Penn State College of Engineering Research Experience for Undergraduates

Sam Krug
Penn State University University Park, PA 16802 phone: (717)608-6218

email:sak5474@psu.edu

Academic Preparation I am a junior at Penn State University, studying Chemical Engineering. I have maintained an overall 3.38 GPA, pursued a minor in music performance, become a leader in several university affiliated organizations, and had undergraduate research experience both at Hershey Medical Center, supervised by Dr. Sinisa Dovat, and on campus during the school year, supervised by Dr. Howard Salis. This past summer, I worked as a research technician at the Hershey Medical Center. This was my first experience in a lab and I was trained by professional medical researchers. My role was to assist in the mapping of a signaling pathway involving the Ikaros protein; a failure in this protein's activation has been linked to leukemia. This year, I began learning how to carry out genetic engineering and synthetic biology in Dr. Salis lab. I was responsible for helping to optimize a multi-enzyme pathway in Escherichia coli that will rapidly regenerate NADPH. NADPH is an important cofactor that acts as a reducing agent in microbial production of many high-value industrial chemicals. During this time, I have become proficient in many molecular cloning techniques, and I am excited to begin a more independent project. I have decided to construct a new metabolic pathway that uses NADPH to produce 3hydroxypropionic acid. My project will demonstrate how an increased NADPH regeneration rate will impact the production of biorenewable chemicals. Research Plan Introduction 3-hydroxypropionic acid (3-HP) is a monomer that can be polymerized to produce a biodegradable plastic, and can also be converted into acrylic acid and nylon. Therefore, there is a large market for 3-HP and its many applications. The metabolic pathway to produce 3-HP contains two enzymes: an acetyl-CoA carboxylase from E. coli and a malonyl-CoA reductase from Chloroflexus aurantiacus. Acetyl-CoA carboxylase is a 4-subunit enzyme that converts acetyl-CoA into malonyl-CoA, using biotin as a cofactor. Malonyl-CoA is then converted to 3HP by the malonyl-CoA reductase, oxidizing NADPH in the process. The 3-HP biosynthesis pathway will be coupled to the NADPH regeneration pathway that has been engineered by ChiamYu Ng, a doctoral candidate in Dr. Salis lab. Objectives & Approach The goal of this research project is to engineer and optimize a six-gene pathway that has, in previous studies, been proven to produce 3-HP. The first five genes are accA, accD, accB, accC, and birA encoding for the acetyl-CoA carboxylase and the biotinilase. These enzymes are naturally found in E. coli, and we will be increasing their expression levels to optimal amounts. The final gene is mcr, encoding the malonyl-CoA reductase. We will optimize this pathway using a newly developed approach that combines computational algorithms with combinatorial DNA mutagenesis to simultaneously vary the expression levels of the 6 enzymes and find the best pathaway variants. To do this, my experimental approach is: 1. Redesign the enzyme coding sequences using codon optimization to ensure that their maximum expression levels can be very high. The DNA fragments will be synthesized.

2. Use the Salis lab's RBS Library Calculator to design a set of ribosome binding site (RBS) sequences that will systematically vary each enzyme's expression level. 3. Assemble the synthesized DNA fragments to construct a plasmid that expresses the six genes in two bacterial operons. The RBS sequences are defined by the RBS Library Calculator results. 4. Introduce the pathway into the E. coli genome using lambda red recombination. 5. Use a directed genome mutagenesis approach, called MAGE, to mutate the ribosome binding site (RBS) sequences and systematically optimize the enzyme expression levels. The expression levels of the five enzymes, accABCD and mcr, will determine the production rate and yield of 3-HP. The best pathway lives somewhere in a 5-dimensional space. This task may seem daunting, but Salis lab members have already optimized a 3-enzyme pathway using computational algorithms. With assistance, I will use the same algorithms to analyze a limited data-set and find the optimal expression levels for the 5 enzymes. I will use the Salis lab's biophysical model of translation to predict how changing the ribosome binding site sequence controls enzyme expression, and I will design new RBS sequences for optimal expression levels. These RBS mutations are made directly to the genome using the MAGE technique, which I have previously carried out. I will use HPLC to measure the concentrations of glucose and 3-HP from my E. coli cultures over several time points. Using this data, I will calculate the production rate of 3-HP and the conversion yield from glucose to 3-HP. Project Timeline My first month will be designated to constructing the six-enzyme pathway on a plasmid and verifying that the plasmid-encoded 3-HP biosynthesis pathway is capable of converting glucose into 3-HP within E. coli cells. I will use HPLC to perform these measurements. I will then cut out the biosynthesis pathway from the plasmid, and integrate it into the E. coli genome using lambda red recombination. This E. coli genome will already have the newly engineered NADPH regeneration pathway in it. During the second month, I will use MAGE to mutate the genomic RBS sequences of 5 enzymes in the pathway. MAGE is an iterative technique, and I will carry out 30 iterations, taking approximately two weeks. I will then isolate colonies, and culture them, to measure the production rate of 3-HP. By the end of the summer, I will analyze my measurement data to determine the optimal enzyme expression levels to maximize production of 3-HP. I also plan on working with a graduate student on a daily basis so that I can become more familiar with using the RBS design software that was created by the lab and so that any question I may have about my design process will be answered. I also plan to meet weekly with Dr. Salis, and to attend the weekly lab meetings. Ultimately, the goal of this research project is to successfully design, characterize, and optimize the 3-HP biosynthesis pathway, while using the computational algorithms to extract more, re-usable information from my experiments.