María-Eugenia Guazzaroni

Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening

There is a growing demand for enzymes with improved catalytic performance or tolerance to process‐specific parameters, and biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation. Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in the genomes of microorganisms found in environmental samples, and provides a set of new technologies directed towards screening for new catalytic activities from environmental samples with potential biotechnology applications. However, biased and low level of expression of heterologous proteins in Escherichia coli together with the use of non‐optimal cloning vectors for the construction of metagenomic libraries generally results in an extremely low success rate for enzyme identification. The bottleneck arising from inefficient screening of enzymatic activities has been addressed from several perspectives; however, the limitations related to biased expression in heterologous hosts cannot be overcome by using a single approach, but rather requires the synergetic implementation of multiple methodologies. Here, we review some of the principal constraints regarding the discovery of new enzymes in metagenomic libraries and discuss how these might be resolved by using synthetic biology methods.

Deciphering the Cis-Regulatory Elements for XYR1 and CRE1 Regulators in Trichoderma reesei

In this work, we report the in silico identification of the cis-regulatory elements for XYR1 and CRE1 proteins in the filamentous fungus Trichoderma reesei, two regulators that play a central role in the expression of cellulase genes. Using four datasets of condition-dependent genes from RNA-seq and RT-qPCR experiments, we performed unsupervised motif discovery and found two short motifs resembling the proposed binding consensus for XYR1 and CRE1. Using these motifs, we analysed the presence and arrangement of putative cis-regulatory elements recognized by both regulators and found that shortly spaced sites were more associated with XYR1- and CRE1-dependent promoters than single, high-score sites. Furthermore, the approach used here allowed the identification of the previously reported XYR1-binding sites from cel7a and xyn1 promoters, and we also mapped the potential target sequence for this regulator at the cel6a promoter that has been suggested but not identified previously. Additionally, seven other promoters (for cel7b, cel61a, cel61b, cel3c, cel3d, xyn3 and swo genes) presented a putative XYR1-binding site, and strong sites for CRE1 were found at the xyr1 and cel7b promoters. Using the cis-regulatory architectures nearly defined for XYR1 and CRE1, we performed genome-wide identification of potential targets for direct regulation by both proteins and important differences on their functional regulons were elucidated. Finally, we performed binding site mapping on the promoters of differentially expressed genes found in T. reesei mutant strains lacking xyr1 or cre1 and found that indirect regulation plays a key role on their signalling pathways. Taken together, the data provided here sheds new light on the mechanisms for signal integration mediated by XYR1 and CRE1 at cellulase promoters.

Mining Novel Constitutive Promoter Elements in Soil Metagenomic Libraries in Escherichia coli

Although functional metagenomics has been widely employed for the discovery of genes relevant to biotechnology and biomedicine, its potential for assessing the diversity of transcriptional regulatory elements of microbial communities has remained poorly explored. Here, we experimentally mined novel constitutive promoter sequences in metagenomic libraries by combining a bi-directional reporter vector, high-throughput fluorescence assays and predictive computational methods. Through the expression profiling of fluorescent clones from two independent soil sample libraries, we have analyzed the regulatory dynamics of 260 clones with candidate promoters as a set of active metagenomic promoters in the host Escherichia coli. Through an in-depth analysis of selected clones, we were able to further explore the architecture of metagenomic fragments and to report the presence of multiple promoters per fragment with a dominant promoter driving the expression profile. These approaches resulted in the identification of 33 novel active promoters from metagenomic DNA originated from very diverse phylogenetic groups. The in silico and in vivo analysis of these individual promoters allowed the generation of a constitutive promoter consensus for exogenous sequences recognizable by E. coli in metagenomic studies. The results presented here demonstrates the potential of functional metagenomics for exploring environmental bacterial communities as a source of novel regulatory genetic parts to expand the toolbox for microbial engineering.

Enhancing Metagenomic Approaches Through Synthetic Biology

Bioactive compounds and enzymes with tolerance to process-specific parameters or improved catalytic performance play a crucial role in the development of applications in the chemical and pharmaceutical industry or energy production. Metagenomics takes advantage of the wealth of biochemical diversity present in the genomes of microorganisms found in environmental samples and provides a set of new technologies directed toward screening for new genes with potential in biotechnological applications. However, despite the vast number of published successful studies using this approach, metagenomic strategies typically have low rates of target discovery, and a number of issues need to be addressed in order to improve the screening efficiency of metagenomic libraries. Current limitations include biases imposed by expression in foreign host organisms, low vector performance in particular hosts, and the absence of suitable screening strategies for many targets. These restrictions cannot be overcome by using a single approach but rather require the synergetic implementation of multiple methodologies. In this chapter, we review some of the principal constraints regarding the discovery of new genes with potential use in biotechnology in metagenomic libraries and discuss how these might be resolved using synthetic biology methods. In addition, we review the state of art of synthetic biology approaches directed to improve the recovery of target genes in metagenomic screenings.

Engineering Complexity in Bacterial Regulatory Circuits for Biotechnological Applications

Engineering microbial systems allows the generation of new technologies having significant impact in the biotechnological industry and on human health. In the past few years, several synthetic biology approaches have been implemented in bacteria to allow precise engineering of novel regulatory circuits for several applications. ABSTRACT Engineering microbial systems allows the generation of new technologies having significant impact in the biotechnological industry and on human health. In the past few years, several synthetic biology approaches have been implemented in bacteria to allow precise engineering of novel regulatory circuits for several applications. The advent of high-throughput technologies and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based DNA editing techniques have been pivotal in this process. Yet, despite the tremendous advances experienced recently, there are still a number of bottlenecks that need to be overcome in order to generate high-performance redesigned living machines, and the use of novel computer-aided approaches would be essential for this task. In this perspective, we discuss some of the main advances in the field of microbial engineering and the new technologies and approaches that should allow the construction of on demand synthetic microbial factories through the redesign of regulatory complexity.

Reverse engineering of an aspirin-responsive regulator in bacteria

Bacterial transcriptional factors (TFs) and their target promoters are key devices for engineering of complex circuits in many biotechnological applications. Yet, there is a dearth of well characterized inducer-responsive TFs that could be used in the context of an animal or human host. In this work we have deciphered the inducer recognition mechanism of two AraC/XylS regulators from Pseudomonas putida (BenR and XylS) for creating a novel expression system responsive to acetyl salicylate (i.e. Aspirin). Using protein homology modeling and molecular docking with the cognate inducer benzoate and a suite of chemical analogues, we identified the conserved binding pocket of these two proteins. Using site directed mutagenesis, we identified a single amino acid position required for efficient inducer recognition and transcriptional activation. While modification of this position in BenR abolishes protein activity, its modification in XylS increases the response to several aromatic compounds, including acetyl salicylic acid to levels close to those achieved by the canonical inducer. Moreover, by constructing chimeric proteins with swapped N-terminal domains, we created novel regulators with mixed promoter and inducer recognition profiles. As a result, a collection of engineered TFs was generated with enhanced response to a well characterized and largely innocuous molecule with a potential for eliciting heterologous expression of bacterial genes in animal carriers.

The art of vector engineering: towards the construction of next-generation genetic tools

When recombinant DNA technology was developed more than 40 years ago, no one could have imagined the impact it would have on both society and the scientific community. In the field of genetic engineering, the most important tool developed was the plasmid vector. This technology has been continuously expanding and undergoing adaptations. Here, we provide a detailed view following the evolution of vectors built throughout the years destined to study microorganisms and their peculiarities, including those whose genomes can only be revealed through metagenomics. We remark how synthetic biology became a turning point in designing these genetic tools to create meaningful innovations. We have placed special focus on the tools for engineering bacteria and fungi (both yeast and filamentous fungi) and those available to construct metagenomic libraries. Based on this overview, future goals would include the development of modular vectors bearing standardized parts and orthogonally designed circuits, a task not fully addressed thus far. Finally, we present some challenges that should be overcome to enable the next generation of vector design and ways to address it.

Engineering the affinity of a family 11 carbohydrate binding module to improve binding of branched over unbranched polysaccharides

Carbohydrate binding modules (CBMs) are non-catalytic domains within larger multidomain polypeptides. The CelH from Ruminoclostridium (Clostridium) thermocellum contains a family 11 CBM (RtCBM11) with high binding affinity for the linear polysaccharide β-glucan, and low affinity for the branched xyloglucan. Screening a random RtCBM11 mutant phage library created by error prone PCR for xyloglucan binding identified RtCBM11 mutants with enhanced xyloglucan affinity. Subsequent recombination of the selected variants by site-directed mutagenesis generated the H102L/Y152F and Y46N/G52D/H102L/Y152F mutants. Fusion of the quadruple RtCBM11 mutant with the xyloglucanase from Aspergillus niveus increased the catalytic efficiency of the enzyme by 38%. Isothermal titration calorimetry demonstrated increased xyloglucan affinity for both mutants and reduced affinity for β-glucan in the H102L/Y152F mutant. Molecular dynamics simulations indicated that the increased xyloglucan specificity results both from formation of a xylosyl binding pocket in the carbohydrate binding cleft, and via modulation of a hydrogen bond network between the oligosaccharide ligand and the protein. These results explain the improved xyloglucan binding in the RtCBM11 H102L/Y152F mutant and advance the understanding of the structural determinants of CBMs binding that discriminate between branched and unbranched polysaccharides.

Boosting Secondary Metabolite Production and Discovery through the Engineering of Novel Microbial Biosensors

Bacteria are a source of a large number of secondary metabolites with several biomedical and biotechnological applications. In recent years, there has been tremendous progress in the development of novel synthetic biology approaches both to increase the production rate of secondary metabolites of interest in native producers and to mine and reconstruct novel biosynthetic gene clusters in heterologous hosts. Here, we present the recent advances toward the engineering of novel microbial biosensors to detect the synthesis of secondary metabolites in bacteria and in the development of synthetic promoters and expression systems aiming at the construction of microbial cell factories for the production of these compounds. We place special focus on the potential of Gram-negative bacteria as a source of biosynthetic gene clusters and hosts for pathway assembly, on the construction and characterization of novel promoters for native hosts, and on the use of computer-aided design of novel pathways and expression systems for secondary metabolite production. Finally, we discuss some of the potentials and limitations of the approaches that are currently being developed and we highlight new directions that could be addressed in the field.

Big trouble with little inserts: boundaries in metagenomic screenings using lacZα based vectors

The vast biochemical repertoire found in microbial communities from a wide-range of environments allows screening and isolation of novel enzymes with improved catalytic features. In this sense, metagenomics approaches have been of high relevance for providing enzymes used in diverse industrial applications. For instance, glycosyl hydrolases, which catalyze the hydrolysis of carbohydrates to sugars, are essential for bioethanol production from renewable resources. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities from microbial communities inhabiting a soil sample by using the lacZα-based plasmid pSEVA232 in the generation of a screenable metagenomic library. For this, we used a functional screen based on skimmed milk agar and a pH indicator dye as previously reported in literature. Although we effectively identified nine positive clones in the screenings, subsequent experiments revealed that this phenotype was not because of the hydrolytic activity encoded in the metagenomic fragments, but rather due to the insertion of small metagenomic DNA fragments in frame within the coding region of the lacZα alpha gene present in the original vector. We concluded that the current method has a higher tendency for false positive recovery of clones, when used in combination with a lacZα-based vector. Finally, we discuss the molecular explanation for positive phenotype recovering and highlight the importance of reporting boundaries in metagenomic screenings methodologies.