Production of novel Rieske dioxygenase metabolites enabled by enzyme engineering

Rieske dioxygenases are multi-component enzyme systems, naturally found in many soil bacteria, that have been widely applied in the production of fine chemicals, owing to the unique and valuable oxidative dearomatization reactions they catalyze. The range of practical applications for these enzymes in this context has historically been limited, however, due to their limited substrate scope and strict selectivity. In an attempt to overcome these limitations, our research group has employed the tools of enzyme engineering to expand the substrate scope or improve the reactivity of these enzyme systems in specific contexts. Traditionally, enzyme engineering campaigns targeting metalloenzymes have avoided mutations to metal-coordinating residues, based on the assumption that these residues are essential for enzyme activity. Inspired by the success of other recent enzyme engineering reports, our research group investigated the potential to alter or improve the reactivity of Rieske dioxygenases by altering or eliminating iron coordination in the active site of these enzymes. Herein, we report the modification of all three iron-coordinating residues in the active site of toluene dioxygenase both to alternate residues capable of coordinating iron, and to a residue that would eliminate iron coordination. The enzyme variants produced in this way were tested for their activity in the cis-dihydroxylation of a small library of potential aromatic substrates. The results of these studies demonstrated that all three iron-coordinating residues, in their natural state, are essential for enzyme activity in toluene dioxygenase, as the introduction of any mutations at these sites resulted in a complete loss of cis-dihydroxylation activity.

Biotic stress nexus: Integrating various physiological processes in medicinal and aromatic plants

Rieske dioxygenases have a history of utility in organic synthesis, owing to their ability to catalyze the asymmetric dihydroxylation of aromatics to produce chiral diene‐diol metabolites. However, their utility as green‐chemical tools has been limited by steric and electronic constraints on their substrate scopes and their activity. Herein we report the rational engineering of a widely used Rieske dioxygenase, toluene dioxygenase (TDO), to improve the activity of this enzyme system for the dihydroxylation of a synthetically valuable substrate class for which the wild‐type enzyme possesses low activity, the ester‐functionalized aromatics. Through active site targeted mutagenesis and application of a recently reported high throughput screening platform, engineered TDO variants with significantly increased activity in the dihydroxylation of these valuable substrates were identified and characterized, revealing key active site residues that modulate the enzyme's activity and selectivity.

Since prehistoric times, medicinal and aromatic plants (MAPs) have been employed for various therapeutic purposes due to their varied array of pharmaceutically relevant bioactive compounds, i.e. secondary metabolites. However, when secondary metabolites are isolated directly from MAPs, there is occasionally very poor yield and limited synthesis of secondary metabolites from particular tissues and certain developmental stages. Moreover, many MAPs species are in danger of extinction, especially those used in pharmaceuticals, as their natural populations are under pressure from overharvesting due to the excess demand for plant‐based herbal remedies. The extensive use of these metabolites in a number of industrial and pharmaceutical industries has prompted a call for more research into increasing the output via optimization of large‐scale production using plant tissue culture techniques. The potential of plant cells as sources of secondary metabolites can be exploited through a combination of product recovery technology research, targeted metabolite production, and in vitro culture establishment. The plant tissue culture approach provides low‐cost, sustainable, continuous, and viable secondary metabolite production that is not affected by geographic or climatic factors. This study covers recent advancements in the induction of medicinally relevant metabolites, as well as the conservation and propagation of plants by advanced tissue culture technologies.

Rational and mechanistic approaches for improving biocatalyst performance

Herbs, Medicinal and Aromatic Plants (MAPs) have maintained their traditional basic curative role while new trends seek natural alternatives with lesser side effects to using conventional drugs. Besides their traditional culinary and food industry uses, MAPs are intensively consumed as food supplements (food additives). In animal husbandry, feed-additives are used to replace synthetic chemicals and production increasing hormones. Nearly unlimited and increasing huge demand have led to the overexploitation of natural resources, thus endangering not only plant species but incomes, even livelihoods, especially in developing countries. A New Look, a different holistic focus and R+D action is needed to sustain an energetic and socio-economically sound MAPs sector. Guided by international standards (e.g., ISSC-MAP, GA(C)P, FairWild), the sustainable exploitation and management of MAP natural resources have become an imperative from both environment protection and socio-economic points of view. The raw material supplies should be secured by conserving and improving the germplasm of cultivated species, and using both in situ and/or ex-situ technologies. There is a strong demand on the domestication/introduction into cultivation of presently wildcrafted species. Sophisticated in vitro propagation and breeding (selection) technologies aided by advanced phytochemical and molecular biological analytical techniques can further assist this progress. There is also a need to ensure the quality of medicinal plant products by using modern control techniques and applying suitable standards. Based on the already available modern sample preparation techniques (e.g., SPME, SFE, PLE, MAE and SME), the study of the plant metabolome has already yielded successes. Advances in plant genomics and metabolite profiling, also seem to offer unprecedented possibilities in exploring the extraordinary complexity of plant biochemical capacity. State-of-the-art genomics tools can be used to enhance the production of known target metabolites and/or to synthesize entire novel compounds in cultivated plant cells by the so-called combinatorial biochemistry. Ultimate goal of these efforts should be to help improve the traceability and safety (reliability) of natural products, as well as the appropriate policies and legal frameworks to guide the protection, production (including organic production), trade, and applications of medicinal and aromatic plant materials.

The demand for medicinal and aromatic plants (MAPs) has grown significantly in recent years, due to their therapeutic value. Among these, Sideritis cypria Post is a promising yet under-evaluated species. Existing research assessing the effects of nitrogen (N) fertilization, zinc (Zn) foliar applications, and toxic copper (Cu) concentrations often overlooks MAPs such as S. cypria. Additionally, the interactions among these parameters, as well as their combined roles in MAPs plant physiology and secondary metabolite biosynthesis, have yet to be fully elucidated. In this study, hydroponically grown S. cypria plants were cultivated using nutrient solutions (NSs) with different N (75, 150, and 300 mg L-1) and Cu (5 and 100 μM) levels, combined with foliar spraying (0 and 1.74 mM Zn), to evaluate the growth, mineral uptake, secondary metabolites production and stress response. N levels at 75 and 150 mg L-1 resulted in increased dry matter content, whereas fresh biomass production was preserved. Foliar Zn applications enhanced chlorophylls and antioxidants, contingent upon N and Cu in the NS. Increased N accumulation was observed via the increase in N in the NS, while foliar Zn enhanced its uptake at moderate N levels. Excess Cu stimulated its accumulation, while a reduction was observed with foliar Zn at low and high N levels. Excess Cu increased lipid peroxidation (MDA) at low and moderate N in the NS, while foliar Zn decreased both MDA and hydrogen peroxide, contingent upon Cu and N levels. Low-to-moderate N in the NS can be applied under excess Cu without compromising the yield, quality, and safety of S. cypria plants, while foliar Zn can modulate the stress response of plants under excess Cu and the production of secondary metabolites. These results may be utilized for optimizing nutrient management strategies for the cultivation of MAPs, contributing to conservation efforts by supporting the cultivation of endemic species like S. cypria, considering the potential benefits of Zn foliar applications under Cu-contaminated conditions.

In recent decades, the increasing number of degraded lands worldwide makes their rehabilitation essential and crucial. Various techniques have emerged to fulfill these needs but most of them are expensive and difficult to be applied. Revegetation is a cost effective, environmental friendly, and aesthetically pleasing approach suitable for degraded areas. However, the use of edible crops, especially for areas with heavy metals (HM) contamination, is not ecologically suitable because the HM may enter the food chain. Alternatively, non-edible, fast-growing, deep-rooting, and metal-stabilizing plants with high biomass, which can produce high-value products hold a great potential and have been regarded as potential candidates of edible crops. This current review presents the benefits of using aromatic and medicinal plants (AMPs) and their associated microorganisms for revegetation of degraded sites as they are high-value economic crops. We discussed the effect of various stress on productivity of secondary metabolites in AMPs in addition to the potential health risk with human consumption of these plants and their products. A focus was also given to the effect of HM stress on the essential oil (EO) content of certain AMPs. Reported data showed that AMPs growing on HM-contaminated soils are safe products to use as they are not significantly contaminated themselves by HM.

Elucidating the mechanisms, responses and future prospects of medicinal and aromatic plants to elevated CO2 and elevated temperature

Although people's interest in green and healthy plant-based products and natural active ingredients in the cosmetic, pharmaceutical, and food industries is steadily increasing, medicinal and aromatic plants (MAPs) represent a niche crop type.It is possible to increase cultivation and sales of MAPs, by utilizing plant components that are usually discarded. This chapter provides an overview of studies concerning material flows and methods used for sustainable production of valuable metabolites from MAPs between 2018 and 2023. Additionally, it describes new developments and strategies for extraction and isolation, as well as innovative applications. In order to use these valuable resources almost completely, a systematic recycling of the plant material is recommended. This would be a profitable way to increase sustainability in the cultivation and usage of MAPs and provide new opportunities for extraction in plant science.

Heterologous production of Cannabis sativa-derived specialised metabolites of medicinal significance – Insights into engineering strategies

Enzyme-based biocatalysts have a wide range of industrial applications for production, processing and improving the quality attributes of animal feed, beverages, detergent, food, pharmaceutical and textile products. Microorganisms are the major sources of enzymes but natural enzymes as biocatalysts have limitations such as lower catalytic efficiency at ambient conditions, enzyme denaturation due to inability to withstand the pressure of large-scale industrial fermenters and poor productivity in native microbial cultures. Therefore, to meet their increasing demand at the industrial level, native enzymes are often engineered to work under nonphysiological reactions, to design innovative and efficient enzyme catalyzed pathways, and for the production of novel metabolites. Commonly employed enzyme engineering strategies include directed evolution, site-directed mutagenesis, truncation, and terminal fusion. These powerful and revolutionary techniques of enzyme engineering provide excellent opportunities for tailoring existing enzymes and constructing highly efficient novel industrial enzymes for the cost-effective production of value-added products. The present review highlights the major enzyme engineering strategies and methods to modify and optimize key catalytic properties, structure stabilization and introducing novel features to cater growing requirement of enzyme regulated industrial bioprocesses.

CULTIVATION OF MEDICINAL AND AROMATIC PLANTS IN INDIA - A COMMERCIAL APPROACH

The use of human cytochrome P450 (CYP) enzymes is increasing for the production of drug metabolites used for drug safety testing and doping analysis. Major challenges are high-priced cofactors, poor stability, and comparatively low activities. We have shown previously that production of specific metabolites in milligrams to gram scale is feasible using human CYPs recombinantly expressed in fission yeast. In this study, we sought to improve the activities of human CYP3A enzymes by genetic engineering. Two side chains (Pro293 and Arg409) of known activating human CYP3A polymorphic variants were--separately or together--introduced into the wild-type forms of each of the three enzymes CYP3A4, CYP3A5, and CYP3A7, respectively. Different effects of the two mutations and their combination on enzyme activity were monitored using both polar and nonpolar substrates. Interestingly, the CYP3A7 double mutant displayed a strong increase in activity with respect to testosterone 6β-hydroxylation (300 % of wild-type activity) and luciferin-6'-pentafluoro-benzyl ether turnover (400 % compared to wild type), while the single mutant CYP3A5(Pro293) showed 370 and 400 % of wild-type activity towards 6β-hydroxylation of testosterone and 16α-hydroxylation of dehydroepiandrosterone, respectively. Overall, six out of seven newly created mutants displayed increased activity with at least one of the tested substrates. These results support the notion that pharmacogenetic knowledge can directly contribute to the improvement of biotechnological processes.

This study employed shotgun metagenomics to investigate microbial dynamics, phage-bacteria interactions, and functional genes throughout a three-month apple vinegar fermentation process. A total of 5621 microbial species were identified, revealing three distinct phases: (i) Enterobacteria and non-Saccharomyces species dominated the initial substrate; (ii) S. cerevisiae and Leuconostoc pseudomesenteroides prevailed in the intermediate phase; and (iii) acetic acid bacteria (Acetobacter ghanesis and Gluconobacter spp.), alongside non-Saccharomyces species (Pichia kudriavzevii and Malassezia restricta), dominated the final stages. Bacteriophage analysis revealed the presence of phages targeting spoilage bacteria, such as Pseudomonas and Erwinia, suggesting a role in regulating microbial stability and enhancing fermentation control. Functional metagenomic analysis highlighted key pathways associated with microbial growth and metabolite production, including carbohydrate and amino acid metabolism, energy production, and glycan biosynthesis. Enzymes involved in stress adaptation and secondary metabolism, including oxidative phosphorylation and phenolic compound synthesis, demonstrated microbial resilience and their potential role in shaping the product’s sensory and functional properties. Moreover, Enterobacteriaceae species were associated with pectin degradation during the early stages, aiding substrate breakdown. These findings are crucial for microbial and phage management in fermentation technology, offering valuable insights for innovation in the vinegar industry.