Combinatorial search strategies for the metabolic engineering of microorganisms

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2010.Cataloged from student submitted PDF version of thesis.Includes bibliographical references (p. 231-246).Although the field of microbial metabolic engineering has traditionally been dominated by rational and knowledge-driven approaches, recent advances in genetic engineering have led to the emergence of a new methodology based on phenotypic diversification and screening. Unlike "classical strain improvement," which requires the use of general mutagens to introduce nonspecific chromosomal substitutions, these novel combinatorial methods enable sampling of a wider range of phenotypic space and, additionally, offer the important feature of genetic traceability. As an example, the use of transposon mutagenesis allows for the random integration of a genetic cassette within the chromosome for the generation of gene knockout libraries. More recently, the mutagenesis of cellular transcriptional components (global transcription machinery engineering, gTME) has enabled a complete reprogramming of the transcriptome, a useful feature for eliciting a broad array of phenotypes. Despite these advances in library generation, however, the application of these combinatorial approaches has surprisingly been limited to the engineering of only a handful of cellular properties. Thus, there remains a pressing need for a full evaluation of these techniques and, more specifically, an objective comparison of their relative strengths and weaknesses when applied towards strain improvement endeavors. We decided to explore these specific issues using the metabolic engineering framework of L-tyrosine overproduction in Escherichia coli. Although this particular strain optimization problem merely represents a "model system" for these studies, such endeavors do have important industrial implications, as L-tyrosine serves both as a dietary supplement and a valuable precursor for a myriad of polymers, adhesives and coatings, pharmaceuticals, biocosmetics, and flavonoid products. To establish the early foundations for a combinatorial approach, we began with the construction of a "parental" or starting strain for the generation of these genetic libraries. This was achieved by utilizing several common rational engineering strategies to both deregulate and increase the flux through the aromatic amino acid biosynthetic pathway. The resulting strains, P1 and P2, exhibited L-tyrosine production levels of 358 mg/l and 418 mg/l, respectively, thus establishing an already high base line for this study. In a parallel investigation, we also worked on developing a simple high-throughput screen for Ltyrosine production in E. coli, another prerequisite for the use of these combinatorial approaches. This was accomplished through the heterologous expression of a bacterial tyrosinase which provided a visual link between L-tyrosine production and the synthesis of the colored pigment, melanin. When implemented on a solid agar format, this assay allowed for the identification and isolation of high L-tyrosine producers from combinatorial libraries of more than 106 mutants. Having established the basis for a combinatorial study, these strains and tools were subsequently applied for the generation and screening of three separate libraries - a random knockout library constructed through transposon integration and two plasmid-encoded gTME libraries based on the mutagenesis of the a subunit and the s70 sigma factor of RNA polymerase (rpoA and rpoD, respectively). Several strains were isolated, with some gTME mutants exhibiting impressive titers of up to ~900 mg/l L-tyrosine, a 114% increase over the parental. Upon further examination, however, we discovered that phenotypic transferability was somewhat hampered in these strains due to an unusual requirement for both the plasmidencoded rpoA/rpoD and a mutated chromosomal background to achieve the desired phenotype. Furthermore, the biochemical mechanisms triggered by these factors appeared to be nonspecific, as several plasmid-background combinations were found to lead to the same cellular behaviors. To elucidate the biochemical underpinnings for these phenomena, we decided to conduct a full characterization of three isolated gTME strains through both microarray analysis and whole genome sequencing. Interestingly, whole genome sequencing revealed the presence of a separate unique mutation within each strain in two biochemically-related loci (hisH, purF). Although microarray experiments generally yield intractable results, we were also fortunate to find patterns of expression linking this phenotype to two different cellular responses -- the acid stress resistance pathway and the stringent response. Indeed, the overexpression of two transcriptional regulators for these pathways (evgA, relA) was able to supplant the need for the mutant rpoA or rpoD plasmids, thus validating the contributions of these specific mechanisms towards determining cellular phenotype. The successful identification of these critical genetic factors led us to the construction of a novel, genetically-defined strain (rpoA14R) exhibiting a titer of 902 mg/l L-tyrosine and a yield of 0.18 g L-tyrosine/g glucose in 50 ml cultures. To put these numbers into perspective, this yield on glucose is more than 150% greater than a classically-improved strain (DPD4195) currently used for the industrial production of L-tyrosine and, when excluding biomass-related glucose utilization, represents 85% of the maximum theoretical yield. As an added feature, further engineering of this strain has established its capacity to produce the flavonoid precursor naringenin at competitive levels, thus providing a route for the synthesis of other important Ltyrosine derivatives. During this study, we have successfully applied a combinatorial engineering approach for both eliciting a complex phenotype and identifying novel biochemical and genetic avenues by which to engineer future strains. As such, these combinatorial techniques have certainly proven to be valuable tools within the metabolic engineer's ever-expanding arsenal.by Christine Nicole S. Santos.Ph.D

Measurement of the cosmic ray spectrum above 4×10184{\times}10^{18} eV using inclined events detected with the Pierre Auger Observatory

A measurement of the cosmic-ray spectrum for energies exceeding $4{\times}10^{18}$ eV is presented, which is based on the analysis of showers with zenith angles greater than $60^{\circ}$ detected with the Pierre Auger Observatory between 1 January 2004 and 31 December 2013. The measured spectrum confirms a flux suppression at the highest energies. Above $5.3{\times}10^{18}$ eV, the "ankle", the flux can be described by a power law $E^{-\gamma}$ with index $\gamma=2.70 \pm 0.02 \,\text{(stat)} \pm 0.1\,\text{(sys)}$ followed by a smooth suppression region. For the energy ($E_\text{s}$) at which the spectral flux has fallen to one-half of its extrapolated value in the absence of suppression, we find $E_\text{s}=(5.12\pm0.25\,\text{(stat)}^{+1.0}_{-1.2}\,\text{(sys)}){\times}10^{19}$ eV.Comment: Replaced with published version. Added journal reference and DO

Energy Estimation of Cosmic Rays with the Engineering Radio Array of the Pierre Auger Observatory

The Auger Engineering Radio Array (AERA) is part of the Pierre Auger Observatory and is used to detect the radio emission of cosmic-ray air showers. These observations are compared to the data of the surface detector stations of the Observatory, which provide well-calibrated information on the cosmic-ray energies and arrival directions. The response of the radio stations in the 30 to 80 MHz regime has been thoroughly calibrated to enable the reconstruction of the incoming electric field. For the latter, the energy deposit per area is determined from the radio pulses at each observer position and is interpolated using a two-dimensional function that takes into account signal asymmetries due to interference between the geomagnetic and charge-excess emission components. The spatial integral over the signal distribution gives a direct measurement of the energy transferred from the primary cosmic ray into radio emission in the AERA frequency range. We measure 15.8 MeV of radiation energy for a 1 EeV air shower arriving perpendicularly to the geomagnetic field. This radiation energy -- corrected for geometrical effects -- is used as a cosmic-ray energy estimator. Performing an absolute energy calibration against the surface-detector information, we observe that this radio-energy estimator scales quadratically with the cosmic-ray energy as expected for coherent emission. We find an energy resolution of the radio reconstruction of 22% for the data set and 17% for a high-quality subset containing only events with at least five radio stations with signal.Comment: Replaced with published version. Added journal reference and DO

Measurement of the Radiation Energy in the Radio Signal of Extensive Air Showers as a Universal Estimator of Cosmic-Ray Energy

We measure the energy emitted by extensive air showers in the form of radio emission in the frequency range from 30 to 80 MHz. Exploiting the accurate energy scale of the Pierre Auger Observatory, we obtain a radiation energy of 15.8 \pm 0.7 (stat) \pm 6.7 (sys) MeV for cosmic rays with an energy of 1 EeV arriving perpendicularly to a geomagnetic field of 0.24 G, scaling quadratically with the cosmic-ray energy. A comparison with predictions from state-of-the-art first-principle calculations shows agreement with our measurement. The radiation energy provides direct access to the calorimetric energy in the electromagnetic cascade of extensive air showers. Comparison with our result thus allows the direct calibration of any cosmic-ray radio detector against the well-established energy scale of the Pierre Auger Observatory.Comment: Replaced with published version. Added journal reference and DOI. Supplemental material in the ancillary file

An inclusive Research and Education Community (iREC) model to facilitate undergraduate science education reform

Funding: This work was supported by Howard Hughes Medical Institute grants to DIH is GT12052 and MJG is GT15338.Over the last two decades, there have been numerous initiatives to improve undergraduate student outcomes in STEM. One model for scalable reform is the inclusive Research Education Community (iREC). In an iREC, STEM faculty from colleges and universities across the nation are supported to adopt and sustainably implement course-based research – a form of science pedagogy that enhances student learning and persistence in science. In this study, we used pathway modeling to develop a qualitative description that explicates the HHMI Science Education Alliance (SEA) iREC as a model for facilitating the successful adoption and continued advancement of new curricular content and pedagogy. In particular, outcomes that faculty realize through their participation in the SEA iREC were identified, organized by time, and functionally linked. The resulting pathway model was then revised and refined based on several rounds of feedback from over 100 faculty members in the SEA iREC who participated in the study. Our results show that in an iREC, STEM faculty organized as a long-standing community of practice leverage one another, outside expertise, and data to adopt, implement, and iteratively advance their pedagogy. The opportunity to collaborate in this manner and, additionally, to be recognized for pedagogical contributions sustainably engages STEM faculty in the advancement of their pedagogy. Here, we present a detailed pathway model of SEA that, together with underpinning features of an iREC identified in this study, offers a framework to facilitate transformations in undergraduate science education.Peer reviewe

Diving into the vertical dimension of elasmobranch movement ecology

Knowledge of the three-dimensional movement patterns of elasmobranchs is vital to understand their ecological roles and exposure to anthropogenic pressures. To date, comparative studies among species at global scales have mostly focused on horizontal movements. Our study addresses the knowledge gap of vertical movements by compiling the first global synthesis of vertical habitat use by elasmobranchs from data obtained by deployment of 989 biotelemetry tags on 38 elasmobranch species. Elasmobranchs displayed high intra- and interspecific variability in vertical movement patterns. Substantial vertical overlap was observed for many epipelagic elasmobranchs, indicating an increased likelihood to display spatial overlap, biologically interact, and share similar risk to anthropogenic threats that vary on a vertical gradient. We highlight the critical next steps toward incorporating vertical movement into global management and monitoring strategies for elasmobranchs, emphasizing the need to address geographic and taxonomic biases in deployments and to concurrently consider both horizontal and vertical movements

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure