Optimisation of coumaric acid production from aromatic amino acids in Kluyveromyces marxianus

Yeasts are attractive hosts for the production of heterologous products due to their genetic tractability and relative ease of growth. While the baker's yeast Saccharomyces cerevisiae is a powerful workhorse of the biotechnology industry, the species has metabolic limitations and it is critical that we develop alternative platforms that will facilitate the development of bioprocesses that rely on sustainable feedstocks. In this study, we used synthetic biology tools to construct coumaric acid-producing strains of Kluyveromyces marxianus, a yeast whose physiological traits render it attractive for biotechnology applications. Coumaric acid is a building block in the synthesis of many different families of aromatics and is a key precursor for the synthesis of complect phenylpropanoid molecules, including many flavours and aromas. The starting point for this work was a K. marxianus chassis strain that has increased flux towards the synthesis of tyrosine and phenylalanine, the aromatic amino acids that can serve as starting points for coumaric acid synthesis. Following principles of synthetic biology, a modular approach was taken to identify the best solution to different metabolic possibilities and these were then combined in different ways. For the first step, it was established that the route from phenylalanine was superior to that from tyrosine and the combined overexpression of PlPAL, AtC4H and AtCPR1 delivered the highest yield of coumaric acid. Next, it was established that while Pdc5 and Aro10 both had phenylpyruvate decarboxylase activity, inactivation of ARO10 was sufficient to prevent flux loss in the pathway. Since phenylalanine is the starting point, efforts were made to improve efficiency of its production. It was found that glutamate was a preferred nitrogen source for coumaric acid production, and this knowledge was used to engineer a strain that overexpressed S. cerevisiae GDH1 and delivered higher yields of coumaric acid. Ultimately, this strategy led to the development of strains that has yields of up to 48 mg coumaric acid /g glucose. Strains were evaluated in bioreactors to investigate the effects of different process parameters. These analyses indicated that engineered strains face some redox balance challenges and further work will be required overcome these to develop strains that can perform well under industrial conditions.