Boundaries in metagenomic screenings using lacZα-based vectors.

Metagenomics approaches have been of high relevance for providing enzymes used in diverse industrial applications. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities from a soil sample by using the lacZα -based plasmid pSEVA232. For this, we used a functional screen based on skimmed milk agar and a pH indicator dye for detection of both enzymes, as previously reported in literature. Although we effectively identified 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 lacZ gene present in the original vector. Analyses of the thermodynamic stability of mRNA secondary structures indicated that recovering of positive clones was probably due to higher expression levels of the chimeric lacZα-genes in respect to the original from empty vector. We concluded that this method has a higher tendency for recovery false positive clones, when used in combination with a lacZα-based vector. As these vectors are massively used in functional metagenomic screenings, we highlight the importance of reporting boundaries in established metagenomic screenings methodologies.

Genetically Engineered Proteins to Improve Biomass Conversion: New Advances and Challenges for Tailoring Biocatalysts.

Protein engineering emerged as a powerful approach to generate more robust and efficient biocatalysts for bio-based economy applications, an alternative to ecologically toxic chemistries that rely on petroleum. On the quest for environmentally friendly technologies, sustainable and low-cost resources such as lignocellulosic plant-derived biomass are being used for the production of biofuels and fine chemicals. Since most of the enzymes used in the biorefinery industry act in suboptimal conditions, modification of their catalytic properties through protein rational design and in vitro evolution techniques allows the improvement of enzymatic parameters such as specificity, activity, efficiency, secretability, and stability, leading to better yields in the production lines. This review focuses on the current application of protein engineering techniques for improving the catalytic performance of enzymes used to break down lignocellulosic polymers. We discuss the use of both classical and modern methods reported in the literature in the last five years that allowed the boosting of biocatalysts for biomass degradation.

Improving Saccharomyces cerevisiae acid and oxidative stress resistance using a prokaryotic gene identified by functional metagenomics.

Innovations in obtaining products from lignocellulosic biomass have been largely based on the improvement of microorganisms and enzymes capable of degrading these materials. To complete the whole process, microorganisms must be able to ferment the resulting sugars and tolerate high concentrations of product, osmotic pressure, ion toxicity, temperature, toxic compounds from lignocellulose pretreatment, low pH, and oxidative stress. In this work, we engineered laboratory and industrial Saccharomyces cerevisiae strains by combining a gene (hu) recovered from a metagenomic approach with different native and synthetic promoters to obtain improved acid and oxidative stress resistance. Laboratorial strains harboring hu gene under the control of the synthetic stress responsive PCCW14v5 showed increased survival rates after 2 h exposure to pH 1.5. The hu gene was also able to significantly enhance the tolerance of the industrial strain to high concentrations of H2O2 when combined with PTEF1, PYGP1 or PYGP1v7 after 3 h exposure.

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.

Reverse Engineering of an Aspirin-Responsive Transcriptional Regulator in Escherichia coli.

Bacterial transcription factors (TFs) are key devices for the engineering of complex circuits in many biotechnological applications, yet there are few well-characterized inducer-responsive TFs that could be used in the context of an animal or human host. 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 BenR and XylS. By means of site-directed mutagenesis, we identified a single amino acid position required for efficient inducer recognition and transcriptional activation. Whereas this modification in BenR abolishes protein activity, in XylS, it increases the response to several inducers, 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 an enhanced response to benzoate, 3-methylbenzoate, 2-methylbenzoate, 4-methylbenzoate, salicylic acid, aspirin, and acetylsalicylic acid molecules for eliciting gene expression in E. coli.

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.

Novel Ethanol- and 5-Hydroxymethyl Furfural-Stimulated ß-Glucosidase Retrieved From a Brazilian Secondary Atlantic Forest Soil Metagenome.

Beta-glucosidases are key enzymes involved in lignocellulosic biomass degradation for bioethanol production, which complete the final step during cellulose hydrolysis by converting cellobiose into glucose. Currently, industry requires enzymes with improved catalytic performance or tolerance to process-specific parameters. In this sense, metagenomics has become a powerful tool for accessing and exploring the biochemical biodiversity present in different natural environments. Here, we report the identification of a novel ß-glucosidase from metagenomic DNA isolated from soil samples enriched with decaying plant matter from a Secondary Atlantic Forest region. For this, we employed a functional screening approach using an optimized and synthetic broad host-range vector for library production. The novel ß-glucosidase - named Lfa2 - displays three GH3-family conserved domains and conserved catalytic amino acids D283 and E487. The purified enzyme was most active in pH 5.5 and at 50°C, and showed hydrolytic activity toward several pNP synthetic substrates containing ß-glucose, ß-galactose, ß-xylose, ß-fucose, and α-arabinopyranose, as well as toward cellobiose. Lfa2 showed considerable glucose tolerance, exhibiting an IC50 of 300 mM glucose and 30% of remaining activity in 600 mM glucose. In addition, Lfa2 retained full or slightly enhanced activity in the presence of several metal ions. Further, ß-glucosidase activity was increased by 1.7-fold in the presence of 10% (v/v) ethanol, a concentration that can be reached in conventional fermentation processes. Similarly, Lfa2 showed 1.7-fold enhanced activity at high concentrations of 5-hydroxymethyl furfural, one of the most important cellulase inhibitors in pretreated sugarcane bagasse hydrolysates. Moreover, the synergistic effect of Lfa2 on Bacillus subtilis GH5-CBM3 endoglucanase activity was demonstrated by the increased production of glucose (1.6-fold). Together, these results indicate that ß-glucosidase Lfa2 is a promissory enzyme candidate for utilization in diverse industrial applications, such as cellulosic biomass degradation or flavor enhancement in winemaking and grape processing.

Metagenomic Approaches for Understanding New Concepts in Microbial Science.

Over the past thirty years, since the dawn of metagenomic studies, a completely new (micro) universe was revealed, with the potential to have profound impacts on many aspects of the society. Remarkably, the study of human microbiome provided a new perspective on a myriad of human traits previously regarded as solely (epi-) genetically encoded, such as disease susceptibility, immunological response, and social and nutritional behaviors. In this context, metagenomics has established a powerful framework for understanding the intricate connections between human societies and microbial communities, ultimately allowing for the optimization of both human health and productivity. Thus, we have shifted from the old concept of microbes as harmful organisms to a broader panorama, in which the signal of the relationship between humans and microbes is flexible and directly dependent on our own decisions and practices. In parallel, metagenomics has also been playing a major role in the prospection of "hidden" genetic features and the development of biotechnological applications, through the discovery of novel genes, enzymes, pathways, and bioactive molecules with completely new or improved biochemical functions. Therefore, this review highlights the major milestones over the last three decades of metagenomics, providing insights into both its potentialities and current challenges.