How to prepare Vanillin from plastic?

Vanillin is an aromatic organic compound widely used in food, beverages, daily necessities, medicine and other fields. It is one of the most widely used flavoring agents in the world, and the demand far exceeds the supply of natural vanilla beans. Currently, about 85% of vanillin is synthesized from fossil fuel-derived chemicals. Researchers have found that plastic can be converted into Vanillin using genetically modified bacteria, which not only solves plastic pollution but also moves towards a circular economy.

The synthesis of Vanillin is as follows:

(1) Construction of a new enzyme pathway for the production of vanillin from terephthalate (TA)

Since E. coli has the ability to reduce aldehydes to the corresponding alcohols, E. coli MG1655 RARE (reduced aromatic aldehydes) was selected as the host microorganism. It has previously been used to biosynthesize vanillin from glucose. It was hypothesized that TA could be converted to vanillin via a de novo pathway involving: (i) terephthalate 1,2-dioxygenase (TPADO), (ii) dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase (DCDDH), (iii) carboxylic acid reductase (CAR) and (iv), catechol O-methyltransferase (COMT) (Fig. 2a). Notably, both CAR39 and O-MT40 were reported to accept PC, dihydroxybenzaldehyde (DHBAl) and vanillic acid (VA) as substrates, thus this pathway could potentially produce vanillin via two possible intermediates (VA or DHBAl).

(2) Conversion of TA to vanillin

Resuspended E. coli RARE cells expressing pVan1, pVan2 and pSfp (E. coli RARE_pVanX) were used in a screening-scale bioconversion reaction to convert TA to vanillin. While vanillin was not detected in cells expressing pVan1 alone or in experiments lacking TA substrate, cells expressing all three plasmids supplemented with TA (5 mM) gave detectable amounts of the target compound vanillin, and the intermediates PC, DHBAl, and vanillic acid were detected at concentrations of 18 μM, 10 μM, and 2 μM, respectively.

(3) Process optimization to improve vanillin yield

Screening of protein expression culture media showed that M9 basal medium supplemented with casamino acids (M9-CA) gave the highest combined conversion of vanillin and the key intermediate PC. It was shown that resuspending whole cells in fresh M9 medium during the exponential growth phase was superior to adding TA to the expression culture, increasing the vanillin titer by 4-fold (77 μM ± 11 μM). Lowering the reaction temperature from 30 °C to 22 °C increased vanillin production by 5-fold (577 μM ± 22 μM compared to 117 μM ± 40 μM at 30 °C). E. coli RARE_pVanX cells were resuspended in M9-glucose supplemented with l-Met and nBuOH (pH 5.5), incubated with TA for 24 h at room temperature, and covered with oleyl alcohol. Product formation under the optimized conditions was confirmed after scaling up the biotransformation to 40 mL scale and analyzing the reaction products by NMR spectroscopy.

The reaction is mild, uses a whole-cell catalyst produced from renewable feedstocks, occurs in aqueous media under ambient conditions (room temperature, pH 5.5-7), does not require additional cofactors or reagents, and does not generate hazardous waste. The maximum vanillin titer was 785 μM (119 mg L−1, 79% conversion), a 157-fold increase over the titer in the pre-optimization experiment. The keys to this high conversion were the bioconversion temperature, cell permeabilization, and the use of ISPR to increase the flux to vanillin.

References:

[1] SADLER J C, WALLACE S. Microbial synthesis of vanillin from waste poly(ethylene terephthalate)†[J]. Green Chemistry, 2021, 13: 4665-4672. DOI:10.1039/D1GC00931A.

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