Non Technical Summary
Plants play a vital role in our society and environment by producing various phenolic chemicals, called phenylpropanoids, which not only act as a significant carbon sink (e.g., lignin) but also provide essential nutrients, medicines, and valuable biomaterials. Through advanced plant gene editing and synthetic biology technologies, we now have the potential to enhance the nutritional and medicinal properties of crops and develop sustainable plant systems for producing a variety of phenolic chemicals using sunlight energy and carbon dioxide (CO2) from the air.Plants produce these phenolic chemicals primarily from an amino acid called phenylalanine. The PD's recent research has identified genetic mutations that greatly increase the production of phenylalanine from CO2 in plants. However, the production of phenylpropanoid compounds remained largely unchanged due to limitations in the metabolism of phenylalanine via the gate keeping enzyme called phenylalanine ammonia lyase (PAL). To resolve this critical bottleneck, this project aims to understand how PAL enzymes are regulated, which will allow us to redirect carbon flow from the accumulated phenylalanine towards the production of these valuable phenylpropanoid compounds in plants. By gaining fundamental insights into PAL regulation through detailed biochemical and protein structure analyses, this project further aims to develop new strategies for enhancing the production of phenylpropanoids in crops. This could lead to the generation of nutrient-enriched foods, natural dyes, and flavors by modulating native plant genes. Additionally, plants engineered to have improved production of phenylalanine and phenylpropanoid compounds could serve as efficient sources for a wide range of phenolic chemicals with nutritional, medicinal, and industrial applications.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	2499	1000	50%
206	1820	2020	30%
206	1999	2020	20%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1999 - Tobacco, general/other; 1820 - Soybean; 2499 - Plant research, general;

Field Of Science
2020 - Engineering; 1000 - Biochemistry and biophysics;

Goals / Objectives
This project has two major goals. The first goal is to elucidate the regulation of the interface of the AAA and phenylpropanoid pathways mediated by PALs, which can potentially provide useful enzyme variants and gene editing targets to efficiently direct carbon flow through the entry step of phenylpropanoid biosynthesis. The second goal is to test the impact of deregulating the entry steps of both AAA and phenylpropanoid biosynthesis in planta, with the eventual goal of building crop chemical production platforms with enhanced carbon flow from CO2 to the phenylpropanoid pathway. Metabolic engineering experiments will also be carried out in different plants to gain foundational knowledge for generating crop chemical production platforms with the improved entry step into the phenylpropanoid biosynthesis.Our specific objectives are to:Aim 1: Determine the molecular basis of PAL regulation, through phylogeny-guided sequence-structure-function analyses.Aim 2: Examine in planta functions of various PAL enzymes having altered regulation.Aim 3: Test impacts of simultaneously deregulating the entry steps of AAA and phenylpropanoid biosynthesis in crop plants.

Project Methods
The PD's group recently identified genetic mutations, called sota, that can enhance conversion of CO2 into aromatic amino acids (AAAs), especially L-phenylalanine (Phe); however, this study indicated that tight regulation of the committed enzyme, Phe ammonia lyase (PAL), limits phenylpropanoid production. This project will further elucidate the regulatory properties of PALs with the goal of efficiently directing carbon flow towards the phenylpropanoid pathway from AAAs. While many steps of the phenylpropanoid pathway are regulated at multiple levels, this project focuses on the feedback regulation of the PAL enzymes that connect AAA and phenylpropanoid biosynthesis. Based on our preliminary data, these committed enzymes are limiting phenylpropanoid production in the plant tissues when AAAs are highly accumulated. In this project we will:Aim 1: Determine the molecular basis of PAL feedback regulation through structure, biochemical, and phylogeny-guided sequence-structure-function analyses. The identified residues critical to PAL enzyme properties, especially feedback regulation, will be further evaluated by site-directed mutagenesis to experimentally test the role of these residues.Aim 2: Examine in planta functions of PAL enzyme variants and mutants having altered regulation by transiently expressing in the Nicotiana benthamiana leaves. The effects of various PAL expressions will be experimentally evaluated by extracting metabolites from plant tissues and metabolite profiling experiments will be conducted using liquid chromatography-mass spectrometry (LC-MS).Aim 3: Test impacts of simultaneously deregulating the entry steps of AAA and phenylpropanoid biosynthesis in soybean (Glycine max), a major crop in the US that can be efficiently transformed. These transgenic soybean plants will be generated at the Wisconsin Crop Innovation Center (WCIC), who will also conduct screening for successful transgene insertions. Then, metabolite profiling will be carried out by PD's group to evaluate the impacts of expressing deregulated enzymes, including PALs, by carrying out metabolic profiling as well as 13CO2 labeling experiments to trace the carbon flow through the AAA and phenylpropanoid pathways.