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

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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Development of novel whole-cell immunoadsorbents by yeast surface display of the IgG-binding domain

Applied Microbiology and Biotechnology ◽

10.1007/s002530100802 ◽

2001 ◽

Vol 57 (4) ◽

pp. 500-505 ◽

Cited By ~ 38

Author(s):

Nakamura Y. ◽

Shibasaki S. ◽

Ueda M. ◽

Tanaka A. ◽

Fukuda H. ◽

...

Keyword(s):

Surface Display ◽

Binding Domain ◽

Yeast Surface Display ◽

Whole Cell ◽

Igg Binding ◽

Yeast Surface

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Construction and characterization of a thermostable whole-cell chitinolytic enzyme using yeast surface display

World Journal of Microbiology and Biotechnology ◽

10.1007/s11274-014-1681-5 ◽

2014 ◽

Vol 30 (10) ◽

pp. 2577-2585 ◽

Cited By ~ 7

Author(s):

Xiaobo Li ◽

Xiaobao Jin ◽

Xuemei Lu ◽

Fujiang Chu ◽

Juan Shen ◽

...

Keyword(s):

Surface Display ◽

Yeast Surface Display ◽

Chitinolytic Enzyme ◽

Whole Cell ◽

Yeast Surface

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

Applied and Environmental Microbiology ◽

10.1128/aem.01466-14 ◽

2014 ◽

Vol 80 (18) ◽

pp. 5732-5742 ◽

Cited By ~ 17

Author(s):

Michael L. Burns ◽

Thomas M. Malott ◽

Kevin J. Metcalf ◽

Benjamin J. Hackel ◽

Jonah R. Chan ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Directed Evolution ◽

Neurotrophic Factor ◽

Surface Display ◽

Brain Derived Neurotrophic Factor ◽

Binding Activity ◽

Yeast Surface Display ◽

Site Directed Mutagenesis ◽

Secretion Systems ◽

Yeast Surface

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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Expression cassette and plasmid construction for Yeast Surface Display in Saccharomyces cerevisiae

Biotechnology Letters ◽

10.1007/s10529-021-03142-w ◽

2021 ◽

Author(s):

Renan Eugênio Araujo Piraine ◽

Vitória Sequeira Gonçalves ◽

Alceu Gonçalves dos Santos Junior ◽

Rodrigo Casquero Cunha ◽

Pedro Machado Medeiros de Albuquerque ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Surface Display ◽

Yeast Surface Display ◽

Expression Cassette ◽

Plasmid Construction ◽

Yeast Surface

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Consolidated bioprocessing of corn cob-derived hemicellulose: engineered industrial Saccharomyces cerevisiae as efficient whole cell biocatalysts

10.1101/2020.07.01.182345 ◽

2020 ◽

Author(s):

Joana T. Cunha ◽

Aloia Romaní ◽

Kentaro Inokuma ◽

Björn Johansson ◽

Tomohisa Hasunuma ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Ethanol Production ◽

Large Scale ◽

Consolidated Bioprocessing ◽

Surface Display ◽

Total Biomass ◽

Economic Costs ◽

Corn Cob ◽

Direct Production ◽

Whole Cell

AbstractConsolidated bioprocessing, which combines saccharolytic and fermentative abilities in a single microorganism, is receiving increased attention to decrease environmental and economic costs in lignocellulosic biorefineries. Nevertheless, the economic viability of lignocellulosic ethanol is also dependent of an efficient utilization of the hemicellulosic fraction, which is mainly composed of xylose and may comprise up to 40 % of the total biomass. This major bottleneck is mainly due to the necessity of chemical/enzymatic treatments to hydrolyze hemicellulose into fermentable sugars and to the fact that xylose is not readily consumed by Saccharomyces cerevisiae – the most used organism for large-scale ethanol production. In this work, industrial S. cerevisiae strains, presenting robust traits such as thermotolerance and improved resistance to inhibitors, were evaluated as hosts for the cell-surface display of hemicellulolytic enzymes and optimized xylose assimilation, aiming at the development of whole-cell biocatalysts for consolidated bioprocessing of corn cob-derived hemicellulose. These modifications allowed the direct production of ethanol from non-detoxified hemicellulosic liquor obtained by hydrothermal pretreatment of corn cob, reaching an ethanol titer of 11.1 g/L corresponding to a yield of 0.328 gram per gram of potential xylose and glucose, without the need for external hydrolytic catalysts. Also, consolidated bioprocessing of pretreated corn cob was found to be more efficient for hemicellulosic ethanol production than simultaneous saccharification and fermentation with addition of commercial hemicellulases. These results show the potential of industrial S. cerevisiae strains for the design of whole-cell biocatalysts and paves the way for the development of more efficient consolidated bioprocesses for lignocellulosic biomass valorization, further decreasing environmental and economic costs.

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