Research Highlight 1: Production of Vanillin from Waste PET Bottle.

Vanillin – the compound responsible for the aroma and taste of vanilla (in our ice cream, chocolates and so on) – can now be produced from waste plastic bottles. Yes, you heard it right. Researchers from the University of Edinburgh, Dr. Stephen Wallace (Senior Lecturer at Institute for Quantitative Biology, Biochemistry and Biotechnology (IQB3) and also a UKRI Future Leaders Fellow) and Dr. Joanna Sadler (Post-doctoral Fellow in Wallace Lab) have found a way to directly upcycle poly(ethylene terephthalate) (PET) plastic bottle into vanillin – via enzymatic biotransformation. The work is published in the journal ‘Green Chemistry’ as Open Access [1].

PET has been widely used in textiles and packaging applications, and manufactured 70 million tonnes annually [2]. However, most of them ends up in landfill, oceans or littering in the cities after a single-use application. From a ‘Circular Economy’ perspective, most of the research has been focused on recycling of PET into its starting monomers, terephthalic acid and ethylene glycol or bis(2-hydroxyethyl) terephthalate to ‘Close-the-Loop’ and produce pristine quality PET [3,4]. Wallace and Sadler instead focused on upcycling it into a chemical that already has a high market value. Their strategy contributes to the ‘Open-Loop-Recycling’ of Circular Economy [5] – meaning “upcycling of a material into a high or similar value material after its end-of-life” – by fully utilizing the inherent value embedded in it – so that it does not enter into the waste stream and stays in the economy!

Vanillin is a naturally occurring phenol (3-methoxy-4-hydroxy benzaldehyde) and is extracted from the cured seed pods of the orchid ‘vanilla planifolia’– which has an expected market value of USD 734.5 million in 2025 (CAGR of 7.9% from 2019), accounting to its uses as flavouring agent in food and beverage industry, fragrances in cosmetic and detergent industry, and pharmaceuticals [1,6,7]. It is also a value-added chemical for the production of a wide range of thermoplastic and thermosetting polymers. Vanillin has a very high demand (exceeded 37 000 tonnes in 2018) which cannot meet solely by its natural supply. 1 kg of vanillin production requires 500 kg cured vanilla pods – meaning an approximate of 40,000 pollinated flowers, and the whole process takes more than a year. Thus, vanillin is also produced industrially – approximately 85% of market is based on petro-based route such as reaction of guaiacol with glyoxylic acid or formaldehyde and rest 15% is produced via oxidation of lignosulfonates, a byproduct of wood-pulping industry. These comes under the ‘synthetic vanillin’ grade. Growing environmental concerns and the need for a ‘natural grade’ vanillin directed towards its synthesis via biotechnological conversion using microorganisms such as fungi or bacteria. In this regard, fermentation of vanillin using ferulic acid as substrate has been successful industrially. Other substrates such as glucose, glycerol, xylose, L-tyrosine, eugenol, isoeugenol, curcumin and so on, were also explored.

The featured article is the first report on producing vanillin from waste plastic bottle! The authors have used an empty sparkling water bottle found on the road side. They simply cleaned the bottle by washing with ethanol and cut it into pieces. The biotransformation is a one-pot process involving hydrolytic breakdown of PET into terephthalic acid (TPA) {process (i)} using Leaf-branch Compost Cutinanase (LCC), followed by de novo conversion of TPA into vanillin using Escherichia Coli (E. coli) – MG1655 RARE (Reduced Aromatic Aldehyde Reduction) {process (ii)} as the host microorganism. The process (ii) undertakes in different plasmids of E. coli RARE – mainly pVan1, and pVan2, within the time period of 24h at room temperature (see Scheme 1). In pVan1, the conversion of TPA into protocatechuic acid (PC) takes place via dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD) intermediate using oxidase (TphA1, TphA2, TphB2) and dehydrogenase (DCDDH) enzymes from Comamonas sp. Then, the conversion of PC into vanillin in pVan2 plasmid, which can proceed via two different pathways – methylation of m-phenolic hydroxyl (using catechol methyl transferase (S-COMT) from Rattus norvegicus) and then carboxylic acid reduction to aldehyde (using carboxylic acid reductase (CAR) from Nocardia iowensis) {pathway 1} or at first reduction of carboxylic acid and then methylation of m-phenolic hydroxyl {pathway 2}. The key intermediates, vanillic acid (VA) and dihydroxybenzaldehyde (DBAl) are produced in the case of pathway 1 and pathway 2, respectively.

Before direct upcycling of PET bottle into vanillin, the authors conducted systematic experiments by using the major intermediate ‘TPA’ as substrate to optimize the conditions that provide the highest vanillin titers. They have found that the utilization of M9-glucose as expression media, enhancing the E. coli cell membrane permeability by using n-butanol, addition of L-methionine, balancing the pH at 5.5 for maximum diffusion of TPA into the cell without causing stress, and decreasing biotransformation temperature from 30 ºC to 22 ºC yielded the highest vanillin concentration. The authors have stated in the article that their next step is to intensify this methodology using further strain engineering and process optimization. For example, one of their strategy is to control the different pathways happening during conversion of PC into vanillin. So far, they were able to favor pathway 2 over pathway 1 by decreasing the biotransformation temperature (22 ºC) and by using in situ product removal reagent (ISPR) such as oleyl alcohol. Their next focus is to improve the conversion of DBAl to vanillin by screening alternative methyl transferase as it was found to be the bottleneck in pathway 2. They also have plans to extend this biotransformation of PET bottle into other metabolites – high value-added chemicals other than vanillin. A wide range of chemicals such as muconic acid, gallic acid, catechol, and pyrogallol can be produced from ‘TPA’ [8] – the major hydrolyzed product of PET – hence, waste PET bottles could be the ‘next-generation feedstock’ for the chemical industry.

References:

[1]. Sadler, J. C.; Wallace, S. Microbial synthesis of vanillin from waste poly(ethylene terephthalate). Green Chem. 2021, 23, 4665-4672.

[2]. Tournier, V.; Topham, C. M.; Gilles, A. et al. An engineered PET depolymerase to break down and recycle plastic bottles. Nature 2020, 580, 216-219.

[3]. Jehanno, C.; Flores, I.; Dove, A. P.; Müller, A. J.; Ruipérez, F.; Sardon, H. Organocatalysed depolymerisation of PET in a fully sustainable cycle using thermally stable protic ionic salt. Green Chem. 2018, 20, 1205-1212.

[4]. Q. Hou.; M. Zhen.; H. Qian.; Y. Nie.; X. Bai.; T. Xia.; M. L. Ur Rehman.; Q. Li.; M. Ju. Upcycling and catalytic degradation of plastic wastes. Cell Rep. 2021, 2, 100514.

[5]. The new plastics economy: Rethinking the future of plastics & catalysing action, Ellen MacArthur Foundation 2017, http://www.ellenmacarthurfoundation.org/publications.

[6]. Banerjee, G.; Chattopadhyay, P. Vanillin biotechnology: The perspectives and future. J. Sci. Food Agric. 2019, 99, 499-506.

[7]. Fache, M.; Boutevin, B.; Caillol, S. Vanillin production from lignin and its use as a renewable chemical. ACS Sustainable Chem. Eng. 2016, 4, 35-46.

[8]. Kim, H. T.; Kim, J. K.; Cha, H. G. et al. Biological valorization of poly(ethylene terephthalate) monomers for upcycling waste PET. ACS Sustainable Chem. Eng. 2019, 7, 19396-19406.

[9]. All articles cited in the featured article, Ref. 1

Created by: Sini Nalakathu Kolanadiyil