Integrating bioprocesses for the production of aromatics

Aromatic compounds include valuable flavors, nutritional supplements, and precursors to polymers and pharmaceuticals. Current methods for production of aromatics include agricultural biomass extraction and petrochemical synthesis, but these methods have issues in some cases with impurities and with not being labeled natural. Bioprocessing is a promising alternative approach for the production of aromatics without these issues, but with its’ own limitations including product toxicity and product inhibition. Using p-coumaric acid as a model aromatic target molecule, a bioprocess was developed and tested to overcome these limitations. Four variants of the key enzyme tyrosine ammonia lyase (TAL) were cloned, expressed, and characterized to identify TAL from Flavobacterium Johnsoniae as the preferred variant due to its’ relatively high catalytic rate and low degree of product inhibition. This variant was also found to be highly active at pH 10 and 45°C, increasing tyrosine solubility. Four different TAL expression systems were compared, and an arabinose inducible system in E. coli was found to be preferable due to its’ tight yet strong expression, and due to the relatively low cost of arabinose as an inducer compared to IPTG. TAL expression was optimized in a microbioreactor, identifying the growth rate during the induction phase as the most important variable, with the inducer concentration also being significant. The fed-batch fermentation process was transferred to liter scale, where 40 grams cell dry weight per liter expressing TAL was produced within 24 hours. These whole-cells were immobilized in an alkali-stable k-carrageenan hydrogel to improve biocatalyst stability and ease of biocatalyst recycle. Several strategies of mitigating product inhibition were considered and tested, including the use of co-solvents to increase substrate solubility, as well as in-situ product removal using ion-exchange or liquid-liquid extraction. The use of a polymer/salt aqueous two-phase system was selected as the preferred strategy due to its’ high partition coefficient of p-coumaric acid into the polymer phase. The aqueous two-phase system was optimized to increase compatibility with the biocatalyst, and the two were successfully integrated in a rotating bed reactor. Finally, tyrosine was converted into 33 grams per liter p-coumaric acid within 24 hours using this setup, almost double the product concentration without the aqueous two-phase system. This work advances the development of bioprocesses for the production of aromatics.