Biorefinery Applications Research Articles

Adaptive laboratory evolution of Lipomyces starkeyi for high production of lignin derivative alcohol and lipids with comparative untargeted metabolomics-based analysis.

Adaptive laboratory evolution (ALE) is an impactful technique for cultivating microorganisms to adapt to specific environmental circumstances or substrates through iterative growth and selection. This study utilized an adaptive laboratory evolution method on Lipomyces starkeyi for high tolerance in producing lignin derivative alcohols and lipids from syringaldehyde. Afterward, untargeted metabolomics analysis was employed to find the key metabolites that play important roles in the better performance of evolved strains compared to the wild type. Lignin, a prominent constituent of plant biomass, is a favorable source material for the manufacture of biofuel and lipids. Nevertheless, the effective transformation of chemicals produced from lignin into products with high economic worth continues to be a difficult task. In this study, we exposed L. starkeyi to a series of flask passaging experiments while applying selective pressure to facilitate its adaptation to syringaldehyde, a specific type of lignin monomeric aldehyde. Using ALE, we successfully developed a new strain, DALE-22, which can synthesize syringyl alcohol up to 18.74 mM from 22.28 mM syringaldehyde with 41.9% lipid accumulation. In addition, a comprehensive examination of untargeted metabolomics identified six specific crucial metabolites linked to the improved tolerance of the evolved strain in the utilization of syringaldehyde, including 2-aminobutyric acid, allantoin, 4-hydroxyphenethyl alcohol, 2-aminoethanol, tryptophan, and 5-aminovaleric acid. The results of our study reveal how L. starkeyi adapts to using substrates produced from lignin. These findings offer important information for developing strategies to improve the process of converting lignin into valuable products for sustainable biorefinery applications.

N:P ratio and salinity as keys: A study on optimizing biomass and lipid production in marine Chlorella sp. NITT 02 and Picochlorum sp. NITT 04 for biodiesel production

A first study on nitrogen to phosphorus (N:P) supply ratio with different N sources (NaNO3, urea, and L-Asparagine) and salinity within the cultivation medium of microalgal species towards biomass and lipid enhancement is demonstrated. Among the three nitrogen sources, L-Asparagine at N:P supply ratio of 75:1 and 100:1 gave the highest biomass concentration for the tested microalgal species. For chlorella sp. NITT 02, the DCW of 1.257 ± 0.003 g L−1 at N:P = 100:1 and 1.256 ± 0.005 g L−1 at N:P = 75:1 was obtained for L-Asaparagine, followed by urea (N:P = 100:1 → 0.842 ± 0.002 g L−1; N:P = 75:1 → 0.843 ± 0.004 g L−1). The DCW of Picochlorum sp. NITT 04 with L-Asparagine (N:P = 100:1 → 0.964 ± 0.026 g L−1; N:P = 75:1 → 0.965 ± 0.034 g L−1) and urea (N:P = 100:1 → 0.911 ± 0.012 g L−1; N:P = 75:1 → 0.908 ± 0.01 g L−1) was attained maximum than NaNO3. The systematic exploration of the interaction between N:P ratio and salinity towards lipid content enhancement were performed by RSM-CCD approach. The optimal N:P ratio of 1.4:0.6 and salinity of 7.5 ppt resulted in maximum lipid content of 49.252 ± 0.187 % for Chlorella sp. NITT 02 with the total FAME content of 93.49 %. For Picochlorum sp. NITT 04, the optimal N:P ratio of 1.4:0.6 and salinity of 24 ppt resulted in maximum lipid content of 49.717 ± 0.073 % with the total FAME content of 91.71 %. The FAME analysis confirms the use of tested microalgal species towards biodiesel production and concurrent use of residual biomass for biorefinery applications.

Room-temperature [C2mim]OAc crystal

1-Ethyl-3-methylimidazolium acetate ([C2mim]OAc) is the most commonly used cellulose-dissolving ionic liquid and has been extensively studied for biorefinery applications. Although [C2mim]OAc is generally considered to be a liquid, we found [C2mim]OAc crystals at room temperature (around 25°C) with a melting point of 44°C. A plausible reason for the crystallization is shearing stress. [C2mim]OAc seed crystals led to the crystallization of liquid [C2mim]OAc.

Biorefinery-oriented pyrolysis and anaerobic fermentation synergies: Leveraging biomass tar for enhanced chemical production from food waste

The carboxylic platform via mixed culture anaerobic fermentation holds significant potential for biorefinery applications. However, effective and economical methanogenesis inhibition remains crucial for maximizing production efficiency. This study investigates the use of biomass tar, derived from the pyrolysis of rice husk, as a methanogenesis inhibitor to enhance the anaerobic fermentation of food waste and boost the production of reduced fatty acids. The experimental results demonstrated that adding 5.0 g/L of tar achieved the highest hydrogen production, reaching 65.0 mL/g VS. Furthermore, increasing tar concentrations led to higher yields of reduced and longer-chain fatty acids, with butyrate production peaking at 726.8 mg COD (chemical oxygen demand)/g VS (volatile solid) with 30.0 g/L tar, and caproate at 128.6 mg COD/g VS with 10.0 g/L tar. GC-MS analysis identified phenolic compounds and furan derivatives as the primary inhibitory agents, which gradually degraded during fermentation. Microbial community analysis revealed that the tar-based inhibitors effectively suppressed dominant methanogenic archaea, including Methanothrix, Methanobacterium, and Methanosarcina, while promoting the enrichment of chain-elongating bacteria such as Clostridium_sensu_stricto and Clostridium_IV. This study suggests that integrating biomass tar from pyrolysis with anaerobic fermentation can serve as a highly efficient biorefinery approach, fully utilizing diverse organic wastes for sustainable energy production.

Lignocellulose Treatment Using a Flow-Through Variant of OrganoCat Process.

This study adapts the biphasic OrganoCat system into a flow-through (FT) reactor, using a heated tubular setup where a mixture of oxalic acid and 2-methyltetrahydrofuran (2-MTHF) is pumped through beech wood biomass. This method minimizes solvent-biomass contact time, facilitating rapid product removal and reducing the risk of secondary reactions. A comparative analysis with traditional batch processes reveals that the FT system, especially under severe conditions, significantly enhances extraction efficiency, yielding higher amounts of lignin and sugars with reduced solid residue. Notably, the FT system shows partial hydrolysis of the cellulose, which increases with temperature while not producing significant amounts of furfural or 5-HMF, indicating more efficient depolymerization of polysaccharides without substantial sugar degradation. A statistical design of experiments (DOE) using a Box-Behnken design elucidates the influence of process variables (time, solvent flow rate, temperature) on the yield. Key findings highlight reactor temperature as the dominant factor affecting yields, with process time showing a significant but less pronounced impact. This study demonstrates the potential of the FT OrganoCat system for efficient lignocellulosic biomass fractionation and represents an advancement towards continuous lignocellulose processing, contributing to our knowledge of process optimization for improved biorefinery applications.

Role of cultivation parameters in carbohydrate accretion for production of bioethanol and C-phycocyanin from a marine cyanobacterium Leptolyngbya valderiana BDU 41001: A sustainable approach

The investigation aimed to augment carbohydrate accumulation in the marine cyanobacterium Leptolyngbya valderiana BDU 41001 to facilitate bioethanol production. Under the standardised physiochemical condition (SPC), i.e. 90 µmol photon m−2 s−1 light intensity, initial culture pH 8.5, 35 °C temperature and mixing at 150 rpm increased the carbohydrate productivity ∼70 % than the control, while a 47 % rise in content was obtained under the nitrate (N)-starved condition. Therefore, a two-stage cultivation strategy was implemented, combining SPC at the 1st stage and N starvation at the 2nd stage, resulting in 80 % augmentation of carbohydrate yield, which enhanced the bioethanol yield by ∼86 % as compared to the control employing immobilised yeast fermentation. Moreover, biomass utilisation was maximised by extracting C-phycocyanin, where a ∼77 % rise in productivity was recorded under the SPC. This study highlights the potential of L. valderiana for pilot-scale biorefinery applications, advancing the understanding of sustainable biofuel production.

Preliminary assessment of astaxanthin production in a new Chlamydomonas strain

Green microalgae are increasingly valuable in industries such as food, cosmetics, animal feed, functional foods, and pharmaceuticals due to their ability to produce significant secondary metabolites like carotenoid pigments. Despite the growing demand for microalgae-derived carotenoids, the identification of robust wild-type strains with high biomass productivity under specific growth conditions remains limited. This study introduces Chlamydomonas sp. KIOST-2 (accession number: PP532860), a newly identified wild-type microalgal strain with 99.9 % genetic similarity to Chlamydomonas callosa, characterized through 18S rRNA gene sequence analysis. Notably, Chlamydomonas sp. KIOST-2 demonstrates considerable biomass productivity at 20–30 °C under alkaline (pH 8–10) and freshwater conditions, making it suitable for large-scale cultivation. Under drought stress, this strain forms orange cysts with high concentrations of astaxanthin (5.7 ± 0.6 mg/g) and notable lipid accumulation, primarily of oleic acid (C18:1 n9c), palmitic acid (C16:0), and linoleic acid (C18:2 n6c). The ability of Chlamydomonas sp. KIOST-2 to produce substantial amounts of astaxanthin under drought conditions without genetic modification highlights its potential for biorefinery applications and industrial exploitation. This discovery underscores the strain's unique combination of drought resistance and high astaxanthin productivity, positioning it as a valuable source of bioactive compounds for various industries.

Machine learning-supported solvent design for lignin-first biorefineries and lignin upgrading

Solvent selection is a difficult task for lignin-first biorefineries and lignin upgrading as the solvent must satisfy multiple complex technical requirements, while remaining extremely stable to allow recycling. High lignin solubility is a common selection criterion, but the ideal solvent for lignin-first biorefineries also requires non-reactivity towards acids and stabilising reagents encountered in the reaction liquor. To facilitate the search for promising solvents, we developed a computational solvent design framework. The framework consists of a graph-based genetic algorithm for molecular design wherein a graph neural network is used for lignin solubility predictions. Based on these predictions, the genetic algorithm iteratively optimises the molecular structures, inspired by evolutionary strategies, such as selection, cross-over, and mutation. The developed framework designed numerous solvents with high potential for application in lignin-first biorefineries and lignin upgrading. For these solvents, experiments confirmed solubilities between 20 and 60 wt.% across different types of lignin. Notably, several solvents were stable under typical biorefinery process conditions. Furthermore, the explainability of graph neural networks enabled us to link the lignin solubility predictions with structural features of the solvents, providing a clear rationale for solvent selection.

Unraveling the unique structural motifs of glucuronoxylan from hybrid aspen wood

Xylan is a fundamental structural polysaccharide in plant secondary cell walls and a valuable resource for biorefinery applications. Deciphering the molecular motifs of xylans that mediate their interaction with cellulose and lignin is fundamental to understand the structural integrity of plant cell walls and to design lignocellulosic materials. In the present study, we investigated the pattern of acetylation and glucuronidation substitution in hardwood glucuronoxylan (GX) extracted from aspen wood using subcritical water and alkaline conditions. Enzymatic digestions of GX with β-xylanases from glycosyl hydrolase (GH) families GH10, GH11 and GH30 generated xylo-oligosaccharides with controlled structures amenable for mass spectrometric glycan sequencing. We identified the occurrence of intramolecular motifs in aspen GX with block repeats of even glucuronidation (every 2 xylose units) and consecutive glucuronidation, which are unique features for hardwood xylans. The acetylation pattern of aspen GX shows major domains with evenly-spaced decorations, together with minor stretches of highly acetylated domains. These heterogenous patterns of GX can be correlated with its extractability and with its potential interaction with lignin and cellulose. Our study provides new insights into the molecular structure of xylan in hardwood species, which has fundamental implications for overcoming lignocellulose recalcitrance during biochemical conversion.

Tandem hydroformylation-amine condensation and reduction of canola fatty acid methyl esters for production of diesel renewable antioxidant additives

A canola fatty acid methyl esters (FAME) mixture was synthesized through transesterification and modified by rhodium homogeneous tandem hydroformylation-amine condensation, with primary amines, yielding imine products. The obtained imines were reduced by maceration with sodium borohydride and alumina, yielding secondary amine products. The synthesized fatty imines and amines were evaluated as additives for commercial diesel and biodiesel on concentrations of 0.5 and 1.0 %, while tested for oxidative stability fallowing the EN14112 standard method. The tested amino products exhibited higher oxidative stability, which justifies its application as antioxidant additives, while the diesel blends did not show expressive physicochemical modifications. The modified amino-FAME antioxidant performance, justifies a renewable biorefinery application, extend the biodiesel shelf-life prior to diesel blending, complies with UN’s Sustainable Development Goals (SDGs) and if prepared from residues, enhance the circular economy appeal.

Development of a Microwave-Assisted Bench Reactor for Biomass Pyrolysis Using Hybrid Heating.

Microwave-assisted pyrolysis (MAP) is a cutting-edge technology that converts biomass into fuels, chemicals, and materials. In this study, an Arduino was used to control and automate a MAP system built from a microwave oven with a cordierite chamber filled with silicon carbide. Sugar cane bagasse was pyrolyzed at 250, 350, 450, and 550 °C to validate the MAP system and obtain pyrolytic products with different yields and chemical compositions. Lower temperatures led to high biochar yields, but the highest surface area of 25.14 m2 g-1 was only achieved at 550 °C. By contrast, higher temperatures favored the recovery of pyrolysis liquids. BET and scanning electron microscopy analyses revealed a porous biochar structure, while Fourier transform infrared spectroscopy analysis showed that the availability of functional groups on the biochar surface decreased with an increase in pyrolysis temperature. GC-MS analysis quantified valuable low molecular mass compounds in pyrolysis liquids, including aldehydes, ketones, phenols, and alcohols. With its unprecedented hybrid heating device, the MAP system promoted suitable heating rates (31.9 °C min-1) and precise temperature control (only 19 °C of set point variation), generating pyrolysis products devoid of microwave susceptor interferences. Therefore, MAP provided a rapid, safe, and efficient means of depolymerizing biomass, thus holding promise for biorefinery applications.

Characterization and utilization of industrial wastewater in biorefinery systems: A comprehensive approach

AbstractThis review explores the innovative utilization of industrial effluents in biorefinery applications, addressing the environmental challenges posed by the complex mixture of pollutants in industrial wastewater. It emphasizes the transformation of these effluents into valuable resources, such as biofuels, biochemicals, and bioplastics, through advanced biotechnological processes including anaerobic digestion, fermentation, enzymatic conversion, and microbial biomass production. The study highlights the critical role of microbial biocatalysts in breaking down diverse pollutants and transforming waste into wealth, thereby contributing to sustainable industrial practices and a circular economy. Challenges such as variability in effluent composition, inhibitory substances, and the necessity for robust bioprocesses are discussed, along with suggestions for future research directions like effluent characterization, development of specialized microbial consortia, and effective monitoring and control strategies. The review underscores the importance of collaboration between industry, academia, and government to advance biorefinery technologies, ultimately advocating for a sustainable and resource‐efficient future through the innovative treatment of industrial wastewaters.

Natural deep eutectic solvents (NADES) for the extraction of bioactives: emerging opportunities in biorefinery applications

Natural deep eutectic solvents (NADES) have emerged as an eco-friendly alternative for extracting bioactives, avoiding the use of flammable organic solvents and extreme temperatures and pH conditions. NADES rely on intermolecular interactions between hydrogen bonding donors (HBD) and hydrogen bonding acceptors (HBA) to form eutectic mixtures with significantly lower melting points than their individual components. These matrices are influenced by factors like water content, temperature, and component ratios. NADES high viscosity can hinder extractive efficiency, which can be mitigated by adding water or working at higher temperatures. However, excessive dilution with water may disrupt the supramolecular structure of NADES, reducing extraction efficiency. A notable feature of NADES is their fine-tunability for specific purposes. Adjusting physicochemical properties such as polarity, pH, and viscosity optimizes extraction efficiency by promoting the solubility of target molecules and interactions between the NADES and target molecules. NADES, unlike organic solvents, can partially disrupt plant and microalgae cell walls, enhancing permeability and extraction efficiency. Moreover, NADES can have a stabilising effect on bioactives and can enhance their biological activity and bioavailability. These attributes, coupled with their low environmental impact in terms of low toxicity and high biodegradability, make NADES attractive for biorefinery applications.

Screening of efficient microalgae strains isolated from Tunisian ecosystems: Assessment of algal growth rate and added-value bioproducts for biorefinery applications

Microalgae are highly diverse organisms with significant applications in the production of high-value molecules and biodiesel from microalgae rich in oils. In this study, our main objectives were to evaluate the potential applications of four isolated microalgae strains, namely Scenedesmus sp., Nannochloris sp., Oscillatoria sp., and Amphora sp., which were selected from the natural environment. These strains were batch cultured, and their growth was monitored. We determined the content of chlorophyll a, carotenoids, and lipids, and analyzed the fatty acid profile of each cultivated strain to assess their lipid potential for biodiesel production. Our findings revealed that these strains exhibited high biomass production, ranging from 3000 mg. L−1 for Amphora sp. to 3800 mg. L−1 for Oscillatoria sp. Notably, Oscillatoria sp. and Scenedesmus sp. were found to be the richest in saturated fatty acids, with 10.68% and 9.36% of dry cell weight, respectively, suggesting their potential use in the production of biodiesel from microalgal oils with possibility of blending with other oils feedstock to improve biodiesel final quality. Furthermore, our results showed that the Nannochloris sp. strain could be cultivated to extract pigments for various applications, as it exhibited high levels of chlorophyll a (38.43 mg. L−1) and carotenoids (4.153 μg. mL−1).

Ecological insights and environmental threats of invasive alien plant Chromolaena odorata: Prospects for sustainable management

AbstractThe Anthropocene witnessed the landscape spread of several invasive alien plants which can remarkably influence the ecosystem services and environmental sustainability. To this end, Chromolaena odorata (Asteraceae) is widely identified among top hundred pervasive invaders in view of its adverse influence on environment, agriculture, and wildlife. The adverse effects of C. odorata can further be exacerbated under the event of invasion interaction with other anthropogenic stressors such as climate change. Nevertheless, there exists dearth of pragmatic studies on invasion ecology, socio‐economic impacts, ecological economics, and sustainable management prospects of C. odorata. The present review discusses the invasion ecology, distribution, mechanisms, impacts, and management strategies of C. odorata invasion. The side‐effects and unsustainable use of traditional control measures (e.g., controlled fire and chemical herbicides) of C. odorata paves the way to biological control methods (e.g., gall fly). However, the control measures of C. odorata need to be assessed for their long‐term ecosystem level effects to mitigate the possible adverse environmental responses. The wise application of C. odorata biomass in marginal lands for soil fertility restoration, biosorption, biorefinery, bio‐medicine, and bioenergy in conjunction with ecological control measures can cover economic incentive to facilitate sustainable management of C. odorata in forestry/agroforestry systems. Nevertheless, the use of weed biomass as feedstock in biorefinery and ethno‐medicine may be inadequate in reducing its invasion. Future studies should explicitly elucidate the ecological mechanisms of C. odorata in concert with the quest for its field‐scale application in biorefinery to augment sustainable management.

Enzymatic hydrolysis of cellulose banana stem (alkaline microwave-assisted pre-treatment)

The banana stem waste holds immense promise as a readily available and abundant source of lignocellulosic biomass, making it a compelling alternative for biofuel and biochemical applications. Therefore, this study focuses on investigating the impact of both time and substrate loading on the enzymatic hydrolysis of banana stem cellulose that has undergone alkaline microwave-assisted pre-treatment. The pre-treatment method involves subjecting the biomass to 5% KOH for 30 min, followed by microwave exposure at 300 W for 5 min, a process aimed at enhancing the accessibility of cellulose. Enzymatic hydrolysis experiments were carried out utilizing cellulase enzymes derived from Aspergillus niger, with variations in hydrolysis times (ranging from 5 to 45 h) and enzyme-to-substrate ratios (ranging from 1:1 to 1:10). The results of this investigation revealed a substantial improvement in hydrolysis efficiency, owing to the synergistic effects of alkaline microwave-assisted pre-treatment, signifying enhanced cellulose accessibility. Notably, the highest concentration of reducing sugars (1.3 mg mL−1) was achieved at a substrate-to-enzyme ratio of 1:1 and a hydrolysis duration of 45 h. These findings provide valuable insights into the conversion of lignocellulosic biomass, emphasizing the potential of integrated pre-treatment strategies for sustainable biorefinery applications. This research contributes to advancing our understanding of lignocellulosic biomass utilization, offering a promising avenue for biofuel and biochemical production from banana stem waste.

Novel strategy to elevate solid state fermentation to produce alkilophilic endoglucanase using date waste feedstocks and peapod extract based nutrient media and expired probiotic strain: Application in fermentable sugar production

Solid waste generation and its impactful management are among the most raised environmental concerns seeking sustainable alternatives in the form of value addition. In this study, a novel strategy has been presented towards enhanced bacterial endoglucanase (EG) production under solid state fermentation (SSF). The EG was produced using the waste probiotic juice of the bacteria Lactobacillus casei strain Shirota on a substrate of waste date pulp and seeds in co-fermentation mode. Additionally, peapod extract has been applied as a nutrient medium to maintain moisture in place of commercial media in SSF. Further, for enhancement in enzyme production, the impact of incubation temperature and pH of the SSF medium have been studied to obtain optimum enzyme activity. Under optimum conditions, bacteria produced 17.2 U/mL EG at a temperature of 37 °C and a pH of 8.0. The raw EG showed pH and thermal stability up to 5 h at 60 °C and pH 8.0. Furthermore, the enzymatic hydrolysis of date pulp released 67.71 g/L fermentable glucose obtained in 24 h of the reaction at 60 °C and pH 8.0. This work may have great significance in the areas of solid waste management and microbe-mediated value addition of waste biomass in the context of hydrolytic enzyme production for biorefinery applications.

Fungal bioremediation approaches for the removal of toxic pollutants: Mechanistic understanding for biorefinery applications

Pollution is a global menace that poses harmful effects on all the living ecosystems and to the Earth. As years pass by, the available and the looming rate of pollutants increases at a faster rate. Although many treatments and processing strategies are waged for treating such pollutants, the by-products and the wastes or drain off generated by these treatments further engages in the emission of hazardous waste. Innovative and long-lasting solutions are required to address the urgent global issue of hazardous pollutant remediation from contaminated environments. Myco-remediation is a top-down green and eco-friendly tool for pollution management. It is a cost-effective and safer practice of converting pernicious substances into non-toxic forms by the use of fungi. But these pollutants can be transformed into useable products along with multiple benefits for the environment such as sequestration of carbon emissions and also to generate high valuable bioactive materials that fits as a sustainable economic model. The current study has examined the possible applications of fungi in biorefineries and their critical role in the transformation and detoxification of pollutants. The paper offers important insights into using fungal bioremediation for both economically and environmentally sound solutions in the domain of biorefinery applications by combining recent research findings.

Regulation of genes encoding polysaccharide-degrading enzymes in Penicillium.

Penicillium fungi, including Penicillium oxalicum, can secrete a range of efficient plant-polysaccharide-degrading enzymes (PPDEs) that is very useful for sustainable bioproduction, using renewable plant biomass as feedstock. However, the low efficiency and high cost of PPDE production seriously hamper the industrialization of processes based on PPDEs. In Penicillium, the expression of PPDE genes is strictly regulated by a complex regulatory system and molecular breeding to modify this system is a promising way to improve fungal PPDE yields. In this mini-review, we present an update on recent research progress concerning PPDE distribution and function, the regulatory mechanism of PPDE biosynthesis, and molecular breeding to produce PPDE-hyperproducing Penicillium strains. This review will facilitate future development of fungal PPDE production through metabolic engineering and synthetic biology, thereby promoting PPDE industrial biorefinery applications. KEY POINTS: • This mini review summarizes PPDE distribution and function in Penicillium. • It updates progress on the regulatory mechanism of PPDE biosynthesis in Penicillium. • It updates progress on breeding of PPDE-hyperproducing Penicillium strains.

Development of an Haa1-Based Biosensor for Acetic Acid Sensing in Saccharomyces cerevisiae.

Transcription factor (TF)-based biosensors are important tools in strain development and screening as they can allow accurate monitoring of intracellular concentrations of a molecule. Acetic acid is one of the main inhibitors in lignocellulosic biomass and a major challenge when using yeast cell factories for biorefinery applications. Thus, developing acetic acid tolerant strains is of great importance. The acetic acid sensing biosensor developed relies on the endogenous Saccharomyces cerevisiae TF Haa1 that upon binding of acetic acid translocates to the nucleus. The acetic acid biosensor can be used as a tool for strain development and evaluation, as well as for screening of acetic acid-producing strains and for dynamic monitoring of acetic acid accumulation. This chapter describes a methodology for developing a TF-based biosensor for acetic acid sensing. Protocols for design considerations, part construction, and characterization procedures are included. The approach can potentially be adapted to any molecule where a suitable TF can be identified.