Insights into enhancing Centella asiatica organ cell biofactories via hairy root protein profiling.

Recent advancements in plant biotechnology have highlighted the potential of hairy roots as a biotechnological platform, primarily due to their rapid growth and ability to produce specialized metabolites. This study aimed to delve deeper into hairy root development in C. asiatica and explore the optimization of genetic transformation for enhanced bioactive compound production. Previously established hairy root lines of C. asiatica were categorized based on their centelloside production capacity into HIGH, MID, or LOW groups. These lines were then subjected to a meticulous label-free proteomic analysis to identify and quantify proteins. Subsequent multivariate and protein network analyses were conducted to discern proteome differences and commonalities. Additionally, the quantification of rol gene copy numbers was undertaken using qPCR, followed by gene expression measurements. From the proteomic analysis, 213 proteins were identified. Distinct proteome differences, especially between the LOW line and other lines, were observed. Key proteins related to essential processes like photosynthesis and specialized metabolism were identified. Notably, potential biomarkers, such as the Tr-type G domain-containing protein and alcohol dehydrogenase, were found in the HIGH group. The presence of ornithine cyclodeaminase in the hairy roots emerged as a significant biomarker linked with centelloside production capacity lines, indicating successful Rhizobium-mediated genetic transformation. However, qPCR results showed an inconsistency with rol gene expression levels, with the HIGH line displaying notably higher expression, particularly of the rolD gene. The study unveiled the importance of ornithine cyclodeaminase as a traceable biomarker for centelloside production capacity. The strong correlation between this biomarker and the rolD gene emphasizes its potential role in optimizing genetic transformation processes in C. asiatica.

The colors of peroxygenase activity: Colorimetric high-throughput screening assays for directed evolution.

Fungal unspecific peroxygenases (UPOs) are arising as versatile biocatalysts for C-H oxyfunctionalization reactions. In recent years, several directed evolution studies have been conducted to design improved UPO variants. An essential part of this protein engineering strategy is the design of reliable colorimetric high-throughput screening (HTS) assays for mutant library exploration. Here, we present a palette of 12 colorimetric HTS assays along with their step-by-step protocols, which have been validated for directed UPO evolution campaigns. This array of colorimetric assays will pave the way for the discovery and design of new UPO variants.

Enhancing Centelloside Production in Centella asiatica Hairy Root Lines through Metabolic Engineering of Triterpene Biosynthetic Pathway Early Genes.

Centella asiatica is a medicinal plant with a rich tradition of use for its therapeutic properties. Among its bioactive compounds are centellosides, a group of triterpenoid secondary metabolites whose potent pharmacological activities have attracted significant attention. Metabolic engineering has emerged as a powerful biotechnological tool to enhance the production of target compounds. In this study, we explored the effects of overexpressing the squalene synthase (SQS) gene and transcription factor TSAR2 on various aspects of C. asiatica hairy root lines: the expression level of centelloside biosynthetic genes, morphological traits, as well as squalene, phytosterol, and centelloside content. Three distinct categories of transformed lines were obtained: LS, harboring At-SQS; LT, overexpressing TSAR2; and LST, simultaneously carrying both transgenes. These lines displayed noticeable alterations in morphological traits, including changes in branching rate and biomass production. Furthermore, we observed that the expression of T-DNA genes, particularly aux2 and rolC genes, significantly modulated the expression of pivotal genes involved in centelloside biosynthesis. Notably, the LS lines boasted an elevated centelloside content but concurrently displayed reduced phytosterol content, a finding that underscores the intriguing antagonistic relationship between phytosterol and triterpene pathways. Additionally, the inverse correlation between the centelloside content and morphological growth values observed in LS lines was countered by the action of TSAR2 in the LST and LT lines. This difference could be attributed to the simultaneous increase in the phytosterol content in the TSAR2-expressing lines, as these compounds are closely linked to root development. Overall, these discoveries offer valuable information for the biotechnological application of C. asiatica hairy roots and their potential to increase centelloside production.

Toward Kilogram-Scale Peroxygenase-Catalyzed Oxyfunctionalization of Cyclohexane.

Mol-scale oxyfunctionalization of cyclohexane to cyclohexanol/cyclohexanone (KA-oil) using an unspecific peroxygenase is reported. Using AaeUPO from Agrocybe aegerita and simple H2O2 as an oxidant, cyclohexanol concentrations of more than 300 mM (>60% yield) at attractive productivities (157 mM h-1, approx. 15 g L-1 h-1) were achieved. Current limitations of the proposed biooxidation system have been identified paving the way for future improvements and implementation.

Robust Light Driven Enzymatic Oxyfunctionalization via Immobilization of Unspecific Peroxygenase.

Unspecific peroxygenases have attracted interest in synthetic chemistry, especially for the oxidative activation of C-H bonds, as they only require hydrogen peroxide (H2 O2 ) instead of a cofactor. Due to their instability in even small amounts of H2 O2 , different strategies like enzyme immobilization or in situ H2 O2 production have been developed to improve the stability of these enzymes. While most strategies have been studied separately, a combination of photocatalysis with immobilized enzymes was only recently reported. To show the advantages and limiting factors of immobilized enzyme in a photobiocatalytic reaction, a comparison is made between free and immobilized enzymes. Adjustment of critical parameters such as (i) enzyme and substrate concentration, (ii) illumination wavelength and (iii) light intensity results in significantly increased enzyme stabilities of the immobilized variant. Moreover, under optimized conditions a turnover number of 334,500 was reached.

Peroxygenase-Catalysed Selective Oxidation of Silanes to Silanols.

A peroxygenase-catalysed hydroxylation of organosilanes is reported. The recombinant peroxygenase from Agrocybe aegerita (AaeUPO) enabled efficient conversion of a broad range of silane starting materials in attractive productivities (up to 300 mM h-1 ), catalyst performance (up to 84 s-1 and more than 120 000 catalytic turnovers). Molecular modelling of the enzyme-substrate interaction puts a basis for the mechanistic understanding of AaeUPO selectivity.

Genetic approaches in improving biotechnological production of taxanes: An update.

Paclitaxel (PTX) and its derivatives are diterpene alkaloids widely used as chemotherapeutic agents in the treatment of various types of cancer. Due to the scarcity of PTX in nature, its production in cell cultures and plant organs is a major challenge for plant biotechnology. Although significant advances have been made in this field through the development of metabolic engineering and synthetic biology techniques, production levels remain insufficient to meet the current market demand for these powerful anticancer drugs. A key stumbling block is the difficulty of genetically transforming the gymnosperm Taxus spp. This review focuses on the progress made in improving taxane production through genetic engineering techniques. These include the overexpression of limiting genes in the taxane biosynthetic pathway and transcription factors involved in its regulation in Taxus spp. cell cultures and transformed roots, as well as the development and optimization of transformation techniques. Attempts to produce taxanes in heterologous organisms such as bacteria and yeasts are also described. Although promising results have been reported, the transfer of the entire PTX metabolic route has not been possible to date, and taxane biosynthesis is still restricted to Taxus cells and some endophytic fungi. The development of a synthetic organism other than Taxus cells capable of biotechnologically producing PTX will probably have to wait until the complete elucidation of its metabolic pathway.

Repertoire of Computationally Designed Peroxygenases for Enantiodivergent C-H Oxyfunctionalization Reactions.

The generation of enantiodivergent biocatalysts for C-H oxyfunctionalizations is ever more important in modern synthetic chemistry. Here, we have applied the FuncLib algorithm based on phylogenetic and Rosetta calculations to design a diverse repertoire of active, stable, and enantiodivergent fungal peroxygenases. 24 designs, each carrying 4-5 mutations in the catalytic core, were expressed functionally in yeast and benchmarked against characteristic model compounds. Several designs were active and stable in a range of temperature and pH, displaying unprecedented enantiodivergence, changing regioselectivity from alkyl to aromatic hydroxylation, and increasing catalytic efficiencies up to 10-fold, with 15-fold improvements in total turnover numbers over the parental enzyme. We find that this dramatic functional divergence stems from beneficial epistasis among the mutations and an extensive reorganization of the heme channel. Our work demonstrates that FuncLib can rapidly design highly functional libraries enriched in enantioselective peroxygenases not seen in nature for a range of biotechnological applications.

Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids.

The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g L-1 in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (ω-1)-hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer.

Designed High-Redox Potential Laccases Exhibit High Functional Diversity.

White-rot fungi secrete an impressive repertoire of high-redox potential laccases (HRPLs) and peroxidases for efficient oxidation and utilization of lignin. Laccases are attractive enzymes for the chemical industry due to their broad substrate range and low environmental impact. Since expression of functional recombinant HRPLs is challenging, however, iterative-directed evolution protocols have been applied to improve their expression, activity, and stability. We implement a rational, stabilize-and-diversify strategy to two HRPLs that we could not functionally express. First, we use the PROSS stability-design algorithm to allow functional expression in yeast. Second, we use the stabilized enzymes as starting points for FuncLib active-site design to improve their activity and substrate diversity. Four of the FuncLib-designed HRPLs and their PROSS progenitor exhibit substantial diversity in reactivity profiles against high-redox potential substrates, including lignin monomers. Combinations of 3-4 subtle mutations that change the polarity, solvation, and sterics of the substrate-oxidation site result in orders of magnitude changes in reactivity profiles. These stable and versatile HRPLs are a step toward generating an effective lignin-degrading consortium of enzymes that can be secreted from yeast. The stabilize-and-diversify strategy can be applied to other challenging enzyme families to study and expand the utility of natural enzymes.

Using machine learning to link the influence of transferred Agrobacterium rhizogenes genes to the hormone profile and morphological traits in Centella asiatica hairy roots.

Hairy roots are made after the integration of a small set of genes from Agrobacterium rhizogenes in the plant genome. Little is known about how this small set is linked to their hormone profile, which determines development, morphology, and levels of secondary metabolite production. We used C. asiatica hairy root line cultures to determine the putative links between the rol and aux gene expressions with morphological traits, a hormone profile, and centelloside production. The results obtained after 14 and 28 days of culture were processed via multivariate analysis and machine-learning processes such as random forest, supported vector machines, linear discriminant analysis, and neural networks. This allowed us to obtain models capable of discriminating highly productive root lines from their levels of genetic expression (rol and aux genes) or from their hormone profile. In total, 12 hormones were evaluated, resulting in 10 being satisfactorily detected. Within this set of hormones, abscisic acid (ABA) and cytokinin isopentenyl adenosine (IPA) were found to be critical in defining the morphological traits and centelloside content. The results showed that IPA brings more benefits to the biotechnological platform. Additionally, we determined the degree of influence of each of the evaluated genes on the individual hormone profile, finding that aux1 has a significant influence on the IPA profile, while the rol genes are closely linked to the ABA profile. Finally, we effectively verified the gene influence on these two specific hormones through feeding experiments that aimed to reverse the effect on root morphology and centelloside content.

Biotic Elicitors in Adventitious and Hairy Root Cultures: A Review from 2010 to 2022.

One of the aims of plant in vitro culture is to produce secondary plant metabolites using plant cells and organ cultures, such as cell suspensions, adventitious, and hairy roots (among others). In cases where the biosynthesis of a compound in the plant is restricted to a specific organ, unorganized systems, such as plant cell cultures, are sometimes unsuitable for biosynthesis. Then, its production is based on the establishment of organ cultures such as roots or aerial shoots. To increase the production in these biotechnological systems, elicitors have been used for years as a useful tool since they activate secondary biosynthetic pathways that control the flow of carbon to obtain different plant compounds. One important biotechnological system for the production of plant secondary metabolites or phytochemicals is root culture. Plant roots have a very active metabolism and can biosynthesize a large number of secondary compounds in an exclusive way. Some of these compounds, such as tropane alkaloids, ajmalicine, ginsenosides, etc., can also be biosynthesized in undifferentiated systems, such as cell cultures. In some cases, cell differentiation and organ formation is necessary to produce the bioactive compounds. This review analyses the biotic elicitors most frequently used in adventitious and hairy root cultures from 2010 to 2022, focusing on the plant species, the target secondary metabolite, the elicitor and its concentration, and the yield/productivity of the target compounds obtained. With this overview, it may be easier to work with elicitors in in vitro root cultures and help understand why some are more effective than others.

Efficient Base-Catalyzed Kemp Elimination in an Engineered Ancestral Enzyme.

The routine generation of enzymes with completely new active sites is a major unsolved problem in protein engineering. Advances in this field have thus far been modest, perhaps due, at least in part, to the widespread use of modern natural proteins as scaffolds for de novo engineering. Most modern proteins are highly evolved and specialized and, consequently, difficult to repurpose for completely new functionalities. Conceivably, resurrected ancestral proteins with the biophysical properties that promote evolvability, such as high stability and conformational diversity, could provide better scaffolds for de novo enzyme generation. Kemp elimination, a non-natural reaction that provides a simple model of proton abstraction from carbon, has been extensively used as a benchmark in de novo enzyme engineering. Here, we present an engineered ancestral ß-lactamase with a new active site that is capable of efficiently catalyzing Kemp elimination. The engineering of our Kemp eliminase involved minimalist design based on a single function-generating mutation, inclusion of an extra polypeptide segment at a position close to the de novo active site, and sharply focused, low-throughput library screening. Nevertheless, its catalytic parameters (kcat/KM~2·105 M-1 s-1, kcat~635 s-1) compare favorably with the average modern natural enzyme and match the best proton-abstraction de novo Kemp eliminases that are reported in the literature. The general implications of our results for de novo enzyme engineering are discussed.

A Biocatalytic Platform for the Synthesis of Enantiopure Propargylic Alcohols and Amines.

Propargylic alcohols and amines are versatile building blocks in organic synthesis. We demonstrate a straightforward enzymatic cascade to synthesize enantiomerically pure propargylic alcohols and amines from readily available racemic starting materials. In the first step, the peroxygenase from Agrocybe aegerita converted the racemic propargylic alcohols into the corresponding ketones, which then were converted into the enantiomerically pure alcohols using the (R)-selective alcohol dehydrogenase from Lactobacillus kefir or the (S)-selective alcohol dehydrogenase from Thermoanaerobacter brokii. Moreover, an enzymatic Mitsunobu-type conversion of the racemic alcohols into enantiomerically enriched propargylic amines using (R)-selective amine transaminase from Aspergillus terreus or (S)-selective amine transaminase from Chromobacterium violaceum was established. The one-pot two-step cascade reaction yielded a broad range of enantioenriched alcohol and amine products in 70-99% yield.

Surfing the wave of oxyfunctionalization chemistry by engineering fungal unspecific peroxygenases.

The selective insertion of oxygen into non-activated organic molecules has to date been considered of utmost importance to synthesize existing and next generation industrial chemicals or pharmaceuticals. In this respect, the minimal requirements and high activity of fungal unspecific peroxygenases (UPOs) situate them as the jewel in the crown of C-H oxyfunctionalization biocatalysts. Although their limited availability and development has hindered their incorporation into industry, the conjunction of directed evolution and computational design is approaching UPOs to practical applications. In this review, we will address the most recent advances in UPO engineering, both of the long and short UPO families, while discussing the future prospects in this fast-moving field of research.

Stable and Functionally Diverse Versatile Peroxidases Designed Directly from Sequences.

White-rot fungi secrete a repertoire of high-redox potential oxidoreductases to efficiently decompose lignin. Of these enzymes, versatile peroxidases (VPs) are the most promiscuous biocatalysts. VPs are attractive enzymes for research and industrial use but their recombinant production is extremely challenging. To date, only a single VP has been structurally characterized and optimized for recombinant functional expression, stability, and activity. Computational enzyme optimization methods can be applied to many enzymes in parallel but they require accurate structures. Here, we demonstrate that model structures computed by deep-learning-based ab initio structure prediction methods are reliable starting points for one-shot PROSS stability-design calculations. Four designed VPs encoding as many as 43 mutations relative to the wildtype enzymes are functionally expressed in yeast, whereas their wildtype parents are not. Three of these designs exhibit substantial and useful diversity in their reactivity profiles and tolerance to environmental conditions. The reliability of the new generation of structure predictors and design methods increases the scale and scope of computational enzyme optimization, enabling efficient discovery and exploitation of the functional diversity in natural enzyme families directly from genomic databases.

A Peroxygenase-Alcohol Dehydrogenase Cascade Reaction to Transform Ethylbenzene Derivatives into Enantioenriched Phenylethanols.

In this study, we developed a new bienzymatic reaction to produce enantioenriched phenylethanols. In a first step, the recombinant, unspecific peroxygenase from Agrocybe aegerita (rAaeUPO) was used to oxidise ethylbenzene and its derivatives to the corresponding ketones (prochiral intermediates) followed by enantioselective reduction into the desired (R)- or (S)-phenylethanols using the (R)-selective alcohol dehydrogenase (ADH) from Lactobacillus kefir (LkADH) or the (S)-selective ADH from Rhodococcus ruber (ADH-A). In a one-pot two-step cascade, 11 ethylbenzene derivatives were converted into the corresponding chiral alcohols at acceptable yields and often excellent enantioselectivity.

Corrigendum: Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase From Agrocybe aegerita.

[This corrects the article DOI: 10.3389/fbioe.2021.741282.].

Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase from Agrocybe aegerita.

Fungal unspecific peroxygenases (UPOs) are hybrid biocatalysts with peroxygenative activity that insert oxygen into non-activated compounds, while also possessing convergent peroxidative activity for one electron oxidation reactions. In several ligninolytic peroxidases, the site of peroxidative activity is associated with an oxidizable aromatic residue at the protein surface that connects to the buried heme domain through a long-range electron transfer (LRET) pathway. However, the peroxidative activity of these enzymes may also be initiated at the heme access channel. In this study, we examined the origin of the peroxidative activity of UPOs using an evolved secretion variant (PaDa-I mutant) from Agrocybe aegerita as our point of departure. After analyzing potential radical-forming aromatic residues at the PaDa-I surface by QM/MM, independent saturation mutagenesis libraries of Trp24, Tyr47, Tyr79, Tyr151, Tyr265, Tyr281, Tyr293 and Tyr325 were constructed and screened with both peroxidative and peroxygenative substrates. These mutant libraries were mostly inactive, with only a few functional clones detected, none of these showing marked differences in the peroxygenative and peroxidative activities. By contrast, when the flexible Gly314-Gly318 loop that is found at the outer entrance to the heme channel was subjected to combinatorial saturation mutagenesis and computational analysis, mutants with improved kinetics and a shift in the pH activity profile for peroxidative substrates were found, while they retained their kinetic values for peroxygenative substrates. This striking change was accompanied by a 4.5°C enhancement in kinetic thermostability despite the variants carried up to four consecutive mutations. Taken together, our study proves that the origin of the peroxidative activity in UPOs, unlike other ligninolytic peroxidases described to date, is not dependent on a LRET route from oxidizable residues at the protein surface, but rather it seems to be exclusively located at the heme access channel.

Ether Oxidation by an Evolved Fungal Heme-Peroxygenase: Insights into Substrate Recognition and Reactivity.

Ethers can be found in the environment as structural, active or even pollutant molecules, although their degradation is not efficient under environmental conditions. Fungal unspecific heme-peroxygenases (UPO were reported to degrade low-molecular-weight ethers through an H2O2-dependent oxidative cleavage mechanism. Here, we report the oxidation of a series of structurally related aromatic ethers, catalyzed by a laboratory-evolved UPO (PaDa-I) aimed at elucidating the factors influencing this unusual biochemical reaction. Although some of the studied ethers were substrates of the enzyme, they were not efficiently transformed and, as a consequence, secondary reactions (such as the dismutation of H2O2 through catalase-like activity and suicide enzyme inactivation) became significant, affecting the oxidation efficiency. The set of reactions that compete during UPO-catalyzed ether oxidation were identified and quantified, in order to find favorable conditions that promote ether oxidation over the secondary reactions.