Biocatalysis with in-situ product removal improves p-coumaric acid production.

Natural and pure p-coumaric acid has valuable applications, and it can be produced via bioprocessing. However, fermentation processes have so far been unable to provide sufficient production metrics, while a biocatalytic process decoupling growth and production historically showed much promise. This biocatalytic process is revisited in order to tackle product inhibition of the key enzyme tyrosine ammonia lyase. In situ product removal is proposed as a possible solution, and a polymer/salt aqueous two-phase system is identified as a suitable system for extraction of p-coumaric acid from an alkaline solution, with a partition coefficient of up to 13. However, a 10% salt solution was found to reduce tyrosine ammonia lyase activity by 19%, leading to the need for a more dilute system. The cloud points of two aqueous two-phase systems at 40°C and pH 10 were found to be 3.8% salt and 9.5% polymer, and a 5% potassium phosphate and 12.5% poly(ethylene glycol-ran-propylene glycol) mW~2500 system was selected for in situ product removal. An immobilized tyrosine ammonia lyase biocatalyst in this aqueous two-phase system produced up to 33 g/L p-coumaric acid within 24 hours, a 1.9-fold improvement compared to biocatalysis without in situ product removal.

Characterization of tyrosine ammonia lyases from Flavobacterium johnsonian and Herpetosiphon aurantiacus.

p-Coumaric acid (pCA) can be produced via bioprocessing and is a promising chemical precursor to making organic thin film transistors. However, the required tyrosine ammonia lyase (TAL) enzyme generally has a low specific activity and suffers from competitive product inhibition. Here we characterized the purified TAL variants from Flavobacterium johnsoniae and Herpetosiphon aurantiacus in terms of their susceptibility to product inhibition and their activity and stability across pH and temperature via initial rate experiments. FjTAL was found to be more active than previously described and to have a relatively weak affinity for pCA, but modeling revealed that product inhibition would still be problematic at industrially relevant product concentrations, due to the low solubility of the substrate tyrosine. The activity of both variants increased with temperature when tested up to 45°C, but HaTAL1 was more stable at elevated temperature. FjTAL is a promising biocatalyst for pCA production, but enzyme or bioprocess engineering are required to stabilize FjTAL and reduce product inhibition.

Combining genetic engineering and bioprocess concepts for improved phenylpropanoid production.

The group of natural aromatic compounds known as phenylpropanoids has diverse applications, but current methods of production which are largely based on synthesis from petrochemicals or extraction from agricultural biomass are unsustainable. Bioprocessing is a promising alternative, but improvements in production titers and rates are required to make this method profitable. Here the recent advances in genetic engineering and bioprocess concepts for the production of phenylpropanoids are presented for the purpose of identifying successful strategies, including adaptive laboratory evolution, enzyme engineering, in-situ product removal, and biocatalysis. The pros and cons of bacterial and yeast hosts for phenylpropanoid production are discussed, also in the context of different phenylpropanoid targets and bioprocess concepts. Finally, some broad recommendations are made regarding targets for continued improvement and areas requiring specific attention from researchers to further improve production titers and rates.