EBRC Seminar Series – May 21, 2021 (4:00 PM ET)
Please join us for an exciting seminar on May 21, 2021, from 4-5:00 PM ET. This is the final seminar in the 2021 EBRC Seminar Series.
Speaker abstracts are below. The seminar is open to all, so please feel free to share this information with your colleagues.
The seminar will be held on Zoom using the following link for all sessions:
Zoom link: https://berkeley.zoom.us/j/97626552307?pwd=alVlS3dXM0lZYklYeE9zVXljWUI0UT09
Meeting ID: 976 2655 2307
“Sequence-function analysis helps identify multiple pathways to enhance Phenylalanine Ammonia-Lyase (PAL) activity”
Nikhil Unni Nair (Tufts University)
Phenylalanine ammonia-lyases (PALs) non-oxidatively deaminate L-phenylalanine to trans-cinnamic acid (tCA) and are widely found associated with secondary metabolism in plants, bacteria, and fungi. Biocatalytic applications for natural product and fine chemical synthesis has driven the discovery, expression, characterization, and engineering of PALs. More recently, development of PALs for phenylketonuria (PKU) management and cancer therapy has further increased interest in engineering this class of enzymes. While there is a general understanding of how residues in the substrate-binding pocket contribute to specificity and turnover, led by rational mutagenesis studies, there is generally a poor understanding of how distal residues affect function. In general, outcomes from directed evolution can identify distal hotspots but there have only been two such studies with this enzyme. The first study resulted in modest improvement in activity whereas the other, conducted by us, identified residues within the active site only. Deep mutational scanning (DMS) can identify functional hotspots, and when coupled with directed
evolution can accelerate engineering campaigns, although there are few examples of this approach. Further, DMS can provide a comprehensive map of sequence–function relationships to explore the protein fitness landscapes, uncover functionally relevant sites, improve molecular energy functions, and identify beneficial combinations of mutations for protein engineering. Though extensive body of research exists on function, structure, mechanism of PAL, a systematic study exploring the sequence-function space has not been attempted.
Previously, we developed a growth-coupled enrichment for rapid screening of high-activity variants of AvPAL* (used to formulate the PKU drug Pegvaliase®) in E. coli. After a single round of directed evolution using this growth-coupled enrichment, we identified 2 active site mutations improved kcat < 2-fold. However, the sequence-function fitness landscape of AvPAL* remains to be explored. In this study, we achieve several outcomes. First, we obtained the detailed sequence-function landscape of PAL, to date, using DMS, identifying >60 mutational hotspots. Next, we picked seven sites for comprehensive single and multi-site saturation mutagenesis and we identified multi-site mutations with ~2.5-fold improvement in the kcat (and >3-fold increase in catalytic efficiency). We then explored the epistatic effect of these mutations, uncovering positive, neutral, and negative interactions among distal and proximal sites. Finally, to understand the mechanistic role of key mutations in hyperactive variants, we performed modelling studies and concluded that there are multiple pathways to enhance PAL catalytic activity, including, decreased root mean square fluctuation (RMSF) of substrate in the active site, greater proximity of the substrate to catalytic residues, and facilitated diffusion of the substrate to the active site, among others. In summary, this study significantly advances basic and applied enzymology of PALs, a heretofore understudied class of enzymes with a wide array of applications.
Development of a yeast-based assay for bioavailable phosphorous
Heather Shepherd (University of Notre Dame)
Preventing eutrophication of inland freshwater ecosystems requires quantifying the phosphorous (P) content of the streams and rivers that feed them. Typical methods for measuring P assess soluble reactive P (SRP) or total P (TP) and require expensive analytical techniques that produce hazardous waste. Here we present a novel method for measuring the more relevant bioavailable P (BAP); this assay utilizes the growth of familiar baker’s yeast, avoids production of hazardous waste, and reduces cost relative to measurements of SRP and TP. The yeast BAP (yBAP) assay takes advantage of the observation that yeast density at saturating growth increases linearly with provided P. We show that this relationship can be used to measure P in freshwater in concentration ranges relevant to eutrophication. In addition, we measured yBAP in water containing known amount of fertilizer and in samples from agricultural waterways. We observed that the majority of yBAP values were between those obtained from standard SRP and TP measurements, demonstrating that the assay is compatible with real-world settings. The cost-effective and nonhazardous nature of the yeast-based assay suggests that it could have utility in a range of settings, offering added insight to identify water systems at risk of eutrophication from excess phosphorus.
Engineering alternative degradation tags for synthetic circuits
Prajakta Jadhav (South Dakota State University)
The goal in synthetic biology is to build robust synthetic circuits in bacteria that are dynamic, highly regulated, and result in a unified response. In the last 20 years, the synthetic biology field has effectively leveraged transcriptional (RNA production) and translational (protein production) controls. However, protein degradation plays an important role in determining the half-life of proteins and regulating biological systems. Amino acid degradation tags are exploited to avoid a reliance on cell division for protein dilution and to build dynamic circuits. The most leveraged
degradation tag in E. coli is the ssrA-tag, which is an 11-amino acid sequence that primarily target proteins for degradation by the ClpXP proteolytic system. However, the use of the ssrA-tag limits scalability and complexity especially in synthetic oscillators in bacteria due to itsmultiple proteolytic target recognition signals. Our goal is to build orthogonal oscillators that utilize degradation tags targeting to multiple proteases with no crosstalk. In this study, we aremodifying the ssrA-tag to change the substrate affinity and degradation rate and produce new
synthetic oscillators. We hypothesize that changing the degradation rate can alter the output signal of the oscillator. We have tailored the ssrA-tag to reduce crosstalk between proteolytic systems to increase robustness and developed a variety of degradation tags. While many of these tags exhibited decreased or no change in degradation rates, two depicted significant increase. We further tested interesting candidates for crosstalk between proteolytic systems and identified a tag that display minimum to no crosstalk with other proteolytic systems. We aim to build and compare the output signals of different oscillators with novel tags in batch cultures and at the single-cell level. The design and implementation of these novel degradation tags will enable development of biological building blocks for increased complexity in synthetic circuits.