Combinatorial biosynthesis of plant natural products in microorganisms

Abstract

Plant natural products (PNPs) are among the most significant compounds used in pharmaceutical and cosmetic industries. However, most PNPs accumulate at low quantities in plants and are difficult and expensive to isolate. Additionally, a high investment of land, water and time is required and pests and extreme weather cause insecurity in the supply chain. Moreover, their chemical synthesis is challenging due to their stereochemical complexity. Therefore, in the last decades, PNPs pathways have been engineered in microbial hosts using combinatorial biosynthesis. In this approach, genes from different species are assembled to construct complex biosynthetic pathways. Curcuminoids are PNPs whose biosynthetic pathways have been extensively explored in the last years due to their applications. They have been used in traditional food, cosmetic and medicine for centuries. Their therapeutic properties include anti-cancer, antiinflammatory, anti-oxidant, anti-Alzheimers, and anti-HIV, among others. Curcumin, the most promising curcuminoid, has a projected market size of USD 130 million by 2025. Herein, we propose an optimized artificial biosynthetic pathway to produce curcuminoids. This pathway involves 6 enzymes and produces ferulic acid as an intermediate using caffeic acid Omethyltransferase. Starting from tyrosine, 1325 M of ferulic acid were obtained, comprising the first part of the pathway. Then, the second part of the pathway was also optimized. From ferulic acid we obtained the highest concentration of curcumin reported (1212.7 M) so far, corresponding to a 26% increase [1]. Subsequently, curcumin was produced from tyrosine (the whole pathway) using a mono-culture. Production increased comparing to a previously reported pathway that used a caffeoyl-CoA O-methyltransferase [2]. Additionally, a co-culture strategy was evaluated to further improve the production by reducing cells metabolic burden. We used one E. coli strain able to convert tyrosine to hydroxycinnamic acids and another able to convert them to curcuminoids. Using CRISPR-Cas9 method we disrupted lacZ gene in one of the strains which allowed to follow co-culture population composition using the blue-white screening method. This co-culture strategy increased 8.8 times the curcuminoids production (126 M) as compared to the mono-culture production. These results comprise a significant step towards the large-scale production of these valuable compounds. References [1] Couto MR, Rodrigues JL, Rodrigues LR (2017) Optimization of fermentation conditions for the production of curcumin by engineered Escherichia coli, Journal of the Royal Society Interface, 14, 1-8. [2] Rodrigues JL, Araújo RG, Prather KLJ, Kluskens LD, Rodrigues LR (2015) Production of curcuminoids from tyrosine by a metabolically engineered Escherichia coli using caffeic acid as an intermediate, Biotechnology Journal, 10, 599-609.