4-hydroxy-3-methoxybenzaldehyde, also known as Vanillin, is an aromatic aldehyde used in industrial applications as a flavoring agent, aromatic ingredient, and pharmaceutical development (1,2). It is also the primary ingredient for vanilla, commonly used in food products and perfumes due to its sweet fragrance (2). Aside from this, it is also used in cosmetics due to its antimicrobial and antioxidant properties (5).
Traditional methods for vanilla extraction
Although the worldwide consumption of vanillin exceeds 16,000 Tons yearly, only 0.25% comes from natural extraction using ethanol as a solvent from vanilla pods of two species of vanilla orchid: Vanilla planifolia and Vanilla tahitensis (1,4). This process is known to have several drawbacks, such as slow growth of the vanilla orchid and extracting low yield from these plants (1,3). It requires approximately 500 kilograms of vanilla pods, only to produce 1 kilogram of vanillin which makes this method lead to high market costs compared to other extraction processes (4).
Biomanufacturing as a Sustainable Alternative
The biotechnological production of vanillin using microbial systems, such as recombinant E. coli , offers a promising alternative to both natural extraction and chemical synthesis by utilizing simple and cheap carbon sources which costs less than 30 cents per kilogram (1). This approach leverages the natural biosynthetic capabilities of microbes (1).
Background
Recent research has concentrated on creating novel synthetic pathways in microbial systems by merging plant and microbial pathways. For instance, Ni and colleagues devised a unique synthetic route in E. coli , integrating the plant-derived phenylpropanoid pathway with a microbial coenzyme-dependent non-β-oxidative pathway. This innovative pathway successfully utilized various inexpensive substrates, including tyrosine, glucose, xylose, and glycerol, achieving vanillin production yields ranging from 13.3 mg/L to 97.2 mg/L. This approach provides valuable insights into the development of synthetic de novo pathways (1).
Additionally, Li and his team capitalized on E. coli's inherent shikimate pathway to establish a novel synthetic route for vanillin production. By channeling metabolic flux through protocatechuic acid, they advanced the biosynthesis of vanillin, showcasing the potential of leveraging native microbial pathways in engineered systems (4).
Ferulic acid is the most extensively studied substrate for vanillin production due to its abundance in nature and structural similarity to vanillin. This compound, also known as 4-hydroxy-3-methoxycinnamic acid, is a prevalent phenylpropanoid found in many plants. In plants, ferulic acid is synthesized from the aromatic amino acids phenylalanine and tyrosine, which are generated through the shikimic acid pathway (4).
In experiments involving recombinant E. coli strains, those containing the plasmids sam8-sam5/pACYC and tal-hpaBC/pACYC were cultured under identical conditions. After 48 hours, the strain with sam8-sam5/pACYC produced 136 mg/L of caffeic acid, whereas the strain with tal-hpaBC/pACYC produced only 44 mg/L. This significant difference is likely due to the difficulty in expressing actinomycete genes in E. coli , as these genes often include rare codons and structural variations in mRNA. Additionally, the enzyme encoded by hpaBC can convert tyrosine to l-DOPA, which may interfere with the production of 4-coumaric acid, thereby reducing overall caffeic acid production (1).
Further enhancement of this pathway was achieved by adding the comt gene to the plasmids. This modification led to the production of 156 mg/L of ferulic acid in the VT-1 strain (sam8-sam5-comt/pACYC) and 43 mg/L in the VT-2 strain (tal-hpaBC-comt/pACYC) under the same culture conditions. The VT-1 strain demonstrated superior efficiency in producing ferulic acid, making the sam8-sam5-comt/pACYC plasmid a promising candidate for further research (1).
Our Method
This project aims to develop and optimize a de novo synthetic pathway in E. coli for the sustainable production of vanillin. The approach focuses on integrating plant-derived pathways with E. coli's native metabolic processes to efficiently convert simple carbon sources, specifically tyrosine into vanillin. Our method involves creating four different constructs as shown bellow using different gene combinations to produce vanillin.
Ni J, Tao F, Du H, Xu P. Mimicking a natural pathway for de novo biosynthesis: natural vanillin production from accessible carbon sources. Scientific Reports. 2015 Sep 2;5(1).
Kunjapur A, Hyun J, Prather K. Deregulation of S-adenosylmethionine biosynthesis and regeneration improves methylation in the E. coli de novo vanillin biosynthesis pathway. Microbial Cell Factories. 2016 Apr 11;15(1).
Liu Y, Sun L, Huo Y, Guo S. Strategies for improving the production of bio-based vanillin. Microbial Cell Factories. 2023 Aug 5;22(1).
Gallage J, Moller B. Vanillin-Bioconversion and Bioengineering of the Most Popular Plant Flavor and Its De Novo Biosynthesis in the Vanilla Orchid. Molecular Plant. 2015 Jan;8(1):40-57.
Zhang X, He Y, Wu Z, Liu G, Tao Y, Jing J, et al. Whole-Cell Biosensors Aid Exploration of Vanillin Transmembrane Transport. Journal of Agricultural and Food Chemistry [Internet]. 2021;69(10):3114-23. Available from: https://pubs.acs.org/doi/full/10.1021/acs.jafc.0c07886
