Enhancement of Protocatechuate Decarboxylation for the Effective Production of Muconic Acid from Renewable Feedstocks

Protocatechuic acid (PCA, 3,4-dihydroxybenzoic acid) is an intermediate to produce a valuable platform chemical, cis,cis-muconic acid (ccMA), from renewable feedstocks such as sugar. For the effective production of ccMA, PCA decarboxylation (Pdc) step has been described as a bottleneck, and it has also been required to find out some methods to enhance the enzymatic activity for developing the efficient ccMA production. The Pdc from Klebsiella pneumoniae subsp. pneumoniaeA170-40 (AroY) had been identified, and it has been mainly used to produce ccMA from sugar, however, no reports on the enhancement of Pdc activity and biochemical characteristics of AroY have been published. In addition to improving the Pdc activity, our current work focuses on ccMA production in particular, from lignin-related aromatic compounds because sugars are already in high demand in the development of many bio-based products.

We identified a way to increase Pdc activity in Escherichia coli through the expression of both AroY and the B subunit (KpdB) of 4-hydroxybenzoate decarboxylase complex. The Pdc activity increased approximately 20-fold higher than that without the KpdB expression. The effects of pH and temperature on Pdc activity under the condition that AroY was expressed either with or without KpdB. Regardless of the KpdB expression, the optimal pH and temperature for Pdc activity were 5.5 and 35^oC, respectively. AroY could decarboxylate not only PCA but also Gallic acid (3,4,5-trihydrroxybenzoic acid) and 3-O-methylgallic acid (4,5-dihydroxy-3-methoxybenzoic acid) although the decarboxylated products from p-hydroxybenzoic acid, vanillic acid (4-hydroxy-3-methoxybenzoic acid), syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) were not detected. We are now developing a new transformed bacterial strain using aroY , kpdB and other required genes to produce ccMA from variety of aromatic compounds, vanillic acid, p-hydroxybenzoic acid, ferullic acid (trans-ferullic acid, 4-hydroxy-3-methoxycinnamic acid), and p-coumaric acid (trans-p-coumaric acid, 4-hydroxycinnnamic acid) etc, with high yields.

This work is supported in part by a Grant-in-Aid for Scientific Research (JSPS KAKENHI 26850041), and Advanced Low Carbon Research and Development Program, Japan Agency for Science and Technology (JST-ALCA).