the form of disulfide-bonded aggregates. Thus, for the purpose of increasing the compatibility of yeast as an expression host for BDNF, directed-evolution approaches were employed to improve BDNF folding and expression levels. Yeast surface display was combined with two rounds of directed evolution employing random mutagenesis and shuffling to identify BDNF mutants that had 5-fold improvements in expression, 4-fold increases in specific TrkB binding activity, and restored p75 binding activity, both as displayed proteins and as secreted proteins. Secreted BDNF mutants were found largely in the form of soluble homodimers that could stimulate TrkB phosphorylation in transfected PC12 cells. Site-directed mutagenesis studies indicated that a particularly important mutational class involved the introduction of cysteines proximal to the native cysteines that participate in the BDNF cysteine knot architecture. Taken together, these findings show that yeast is now a viable alternative for both the production and the engineering of BDNF.

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Development and Characterization of Two Types of Surface Displayed Levansucrases for Levan Biosynthesis

Catalysts ◽

10.3390/catal11070757 ◽

2021 ◽

Vol 11 (7) ◽

pp. 757

Author(s):

Huiyi Shang ◽

Danni Yang ◽

Dairong Qiao ◽

Hui Xu ◽

Yi Cao

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Weight ◽

Large Scale ◽

Surface Display ◽

Yeast Surface Display ◽

Fusion Partner ◽

Whole Cell ◽

Food Industries ◽

Yeast Surface ◽

The Stability

Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.

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Directed Evolution of a Bond‐Forming Enzyme: Ultrahigh‐Throughput Screening of Microbial Transglutaminase Using Yeast Surface Display

Chemistry - A European Journal ◽

10.1002/chem.201803485 ◽

2018 ◽

Vol 24 (57) ◽

pp. 15195-15200 ◽

Cited By ~ 12

Author(s):

Lukas Deweid ◽

Lara Neureiter ◽

Simon Englert ◽

Hendrik Schneider ◽

Jakob Deweid ◽

...

Keyword(s):

Directed Evolution ◽

Surface Display ◽

Microbial Transglutaminase ◽

Yeast Surface Display ◽

Bond Forming ◽

Yeast Surface

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Development of High Affinity and High Specificity Inhibitors of Matrix Metalloproteinase 14 through Computational Design and Directed Evolution

Journal of Biological Chemistry ◽

10.1074/jbc.m116.756718 ◽

2017 ◽

Vol 292 (8) ◽

pp. 3481-3495 ◽

Cited By ~ 30

Author(s):

Valeria Arkadash ◽

Gal Yosef ◽

Jason Shirian ◽

Itay Cohen ◽

Yuval Horev ◽

...

Keyword(s):

Directed Evolution ◽

Surface Display ◽

High Specificity ◽

Computational Design ◽

Computational Method ◽

Yeast Surface Display ◽

Mmp Inhibitor ◽

Starting Point ◽

Yeast Surface

Degradation of the extracellular matrices in the human body is controlled by matrix metalloproteinases (MMPs), a family of more than 20 homologous enzymes. Imbalance in MMP activity can result in many diseases, such as arthritis, cardiovascular diseases, neurological disorders, fibrosis, and cancers. Thus, MMPs present attractive targets for drug design and have been a focus for inhibitor design for as long as 3 decades. Yet, to date, all MMP inhibitors have failed in clinical trials because of their broad activity against numerous MMP family members and the serious side effects of the proposed treatment. In this study, we integrated a computational method and a yeast surface display technique to obtain highly specific inhibitors of MMP-14 by modifying the natural non-specific broad MMP inhibitor protein N-TIMP2 to interact optimally with MMP-14. We identified an N-TIMP2 mutant, with five mutations in its interface, that has an MMP-14 inhibition constant (Ki) of 0.9 pm, the strongest MMP-14 inhibitor reported so far. Compared with wild-type N-TIMP2, this variant displays ∼900-fold improved affinity toward MMP-14 and up to 16,000-fold greater specificity toward MMP-14 relative to other MMPs. In an in vitro and cell-based model of MMP-dependent breast cancer cellular invasiveness, this N-TIMP2 mutant acted as a functional inhibitor. Thus, our study demonstrates the enormous potential of a combined computational/directed evolution approach to protein engineering. Furthermore, it offers fundamental clues into the molecular basis of MMP regulation by N-TIMP2 and identifies a promising MMP-14 inhibitor as a starting point for the development of protein-based anticancer therapeutics.

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