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Abstract

Lignin is the second most abundant organic polymer on earth. It is heterogeneous, consists of a variety of phenolic groups and is highly branched. Every year the paper industry accumulates approximately 50 Mtons of lignin as a waste product [1], which is immediately combusted. Studies have shown that the potential degradation products and monomers of lignin can be used as building blocks for further applications. This project aims to generate a multi-enzyme system, based on characteristics of wood-decaying fungi, to efficiently process lignin into high-value chemicals. To achieve this, Pichia pastoris is engineered to express horseradish peroxidase (HRP) and aryl-alcohol oxidase (AAO). HRP degrades lignin using hydrogen peroxide substrate. AAO, in turn, utilizes parts of the degradation products to create more hydrogen peroxide, forming a self-sustaining system. Both enzymes are fused with a secretion tag to create a continuous cell factory, where the supernatant contains the desired enzyme mixture.

Introduction

When documents are printed out, hygiene products are used or textbooks are opened, a deceptively simple commodity is being enjoyed: paper. In reality, paper is the end result of a lengthy and complex process. During one step in this process, called wood pulping, a specific heterogeneous polymer called lignin, is separated out from other wood components. As there is no direct application for industrial use, the isolated lignin is treated as a waste product and subsequently burned as a low-grade fuel. However, lignin is an untapped resource, and its degradation products have many potential high-value applications [1].

**Figure 1. Structure of the wood cell wall.** A structural depiction of a wood cell wall which is made of interconnected ligno-cellulitic units (Adapted from Mathews et al. [7]).

**Figure 2. A partial structural depiction of softwood lignin** (Adapted from [8])

What is lignin?

The total lignin availability in the biosphere exceeds 300 billion tonnes and annually around 20 billion tonnes are produced through photosynthesis, making it the second most common organic substance on earth [1,2,3], and one of the most abundant natural sources of aromatic structures [4,5]. It serves many roles in higher plants, for example, it can act as an adhesive to hold and strengthen cellulose and hemicellulose in plant cell walls, and as an important structural component enabling water transport (Figure 1) [3]. However, lignin’s most noteworthy characteristic is its resistance to degradation. Lignin is broken down at a much slower rate in comparison to cellulose or non-cellulosic polysaccharides [6], functioning as a barrier to enzymatic degradation by fungal and microbial pathogens (below this section, you can find a video made by Uppsala_iGEM_2019 showcasing kraft lignin).

Two reasons for inefficient lignin degradation

Lignin contains many interconnected phenylpropanoid units (Figure 2). Consequently, complete breakdown requires the degradation of resonance bonds in these phenolic rings.
The composition of lignin is heterogeneous and highly variable and may differ depending on the isolation procedure. This complex structure of lignin leads to a low accessibility for potential degradative enzymes [5].

Why is lignin degradation important?

The use of degraded lignin has many potential applications as emulsifiers, dyes, binding agents, thermosets, paints, fuels, among others [9]. However, the most promising area of future research is the production of bio-based chemicals and fuels. Pulp-mills will continue to produce large quantities of lignin waste in order to produce paper (currently at 50 million tons/year worldwide [10]), which means that there will continue to be an excess of lignin for the foreseeable future. Since wood-based biofuels and chemicals are generally considered to be carbon neutral, advances in degradation methods could lead to the emergence of a reliable source of green energy and green materials [11].

**Figure 3. A piece of red oak trunk affected by tree-degrading fungi** (Photo by Kevin T. Smith, USDA Forest Service [14]).

What inspired us?

Sixty-eight percent [12] of the total land area of Sweden is forested. The forest has thus played an important cultural, recreational and economic role in Sweden’s history and will continue to do so in the future. As a result, many of the team’s initial ideas were related to forests and the problems they face.

One of these problems is the invasive growth of the fungal species H. annosum. The fungus sustains itself by growing inside tree trunks, using enzymes to break down the tree’s structural components, including lignin, and causing the tree to rot [11]. Photo needed What if the Uppsala iGEM 2019 team could instead harness the lignin-degrading properties of fungi and use it as a part of the solution to degrade excess lignin waste into smaller structural units, which in turn would allow other industries to build up high-value products?

Current status of the field

Currently, constant ongoing efforts are being made to industrialize lignin-derived products, mostly through chemical methods [5], however, the majority have yet become commercially competitive enough to rule the market. For example, vanillin values 40 times more than lignin from Kraft processes, but only 20% of vanillin on the market is produced from lignin (other 80% all from crude oil) [2]. Not to say that chemical approaches pose a threat to the environment due to the excessive use of organic solvents and strong and corrosive acidic or alkaline solutions [5].

The amount of unused lignin and its underlying potential have also incited studies of lignocellulolytic organisms and heterologously expressed enzymes. Multiple bacteria and fungi strains have been engineered or isolated to degrade lignin, and have managed to produce chemicals such as L-lactate for bioplastics and food, vanillin for flavors and triacylglycerols for biodiesel applications [20]. Despite their success over the benchtop, several factors prevent these projects' to be adopted by the industry. Firstly, these particular strains are difficult to culture, lowering these projects' economic feasibility; Secondly, selling GMOs or using them in the food industry will always raise bio-safety concerns. In terms of heterologous expression of lignocellulolytic enzymes, laccase, manganese peroxidase (MnP), lignin peroxidase (LiP), and versatile peroxidase (VP) have all been studied a lot [20]. Although laccase is the most promising out of the above, expressing it in two months may prove difficult according to yeast experts whom we've spoken to. Peroxidases are another direction of research, one of them, HRP, is widely studied and expressed heterologously due to its relative stability, high catalytic activity and usage in other biological applications (conjugated antibodies) [15]. However, previously no one has considered to combine it with an oxidase to create a lignin-degrading system (refer to “aim” part for detailed mechanism). Out of the oxidases, AAO has been successfully expressed and is an important enzyme in natural occurring lignin-degrading fungi [16].

**Figure 4. Mechanism of lignin degradation.** HRP catalyzes the generation of free radicals from hydrogen peroxide, which in turn attack hydroxyl-groups, generating more radicals. This leads to a chain reaction of degradation in lignin structures (Figure adapted from https://www.uniprot.org/uniprot/P00433).

Aim

The multitude of bond types in lignin requires a system of non-specific enzymes to achieve efficient degradation. In this project, the Uppsala iGEM 2019 team aimed to degrade lignin using a two-enzyme system consisting of HRP and AAO. In this system, HRP would be the main lignin attacker while AAO would be the supplier of hydrogen peroxide [17,18]. The hydrogen peroxide would be used by HRP to perform oxidative catalysis to fragmentize lignin (Figure 4) [19].

As the degradation of lignin would produce a variety of aromatic alcohols, these would be further utilized by AAO to generate more hydrogen peroxide. Consequently, this would result in a bi-enzyme system with a closed energy cycle, which would maintain a continuous catalysis until the molecule was fully degraded into relatively small aromatic compounds.

To summarize, we want to build a two-enzyme system consisting of HRP and AAO to degrade lignin into compounds which can be further processed into value-added products, such as speciality chemicals and biofuel.

How this was achieved

To efficiently express HRP and AAO, the protein expression system of the yeast Pichia pastoris was chosen. Using this eukaryotic system ensured that the folding, as well as post-translational modifications, would occur properly for these eukaryotic enzymes [15,16]. Expression was achieved for both HRP and AAO in their active forms. The activity of those enzymes in terms of lignin degradation was then examined by various analytic methods.

References

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[2] Hodásová, Ľudmila, et al. “Lignin, Potential Products and Their Market Value.” Wood Research, vol. 60, Dec. 2015, pp. 973–86.

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[4] Ayyachamy, Manimaran, et al. "Lignin: Untapped Biopolymers in Biomass Conversion Technologies." Biomass Conversion and Biorefinery, vol. 3, no. 3, 2013, pp. 255-269.

[5] Duval, Antoine, and Martin Lawoko. “A Review on Lignin-Based Polymeric, Micro- and Nano-Structured Materials.” Reactive and Functional Polymers, vol. 85, Dec. 2014, pp. 78–96, doi:10.1016/j.reactfunctpolym.2014.09.017.

[6] Haider, K. M., and G. Guggenberger. “ORGANIC MATTER | Genesis and Formation.” Encyclopedia of Soils in the Environment, edited by Daniel Hillel, Elsevier, 2005, pp. 93–101, doi:10.1016/B0-12-348530-4/00510-5.

[7] Mathews, Stephanie L., et al. “Bacterial Biodegradation and Bioconversion of Industrial Lignocellulosic Streams.” Applied Microbiology and Biotechnology, vol. 99, no. 7, Apr. 2015, pp. 2939–54. DOI.org (Crossref), doi:10.1007/s00253-015-6471-y

[8] Kungliga tekniska högskolan Institutionen för fiber-och polymer. The Ljungberg Textbook: Wood Chemistry and Wood Biotechnology, KF 2010, 2008. Fibre and Polymer Technology, KTH, 2008, https://books.google.se/books?id=XtHQPgAACAAJ.

[9] Vanholme, Ruben, et al. “Lignin Biosynthesis and Structure.” Plant Physiology, vol. 153, no. 3, July 2010, pp. 895–905. www.plantphysiol.org, doi:10.1104/pp.110.155119.

[10] Ahmad, Ejaz, and Kamal K. Pant. “Chapter 14 - Lignin Conversion: A Key to the Concept of Lignocellulosic Biomass-Based Integrated Biorefinery.” Waste Biorefinery, edited by Thallada Bhaskar et al., Elsevier, 2018, pp. 409–44. ScienceDirect, doi:10.1016/B978-0-444-63992-9.00014-8.

[11] Isikgor, Furkan, and C. Remzi Becer. “Lignocellulosic Biomass: A Sustainable Platform for the Production of Bio-Based Chemicals and Polymers.” Polymer Chemistry, vol. 6, no. 25, 2015, pp. 4497–559. arXiv.org, doi:10.1039/C5PY00263J.

[12] Statistics Sweden, Agriculture, forestry and fishery, Retrieved 25 June 2019, www.scb.se/en/finding-statistics/statistics-by-subject-area/agriculture-forestry-and-fishery/ 13) Interagroskog, Biologi., Published 23 Feb. 2017. Retrieved 2018-04-20. www.interagroskog.se/rotroeta-biologi/

[13] Interagroskog, Biologi., Published 23 Feb. 2017. Retrieved 2018-04-20. www.interagroskog.se/rotroeta-biologi/

[14] Smith K. T., White rot of red oak, Quercus rubra. USDA Forest Service. UCIPM – Statewide Integrated Pest Management Website. Online. Retrieved 2019-08-19. http://ipm.ucanr.edu/PMG/U/D-WO-UNKN-TR.019.html

[15] Spadiut, Oliver, et al. “Purification of a Recombinant Plant Peroxidase Produced in Pichia Pastoris by a Simple 2-Step Strategy.” Protein Expression and Purification, vol. 86, no. 2, Dec. 2012, pp. 89–97, doi:10.1016/j.pep.2012.09.008.

[16] Viña‐Gonzalez, Javier, et al. “Functional Expression of Aryl‐alcohol Oxidase in Saccharomyces Cerevisiae and Pichia Pastoris by Directed Evolution.” Biotechnology and Bioengineering, 1 July 2018, https://onlinelibrary.wiley.com/doi/abs/10.1002/bit.26585.

[17] Ferreira, Patricia, et al. “Kinetic and Chemical Characterization of Aldehyde Oxidation by Fungal Aryl-Alcohol Oxidase.” The Biochemical Journal, vol. 425, no. 3, Jan. 2010, pp. 585–93, doi:10.1042/BJ20091499.

[18] GUILLÉN, Francisco, et al. “Substrate Specificity and Properties of the Aryl-Alcohol Oxidase from the Ligninolytic Fungus Pleurotus Eryngii.” European Journal of Biochemistry, vol. 209, no. 2, Oct. 1992, pp. 603–11, doi:10.1111/j.1432-1033.1992.tb17326.x.

[19] Bergel, Alain, and M. E. Lai. “Catalysis of Oxygen Reduction by Catalase and HRP on Glassy Carbon Electrodes : Comparison of the Mechanisms”. Retrieved 20 June 2019, https://www.electrochem.org/dl/ma/201/pdfs/1270.pdf

[20] Yadav, Deepti, et al. “Secretory Expression of Recombinant Small Laccase from Streptomyces Coelicolor A3(2) in Pichia Pastoris.” International Journal of Biological Macromolecules, vol. 108, Mar. 2018, pp. 642–49, doi:10.1016/j.ijbiomac.2017.11.169.

Team:Uppsala Universitet/test1PD