Engineering of Saccharomyces cerevisiae as a consolidated bioprocessing host to produce cellulosic ethanol: Recent advancements and current challenges.

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.

Yeast Surface Display System: Strategies for Improvement and Biotechnological Applications

ABSTRACT Combating climate change and ensuring energy supply to a rapidly growing global population has highlighted the need to replace petroleum fuels with clean, and sustainable renewable fuels. Biofuels offer a solution to safeguard energy security with reduced ecological footprint and process economics. Over the past years, lignocellulosic biomass has become the most preferred raw material for the production of biofuels, such as fuel, alcohol, biodiesel, and biohydrogen. However, the cost-effective conversion of lignocellulose into biofuels remains an unsolved challenge at the industrial scale. Recently, intensive efforts have been made in lignocellulose feedstock and microbial engineering to address this problem. By improving the biological pathways leading to the polysaccharide, lignin, and lipid biosynthesis, limited success has been achieved, and still needs to improve sustainable biofuel production. Impressive success is being achieved by the retouring metabolic pathways of different microbial hosts. Several robust phenotypes, mostly from bacteria and yeast domains, have been successfully constructed with improved substrate spectrum, product yield and sturdiness against hydrolysate toxins. Cyanobacteria is also being explored for metabolic advancement in recent years, however, it also remained underdeveloped to generate commercialized biofuels. The bacterium Escherichia coli and yeast Saccharomyces cerevisiae strains are also being engineered to have cell surfaces displaying hydrolytic enzymes, which holds much promise for near-term scale-up and biorefinery use. Looking forward, future advances to achieve economically feasible production of lignocellulosic-based biofuels with special focus on designing more efficient metabolic pathways coupled with screening, and engineering of novel enzymes. Graphical Abstract

https://www.tandfonline.com/doi/pdf/10.1080/21655979.2022.2051856?needAccess=true

Recent advances in metabolic engineering of microorganisms for advancing lignocellulose-derived biofuels

Lignocellulosic residues have been receiving growing interest as a promising source of polysaccharides, which can be converted into a variety of compounds, ranging from biofuels to bioplastics. Most of these can replace equivalent products traditionally originated from petroleum, hence representing an important environmental advantage. Lignocellulosic materials are theoretically unlimited, cheaper and may not compete with food crops. However, the conversion of these materials to simpler sugars usually requires cellulolytic enzymes. Being still associated with a high cost of production, cellulases are commonly considered as one of the main obstacles in the economic valorization of lignocellulosics. This work provides a brief overview of some of the most studied strategies that can allow an important reduction of cellulases consumption, hence improving the economy of lignocellulosics conversion. Cellulases recycling is initially discussed regarding the main processes to recover active enzymes and the most important factors that may affect enzyme recyclability. Similarly, the potential of enzyme immobilization is analyzed with a special focus on the contributions that some elements of the process can offer for prolonged times of operation and improved enzyme stability and robustness. Finally, the emergent concept of consolidated bioprocessing (CBP) is also described in the particular context of a potential reduction of cellulases consumption.

https://repositorium.sdum.uminho.pt/bitstream/1822/72488/1/document_54402_1.pdf

Strategies towards Reduction of Cellulases Consumption: Debottlenecking the Economics of Lignocellulosics Valorization Processes

Cellulose- and xylan-degrading yeasts: Enzymes, applications and biotechnological potential.

Engineering yeast as a consolidated bioprocessing (CBP) microorganism by surface assembly of cellulosomes has been aggressively utilized for cellulosic ethanol production. However, most of the previous studies focused on Saccharomyces cerevisiae, achieving efficient conversion of phosphoric acid-swollen cellulose (PASC) or microcrystalline cellulose (Avicel) but not carboxymethyl cellulose (CMC) to ethanol, with an average titer below 2 g/L. Harnessing an ultra-high-affinity IM7/CL7 protein pair, here we describe a method to engineer Pichia pastoris with minicellulosomes by in vitro assembly of three recombinant cellulases including an endoglucanase (EG), an exoglucanase (CBH) and a β-glucosidase (BGL), as well as a carbohydrate-binding module (CBM) on the cell surface. For the first time, the engineered yeasts enable efficient and direct conversion of CMC to bioethanol, observing an impressive ethanol titer of 5.1 g/L. The research promotes the application of P. pastoris as a CBP cell factory in cellulosic ethanol production and provides a promising platform for screening the cellulases from different species to construct surface-assembly celluosome.

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Lixia Chen

Papers

Engineering Pichia pastoris with surface-display minicellulosomes for carboxymethyl cellulose hydrolysis and ethanol production.