An automated data-driven pipeline for improving heterologous enzyme expression

A key challenge in cellular biomanufacturing of fuels, chemicals, and pharmaceuticals is that many pathway enzymes have very low activity, limiting overall titers and productivities. One reason is that enzymes are marginally stable under their native conditions, and expression in a different environment can thermodynamically favor the unfolded state. Additionally, overexpression can result in aggregation because natively expressed proteins are close to their solubility limit. This challenge suggests an engineering solution: engineer pathways enzymes to be stable in their biomanufacturing chassis. However, this is difficult because: (a.) many enzymes do not have high-throughput activity screens needed for directed evolution; (b.) there are few or no structures available; (c.) there are often multiple limiting enzymes; (d.) most mutations confer small benefits to stability; and (e.) the plurality of stability-enhancing mutations decrease catalytic efficiency. I will present a culmination of my group\u27s approach to solve the above challenges, in effect automating the design of stable, active enzymes from limited combinatorial datasets. This engineering strategy involves user-defined precise mutagenesis1,2, deep sequencing to evaluate the functional effect of nearly all possible single point mutants on solubility3, Bayesian methods to discriminate stable, catalytically neutral from deleterious mutations3, and computational design to combine up to 50 mutations at once4. I will show recently published work on application of this method to improve the pathway productivity of a medicinal alkaloid pathway in Saccharomyces cerevisiae5, and end with the description of a computational pipeline to automate our process for any enzyme of interest. References 1. Wrenbeck EE, KlesmithJR, AdeniranA, StapletonJA, Tyo KJ, Whitehead TA, (2016) Plasmid-based single-pot saturation mutagenesis, Nature Methods 13(11): 928-930 doi:10.1038/nmeth.4029 2. Medina-Cucurella A, Steiner PJ, Faber MS, Beltran J, Borelli A, Kirby M, Cutler S, Whitehead TA, (2019) “User-defined single pot mutagenesis using unpurified oligo pools”, under review 3. Klesmith JR, Bacik JP, Wrenbeck EE, Michalczyk R, Whitehead TA (2017) Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning, PNAS 114:2265-2270 doi: 10.1073/pnas.1614437114 4. Klesmith JR, Bacik JP, Michalczyk R, Whitehead TA (2015) High-resolution sequence function mapping of a levoglucosan utilization pathway in E. coliâ, ACS Synthetic Biology 4 (11), 1235-1243 DOI: 10.1021/acssynbio.5b001 5. Wrenbeck EE, Bedewitz MA, Klesmith JR, Noshin S, Barry CS, Whitehead TA* (2019) “An automated data-driven pipeline for improving heterologous enzyme expression” ACS Synthetic Biology accepte

Junior Recital:Tyler Mitchell, violin

This recital is presented in partial fulfillment of the requirements for the degree Bachelor of Music in Performance Mr. Mitchell studies violin with Helen Kim.https://digitalcommons.kennesaw.edu/musicprograms/1646/thumbnail.jp

Junior Recital: Dirk Stanfield, flute

This recital is presented in partial fulfillment of requirements for the degree Bachelor of Music in Performance. Mr. Stanfield studies flute with Christina Smith.https://digitalcommons.kennesaw.edu/musicprograms/1435/thumbnail.jp

Guest Artist Rectial: William Johnston, viola and Tim Whitehead, piano

KSU School of Music presents guest artists William Johnston, viola and Tim Whitehead, piano.https://digitalcommons.kennesaw.edu/musicprograms/1149/thumbnail.jp

The small FNR regulon of Neisseria gonorrhoeae: comparison with the larger Escherichia coli FNR regulon and interaction with the NarQ-NarP regulon

BACKGROUND: Neisseria gonorrhoeae can survive during oxygen starvation by reducing nitrite to nitrous oxide catalysed by the nitrite and nitric oxide reductases, AniA and NorB. The oxygen-sensing transcription factor, FNR, is essential for transcription activation at the aniA promoter, and full activation also requires the two-component regulatory system, NarQ-NarP, and the presence of nitrite. The only other gene known to be activated by the gonococcal FNR is ccp encoding a cytochrome c peroxidase, and no FNR-repressed genes have been reported in the gonococcus. In contrast, FNR acts as both an activator and repressor involved in the control of more than 100 operons in E. coli regulating major changes in the adaptation from aerobic to anaerobic conditions. In this study we have performed a microarray-led investigation of the FNR-mediated responses in N. gonorrhoeae to determine the physiological similarities and differences in the role of FNR in cellular regulation in this species. RESULTS: Microarray experiments show that N. gonorrhoeae FNR controls a much smaller regulon than its E. coli counterpart; it activates transcription of aniA and thirteen other genes, and represses transcription of six genes that include dnrN and norB. Having previously shown that a single amino acid substitution is sufficient to enable the gonococcal FNR to complement an E. coli fnr mutation, we investigated whether the gonococcal NarQ-NarP can substitute for E. coli NarX-NarL or NarQ-NarP. A plasmid expressing gonococcal narQ-narP was unable to complement E. coli narQP or narXL mutants, and was insensitive to nitrate or nitrite. Mutations that progressively changed the periplasmic nitrate sensing region, the P box, of E. coli NarQ to the sequence of the corresponding region of gonococcal NarQ resulted in loss of transcription activation in response to the availability of either nitrate or nitrite. However, the previously reported ligand-insensitive ability of gonococcal NarQ, the "locked on" phenotype, to activate either E. coli NarL or NarP was confirmed. CONCLUSION: Despite the sequence similarities between transcription activators of E. coli and N. gonorrhoeae, these results emphasise the fundamental differences in transcription regulation between these two types of pathogenic bacteria

Homology and Derived Series of Groups II: Dwyer's Theorem

We give new information about the relationship between the low-dimensional homology of a group and its derived series. This yields information about how the low-dimensional homology of a topological space constrains its fundamental group. Applications are given to detecting when a set of elements of a group generates a subgroup ``large enough'' to map onto a non-abelian free solvable group, and to concordance and grope cobordism of links. We also greatly generalize several key homological results employed in recent work of Cochran-Orr-Teichner, in the context of classical knot concordance. In 1963 J. Stallings established a strong relationship between the low-dimensional homology of a group and its lower central series quotients. In 1975 W. Dwyer extended Stallings' theorem by weakening the hypothesis on the second homology groups. The naive analogues of these theorems for the derived series are false. In 2003 the second author introduced a new characteristic series, associated to the derived series, called the torsion-free derived series. The authors previously established a precise analogue, for the torsion-free derived series, of Stallings' theorem. Here our main result is the analogue of Dwyer's theorem for the torsion-free derived series. We also prove a version of Dwyer's theorem for the rational lower central series. We apply these to give new results on the Cochran-Orr-Teichner filtration of the classical link concordance group.Comment: 26 pages. In this version, we have included a new proof of part of the main theorem. The new proof is somewhat simpler and stays entirely in the world of group homology and homological algebra rather than using Eilenberg-Mac Lane spaces. Other minor corrections. This is the final version to appear in Geometry & Topolog

Poor choices: the limits of competitive markets in the provision of essential services to low-income consumers

A major study of the problems faced by the poor in the market for seven essential services in the UK - energy, food, housing, water, telecoms, transport, and financial services. Together these represent 60% of spending by the poorest 30% of households

Noncommutative knot theory

The classical abelian invariants of a knot are the Alexander module, which is the first homology group of the the unique infinite cyclic covering space of S^3-K, considered as a module over the (commutative) Laurent polynomial ring, and the Blanchfield linking pairing defined on this module. From the perspective of the knot group, G, these invariants reflect the structure of G^(1)/G^(2) as a module over G/G^(1) (here G^(n) is the n-th term of the derived series of G). Hence any phenomenon associated to G^(2) is invisible to abelian invariants. This paper begins the systematic study of invariants associated to solvable covering spaces of knot exteriors, in particular the study of what we call the n-th higher-order Alexander module, G^(n+1)/G^(n+2), considered as a Z[G/G^(n+1)$-module. We show that these modules share almost all of the properties of the classical Alexander module. They are torsion modules with higher-order Alexander polynomials whose degrees give lower bounds for the knot genus. The modules have presentation matrices derived either from a group presentation or from a Seifert surface. They admit higher-order linking forms exhibiting self-duality. There are applications to estimating knot genus and to detecting fibered, prime and alternating knots. There are also surprising applications to detecting symplectic structures on 4-manifolds. These modules are similar to but different from those considered by the author, Kent Orr and Peter Teichner and are special cases of the modules considered subsequently by Shelly Harvey for arbitrary 3-manifolds.Comment: Published by Algebraic and Geometric Topology at http://www.maths.warwick.ac.uk/agt/AGTVol4/agt-4-19.abs.htm

Exposure of Salmonella enterica Serovar Typhimurium to High Level Biocide Challenge Can Select Multidrug Resistant Mutants in a Single Step

Biocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux.Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide.These data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations

Real-time RFI Mitigation in Radio Astronomy

As the use of wireless technology has increased around the world, Radio Frequency Interference (RFI) has become more and more of a problem for radio astronomers. Preventative measures exist to limit the presence of RFI, and programs exist to remove it from saved data, but the use of algorithms to detect and remove RFI as an observation is occurring is much less common. Such a method would be incredibly useful for observations in which the data must undergo several rounds of processing before being saved, as in pulsar timing studies. Strategies for real-time mitigation have been discussed and tested with simulated data, but ideally the results of any approach would be validated by a detailed comparison of the final data products with and without mitigation applied. The goal of this project is to develop an RFI mitigation approach based on strategies suggested by Buch et al.(2016) and to test this program on real data from the observation of pulsar J1713+0747 at the Green Bank Observatory in West Virginia. We use a Median Absolute Deviation (MAD) filter to identify interference in the observation and replace the compromised data with random Gaussian noise to match a characteristic radio signal from space. In order to verify our results, we analyze the pulsar’s timing residuals obtained both from the mitigated data and from data processed through offline RFI removal software. Comparing the two, our preliminary findings indicate that our program is able to significantly improve the quality of timing results from the observation