Most aromatic chemicals are currently obtained from natural gas and petroleum feedstock and their production processes are often energy intensive and create significant waste streams. Microbial synthesis of aromatic molecules from non-toxic and renewable resources represents an interesting option to replace the existing fossil fuel based production of aromatic hydrocarbons.
Among these aromatics compounds, terephthalic acid (TA) stands out with a wide variety of commercial applications, such as production of polyethylene terephthalate (PET) and high tensile strength fibres (e.g., Kevlar®) used in the fabrication of military protective equipment, making up a total market of $US 41 billion of sales just in Asia. Traditionally this compound has been chemically synthesized from aromatic hydrocarbons, and no biological production has been reported. This project explores the possibility to produce the potential TA precursors p-aminobenzoic acid (pABA) and p-hydroxybenzoic acid (pHBA) from glucose in baker’s yeast. Both molecules are metabolites derived from the aromatic amino acid biosynthesis in microorganisms and plants.
In order to obtain these compounds, S. cerevisiae was modified to redirect the carbon flow from the central metabolism towards the aromatic amino acid biosynthesis, and therefore towards the production of pABA and pHBA. The final titer of pHBA obtained was 0.65 mM using a single knock out strain that overexpress the enzyme chorismate lyase (ubiC) catalysing the reaction from chorismate to pHBA (S. cerevisiae S288 Δaro7 pVC3 UBIC). This strain also lacks the chorismate mutase activity, which is encoded in the gene aro7 that catalyses the reaction from chorismate to prephenate, the first step in the phenylalanine-tyrosine branch. Likewise, 0.25 mM of pABA was obtained using a double knock out strain that overexpresses the enzyme p-aminobenzoate synthase (S. cerevisiae S288 Δaro7 Δtrp3 pVC3 ABZ1) that converts chorismate to 4-amino 4-deoxychorismate, which is a direct precursor of pABA. In addition to be deficient in chorismate mutase activity, this last strain also lacks anthranilate synthase activity (trp3), which is the first step of the tryptophan branch.
Along with the production of pABA and pHBA, accumulation of several intermediaries of the shikimate pathway has been found, such as shikimate, phenylpyruvate, protocatechuate and 4-amino 2-hydroxybenzoic acid. In order to overcome shikimate accumulation, the overexpression of shikimate kinase is proposed. Additionally, with the aim at improving pABA production titer the overexpression of the enzyme encoded in abz2 is also recommended.
The unexpected accumulation of metabolites of the phenylalanine branch, particularly phenylpyruvate, was studied using four different S. cerevisiae strains (Δpha2, Δaro7, Δpha2 Δaro7, and wild-type). Results indicate that this process wasn’t biologically driven by the pha2 encoded 2 enzyme (prephenate dehydratase).This finding leads to the idea that the leak in the phenylalanine branch is carried out chemically rather than biologically.
Even though the level of production of these aromatic molecules is still comparatively low, these results are encouraging, showing that their biotechnological production is a possible process in yeast. There are still several available alternatives for yield improvement, once the limitations that were found herein are alleviated. First, other enzymes of the pathway could be overexpressed and second the combination of ‘in situ’ extraction fermentation processes could be used as well, in order to improve the response towards pHBA and pABA toxicity.