Tuval Ben-Yehezkel

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

BackgroundSelect cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture.ResultsUsing a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins.ConclusionsThe results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production.

Rationally designed, heterologous S. cerevisiae transcripts expose novel expression determinants

Deducing generic causal relations between RNA transcript features and protein expression profiles from endogenous gene expression data remains a major unsolved problem in biology. The analysis of gene expression from heterologous genes contributes significantly to solving this problem, but has been heavily biased toward the study of the effect of 5′ transcript regions and to prokaryotes. Here, we employ a synthetic biology driven approach that systematically differentiates the effect of different regions of the transcript on gene expression up to 240 nucleotides into the ORF. This enabled us to discover new causal effects between features in previously unexplored regions of transcripts, and gene expression in natural regimes. We rationally designed, constructed, and analyzed 383 gene variants of the viral HRSVgp04 gene ORF, with multiple synonymous mutations at key positions along the transcript in the eukaryote S. cerevisiae. Our results show that a few silent mutations at the 5′UTR can have a dramatic effect of up to 15 fold change on protein levels, and that even synonymous mutations in positions more than 120 nucleotides downstream from the ORF 5′end can modulate protein levels up to 160%–300%. We demonstrate that the correlation between protein levels and folding energy increases with the significance of the level of selection of the latter in endogenous genes, reinforcing the notion that selection for folding strength in different parts of the ORF is related to translation regulation. Our measured protein abundance correlates notably(correlation up to r = 0.62 (p=0.0013)) with mean relative codon decoding times, based on ribosomal densities (Ribo-Seq) in endogenous genes, supporting the conjecture that translation elongation and adaptation to the tRNA pool can modify protein levels in a causal/direct manner. This report provides an improved understanding of transcript evolution, design principles of gene expression regulation, and suggests simple rules for engineering synthetic gene expression in eukaryotes.

Processing DNA molecules as text.

Polymerase Chain Reaction (PCR) is the DNA-equivalent of Gutenberg’s movable type printing, both allowing large-scale replication of a piece of text. De novo DNA synthesis is the DNA-equivalent of mechanical typesetting, both ease the setting of text for replication. What is the DNA-equivalent of the word processor? Biology labs engage daily in DNA processing—the creation of variations and combinations of existing DNA—using a plethora of manual labor-intensive methods such as site-directed mutagenesis, error-prone PCR, assembly PCR, overlap extension PCR, cleavage and ligation, homologous recombination, and others. So far no universal method for DNA processing has been proposed and, consequently, no engineering discipline that could eliminate this manual labor has emerged. Here we present a novel operation on DNA molecules, called Y, which joins two DNA fragments into one, and show that it provides a foundation for DNA processing as it can implement all basic text processing operations on DNA molecules including insert, delete, replace, cut and paste and copy and paste. In addition, complicated DNA processing tasks such as the creation of libraries of DNA variants, chimeras and extensions can be accomplished with DNA processing plans consisting of multiple Y operations, which can be executed automatically under computer control. The resulting DNA processing system, which incorporates our earlier work on recursive DNA composition and error correction, is the first demonstration of a unified approach to DNA synthesis, editing, and library construction.

Accurate, Model-Based Tuning of Synthetic Gene Expression Using Introns in S. cerevisiae

Introns are key regulators of eukaryotic gene expression and present a potentially powerful tool for the design of synthetic eukaryotic gene expression systems. However, intronic control over gene expression is governed by a multitude of complex, incompletely understood, regulatory mechanisms. Despite this lack of detailed mechanistic understanding, here we show how a relatively simple model enables accurate and predictable tuning of synthetic gene expression system in yeast using several predictive intron features such as transcript folding and sequence motifs. Using only natural Saccharomyces cerevisiae introns as regulators, we demonstrate fine and accurate control over gene expression spanning a 100 fold expression range. These results broaden the engineering toolbox of synthetic gene expression systems and provide a framework in which precise and robust tuning of gene expression is accomplished.

Mapping the translation initiation landscape of an S. cerevisiae gene using fluorescent proteins.

Accurate and efficient gene expression requires that protein translation initiates from mRNA transcripts with high fidelity. At the same time, indiscriminate initiation of translation from multiple ATG start-sites per transcript has been demonstrated, raising fundamental questions regarding the rate and rationale governing alternative translation initiation. We devised a sensitive fluorescent reporter assay for monitoring alternative translation initiation. To demonstrate it, we map the translation initiation landscape of a Saccharomyces cerevisiae gene (RMD1) with a typical ATG sequence context profile. We found that up to 3%-5% of translation initiation events occur from alternative out-of-frame start codons downstream of the main ATG. Initiation from these codons follows the ribosome scanning model: initiation rates from different start sites are determined by ATG order, rather than their context strength. Genomic analysis of S. cerevisiae further supports the scanning model: ATG codons downstream rather than upstream of the main ATG tend to have higher context scores.

DNA-library assembly programmed by on-demand nano-liter droplets from a custom microfluidic chip

Nanoscale synthetic biology can benefit from programmable nanoliter-scale processing of DNA in microfluidic chips if they are interfaced effectively to biochemical arrays such as microwell plates. Whereas active microvalve chips require complex fabrication and operation, we show here how a passive and readily fabricated microchip can be employed for customizable nanoliter scale pipetting and reaction control involving DNA. This recently developed passive microfluidic device, supporting nanoliter scale combinatorial droplet generation and mixing, is here used to generate a DNA test library with one member per droplet exported to addressed locations on microwell plates. Standard DNA assembly techniques, such as Gibson assembly, compatible with isothermal on-chip operation, are employed and checked using off-chip PCR and assembly PCR. The control of output droplet sequences and mixing performance was verified using dyes and fluorescently labeled DNA solutions, both on-chip and in external capillary channels. Gel electrophoresis of products and DNA sequencing were employed to further verify controlled combination and functional enzymatic assembly. The scalability of the results to larger DNA libraries is also addressed by combinatorial input expansion using sequential injection plugs from a multiwell plate. Hence, the paper establishes a proof of principle of the production of functional combinatorial mixtures at the nanoliter scale for one sequence per well DNA libraries.