Abstract 2309 Using plants as bio-factories to produce high-value fungal-derived medicines

The antibiotic penicillin G, and the immunosuppressant mycophenolic acid (MPA), are critical and widely used medicines with high economical values. They are produced by fungi and their biosynthetic pathways have been well described. Our goal is to produce these essential medicines in a more sustainable way using plants as bio-factories. To prove the concept, we will reproduce both pathways using transient expression in Nicotiana benthamiana. The non-ribosomal peptide synthetase (NRPS) α-aminoadipyl-L-cysteinyl-D-valine synthetase (ACVS) and the non-reducing polyketide synthase (NRPKS; MpaC) are the core multifunctional megasynth(et)ases responsible for penicillin and MPA biosynthesis, respectively. Introducing functional fungal NRPS and NRPKS enzymes into plants will represent a significant scientific and technical advancement to enable the manufacturing of fungal-derived medicines. Development of this technology will enable the study of numerous non-plant biosynthetic pathways that may be challenging to investigate in their natural host. Optimized coding sequence for ACVS, MpaC', and accessory enzymes were cloned into plant expression vectors in-frame with a green fluorescence protein (GFP) tag under the 35S promotor using the Gateway cloning system. The codon-optimized fungal phosphopantethienyl transferase NpgA has also been cloned. Each vector has been transformed into Agrobacterium tumefaciens for delivery of transgenes into N. benthamiana leaf cells. To date, successful individual expression of MpaC, NpgA, MpaB, MpaA, MpaDE, and pbACL in plant leaves has been achieved in the mycophenolic acid pathway, confirmed by fluorescent confocal microscopy and RT-PCR. Additionally, the gene encoding ACVS has successfully been recombined from seven synthetic DNA fragments, and the expression of IPNS, NpgA, IAT, and PaaT in the penicillin G pathway has been successfully achieved. Co-expression of the different metabolic enzymes are expected to lead to intermediate metabolites in N. benthamiana. The production of these metabolites is being confirmed by liquid chromatography-mass spectrometry (LCMS) analysis. Leaf extraction methods have been developed and enable metabolite quantification compared to chemical standards. Tolerance of N. benthamiana towards penicillin G, MPA, and degraded products have been investigated. No toxic effects were observed in N. benthamiana after direct infiltration or exogenous supplementation of either drug. N. benthamiana continuously accumulates penicillin G and MPA over 7 days with exogeneous supplementation of 500 μg/mL of solution. Penillic, penicillic and penicilloic acid are the major degraded products of penicillin G; complete degradation of penicillin G was observed after eight days. However, MPA is a much more stable compound at lower pH and in the plant leaves. The transportation of these drugs to other leaves was also identified over time. Preliminary data from this study indicates that the successful expression and localization of fungal biosynthetic genes in plant hosts is possible and could lead to the synthesis of expected metabolites making it a useful approach for producing essential medicines or investigating fungal metabolic pathways. This work was financially supported by the W. M.