Successful reversal of transgene silencing in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii has been successfully engineered to produce compounds of interest following transgene integration and heterologous protein expression. The advantages of this model include the availability of validated tools for bioengineering, its photosynthetic ability, and its potential use as biofuel. Despite this, breakthroughs have been hindered by its ability to silence transgene expression through epigenetic changes. Histone deacetylases (HDAC) are main players in gene expression. We hypothesized that transgene silencing can be reverted with chemical treatments using HDAC inhibitors. To analyze this, we transformed C. reinhardtii, integrating into its genome the mVenus reporter gene under the HSP70-rbcs2 promoter. From 384 transformed clones, 88 (22.9%) displayed mVenus positive (mVenus+ ) cells upon flow-cytometry analysis. Five clones with different fluorescence intensities were selected. The number of integrated copies was measured by qPCR. Transgene expression levels were followed over the growth cycle and upon SAHA treatment, using a microplate reader, flow cytometry, RT-qPCR, and western blot analysis. First, we observed that expression varies with the cell cycle, reaching a maximum level just before the stationary phase in all clones. Second, we uncovered that supplementation with HDAC inhibitors of the hydroxamate family, such as vorinostat (suberoylanilide-hydroxamic-acid, SAHA) at the initiation of culture increases the frequency (% of mVenus+ cells) and the level of transgene expression per cell over the whole growth cycle, through histone deacetylase inhibition. Thus, we propose a new tool to successfully trigger the expression of heterologous proteins in the green algae C. reinhardtii, overcoming its main obstacle as an expression platform.

Bioengineering of the Marine Diatom Phaeodactylum tricornutum with Cannabis Genes Enables the Production of the Cannabinoid Precursor, Olivetolic Acid.

The increasing demand for novel natural compounds has prompted the exploration of innovative approaches in bioengineering. This study investigates the bioengineering potential of the marine diatom Phaeodactylum tricornutum through the introduction of cannabis genes, specifically, tetraketide synthase (TKS), and olivetolic acid cyclase (OAC), for the production of the cannabinoid precursor, olivetolic acid (OA). P. tricornutum is a promising biotechnological platform due to its fast growth rate, amenability to genetic manipulation, and ability to produce valuable compounds. Through genetic engineering techniques, we successfully integrated the cannabis genes TKS and OAC into the diatom. P. tricornutum transconjugants expressing these genes showed the production of the recombinant TKS and OAC enzymes, detected via Western blot analysis, and the production of cannabinoids precursor (OA) detected using the HPLC/UV spectrum when compared to the wild-type strain. Quantitative analysis revealed significant olivetolic acid accumulation (0.6-2.6 mg/L), demonstrating the successful integration and functionality of the heterologous genes. Furthermore, the introduction of TKS and OAC genes led to the synthesis of novel molecules, potentially expanding the repertoire of bioactive compounds accessible through diatom-based biotechnology. This study demonstrates the successful bioengineering of P. tricornutum with cannabis genes, enabling the production of OA as a precursor for cannabinoid production and the synthesis of novel molecules with potential pharmaceutical applications.

Genome Editing by CRISPR-Cas: A Game Change in the Genetic Manipulation of Chlamydomonas.

Microalgae are promising photosynthetic unicellular eukaryotes among the most abundant on the planet and are considered as alternative sustainable resources for various industrial applications. Chlamydomonas is an emerging model for microalgae to be manipulated by multiple biotechnological tools in order to produce high-value bioproducts such as biofuels, bioactive peptides, pigments, nutraceuticals, and medicines. Specifically, Chlamydomonas reinhardtii has become a subject of different genetic-editing techniques adapted to modulate the production of microalgal metabolites. The main nuclear genome-editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and more recently discovered the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas) nuclease system. The latter, shown to have an interesting editing capacity, has become an essential tool for genome editing. In this review, we highlight the available literature on the methods and the applications of CRISPR-Cas for C. reinhardtii genetic engineering, including recent transformation methods, most used bioinformatic tools, best strategies for the expression of Cas protein and sgRNA, the CRISPR-Cas mediated gene knock-in/knock-out strategies, and finally the literature related to CRISPR expression and modification approaches.

Rapid and Efficient Colony-PCR for High Throughput Screening of Genetically Transformed Chlamydomonas reinhardtii.

Microalgae biotechnologies are rapidly developing into new commercial settings. Several high value products already exist on the market, and biotechnological development is focused on genetic engineering of microalgae to open up future economic opportunities for food, fuel and pharmacological production. Colony-polymerase chain reaction (colony-PCR or cPCR) is a critical method for screening genetically transformed microalgae cells. However, the ability to rapidly screen thousands of transformants using the current colony-PCR method, becomes a very laborious and time-consuming process. Herein, the non-homologous transformation of Chlamydomonas reinhardtii using the electroporation and glass beads methods generated more than seven thousand transformants. In order to manage this impressive number of clones efficiently, we developed a high-throughput screening (HTS) cPCR method to rapidly maximize the detection and selection of positively transformed clones. For this, we optimized the Chlamydomonas transformed cell layout on the culture media to improve genomic DNA extraction and cPCR in 96-well plate. The application of this optimized HTS cPCR method offers a rapid, less expensive and reliable method for the detection and selection of microalgae transformants. Our method, which saves up to 80% of the experimental time, holds promise for evaluating genetically transformed cells and selection for microalgae-based biotechnological applications such as synthetic biology and metabolic engineering.

Effect of a low melting temperature mixture on the surface properties of lignocellulosic flax bast fibers.

Bast flax fibers were treated, with or without ultrasound assistance, using a low melting mixture (LMM) composed of lactic acid, d-glucose and water. This LMM treatment affected both lignin and hemicelluloses contents and modified the fibers properties identified as crucial parameters in an industrial context, i.e. coloration, wettability, crystallinity, fibers diameter and chemical composition. Surface chemistry of the fibers were investigated through fluorescent tagged carbohydrates binding modules revealing macromolecular rearrangements responsible of both a fibers crystallinity enhancement and an unexpected hydrophobicity. It has been found that LMM treatments bleach fibers, which is considered a beneficial effect independent of the treatments.

Determination of optimal biomass pretreatment strategies for biofuel production: investigation of relationships between surface-exposed polysaccharides and their enzymatic conversion using carbohydrate-binding modules.

BACKGROUND: Pretreatment of lignocellulosic biomass (LCB) is a key step for its efficient bioconversion into ethanol. Determining the best pretreatment and its parameters requires monitoring its impacts on the biomass material. Here, we used fluorescent protein-tagged carbohydrate-binding modules method (FTCM)-depletion assay to study the relationship between surface-exposed polysaccharides and enzymatic hydrolysis of LCB. RESULTS: Our results indicated that alkali extrusion pretreatment led to the highest hydrolysis rates for alfalfa stover, cattail stems and flax shives, despite its lower lignin removal efficiency compared to alkali pretreatment. Corn crop residues were more sensitive to alkali pretreatments, leading to higher hydrolysis rates. A clear relationship was consistently observed between total surface-exposed cellulose detected by the FTCM-depletion assay and biomass enzymatic hydrolysis. Comparison of bioconversion yield and total composition analysis (by NREL/TP-510-42618) of LCB prior to or after pretreatments did not show any close relationship. Lignin removal efficiency and total cellulose content (by NREL/TP-510-42618) led to an unreliable prediction of enzymatic polysaccharide hydrolysis. CONCLUSIONS: Fluorescent protein-tagged carbohydrate-binding modules method (FTCM)-depletion assay provided direct evidence that cellulose exposure is the key determinant of hydrolysis yield. The clear and robust relationships that were observed between the cellulose accessibility by FTCM probes and enzymatic hydrolysis rates change could be evolved into a powerful prediction tool that might help develop optimal biomass pretreatment strategies for biofuel production.

Predicting the most appropriate wood biomass for selected industrial applications: comparison of wood, pulping, and enzymatic treatments using fluorescent-tagged carbohydrate-binding modules.

BACKGROUND: Lignocellulosic biomass will progressively become the main source of carbon for a number of products as the Earth's oil reservoirs disappear. Technology for conversion of wood fiber into bioproducts (wood biorefining) continues to flourish, and access to reliable methods for monitoring modification of such fibers is becoming an important issue. Recently, we developed a simple, rapid approach for detecting four different types of polymer on the surface of wood fibers. Named fluorescent-tagged carbohydrate-binding module (FTCM), this method is based on the fluorescence signal from carbohydrate-binding modules-based probes designed to recognize specific polymers such as crystalline cellulose, amorphous cellulose, xylan, and mannan. RESULTS: Here we used FTCM to characterize pulps made from softwood and hardwood that were prepared using Kraft or chemical-thermo-mechanical pulping. Comparison of chemical analysis (NREL protocol) and FTCM revealed that FTCM results were consistent with chemical analysis of the hemicellulose composition of both hardwood and softwood samples. Kraft pulping increased the difference between softwood and hardwood surface mannans, and increased xylan exposure. This suggests that Kraft pulping leads to exposure of xylan after removal of both lignin and mannan. Impact of enzyme cocktails from Trichoderma reesei (Celluclast 1.5L) and from Aspergillus sp. (Carezyme 1000L) was investigated by analysis of hydrolyzed sugars and by FTCM. Both enzymes preparations released cellobiose and glucose from pulps, with the cocktail from Trichoderma being the most efficient. Enzymatic treatments were not as effective at converting chemical-thermomechanical pulps to simple sugars, regardless of wood type. FTCM revealed that amorphous cellulose was the primary target of either enzyme preparation, which resulted in a higher proportion of crystalline cellulose on the surface after enzymatic treatment. FTCM confirmed that enzymes from Aspergillus had little impact on exposed hemicelluloses, but that enzymes from the more aggressive Trichoderma cocktail reduced hemicelluloses at the surface. CONCLUSIONS: Overall, this study indicates that treatment with enzymes from Trichoderma is appropriate for generating crystalline cellulose at fiber surface. Applications such as nanocellulose or composites requiring chemical resistance would benefit from this enzymatic treatment. The milder enzyme mixture from Aspergillus allowed for removal of amorphous cellulose while preserving hemicelluloses at fiber surface, which makes this treatment appropriate for new paper products where surface chemical responsiveness is required.

Characterization of a Novel Alkalophilic Lipase From Aneurinibacillus thermoaerophilus: Lid Heterogeneity and Assignment to Family I.5.

Recent investigations of Aneurinibacillus thermoaerophilus strains have allowed identification of a unique solvent tolerant lipase, distinct from known lipases. This work reports the expression and purification of this lipase (LipAT) and the first characterization of its structure and temperature and pH-dependent behaviour. LipAT has a secondary structural content compatible with the canonical lipase α/ß hydrolase fold, and is dimeric at neutral pH. The protein was folded from pH 5 to 10, and association into folded aggregates at pH 7 and 8 likely protected its secondary structures from thermal unfolding. The enzyme was active from 25 to 65 °C under neutral pH, but its maximal activity was detected at pH 10 and 45 °C. The ability of LipAT to recover from high temperature was investigated. Heating at 70 °C and pH 10 followed by cooling prevented the restoration of activity, while similar treatments performed at pH 8 (where folded aggregates may form) allowed recovery of 50% of the initial activity. In silico analyses revealed a high conservation (85% or more) for the main lipase signature sequences in LipAT despite an overall low residue identity (60% identity compared to family I.5 lipases). In contrast, the active site lid region in LipAT is very distinct showing only 25% amino acid sequence identity to other homologous lipases in this region. Comparison of lids among lipases from the I.5 family members and LipAT reveals that this region should be a primary target for elucidation, optimisation and prediction of structure-function relationships in lipases.

Impact of Salt Concentration and pH on Surface Charged Residues: Controlling Protein Association Pathways in Carboxylesterase EstGtA2.

BACKGROUND: Understanding the relationship between enzymatic stability and the amino acid sequence encoding carboxylesterases is of utmost importance. OBJECTIVES: Here we thoroughly characterized the behavior of the carboxylesterase EstGtA2 from Geobacillus thermodenitrificans during thermal denaturation at different pH with various salt concentrations. METHOD: EstGtA2 was characterized by circular dichroism regarding conformation and thermal stability, by dynamic light scattering for detection of association/aggregation, by enzymatic assays for activity and by monitoring the impact of heat treatments on activity. RESULTS: Our investigation revealed a particular dependence between aggregation/association and preservation of secondary structures upon heating in EstGtA2. At pH 7, 8 and 9, depending on salt concentration, a folded but non-native associated state characterised by an apparent particle size of 300 nm resisted secondary structure unfolding up to 95°C. CONCLUSION: The paths leading to various aggregative states were found to be controlled by pH (depending on proximity to pI) and to a lesser extent, ionic strength, suggesting that ionic interactions at the surface of the protein are responsible for behavior of EstGtA2. The various paths available to EstGtA2 could be important for protection of Geobacillus termodenitrificans when exposed to heat stress. The understanding and/or control of these paths would allow for optimal use of EstGtA2 in industrial processes.

Microbial diversity in various types of paper mill sludge: identification of enzyme activities with potential industrial applications.

This study is the first comprehensive investigation of enzyme-producing bacteria isolated from four sludge samples (primary, secondary, press and machine) collected in a Kraft paper mill. Overall, 41 strains encompassing 11 different genera were identified by 16S rRNA gene analysis and biochemical testing. Both biodiversity and enzymatic activities were correlated with sludge composition. Press sludge hosted the largest variety of bacterial strains and enzymatic activities, which included hydrolytic enzymes and ligninolytic enzymes. In contrast, strains isolated from secondary sludge were devoid of several enzymatic activities. Most strains were found to metabolize Kraft liquor at its alkaline pH and to decolorize industrial lignin-mimicking dyes. Resistance to lignin or the ability to metabolize this substrate is a prerequisite to survival in any paper mill sludge type. We demonstrate here that the bacterial strains found in a typical Kraft paper mill represent a source of potential novel enzymes for both industrial applications and bioremediation.

Structural Characterization of a Novel Antioxidant Pigment Produced by a Photochromogenic Microbacterium oxydans Strain.

The Microbacteriaceae family, such as Microbacterium, is well known for its ability to produce carotenoid-type pigments, but little has been published on the structure of such pigments. Here, we isolated the yellow pigment that is responsible for the yellowish color of a Microbacterium oxydans strain isolated from a decomposing stump of a resinous tree. The pigment, which is synthesized when the bacterium is grown under light, was purified and characterized using several spectroscopic analyses, such as ultraviolet-visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FTIR), 1H and 13C nuclear magnetic resonance (1H NMR, 13C NMR), and high-resolution mass spectrometry (HRMS). From these analysis, a molecular formula (C27H42O2) and a chemical structure (8-hydroxymethyl-2,4,12-trimethyl-14-(2,6,6-trimethyl-cyclohex-2-enyl)-teradeca-3,7,9,11,13-pentan-2-ol) were deduced. The chemical properties of the pigment, such as aqueous stability at different pH, stability in different organic solvents, and antioxidant capacity, are also reported. Together, these data and previous studies have resulted in the identification of a new antioxidant pigment produced by M. oxydans. To the best of our knowledge, this is the first thorough investigation of this carotenoid-like pigment in the Microbacterium genera.

Specific tracking of xylan using fluorescent-tagged carbohydrate-binding module 15 as molecular probe.

BACKGROUND: Xylan has been identified as a physical barrier which limits cellulose accessibility by covering the outer surface of fibers and interfibrillar space. Therefore, tracking xylan is a prerequisite for understanding and optimizing lignocellulosic biomass processes. RESULTS: In this study, we developed a novel xylan tracking approach using a two-domain probe called OC15 which consists of a fusion of Cellvibrio japonicus carbohydrate-binding domain 15 with the fluorescent protein mOrange2. The new probe specifically binds to xylan with an affinity similar to that of CBM15. The sensitivity of the OC15-xylan detection approach was compared to that of standard methods such as X-ray photoelectron spectroscopy (XPS) and chemical composition analysis (NREL/TP-510-42618). All three approaches were used to analyze the variations of xylan content of kraft pulp fibers. XPS, which allows for surface analysis of fibers, did not clearly indicate changes in xylan content. Chemical composition analysis responded to the changes in xylan content, but did not give any specific information related to the fibers surface. Interestingly, only the OC15 probe enabled the highly sensitive detection of xylan variations at the surface of kraft pulp fibers. At variance with the other methods, the OC15 probe can be used in a high throughput format. CONCLUSIONS: We developed a rapid and high throughput approach for the detection of changes in xylan exposure at the surface of paper fibers. The introduction of this method into the lignocellulosic biomass-based industries should revolutionize the understanding and optimization of most wood biomass processes.

Development of a high-throughput liquid state assay for lipase activity using natural substrates and rhodamine B.

A fluorescence-based assay for the determination of lipase activity using rhodamine B as an indicator, and natural substrates such as olive oil, is described. It is based on the use of a rhodamine B-natural substrate emulsion in liquid state, which is advantageous over agar plate assays. This high-throughput method is simple and rapid and can be automated, making it suitable for screening and metagenomics application. Reaction conditions such as pH and temperature can be varied and controlled. Using triolein or olive oil as a natural substrate allows monitoring of lipase activity in reaction conditions that are closer to those used in industrial settings. The described method is sensitive over a wide range of product concentrations and offers good reproducibility.

Effect of commercial cellulases and refining on kraft pulp properties: correlations between treatment impacts and enzymatic activity components.

The importance of enzymes as biotechnological catalysts for paper industry is now recognized. In this study, five cellulase formulations were used for fibre modification. The number of PFI revolutions decreased by about 50% while achieving the same freeness value (decrease in CSF by 200 mL) with the enzymatic pretreatment. The physical properties of handsheets were modified after enzymatic pretreatment followed by PFI refining. A slight decrease in tear strength was observed with enzymes C1 and C4 at pH 7 while the most decrease in tear was observed after C2, C3, C5 treatments. C1 and C4 which had xylanase activity improved paper properties, while other enzymes had a negative impact. Therefore, the intricate balance between cellulolytic and hemicellulolytic activity is the key to optimizing biorefining and paper properties. It was also observed that C1 impact was pH dependent, which supports the importance of pH in developing an enzymatic strategy for refining energy reduction.

A comparison of plate assay methods for detecting extracellular cellulase and xylanase activity.

Identification of microorganisms for the production of carbohydrolytic enzymes is extremely important given the increased demand for these enzymes in many industries. To this end, dye-polysaccharide interactions which provide a visual indication of polymer hydrolysis (clear zones or halos) have been used for decades. For the detection of extracellular cellulase or xylanase activity many laboratories use Gram's iodine as the chromogenic dye, as it is a more rapid initial screening method compared to the use of other dyes. Here, we compared Gram's iodine and Congo red as indicators of polysaccharide hydrolysis. We attempted to detect cellulase activity using carboxymethylcellulose, and xylanase activity using birchwood xylan, in fourteen uncharacterized bacteria isolated from wood chips. Our results indicate that Gram's iodine may lead to identification of false positives in a typical screening protocol and that Congo red allows for avoidance of such pitfall. Congo red allowed detection of cellulase activity from live microbial colonies but not Gram's iodine. To confirm this, detection of enzymatic activity was also assessed using cell-free enzyme preparations. Congo red was found to be reliable in detecting cellulase activity with isolated enzymes preparations. Under the same conditions, neither of these dyes detected xylanase activity, despite independent evidence of xylanase activity for one of the preparations. We detected xylanase activity for this particular enzyme preparation using a coloured derivative of xylan (Remazol Brillant Blue R-xylan adduct) that respond to xylan hydrolysis. Our results suggest that methods that rely on interactions between a dye (Congo red or Gram's iodine) and a polymeric substrate (carboxymethylcellulose or birchwood xylan) for indirect detection of hydrolysis may require the use of relevant controls and independent confirmation of enzymatic activities.

Multi-wavelength dye concentration determination for enzymatic assays: evaluation of chromogenic para-nitrophenol over a wide pH range.

Enzymatic assays need robust, rapid colorimetric methods that can follow ongoing reactions. For this, we developed a highly accurate, multi-wavelength detection method that could be used for several systems. Here, it was applied to the detection of para-nitrophenol (pNP) in basic and acidic solutions. First, we confirmed by factor analysis that pNP has two forms, with unique spectral characteristics in the 240 to 600 nm range: Phenol in acidic conditions absorbs in the lower range, whereas phenolate in basic conditions absorbs in the higher range. Thereafter, the method was used for the determination of species concentration. For this, the intensity measurements were made at only two wavelengths with a microtiter plate reader. This yielded total dye concentration, species relative abundance, and solution pH value. The method was applied to an enzymatic assay. For this, a chromogenic substrate that generates pNP after hydrolysis catalyzed by a lipase from the fungus Yarrowia lipolytica was used. Over the pH range of 3-11, accurate amounts of acidic and basic pNP were determined at 340 and 405 nm, respectively. This method surpasses the commonly used single-wavelength assay at 405 nm, which does not detect pNP acidic species, leading to activity underestimations. Moreover, alleviation of this pH-related problem by neutralization is not necessary. On the whole, the method developed is readily applicable to rapid high-throughput of enzymatic activity measurements over a wide pH range.

High transformation efficiency of Bacillus subtilis with integrative DNA using glycine betaine as osmoprotectant.

Electroporation is an important approach for genetic engineering experiments allowing for introduction of foreign DNA in a selected host. Here, we describe for the first time the use of glycine betaine as an osmoprotectant for electroporation of gram-positive bacteria Bacillus subtilis. High electroporation efficiency (up to 5×10(5) cfu/µg) was obtained using 7.5% glycine betaine. The new method improved the transformation efficiency of B. subtilis with linear integrative DNA nearly 700-fold compared with existing Bacillus transformation techniques.

Identification of thermophilic bacterial strains producing thermotolerant hydrolytic enzymes from manure compost.

Ten thermophilic bacterial strains were isolated from manure compost. Phylogenetic analysis based on 16S rRNA genes and biochemical characterization allowed identification of four different species belonging to four genera: Geobacillus thermodenitrificans, Bacillus smithii, Ureibacillus suwonensis and Aneurinibacillus thermoaerophilus. PCR-RFLP profiles of the 16S-ITS-23S rRNA region allowed us to distinguish two subgroups among the G. thermodenitrificans isolates. Isolates were screened for thermotolerant hydrolytic activities (60-65°C). Thermotolerant lipolytic activities were detected for G. thermodenitrificans, A. thermoaerophilus and B. smithii. Thermotolerant protease, α-amylase and xylanase activities were also observed in the G. thermodenitrificans group. These species represent a source of potential novel thermostable enzymes for industrial applications.

N-terminal purification tag alters thermal stability of the carboxylesterase EstGtA2 from G. thermodenitrificans by impairing reversibility of thermal unfolding.

The novel thermostable carboxylesterase EstGtA2 from G. thermodenitrificans (accession no. AEN92268) was functionally expressed and purified using an N-terminal fusion tag peptide. We recently reported general properties of the recombinant enzyme. Here we report preliminary data on thermal stability of EstGtA2 and of its tagged form. Conformational stability was investigated using circular dichroism and correlated with residual activity measurements using a colorimetric assay. The tag peptide had no considerable impact on the apparent melting temperature: T(m) value = 64.8°C (tagged) and 65.7°C (cleaved) at pH 8. After thermal unfolding, the tag-free enzyme rapidly recovered initial activity at 25°C (1.2 Umg(-1)), which was corroborated by substantial refolding (83%) as determined by far-UV CD transitions. However, after thermal unfolding, the purification tag drastically decreased specific activity at 25°C (0.07 Umg(-1)). This was corroborated by the absence of refolding transition. Although the purification tag has no undesirable impact on activity before thermal unfolding as well as on Tm, it drastically hinders EstGtA2 refolding resulting in a major loss of thermal stability.

A novel thermostable carboxylesterase from Geobacillus thermodenitrificans: evidence for a new carboxylesterase family.

A novel gene encoding an esterase from Geobacillus thermodenitrificans strain CMB-A2 was cloned, sequenced and functionally expressed in Escherichia coli M15. Sequence analysis revealed an open reading frame of 747 bp corresponding to a polypeptide of 249 amino acid residues (named EstGtA2). After purification, a specific activity of 2.58 U mg(-1) was detected using p-NP caprylate (C8) at 50 degrees C and pH 8.0 (optimal conditions). The enzyme catalyses the hydrolysis of triglycerides (tributyrin) and a variety of p-nitrophenyl esters with different fatty acyl chain length (C4-C16). The enzyme has potential for various industrial applications since it is characterized by its activity under a wide range of pH, from 25 to 65 degrees C. Using Geobacillus stearothermophilus Est30 esterase structure as template, a model of EstGtA2 was built using ESyPred3D. Analysis of this structural model allowed identifying putative sequence features that control EstGtA2 enzymatic properties. Based on sequence properties, multiple sequence comparisons and phylogenetic analyses, this enzyme appears to belong to a new family of carboxylesterases.