A Highly Tunable System for the Simultaneous Expression of Multiple Enzymes in <i>Saccharomyces cerevisiae</i>

Control of the expression levels of multiple enzymes in transgenic yeasts is essential for the effective production of complex molecules through fermentation. Here, we propose a tunable strategy for the control of expression levels based on the design of terminator regions and other gene-expression control elements in <i>Saccharomyces cerevisiae</i>. Our genome-integrated system, which is capable of producing high expression levels over a wide dynamic range, will broadly enable metabolic engineering and synthetic biology. We demonstrated that the activities of multiple cellulases and the production of ethanol were doubled in a transgenic yeast constructed with our system compared with those achieved with a standard expression system

Scheme of the combinatorial screening.

<p>Preparation of the 64 (4 × 4 × 4) possible strains. Transformation of cellulase cassettes was performed repeatedly in the order CBH2, EG2, CBH1. Screening of the cellulase activity of the 64 possible strains was also performed.</p

Diagnostic polymerase chain reaction (PCR) assay of the variants obtained from the combinatorial screening.

<p>PCR was performed with the indicated primer sets in Materials and methods. The type of genome-integrated cellulase construct was determined from the lengths of the PCR products. PCR products of the transformants carrying the CBH1 (A, B), CBH2 (C, D), and EG2 constructs (E, F). The PCR products for the core promoter (A, C, E) and terminator regions (B, D, F) were amplified.</p

SDS-PAGE analysis of secreted cellulases.

<p>Lane 1, SW strain; Lane 2, CBH1 strain; Lane 3, CBH2 strain; Lane 4, EG2 strain; Lane 5, CBH1 + CBH2 + CBH3 with <i>TDH3pro</i> + <i>CYC1t</i> strain; Lane 6, HR strain; Lane 7, 3B5 strain; Lane 8, 2D9 strain; Lane 9, 4D4 strain.</p

Cellulase activity of the transgenic strains obtained from the combinatorial screening.

<p>Squares, diamonds, triangles, circles, and inverted triangles represent the HR, 2D9, 3B5, 4D4, and reference strains, respectively. Cellulase secretion was assessed by culturing the cells in yeast extract–peptone–dextrose medium and then measuring the cellulase activity by using Avicel cellulose as the substrate.</p

Relative cellulase activities of 368 combinatorially prepared transformants.

<p>Relative cellulase activity was normalized to the cellulase activity of the HR strain with Avicel cellulose as the substrate. Approximately 15% of the variants had higher cellulase activity than that of the HR strain.</p

Ethanol fermentation by the transgenic strains obtained from the combinatorial screening.

<p>Conversion of Avicel cellulose to ethanol with the external addition of β-glucosidase. The cultures described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144870#pone.0144870.g005" target="_blank">Fig 5</a> legend were used for ethanol fermentation. Symbols are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144870#pone.0144870.g005" target="_blank">Fig 5</a>.</p

A Genome-Wide Activity Assessment of Terminator Regions in <i>Saccharomyces cerevisiae</i> Provides a ″Terminatome″ Toolbox

The terminator regions of eukaryotes encode functional elements in the 3′ untranslated region (3′-UTR) that influence the 3′-end processing of mRNA, mRNA stability, and translational efficiency, which can modulate protein production. However, the contribution of these terminator regions to gene expression remains unclear, and therefore their utilization in metabolic engineering or synthetic genetic circuits has been limited. Here, we comprehensively evaluated the activity of 5302 terminator regions from a total of 5880 genes in the budding yeast <i>Saccharomyces cerevisiae</i> by inserting each terminator region downstream of the P_<i>TDH3</i>- green fluorescent protein (GFP) reporter gene and measuring the fluorescent intensity of GFP. Terminator region activities relative to that of the <i>PGK1</i> standard terminator ranged from 0.036 to 2.52, with a mean of 0.87. We thus could isolate the most and least active terminator regions. The activities of the terminator regions showed a positive correlation with mRNA abundance, indicating that the terminator region is a determinant of mRNA abundance. The least active terminator regions tended to encode longer 3′-UTRs, suggesting the existence of active degradation mechanisms for those mRNAs. The terminator regions of ribosomal protein genes tended to be the most active, suggesting the existence of a common regulator of those genes. The ″terminatome″ (the genome-wide set of terminator regions) thus not only provides valuable information to understand the modulatory roles of terminator regions on gene expression but also serves as a useful toolbox for the development of metabolically and genetically engineered yeast

A Genome-Wide Activity Assessment of Terminator Regions in <i>Saccharomyces cerevisiae</i> Provides a ″Terminatome″ Toolbox

The terminator regions of eukaryotes encode functional elements in the 3′ untranslated region (3′-UTR) that influence the 3′-end processing of mRNA, mRNA stability, and translational efficiency, which can modulate protein production. However, the contribution of these terminator regions to gene expression remains unclear, and therefore their utilization in metabolic engineering or synthetic genetic circuits has been limited. Here, we comprehensively evaluated the activity of 5302 terminator regions from a total of 5880 genes in the budding yeast <i>Saccharomyces cerevisiae</i> by inserting each terminator region downstream of the P_<i>TDH3</i>- green fluorescent protein (GFP) reporter gene and measuring the fluorescent intensity of GFP. Terminator region activities relative to that of the <i>PGK1</i> standard terminator ranged from 0.036 to 2.52, with a mean of 0.87. We thus could isolate the most and least active terminator regions. The activities of the terminator regions showed a positive correlation with mRNA abundance, indicating that the terminator region is a determinant of mRNA abundance. The least active terminator regions tended to encode longer 3′-UTRs, suggesting the existence of active degradation mechanisms for those mRNAs. The terminator regions of ribosomal protein genes tended to be the most active, suggesting the existence of a common regulator of those genes. The ″terminatome″ (the genome-wide set of terminator regions) thus not only provides valuable information to understand the modulatory roles of terminator regions on gene expression but also serves as a useful toolbox for the development of metabolically and genetically engineered yeast

Hybrid Nanocellulosome Design from Cellulase Modules on Nanoparticles: Synergistic Effect of Catalytically Divergent Cellulase Modules on Cellulose Degradation Activity

Cellulosomes, which are assemblies of cellulases with various catalytic functions on a giant scaffoldin protein with a carbohydrate-binding module (CBM), efficiently degrade solid cellulosic biomass by means of synergistically coupled hydrolysis reactions. In this study, we constructed hybrid nanocellulosomes from the biotinylated catalytic domains (CDs) of two catalytically divergent cellulases (an endoglucanase and a processive endoglucanase) and biotinylated CBMs by clustering the domains and modules on streptavidin-conjugated nanoparticles. Nanocellulosomes constructed by separately clustering each type of CD with multiple CBMs on nanoparticles showed 5-fold enhancement in cellulase degradation activity relative to that of the corresponding free CDs, and mixtures of the two types of nanocellulosomes gradually and synergistically enhanced cellulase degradation activity as the CBM valency increased (finally, 2.5 times). Clustering the two types of CD together on the same nanoparticle resulted in a greater synergistic effect that was independent of CBM valency; consequently, nanocellulosomes composed of equal amounts of the endo and endoprocessive CDs clustered on a nanoparticle along with multiple CBMs (CD/CBM = 7:23) showed the best cellulose degradation activity, producing 6.5 and 2.4 times the amount of reducing sugars produced from amorphous and crystalline cellulose, respectively, by the native free CDs and CBMs in the same proportions. Our results demonstrate that hybrid nanocellulosomes constructed from the building blocks of cellulases and cellulosomes modules have the potential to serve as high-performance artificial cellulosomes