Engineering Lactococcus Lactis for the Synthesis of Plant Polyphenols

Gaspar, P., Technical University of Denmark
Dudnik, A., Technical University of Denmark
Neves, A. R., Current address: Chr. Hansen A/S
Forster, J., Technical University of Denmark

Plant polyphenols are powerful antioxidants and have been shown promising effects in the combat of cardiovascular disease, certain types of cancer, neurodegenerative diseases, allergies, diabetes and inflammation. These health-promoting properties have led to the exploitation of this group of compounds for pharmaceutical and/or biotechnological applications. Nevertheless, polyphenols are produced in small amounts, difficult to purify from natural resources and due to their complexity chemical synthesis is a poor option. Hence, the utilization of microbes as platforms for effective production of plant bioactive metabolites emerges as a feasible alternative.

Lactococcus lactis is an industrially relevant microorganism used worldwide in food fermentations. In view of its economical value, L. lactis has been intensively studied and a large body of comprehensive data on their metabolism and genetics was generated. The technological breakthroughs in functional genomics, combined with a GRAS status, a relatively simple metabolism, a small genome, and rapid growth, makes this organism an ideal host for production of valuable food and pharmaceutical compounds [1]. Thus, L. lactis tailored for optimal production of nutraceutical plant-derived polyphenols is a promising tool to fortify fermented dairy products without the need for food supplements.

Here we describe our efforts to engineer L. lactis for the production of plant polyphenols. Synthetic pathways for resveratrol production from L-tyrosine were assembled and functional expressed in a LDH-deficient strain. Engineered strains accumulated resveratrol and the intermediate p-coumaric acid in different amounts depending on gene combination and pathway configuration (operon vs monocistronic). Additionally, cinnamic acid and the stilbene pinosylvin were produced from L-phenylalanine due to substrate unspecificity of enzymes in the pathway. During growth of the producer strains, the profiles of intermediates and stilbene pools indicated malonyl-CoA availability as a bottleneck in stilbene production. This hypothesis was substantiated by a remarkable increase of resveratrol and pinosylvin specific yields in the presence of the fatty acid synthesis inhibitor cerulenin. We are currently attempting to improve the malonyl-CoA levels through modulation of acetyl-CoA carboxylase activity in the engineered strains. In addition, we will report on the potential of L. lactis for the production of flavonoids, such as quercetin and fisetin [2]. Synthetic pathways for the production of these two flavonoids have been assembled and their functional expression in the LDH-deficient strain is being assessed.

[1] Gaspar et al., 2013. Biotechnol Adv, 31:764-88

[2] Stahlhut et al., 2015. Metab Eng 31:84–93