Genetic manipulation strategies for ethanol production from bioconversion of lignocellulose waste.

Cyprinid herpesvirus 2 (CyHV-2) causes herpesviral hematopoietic necrosis (HVHN) disease outbreaks in farmed Cyprinid fish, which leads to serious economic losses worldwide. Although oral vaccination is considered the most suitable strategy for preventing infectious diseases in farmed fish, so far there is no commercial oral vaccine available for controlling HVNN in gibel carp (C. auratus gibelio). In the present study, we developed for the first time an oral vaccine against CyHV-2 by using yeast cell surface display technology and then investigated the effect of this vaccine in gibel carp. Furthermore, the protective efficacy was evaluated by comparing the immune response of a single vaccination with that of a booster vaccination (booster-vaccinated once 2 weeks after the initial vaccination). Critically, the activities of immune-related enzymes and genes expression in vaccine group, especially in the booster vaccine group, were higher than those in the control group. Moreover, strong innate and adaptive immune responses could be elicited in both mucosal and systemic tissues after receipt of the oral yeast vaccine. To further understand the protective efficacy of this vaccine in gibel carp, we successfully developed the challenge model with CyHV-2. Our results showed the relative percent survival was 66.7% in the booster vaccine group, indicating this oral yeast vaccine is a promising vaccine for controlling CyHV-2 disease in gibel carp aquaculture.

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Gut mucosal immune responses and protective efficacy of oral yeast Cyprinid herpesvirus 2 (CyHV-2) vaccine in Carassius auratus gibelio

Microbial cell surface display technology provides a powerful platform for engineering proteins/peptides with enhanced properties. Compared to the classical intracellular and extracellular expression (secretion) systems, this technology avoids enzyme purification, substrate transport processes, and is an effective solution to enzyme instability. Saccharomyces cerevisiae is well suited to cell surface display as a common cell factory for the production of various fuels and chemicals, with the advantages of large cell size, being a Generally Regarded As Safe (GRAS) organism, and post-translational processing of secreted proteins. In this review, we describe various strategies for constructing modified S. cerevisiae using cell surface display technology and outline various applications of this technology in industrial processes, such as biofuels and chemical products, environmental pollution treatment, and immunization processes. The approaches for enhancing the efficiency of cell surface display are also discussed.

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Saccharomyces cerevisiae cell surface display technology: Strategies for improvement and applications

A need in synthetic biology is the ability to precisely and efficiently make flexible fully designed vectors that addresses challenging cloning strategies of single plasmids that rely on combinatorial co-expression of a multitude of target and bait fusion reporters useful in projects like library screens. For these strategies, the regulatory elements and functional components need to correspond perfectly to project specific sequence elements that facilitate easy exchange of these elements. This requires systematic implementation and building on recent improvements in Golden Gate (GG) that ensures high cloning efficiency for such complex vectors. Currently, this is not addressed in the variety of molecular GG cloning techniques in synthetic biology. Here, we present the bottom-up design and plasmid synthesis to prepare 10 kb functional yeast secrete and display plasmids that uses an optimized version of GG in combination with fluorogen-activating protein reporter technology. This allowed us to demonstrate nanobody/target protein interactions in a single cell, as detected by cell surface retention of secreted target proteins by cognate nanobodies. This validates the GG constructional approach and suggests a new approach for discovering protein interactions. Our GG assembly platform paves the way for vector-based library screening and can be used for other recombinant GG platforms.

Bottom-Up Design: A Modular Golden Gate Assembly Platform of Yeast Plasmids for Simultaneous Secretion and Surface Display of Distinct FAP Fusion Proteins.

Marine chitin upcycling with immobilized chitinolytic enzymes: current state and prospects

Yeast surface display (YSD) is a “whole-cell” platform used for the heterologous expression of proteins immobilized on the yeast’s cell surface. YSD combines the advantages eukaryotic systems offer such as post-translational modifications, correct folding and glycosylation of proteins, with ease of cell culturing and genetic manipulation, and allows of protein immobilization and recovery. Additionally, proteins displayed on the surface of yeast cells may show enhanced stability against changes in temperature, pH, organic solvents, and proteases. This platform has been used to study protein-protein interactions, antibody design and protein engineering. Other applications for YSD include library screening, whole-proteome studies, bioremediation, vaccine and antibiotics development, production of biosensors, ethanol production and biocatalysis. YSD is a promising technology that is not yet optimized for biotechnological applications. This mini review is focused on recent strategies to improve the efficiency and selection of displayed proteins. YSD is presented as a cutting-edge technology for the vectorial expression of proteins and peptides. Finally, recent biotechnological applications are summarized. The different approaches described herein could allow for a better strategy cascade for increasing protein/peptide interaction and production.

Karla V. Teymennet-Ramírez

Papers

Yeast Surface Display System: Strategies for Improvement and Biotechnological Applications