Lignin is an organic substance binding the cells, fibres and vessels which constitute wood and the lignified elements of plants, as in straw. After cellulose, it is the most abundant renewable carbon source on Earth. Between 40 and 50 million tons per annum are produced worldwide as a mostly non commercialized waste product.

It is not possible to define the precise structure of lignin as a chemical molecule. All lignins show a certain variation in their chemical composition. However, the definition common to all is a dendritic network polymer of phenyl propene basic units.

There are two principal categories of lignin: those which are sulphur bearing and those which are sulphur-free. It is the sulphur bearing lignins which have to date been commercialized. These include lignosulphonates (world annual production of 500,000 tons) and Kraft lignins (under 100,000 tons p.a.). Due to the lack of suitable industrial processes, the sulphur-free lignins are as yet non-commercialized.

As a natural and renewable raw material, obtainable at an affordable cost, lignin’s substitution potential extends to any products currently sourced from petrochemical substances.

Bioprospecting refers to the search for and discovery of new biological resources that have potential commercial or industrial applications. Using natural based products is advantageous for various reasons, such as the high biocompatibility of natural products, helpful in pharmaceutical, cosmetics and food industries.

In the past, ethnobotany was the basis of bioprospecting: by studying the use of locally available plants in foods and medicines from ancient Egypt, Greece and Rome, many molecules have been discovered and commercialized from plants in use since those times. Nowadays, bioprospecting is focused mostly on microorganisms, due to their abundance and their extreme biodiversity and numerous secondary metabolites, and traditionally, it was thought that it was more probable to find novel compounds or genetic information in underexplored habitats, such as deserts, caves, seas or even animal and plant microbiota. However, only a small percentage of microorganisms thrives in a laboratory setting and the majority of microbes cannot be cultured in a lab, especially those that live in a marine environment or in extreme conditions.
Due to the difficulty of cultivating microorganisms and the advancement of bioinformatics tools, the majority of bioprospecting is now carried out through i) metagenomics: the analysis of all the genetic material present in samples taken from different habitats, to identify genetic sequences linked to the production of specific enzymes or metabolic pathways for the production of secondary metabolites and ii) meta-transcriptomics: the study of gene expression and how it is influenced by the environmental conditions. Both the tools are extremely powerful in giving further insights into microbial biodiversity potential. However, we are still missing out on a large portion of terrestrial biodiversity (namely animal and plant), for which such advanced techniques have not yet been developed. The main bottlenecks today are the scarcity of natural resources, and the difficulty in cultivation, harvesting and supplying.

Bioactive compounds can be defined as nutrients and nonnutrients present in the food matrix (vegetal and animal sources) that can produce physiological effects beyond their classical nutritional properties.

Some examples of bioactive compounds are carotenoids, flavonoids, carnitine, choline, coenzyme Q, dithiolthiones, phytosterols, phytoestrogens, glucosinolates, polyphenols, and taurine. Since vitamins and minerals elicit pharmacological effects, they can be categorized as bioactive compounds as well.

Bioactive compounds are classified in essential (vitamins and minerals) and non-essential (polyphenols, flavonoids, carotenoids, phytosterols, glucosinolates, saponins, alkaloids, and essential oils).

The depolymerisation process – often referred to as chemolysis or solvolysis – uses different combinations of chemistry, solvents and heat to break down polymers into monomers, the building blocks of polymers. Depolymerisation is the opposite of polymerisation, which is the act or process where monomers join together to form a polymer. Polymers are very big molecules made up of many smaller molecules layered together in a repeating pattern. The word polymer is Greek for ‘many parts’. The smaller molecules that come together to form polymers are called monomers – small units that link together over and over to form a large polymer.

An example of a biological depolymerisation is the digestion of food. Macromolecules in food such as carbohydrates and proteins are degraded into simpler forms. The process is often facilitated by the catalytic action of various enzymes. For example, the amylase in the saliva degrades polysaccharides starch into maltose. Maltose (a disaccharide) is further degraded into glucose units through the action of the enzyme maltase.

After a desk-based review and categorisation of the types and sustainable plant sources of lignin to be explored, the consortium will prospect their bioactivity potential using a combination of cutting-edge chemical and enzymatic depolymerisation methodologies.

Lignin chemical depolymerisation (KELADA): KELADA will perform laboratory validation and further optimization to transform chemical depolymerisation into a process which is viable and industrially scalable. The chemical depolymerisation is considered a “low-risk / medium-gain route”, based on: development of 3 tailored catalyst-free chemical reactions for various lignin-containing substrates, targeting up to 70% of cleaved β-O-4 linkages to realise the production of 500-800 mixtures made up of low to medium (350-1500 gmol-1) MW lignin- derived fragments (derived from 9 raw and 9 oxidized lignin species) with chemical features, retained from the parent lignin, as a means to maximise the potential of identifying bioactive components. This is thanks to the varying depolymerization conditions, who will lead to mixtures of different profiles and compound distribution. Through the execution of this project KELADA will develop a new “reagent based” synthetic methodology to operate the cleavage of the 􏰀-O-4 bond for which we have already obtained proof of concept. It has been shown that the ether cleavage required for the lignin depolymerization occurred on small model compounds in minutes and at room temperature. This latter feature is highly desirable as current methods of depolymerization require high temperatures, which favour monoaromatic fragments and can lead to monomers decomposition to form tar. In addition, KELADA’s proprietary methodology is unique and respectful of the stereochemistry of the C-O bond that is liberated, leading to enantioenriched monomeric compounds. The reproducibility of the chemical depolymerisation processes will be reliably achieved by documentation and standardisation of laboratory validation and optimization processes, including detailing of specific reaction conditions, such as temperature, pressure, reaction times, and the precise quantities/ratios of reagents used.

Lignin enzymatic depolymerization (UH): UH will use their extensive expertise in heterologous expression and purification of enzymes to identify and produce novel enzymes with efficient and improved β-etherase activity. Enzymes from different microbial sources will be produced either in bacterial or fungal production hosts. Enzymes will be tested at different reaction conditions to find optimal reaction conditions for individual enzymes and substrates. Enzymatic depolymerisation is considered a “high-risk / high-gain route”, featuring: identification of up to 10 genes encoding enzymes that will have activity towards oxidised lignin substrates, i.e. targeting at least 30% of β-O-4 bonds cleavage. Low molecular weight lignin fractions from different types of lignin sources will be enzymatically treated following oxidation of soluble lignin optimal for β-etherase activity. The enzymatic depolymerisation step is expected to produce 150-200 lignin-derived mixtures (derived from 9 oxidized lignin species). However many liberated fragments will be linked to glutathione due to the specific depolymerisation reaction. The removal or not of the glutathione will be investigated during the project. It has been reported that glutathione conjugation may also lead to pharmacological or toxicological effects through bioactivation reactions. The reason being that glutathione conjugates are often more active or toxic than the parent compound, at least in cancer. With this in mind, the glutathione decorated lignin conjugates obtained from enzymatic depolymerization have a high chance of displaying bioactivity. To ensure reproducibility of enzyme mining and enzymatic depolymerisation methods, UH will implement quality control measures at each stage of the enzyme production and fermentation processes. This will involve monitoring critical parameters such as enzyme activity, purity, and stability to ensure consistent performance. Additionally, UH will establish a well-defined set of fermentation conditions and experimental protocols for enzymatic depolymerization, which will be documented.

The project is going to use high-throughput screening to identify which lignin-derived compounds are bioactive and can therefor find applications in the selected pharmaceuticals, cosmetics and fragrances markets.

High-throughput Screening (HTS) is essentially the use of automated equipment to rapidly test thousands to millions of samples for biological activity at the model organism, cellular, pathway, or molecular level. In its most common form, HTS is an experimental process in which 103–106 small molecule compounds of known structure are screened in parallel. Other substances, such as chemical mixtures, natural product extracts, oligonucleotides and antibodies, may also be screened. Because HTS typically aims to screen 100,000 or more samples per day, relatively simple and automation-compatible assay designs, robotic-assisted sample handling, and automated data processing is critical. HTS is commonly used in pharmaceutical and biotechnology companies to identify compounds (called hits) with pharmacological or biological activity. These are used as starting points for medicinal chemical optimization during pharmacological probe or drug discovery and development. Typically, HTS assays are performed in microtiter plates in 96-, 384-, or 1536-well formats while traditional HTS usually tests each compound in a compound library at a single concentration, most commonly 10 μM.

Multivariate data analysis (MVDA) is a type of statistical analysis that involves more than two dependent variables, resulting in a single outcome. Variables are factors you compare to the control or unchanging component of the experiment. Variables help you compare your findings with the control of the experiment to identify any changes that might occur or trends that may develop. Multivariate analysis aims to identify patterns between multiple variables.

PROSPLIGN will rely on very abundant natural sources: the lignin present in lignocellulosic biomass, the most abundant renewable feedstock consisting in plant or plant-based material that is not used for food or feed and mainly includes agricultural residues, energy crops, forestry residues, and yard trimmings. PROSPLING will manage to unlock the bioactives captured within the lignin matrix by an innovative combination of techniques. The proposed methods will combine solid sourcing, with parallel de-risked routes of chemical and enzymatic depolymerisation. The produced fractions will then be tested for bioactivity via high-throughput screening, following purification, and then validated in the target markets.

The crucial innovation of this approach involves:

• No requirement of microbial strain isolation and cultivation, with over 99% of the natural microbial biodiversity not being harvestable
• No reliance on indirect or inferred genetic information, such as (meta) DNA or RNA that based their identification of potential hits on perspective chemical structures of metabolites that may or may not be expressed in the target organism at the time of actual purification
• No dependence on expensive (sometimes exploitative) exploration of remote areas or rare species for intact pockets of natural biodiversity, given that our feedstock is an immense range of naturally abundant polymers.
• No specific technologies for harvesting and supplying are needed, given that the supply chain is already well developed and established. Also, there will be no need for developing innovative cultivation methods for new organisms, given that the lignocellulosic biomass is already abundant in Europe.
• Minimised risk of biopiracy, as the starting feedstock is normally commercially available in large or medium volumes as part of the existing supply chains, and further samples from unexploited species can be sources under strict protocols if they are to be “unlocked” via the PROSPLIGN bioprospecting approach. The procurement strategy elaborated in Europe will de-risk the project from restrictions under biodiversity use legislation and regulatory environment (e.g.: Nagoya Protocol). Moreover, the large amount of work needed on natural resources coming from well-known and abundant species will be prioritised vs working on rarer species.
• High degree of assurance that a unique reservoir of chemical diversity is tantalizingly close and hence an increased likelihood of bioactive “hits”. Our approach is partially de-risked (vs traditional bioprospecting) by the fact that the “generic” structure of lignin is known, as there is (i) certainty over the fact that there are specific monomeric groups which make up the larger polymer, (ii) there is considerable precedent that monomers with these functionalities have desirable properties as pharma, cosmetic and fragrance compounds and (iii) certainty that the polymeric lignin structure contains numerous stereocentres (i.e. effectively an untapped chiral pool) – each stereoisomer being an additional element of diversity.