Gene source selection as a metabolic engineering tool for naringenin production in Saccharomyces cerevisiae

Abstract

Mankind has evolved from hunting and gathering lifestyle to be a good and service provider society. This cultural and social evolution has demanded fast developments in food science and technology. However dietary risk was still a major death cause in 2017. Functional foods were designed to tackle these dietary risks. Natural plant secondary metabolites, such as flavonoids have great potential in the food industry as functional food components. Flavonoids, a sub-group of the phenolic compounds have high antimicrobial and antioxidant activities, which presents the opportunity to apply them as food additives and preservatives. Metabolic engineering of Saccharomyces cerevisiae is a sustainable flavonoid production technique. However, current naringenin production yield is low due to inefficient enzymatic activity. This thesis has constructed the naringenin pathway in S. cerevisiae BY4741 strain and used gene source screening as a tool to identify the best gene source for enzymes 4-coumarate: coenzyme ligase (4CL) and chalcone synthase (CHS). For the first time, the 4CL gene from Medicago truncatula and CHS gene from Vitis vinifera were expressed in S. cerevisiae and this combination provided the highest yield of naringenin. It was 28-fold higher as compared to the reference strain. The results have demonstrated, that gene source selection has a high impact on naringenin production in S. cerevisiae. Metabolite profiling has studied the cellular and enzymatic behavior during naringenin production. Three metabolites, L-aspartate, glycine and serine were identified as important cellular compounds. Furthermore, site-directed mutagenesis of CHS demonstrated the significance of amino acids Glu61, Lys151, Ser184 and the Ser388-Ala389-Pro390 triplet. Mutation of Glu61/Asp61 and Ser184/Ala184 have significantly decreased naringenin production, due to their location at the CoA-binding tunnel and at the coumaroyl-binding pocket. A genetically modified Y-28 S. cerevisiae strain was also utilized for the expression of the naringenin pathway. This resulted in an increased yield with the highest naringenin production reaching 28.68 mg/L. In addition, the similarity pattern of the combinations’ production yields was compared between the BY4741 and Y-28 strains and were found to be matching. Alternative carbon and nitrogen sources were compared and analyzed in order to decrease the cost of culturing media. The 1% sucrose and 1% glycerol mixture was found to be a potential carbon source. Furthermore, okara was identified as an ideal nitrogen source. The results demonstrated that the selection and combination of enzymes from the correct gene source could greatly improve naringenin production. For the future, this could help commercialize flavonoid production, which would result in natural food preservatives and additives.