Publications by authors named "Lydia M Contreras"

42 Publications

The Tryptophan-Induced Ribosome Stalling Sequence Exposes High Amino Acid Cross-Talk That Can Be Mitigated by Removal of NusB for Higher Orthogonality.

ACS Synth Biol 2021 Apr 9. Epub 2021 Apr 9.

McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, Texas 78714, United States.

A growing number of engineered synthetic circuits have employed biological parts coupling transcription and translation in bacterial systems to control downstream gene expression. One such example, the leader sequence of the tryptophanase () operon, is a transcription-translation system commonly employed as an l-tryptophan inducible circuit controlled by ribosome stalling. While induction of the operon has been well-characterized in response to l-tryptophan, cross-talk of this modular component with other metabolites in the cell, such as other naturally occurring amino acids, has been less explored. In this study, we investigated the impact of natural metabolites and host factors on induction of the leader sequence. To do so, we constructed and biochemically validated an experimental assay using the operon leader sequence to assess differential regulation of transcription elongation and translation in response to l-tryptophan. Operon induction was then assessed following addition of each of the 20 naturally occurring amino acids to discover that several additional amino acids (, l-alanine, l-cysteine, l-glycine, l-methionine, and l-threonine) also induce expression of the leader sequence. Following characterization of dose-dependent induction by l-cysteine relative to l-tryptophan, the effect on induction by single gene knockouts of protein factors associated with transcription and/or translation were interrogated. Our results implicate the endogenous cellular protein, NusB, as an important factor associated with induction of the operon by the alternative amino acids. As such, removal of the gene from strains intended for tryptophan-sensing utilizing the leader region reduces amino acid cross-talk, resulting in enhanced orthogonal control of this commonly used synthetic system.
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http://dx.doi.org/10.1021/acssynbio.0c00547DOI Listing
April 2021

Signal Recognition Particle RNA Contributes to Oxidative Stress Response in by Modulating Catalase Localization.

Front Microbiol 2020 18;11:613571. Epub 2020 Dec 18.

McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, United States.

The proper functioning of many proteins requires their transport to the correct cellular compartment or their secretion. Signal recognition particle (SRP) is a major protein transport pathway responsible for the co-translational movement of integral membrane proteins as well as periplasmic proteins. is a ubiquitous bacterium that expresses a complex phenotype of extreme oxidative stress resistance, which depends on proteins involved in DNA repair, metabolism, gene regulation, and antioxidant defense. These proteins are located extracellularly or subcellularly, but the molecular mechanism of protein localization in to manage oxidative stress response remains unexplored. In this study, we characterized the SRP complex in R1 and showed that the knockdown (KD) of the SRP RNA (Qpr6) reduced bacterial survival under hydrogen peroxide and growth under chronic ionizing radiation. Through LC-mass spectrometry (MS/MS) analysis, we detected 162 proteins in the periplasm of wild-type , of which the transport of 65 of these proteins to the periplasm was significantly reduced in the Qpr6 KD strain. Through Western blotting, we further demonstrated the localization of the catalases in , DR_1998 (KatE1) and DR_A0259 (KatE2), in both the cytoplasm and periplasm, respectively, and showed that the accumulation of KatE1 and KatE2 in the periplasm was reduced in the SRP-defective strains. Collectively, this study establishes the importance of the SRP pathway in the survival and the transport of antioxidant proteins in under oxidative stress.
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http://dx.doi.org/10.3389/fmicb.2020.613571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775534PMC
December 2020

RNA oxidation in chromatin modification and DNA-damage response following exposure to formaldehyde.

Sci Rep 2020 10 6;10(1):16545. Epub 2020 Oct 6.

McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, TX, 78714, USA.

Formaldehyde is an environmental and occupational chemical carcinogen implicated in the damage of proteins and nucleic acids. However, whether formaldehyde provokes modifications of RNAs such as 8-oxo-7,8-dihydroguanine (8-oxoG) and the role that these modifications play on conferring long-term adverse health effects remains unexplored. Here, we profile 8-oxoG modifications using RNA-immunoprecipitation and RNA sequencing (8-oxoG RIP-seq) to identify 343 RNA transcripts heavily enriched in oxidations in human bronchial epithelial BEAS-2B cell cultures exposed to 1 ppm formaldehyde for 2 h. RNA oxidation altered expression of many transcripts involved in chromatin modification and p53-mediated DNA-damage responses, two pathways that play key roles in sustaining genome integrity and typically deregulated in tumorigenesis. Given that these observations were identified in normal cells exhibiting minimal cell stress and death phenotypes (for example, lack of nuclear shrinkage, F-actin alterations or increased LDH activity); we hypothesize that oxidative modification of specific RNA transcripts following formaldehyde exposure denotes an early process occurring in carcinogenesis analogous to the oxidative events surfacing at early stages of neurodegenerative diseases. As such, we provide initial investigations of RNA oxidation as a potentially novel mechanism underlying formaldehyde-induced tumorigenesis.
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http://dx.doi.org/10.1038/s41598-020-73376-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7538935PMC
October 2020

Post-transcriptional air pollution oxidation to the cholesterol biosynthesis pathway promotes pulmonary stress phenotypes.

Commun Biol 2020 Jul 22;3(1):392. Epub 2020 Jul 22.

McKetta Department of Chemical Engineering, University of Texas at Austin, 200 East Dean Keeton Street, Stop C0400, Austin, TX, 78712, USA.

The impact of environmentally-induced chemical changes in RNA has been fairly unexplored. Air pollution induces oxidative modifications such as 8-oxo-7,8-dihydroguanine (8-oxoG) in RNAs of lung cells, which could be associated with premature lung dysfunction. We develop a method for 8-oxoG profiling using immunocapturing and RNA sequencing. We find 42 oxidized transcripts in bronchial epithelial BEAS-2B cells exposed to two air pollution mixtures that recreate urban atmospheres. We show that the FDFT1 transcript in the cholesterol biosynthesis pathway is susceptible to air pollution-induced oxidation. This process leads to decreased transcript and protein expression of FDFT1, and reduced cholesterol synthesis in cells exposed to air pollution. Knockdown of FDFT1 replicates alterations seen in air pollution exposure such as transformed cell size and suppressed cytoskeleton organization. Our results argue of a possible novel biomarker and of an unseen mechanism by which air pollution selectively modifies key metabolic-related transcripts facilitating cell phenotypes in bronchial dysfunction.
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http://dx.doi.org/10.1038/s42003-020-01118-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376215PMC
July 2020

Multiple Small RNAs Interact to Co-regulate Ethanol Tolerance in .

Front Bioeng Biotechnol 2020 4;8:155. Epub 2020 Mar 4.

McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, United States.

sRNAs represent a powerful class of regulators that influences multiple mRNA targets in response to environmental changes. However, very few direct sRNA-sRNA interactions have been deeply studied in any organism. is a bacterium with unique ethanol-producing metabolic pathways in which multiple small RNAs (sRNAs) have recently been identified, some of which show differential expression in ethanol stress. In this study, we show that two sRNAs (Zms4 and Zms6) are upregulated under ethanol stress and have significant impacts on ethanol tolerance and production in . We conducted multi-omics analysis (combining transcriptomics and sRNA-immunoprecipitation) to map gene networks under the influence of their regulation. We confirmed that Zms4 and Zms6 bind multiple RNA targets and regulate their expressions, influencing many downstream pathways important to ethanol tolerance and production. In particular, Zms4 and Zms6 interact with each other as well as many other sRNAs, forming a novel sRNA-sRNA direct interaction network. This study thus uncovers a sRNA network that co-orchestrates multiple ethanol related pathways through a diverse set of mRNA targets and a large number of sRNAs. To our knowledge, this study represents one of the largest sRNA-sRNA direct interactions uncovered so far.
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http://dx.doi.org/10.3389/fbioe.2020.00155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064620PMC
March 2020

Bioinformatic Application of Fluorescence-Based In Vivo RNA Regional Accessibility Data to Identify Novel sRNA Targets.

Methods Mol Biol 2020 ;2113:41-71

Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA.

Data from fluorescence-based methods that measure in vivo hybridization efficacy of unique RNA regions can be used to infer regulatory activity and to identify novel RNA: RNA interactions. Here, we document the step-by-step analysis of fluorescence data collected using an in vivo regional RNA structural sensing system (iRS) for the purpose of identifying potential functional sites that are likely to be involved in regulatory interactions. We also detail a step-by-step protocol that couples this in vivo accessibility data with computational mRNA target predictions to inform the selection of potentially true targets from long lists of thermodynamic predictions.
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http://dx.doi.org/10.1007/978-1-0716-0278-2_5DOI Listing
January 2021

Applying a New REFINE Approach in Identifies Novel sRNAs That Confer Improved Stress Tolerance Phenotypes.

Front Microbiol 2019 10;10:2987. Epub 2020 Jan 10.

McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, United States.

As global controllers of gene expression, small RNAs represent powerful tools for engineering complex phenotypes. However, a general challenge prevents the more widespread use of sRNA engineering strategies: mechanistic analysis of these regulators in bacteria lags far behind their high-throughput search and discovery. This makes it difficult to understand how to efficiently identify useful sRNAs to engineer a phenotype of interest. To help address this, we developed a forward systems approach to identify naturally occurring sRNAs relevant to a desired phenotype: RNA-seq Examiner for Phenotype-Informed Network Engineering (REFINE). This pipeline uses existing RNA-seq datasets under different growth conditions. It filters the total transcriptome to locate and rank regulatory-RNA-containing regions that can influence a metabolic phenotype of interest, without the need for previous mechanistic characterization. Application of this approach led to the uncovering of six novel sRNAs related to ethanol tolerance in non-model ethanol-producing bacterium . Furthermore, upon overexpressing multiple sRNA candidates predicted by REFINE, we demonstrate improved ethanol tolerance reflected by up to an approximately twofold increase in relative growth rate compared to controls not expressing these sRNAs in 7% ethanol (v/v) RMG-supplemented media. In this way, the REFINE approach informs strain-engineering strategies that we expect are applicable for general strain engineering.
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http://dx.doi.org/10.3389/fmicb.2019.02987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6970203PMC
January 2020

Antisense probing of dynamic RNA structures.

Methods 2020 11 25;183:76-83. Epub 2020 Jan 25.

Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, United States; McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712, United States. Electronic address:

RNA regulation is influenced by the dynamic changes in conformational accessibility on the transcript. Here we discuss the initial validation of a cell-free antisense probing method for structured RNAs, using the Tetrahymena group I intron as a control target. We observe changes in signal that qualitatively match prior traditional DMS footprinting experiments. Importantly, we have shown that application of this technique can elucidate new RNA information given its sensitivity for detecting rare intermediates that are not as readily observed by single-hit kinetics chemical probing techniques. Observing changes in RNA accessibility has broad applications in determining the effect that regulatory elements have on regional structures. We speculate that this method could be useful in quickly observing those interactions, along with other phenomena that influence RNA accessibility including RNA-RNA interactions and small molecules.
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http://dx.doi.org/10.1016/j.ymeth.2020.01.015DOI Listing
November 2020

Computational evolution of an RNA-binding protein towards enhanced oxidized-RNA binding.

Comput Struct Biotechnol J 2020 27;18:137-152. Epub 2019 Dec 27.

Artie McFerrin Department of Chemical Engineering, Texas A&M University, 3122 TAMU Room 200, College Station, TX 77843, United States.

The oxidation of RNA has been implicated in the development of many diseases. Among the four ribonucleotides, guanosine is the most susceptible to oxidation, resulting in the formation of 8-oxo-7,8-dihydroguanosine (8-oxoG). Despite the limited knowledge about how cells regulate the detrimental effects of oxidized RNA, cellular factors involved in its regulation have begun to be identified. One of these factors is polynucleotide phosphorylase (PNPase), a multifunctional enzyme implicated in RNA turnover. In the present study, we have examined the interaction of PNPase with 8-oxoG in atomic detail to provide insights into the mechanism of 8-oxoG discrimination. We hypothesized that PNPase subunits cooperate to form a binding site using the dynamic SFF loop within the central channel of the PNPase homotrimer. We evolved this site using a novel approach that initially screened mutants from a library of beneficial mutations and assessed their interactions using multi-nanosecond Molecular Dynamics simulations. We found that evolving this single site resulted in a fold change increase in 8-oxoG affinity between 1.2 and 1.5 and/or selectivity between 1.5 and 1.9. In addition to the improvement in 8-oxoG binding, complementation of K12 Δ with plasmids expressing mutant PNPases caused increased cell tolerance to HO. This observation provides a clear link between molecular discrimination of RNA oxidation and cell survival. Moreover, this study provides a framework for the manipulation of modified-RNA protein readers, which has potential application in synthetic biology and epitranscriptomics.
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http://dx.doi.org/10.1016/j.csbj.2019.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965710PMC
December 2019

Codon Selection Affects Recruitment of Ribosome-Associating Factors during Translation.

ACS Synth Biol 2020 02 10;9(2):329-342. Epub 2019 Dec 10.

McKetta Department of Chemical Engineering , The University of Texas at Austin , 200 E. Dean Keeton Street Stop C0400 , Austin , Texas 78712 , United States.

An intriguing aspect of protein synthesis is how cotranslational events are managed inside the cell. In this study, we developed an bimolecular fluorescence complementation assay coupled to SecM stalling (BiFC-SecM) to study how codon usage influences the interactions of ribosome-associating factors that occur cotranslationally. We profiled ribosomal associations of a number of proteins, and observed differential association of chaperone proteins TF, DnaK, GroEL, and translocation factor Ffh as a result of introducing synonymous codon substitutions that change the affinity of the translating sequence to the ribosomal anti-Shine-Dalgarno (aSD) sequence. The use of pausing sequences within proteins regulates their transit within the translating ribosome. Our results indicate that the dynamics between cellular factors and the new polypeptide chain are affected by how codon composition is designed. Furthermore, associating factors may play a role in processes including protein quality control (folding and degradation) and cellular respiration.
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http://dx.doi.org/10.1021/acssynbio.9b00344DOI Listing
February 2020

Discovery and Characterization of Native Promoters for Tunable Gene Expression.

Appl Environ Microbiol 2019 11 16;85(21). Epub 2019 Oct 16.

McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA

The potential utilization of extremophiles as a robust chassis for metabolic engineering applications has prompted interest in the use of for bioremediation efforts, but current applications are limited by the lack of availability of genetic tools, such as promoters. In this study, we used a combined computational and experimental approach to identify and screen 30 predicted promoters for expression in using a fluorescent reporter assay. The top eight candidates were further characterized, compared to currently available promoters, and optimized for engineering through minimization for use in Of these top eight, two promoter regions, and , were stronger and more consistent than the most widely used promoter sequence in , Furthermore, half of the top eight promoters could be minimized by at least 20% (to obtain final sequences that are approximately 24 to 177 bp), and several of the putative promoters either showed activity in or were specific, broadening the use of the promoters for various applications. Overall, this work introduces a suite of novel, well-characterized promoters for protein production and metabolic engineering in The tolerance of the extremophile, , to numerous oxidative stresses makes it ideal for bioremediation applications, but many of the tools necessary for metabolic engineering are lacking in this organism compared to model bacteria. Although native and engineered promoters have been used to drive gene expression for protein production in , very few have been well characterized. Informed by bioinformatics, this study expands the repertoire of well-characterized promoters for via thorough characterization of eight putative promoters with various strengths. These results will help facilitate tunable gene expression, since these promoters demonstrate strong and consistent performance compared to the current standard, This study also provides a methodology for high-throughput promoter identification and characterization using fluorescence in The promoters identified in this study will facilitate metabolic engineering of and enable its use in biotechnological applications ranging from bioremediation to synthesis of commodity chemicals.
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http://dx.doi.org/10.1128/AEM.01356-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803307PMC
November 2019

Regulatory non-coding sRNAs in bacterial metabolic pathway engineering.

Metab Eng 2019 03 1;52:190-214. Epub 2018 Dec 1.

McKetta Department of Chemical Engineering, University of Texas at Austin, 200 E. Dean Keeton Street Stop C0400, Austin, TX 78712, USA. Electronic address:

Non-coding RNAs (ncRNAs) are versatile and powerful controllers of gene expression that have been increasingly linked to cellular metabolism and phenotype. In bacteria, identified and characterized ncRNAs range from trans-acting, multi-target small non-coding RNAs to dynamic, cis-encoded regulatory untranslated regions and riboswitches. These native regulators have inspired the design and construction of many synthetic RNA devices. In this work, we review the design, characterization, and impact of ncRNAs in engineering both native and exogenous metabolic pathways in bacteria. We also consider the opportunities afforded by recent high-throughput approaches for characterizing sRNA regulators and their corresponding networks to showcase their potential applications and impact in engineering bacterial metabolism.
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http://dx.doi.org/10.1016/j.ymben.2018.11.013DOI Listing
March 2019

High-throughput in vivo mapping of RNA accessible interfaces to identify functional sRNA binding sites.

Nat Commun 2018 10 4;9(1):4084. Epub 2018 Oct 4.

McKetta Department of Chemical Engineering, University of Texas at Austin, 200 E. Dean Keeton St., Stop C0400, Austin, TX, 78712, USA.

Herein we introduce a high-throughput method, INTERFACE, to reveal the capacity of contiguous RNA nucleotides to establish in vivo intermolecular RNA interactions for the purpose of functional characterization of intracellular RNA. INTERFACE enables simultaneous accessibility interrogation of an unlimited number of regions by coupling regional hybridization detection to transcription elongation outputs measurable by RNA-seq. We profile over 900 RNA interfaces in 71 validated, but largely mechanistically under-characterized, Escherichia coli sRNAs in the presence and absence of a global regulator, Hfq, and find that two-thirds of tested sRNAs feature Hfq-dependent regions. Further, we identify in vivo hybridization patterns that hallmark functional regions to uncover mRNA targets. In this way, we biochemically validate 25 mRNA targets, many of which are not captured by typically tested, top-ranked computational predictions. We additionally discover direct mRNA binding activity within the GlmY terminator, highlighting the information value of high-throughput RNA accessibility data.
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http://dx.doi.org/10.1038/s41467-018-06207-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6172242PMC
October 2018

Methods and advances in RNA characterization and design.

Methods 2018 07;143:1-3

McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 E Dean Keeton St. Stop C0400, Austin, Texas 78712-1589.

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http://dx.doi.org/10.1016/j.ymeth.2018.06.003DOI Listing
July 2018

Synthetic Biology of Small RNAs and Riboswitches.

Microbiol Spectr 2018 05;6(3)

Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720.

In bacteria and archaea, small RNAs (sRNAs) regulate complex networks through antisense interactions with target mRNAs in trans, and riboswitches regulate gene expression in based on the ability to bind small-molecule ligands. Although our understanding and characterization of these two important regulatory RNA classes is far from complete, these RNA-based mechanisms have proven useful for a wide variety of synthetic biology applications. Besides classic and contemporary applications in the realm of metabolic engineering and orthogonal gene control, this review also covers newer applications of regulatory RNAs as biosensors, logic gates, and tools to determine RNA-RNA interactions. A separate section focuses on critical insights gained and challenges posed by fundamental studies of sRNAs and riboswitches that should aid future development of synthetic regulatory RNAs.
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http://dx.doi.org/10.1128/microbiolspec.RWR-0007-2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020158PMC
May 2018

Directed Evolution of Heterologous tRNAs Leads to Reduced Dependence on Post-transcriptional Modifications.

ACS Synth Biol 2018 05 25;7(5):1315-1327. Epub 2018 Apr 25.

McKetta Department of Chemical Engineering , The University of Texas at Austin , Austin , Texas 78712 , United States.

Heterologous tRNA:aminoacyl tRNA synthetase pairs are often employed for noncanonical amino acid incorporation in the quest for an expanded genetic code. In this work, we investigated one possible mechanism by which directed evolution can improve orthogonal behavior for a suite of Methanocaldococcus jannaschii ( Mj) tRNA-derived amber suppressor tRNAs. Northern blotting demonstrated that reduced expression of heterologous tRNA variants correlated with improved orthogonality. We suspected that reduced expression likely minimized nonorthogonal interactions with host cell machinery. Despite the known abundance of post-transcriptional modifications in tRNAs across all domains of life, few studies have investigated how host enzymes may affect behavior of heterologous tRNAs. Therefore, we measured tRNA orthogonality using a fluorescent reporter assay in several modification-deficient strains, demonstrating that heterologous tRNAs with high expression are strongly affected by some native E. coli RNA-modifying enzymes, whereas low abundance evolved heterologous tRNAs are less affected by these same enzymes. We employed mass spectrometry to map msiA37 and Ψ39 in the anticodon arm of two high abundance tRNAs (Nap1 and tRNA), which provides (to our knowledge) the first direct evidence that MiaA and TruA post-transcriptionally modify evolved heterologous amber suppressor tRNAs. Changes in total tRNA modification profiles were observed by mass spectrometry in cells hosting these and other evolved suppressor tRNAs, suggesting that the demonstrated interactions with host enzymes might disturb native tRNA modification networks. Together, these results suggest that heterologous tRNAs engineered for specialized amber suppression can evolve highly efficient suppression capacity within the native post-transcriptional modification landscape of host RNA processing machinery.
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http://dx.doi.org/10.1021/acssynbio.7b00421DOI Listing
May 2018

Advances and prospects in metabolic engineering of Zymomonas mobilis.

Metab Eng 2018 11 5;50:57-73. Epub 2018 Apr 5.

Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Environmental Microbial Technology Center of Hubei Province, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan 430062, China. Electronic address:

Biorefinery of biomass-based biofuels and biochemicals by microorganisms is a competitive alternative of traditional petroleum refineries. Zymomonas mobilis is a natural ethanologen with many desirable characteristics, which makes it an ideal industrial microbial biocatalyst for commercial production of desirable bioproducts through metabolic engineering. In this review, we summarize the metabolic engineering progress achieved in Z. mobilis to expand its substrate and product ranges as well as to enhance its robustness against stressful conditions such as inhibitory compounds within the lignocellulosic hydrolysates and slurries. We also discuss a few metabolic engineering strategies that can be applied in Z. mobilis to further develop it as a robust workhorse for economic lignocellulosic bioproducts. In addition, we briefly review the progress of metabolic engineering in Z. mobilis related to the classical synthetic biology cycle of "Design-Build-Test-Learn", as well as the progress and potential to develop Z. mobilis as a model chassis for biorefinery practices in the synthetic biology era.
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http://dx.doi.org/10.1016/j.ymben.2018.04.001DOI Listing
November 2018

Fluorescence-Based Methods for Characterizing RNA Interactions In Vivo.

Methods Mol Biol 2018 ;1737:129-164

McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, TX, USA.

Fluorescence-based tools that measure RNA-RNA and RNA-protein interactions in vivo offer useful experimental approaches to probe the complex and dynamic physiological behavior of bacterial RNAs. Here we document the step-by-step design and application of two fluorescence-based methods for studying the regulatory interactions RNAs perform in vivo: (i) the in vivo RNA Structural Sensing System (iRS) for measuring RNA accessibility and (ii) the trifluorescence complementation (TriFC) assay for measuring RNA-protein interactions.
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http://dx.doi.org/10.1007/978-1-4939-7634-8_9DOI Listing
January 2019

A high-throughput and rapid computational method for screening of RNA post-transcriptional modifications that can be recognized by target proteins.

Methods 2018 07 1;143:34-47. Epub 2018 Feb 1.

Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, TX, United States. Electronic address:

There are over 150 currently known, highly diverse chemically modified RNAs, which are dynamic, reversible, and can modulate RNA-protein interactions. Yet, little is known about the wealth of such interactions. This can be attributed to the lack of tools that allow the rapid study of all the potential RNA modifications that might mediate RNA-protein interactions. As a promising step toward this direction, here we present a computational protocol for the characterization of interactions between proteins and RNA containing post-transcriptional modifications. Given an RNA-protein complex structure, potential RNA modified ribonucleoside positions, and molecular mechanics parameters for capturing energetics of RNA modifications, our protocol operates in two stages. In the first stage, a decision-making tool, comprising short simulations and interaction energy calculations, performs a fast and efficient search in a high-throughput fashion, through a list of different types of RNA modifications categorized into trees according to their structural and physicochemical properties, and selects a subset of RNA modifications prone to interact with the target protein. In the second stage, RNA modifications that are selected as recognized by the protein are examined in-detail using all-atom simulations and free energy calculations. We implement and experimentally validate this protocol in a test case involving the study of RNA modifications in complex with Escherichia coli (E. coli) protein Polynucleotide Phosphorylase (PNPase), depicting the favorable interaction between 8-oxo-7,8-dihydroguanosine (8-oxoG) RNA modification and PNPase. Further advancement of the protocol can broaden our understanding of protein interactions with all known RNA modifications in several systems.
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http://dx.doi.org/10.1016/j.ymeth.2018.01.015DOI Listing
July 2018

Identification and Characterization of 5' Untranslated Regions (5'UTRs) in as Regulatory Biological Parts.

Front Microbiol 2017 8;8:2432. Epub 2017 Dec 8.

Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, United States.

Regulatory RNA regions within a transcript, particularly in the 5' untranslated region (5'UTR), have been shown in a variety of organisms to control the expression levels of these mRNAs in response to various metabolites or environmental conditions. Considering the unique tolerance of to ethanol and the growing interest in engineering microbial strains with enhanced tolerance to industrial inhibitors, we searched natural -regulatory regions in this microorganism using transcriptomic data and bioinformatics analysis. Potential regulatory 5'UTRs were identified and filtered based on length, gene function, relative gene counts, and conservation in other organisms. An fluorescence-based screening system was developed to confirm the responsiveness of 36 5'UTR candidates to ethanol, acetate, and xylose stresses. UTR_ZMO0347 (5'UTR of gene ZMO0347 encoding the RNA binding protein Hfq) was found to down-regulate downstream gene expression under ethanol stress. Genomic deletion of UTR_ZMO0347 led to a general decrease of expression at the transcript level and increased sensitivity for observed changes in Hfq expression at the protein level. The role of UTR_ZMO0347 and other 5'UTRs gives us insight into the regulatory network of in response to stress and unlocks new strategies for engineering robust industrial strains as well as for harvesting novel responsive regulatory biological parts for controllable gene expression platforms in this organism.
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http://dx.doi.org/10.3389/fmicb.2017.02432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5770649PMC
December 2017

Rational Modular RNA Engineering Based on In Vivo Profiling of Structural Accessibility.

ACS Synth Biol 2017 12 10;6(12):2228-2240. Epub 2017 Aug 10.

McKetta Department of Chemical Engineering, University of Texas at Austin , 200 E. Dean Keeton Street Stop C0400, Austin, Texas 78712, United States.

Bacterial small RNAs (sRNAs) have been established as powerful parts for controlling gene expression. However, development and application of engineered sRNAs has primarily focused on regulating novel synthetic targets. In this work, we demonstrate a rational modular RNA engineering approach that uses in vivo structural accessibility measurements to tune the regulatory activity of a multisubstrate sRNA for differential control of its native target network. Employing the CsrB global sRNA regulator as a model system, we use published in vivo structural accessibility data to infer the contribution of its local structures (substructures) to function and select a subset for engineering. We then modularly recombine the selected substructures, differentially representing those of presumed high or low functional contribution, to build a library of 21 CsrB variants. Using fluorescent translational reporter assays, we demonstrate that the CsrB variants achieve a 5-fold gradient of control of well-characterized Csr network targets. Interestingly, results suggest that less conserved local structures within long, multisubstrate sRNAs may represent better targets for rational engineering than their well-conserved counterparts. Lastly, mapping the impact of sRNA variants on a signature Csr network phenotype indicates the potential of this approach for tuning the activity of global sRNA regulators in the context of metabolic engineering applications.
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http://dx.doi.org/10.1021/acssynbio.7b00185DOI Listing
December 2017

Imposed Environmental Stresses Facilitate Cell-Free Nanoparticle Formation by Deinococcus radiodurans.

Appl Environ Microbiol 2017 09 31;83(18). Epub 2017 Aug 31.

McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, Texas, USA

The biological synthesis of metal nanoparticles has been examined in a wide range of organisms, due to increased interest in green synthesis and environmental remediation applications involving heavy metal ion contamination. is particularly attractive for environmental remediation involving metal reduction, due to its high levels of resistance to radiation and other environmental stresses. However, few studies have thoroughly examined the relationships between environmental stresses and the resulting effects on nanoparticle biosynthesis. In this work, we demonstrate cell-free nanoparticle production and study the effects of metal stressor concentrations and identity, temperature, pH, and oxygenation on the production of extracellular silver nanoparticles by R1. We also report the synthesis of bimetallic silver and gold nanoparticles following the addition of a metal stressor (silver or gold), highlighting how production of these particles is enabled through the application of environmental stresses. Additionally, we found that both the morphology and size of monometallic and bimetallic nanoparticles were dependent on the environmental stresses imposed on the cells. The nanoparticles produced by exhibited antimicrobial activity comparable to that of pure silver nanoparticles and displayed catalytic activity comparable to that of pure gold nanoparticles. Overall, we demonstrate that biosynthesized nanoparticle properties can be partially controlled through the tuning of applied environmental stresses, and we provide insight into how their application may affect nanoparticle production in during bioremediation. Biosynthetic production of nanoparticles has recently gained prominence as a solution to rising concerns regarding increased bacterial resistance to antibiotics and a desire for environmentally friendly methods of bioremediation and chemical synthesis. To date, a range of organisms have been utilized for nanoparticle formation. The extremophile , which can withstand significant environmental stresses and therefore is more robust for metal reduction applications, has yet to be exploited for this purpose. Thus, this work improves our understanding of the impact of environmental stresses on biogenic nanoparticle morphology and composition during metal reduction processes in this organism. This work also contributes to enhancing the controlled synthesis of nanoparticles with specific attributes and functions using biological systems.
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http://dx.doi.org/10.1128/AEM.00798-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5583488PMC
September 2017

A Genome-Wide Search for Ionizing-Radiation-Responsive Elements in Deinococcus radiodurans Reveals a Regulatory Role for the DNA Gyrase Subunit A Gene's 5' Untranslated Region in the Radiation and Desiccation Response.

Appl Environ Microbiol 2017 06 31;83(12). Epub 2017 May 31.

McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, Texas,USA

Tight regulation of gene expression is important for the survival of , a model bacterium of extreme stress resistance. Few studies have examined the use of regulatory RNAs as a possible contributing mechanism to ionizing radiation (IR) resistance, despite their proffered efficient and dynamic gene expression regulation under IR stress. This work presents a transcriptome-based approach for the identification of stress-responsive regulatory 5' untranslated region (5'-UTR) elements in R1 that can be broadly applied to other bacteria. Using this platform and an fluorescence screen, we uncovered the presence of a radiation-responsive regulatory motif in the 5' UTR of the DNA gyrase subunit A gene. Additional screens under HO-induced oxidative stress revealed the specificity of the response of this element to IR stress. Further examination of the sequence revealed a regulatory motif of the radiation and desiccation response (RDR) in the 5' UTR that is necessary for the recovery of from high doses of IR. Furthermore, we suggest that it is the preservation of predicted RNA structure, in addition to DNA sequence consensus of the motif, that permits this important regulatory ability. is an extremely stress-resistant bacterium capable of tolerating up to 3,000 times more ionizing radiation than human cells. As an integral part of the stress response mechanism of this organism, we suspect that it maintains stringent control of gene expression. However, understanding of its regulatory pathways remains incomplete to date. Untranslated RNA elements have been demonstrated to play crucial roles in gene regulation throughout bacteria. In this work, we focus on searching for and characterizing responsive RNA elements under radiation stress and propose that multiple levels of gene regulation work simultaneously to enable this organism to efficiently recover from exposure to ionizing radiation. The model we propose serves as a generic template to investigate similar mechanisms of gene regulation under stress that have likely evolved in other bacterial species.
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http://dx.doi.org/10.1128/AEM.00039-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5452802PMC
June 2017

Optimization of a novel biophysical model using large scale in vivo antisense hybridization data displays improved prediction capabilities of structurally accessible RNA regions.

Nucleic Acids Res 2017 May;45(9):5523-5538

McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 E. Dean Keeton St., Stop C0400, Austin, TX 78712, USA.

Current approaches to design efficient antisense RNAs (asRNAs) rely primarily on a thermodynamic understanding of RNA-RNA interactions. However, these approaches depend on structure predictions and have limited accuracy, arguably due to overlooking important cellular environment factors. In this work, we develop a biophysical model to describe asRNA-RNA hybridization that incorporates in vivo factors using large-scale experimental hybridization data for three model RNAs: a group I intron, CsrB and a tRNA. A unique element of our model is the estimation of the availability of the target region to interact with a given asRNA using a differential entropic consideration of suboptimal structures. We showcase the utility of this model by evaluating its prediction capabilities in four additional RNAs: a group II intron, Spinach II, 2-MS2 binding domain and glgC 5΄ UTR. Additionally, we demonstrate the applicability of this approach to other bacterial species by predicting sRNA-mRNA binding regions in two newly discovered, though uncharacterized, regulatory RNAs.
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http://dx.doi.org/10.1093/nar/gkx115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435917PMC
May 2017

Defective Ribonucleoproteins, Mistakes in RNA Processing, and Diseases.

Biochemistry 2017 Mar 28;56(10):1367-1382. Epub 2017 Feb 28.

McKetta Department of Chemical Engineering, University of Texas at Austin , 200 East. Dean Keeton Street, Stop C0400, Austin, Texas 78712, United States.

Ribonucleoproteins (RNPs) are vital to many cellular events. To this end, many neurodegenerative diseases and cancers have been linked to RNP malfunction, particularly as this relates to defective processing of cellular RNA. The connection of RNPs and diseases has also propagated a shift of focus onto RNA targeting from traditional protein targeting treatments. However, therapeutic development in this area has been limited by incomplete molecular insight into the specific contributions of RNPs to disease. This review outlines the role of several RNPs in diseases, focusing on molecular defects in processes that affect proper RNA handling in the cell. This work also evaluates the contributions of recently developed methods to understanding RNP association and function. We review progress in this area by focusing on molecular malfunctions of RNPs associated with the onset and progression of several neurodegenerative diseases and cancer and conclude with a brief discussion of RNA-based therapeutic efforts.
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http://dx.doi.org/10.1021/acs.biochem.6b01134DOI Listing
March 2017

Integrative FourD omics approach profiles the target network of the carbon storage regulatory system.

Nucleic Acids Res 2017 02;45(4):1673-1686

McKetta Department of Chemical Engineering, University of Texas at Austin, 200 E. Dean Keeton Street Stop C0400, Austin, TX 78712, USA.

Multi-target regulators represent a largely untapped area for metabolic engineering and anti-bacterial development. These regulators are complex to characterize because they often act at multiple levels, affecting proteins, transcripts and metabolites. Therefore, single omics experiments cannot profile their underlying targets and mechanisms. In this work, we used an Integrative FourD omics approach (INFO) that consists of collecting and analyzing systems data throughout multiple time points, using multiple genetic backgrounds, and multiple omics approaches (transcriptomics, proteomics and high throughput sequencing crosslinking immunoprecipitation) to evaluate simultaneous changes in gene expression after imposing an environmental stress that accentuates the regulatory features of a network. Using this approach, we profiled the targets and potential regulatory mechanisms of a global regulatory system, the well-studied carbon storage regulatory (Csr) system of Escherichia coli, which is widespread among bacteria. Using 126 sets of proteomics and transcriptomics data, we identified 136 potential direct CsrA targets, including 50 novel ones, categorized their behaviors into distinct regulatory patterns, and performed in vivo fluorescence-based follow up experiments. The results of this work validate 17 novel mRNAs as authentic direct CsrA targets and demonstrate a generalizable strategy to integrate multiple lines of omics data to identify a core pool of regulator targets.
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http://dx.doi.org/10.1093/nar/gkx048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389547PMC
February 2017

Zymomonas mobilis as a model system for production of biofuels and biochemicals.

Microb Biotechnol 2016 11 15;9(6):699-717. Epub 2016 Sep 15.

National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA.

Zymomonas mobilis is a natural ethanologen with many desirable industrial biocatalyst characteristics. In this review, we will discuss work to develop Z. mobilis as a model system for biofuel production from the perspectives of substrate utilization, development for industrial robustness, potential product spectrum, strain evaluation and fermentation strategies. This review also encompasses perspectives related to classical genetic tools and emerging technologies in this context.
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http://dx.doi.org/10.1111/1751-7915.12408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5072187PMC
November 2016

Synthetic chimeras with orthogonal ribosomal proteins increase translation yields by recruiting mRNA for translation as measured by profiling active ribosomes.

Biotechnol Prog 2016 03 18;32(2):285-93. Epub 2016 Feb 18.

McKetta Dept. of Chemical Engineering, Cockrell School of Engineering, The University of Texas at Austin, 200 E. Dean Keeton St. Stop C0400, Austin, TX, 78712.

In addition to their roles in protein biosynthesis, components of cellular ribosomes perform roles that contribute to a number of important cellular processes. Exploitation of processes has led to the use of ribosomal parts as solubility enhancer partners and purification matrices in protein expression. In this work, an engineered version of the E. coli ribosomal protein L29 (L4H2) as a fusion partner for enhancing cellular expression of proteins that are poorly expressed in bacteria was exploited. It was demonstrated that a chimeric fusion of L4H2 with various Fcγ receptors increases total expression up to 3.2-fold, relative to Fcγ receptors expressed without the fusion. Mechanistic insights using a novel application of in vivo ribosome display suggested that, although total cellular mRNA levels of L4H2-Fcγ receptor remained unchanged relative to wild-type Fcγ receptors, mRNA levels of actively translated L4H2-Fcγ transcript increased about 3.8-fold relative to actively translated levels of wild-type Fcγ transcript. Similar increases in protein expression in the context of the other proteins tested, showing the generality of this approach for proteins beyond human receptors was observed. These results extended the number of potential schemes by which orthogonal ribosomal parts can be used to enhance complex protein expression in bacterial platforms. Within a larger scope, this study features the possibility of engineering 5' tags that enhance mRNA affinity to ribosomes as strategies to augment translation. It was envisioned that the successful application of profiling active ribosomes in a highly targeted manner could be beneficial for mechanistic translation studies concerning synthesis of target proteins. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:285-293, 2016.
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http://dx.doi.org/10.1002/btpr.2227DOI Listing
March 2016

Slowing Translation between Protein Domains by Increasing Affinity between mRNAs and the Ribosomal Anti-Shine-Dalgarno Sequence Improves Solubility.

ACS Synth Biol 2016 Feb 16;5(2):133-45. Epub 2015 Dec 16.

McKetta Department of Chemical Engineering, University of Texas at Austin , 200 East Dean Keeton Street, Stop C0400, Austin, Texas 78712, United States.

Recent studies have demonstrated that effective protein production requires coordination of multiple cotranslational cellular processes, which are heavily affected by translation timing. Until recently, protein engineering has focused on codon optimization to maximize protein production rates, mostly considering the effect of tRNA abundance. However, as it relates to complex multidomain proteins, it has been hypothesized that strategic translational pauses between domains and between distinct individual structural motifs can prevent interactions between nascent chain fragments that generate kinetically trapped misfolded peptides and thereby enhance protein yields. In this study, we introduce synthetic transient pauses between structural domains in a heterologous model protein based on designed patterns of affinity between the mRNA and the anti-Shine-Dalgarno (aSD) sequence on the ribosome. We demonstrate that optimizing translation attenuation at domain boundaries can predictably affect solubility patterns in bacteria. Exploration of the affinity space showed that modifying less than 1% of the nucleotides (on a small 12 amino acid linker) can vary soluble protein yields up to ∼7-fold without altering the primary sequence of the protein. In the context of longer linkers, where a larger number of distinct structural motifs can fold outside the ribosome, optimal synonymous codon variations resulted in an additional 2.1-fold increase in solubility, relative to that of nonoptimized linkers of the same length. While rational construction of 54 linkers of various affinities showed a significant correlation between protein solubility and predicted affinity, only weaker correlations were observed between tRNA abundance and protein solubility. We also demonstrate that naturally occurring high-affinity clusters are present between structural domains of β-galactosidase, one of Escherichia coli's largest native proteins. Interdomain ribosomal affinity is an important factor that has not previously been explored in the context of protein engineering.
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http://dx.doi.org/10.1021/acssynbio.5b00193DOI Listing
February 2016

Cellular RNA is chemically modified by exposure to air pollution mixtures.

Inhal Toxicol 2015 Jan;27(1):74-82

McKetta Department of Chemical Engineering, The University of Texas at Austin , Austin TX , USA and.

RNAs are more susceptible to modifications than DNA, and chemical modifications in RNA have an effect on their structure and function. This study aimed to characterize chemical effects on total RNA in human A549 lung cells after exposure to elevated levels of major secondary air pollutants commonly found in urban locations, including ozone (O3), acrolein (ACR) and methacrolein (MACR). Enzyme-linked immunosorbent assays (ELISA) were used to measure levels of interleukin (IL)-8 in the growth media and 8-oxoguanine (8OG) levels in total cellular RNA, and lactate dehydrogenase (LDH) in the growth media was measured by a coupled enzymatic assay. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure levels of microRNA 10b (miR-10b). The study found that 1-h exposure to all tested pollutant mixtures consistently caused significant increases in the levels of 8OG in total RNA. In the case of 4 ppm O3 exposures, measured levels of IL-8, LDH and miR-10b each showed consistent trends between two independent trials, but varied among these three targets. After 1-h exposures to an ACR+MACR mixture, measured levels of IL-8, LDH and miR-10b showed variable results. For mixtures of O3+ACR+MACR, IL-8 measurements showed no change; miR-10b and LDH showed variable results. The results indicate that short-term high-concentration exposures to air pollution can cause RNA chemical modifications. Chemical modifications in RNAs could represent more consistent markers of cellular stress relative to other inflammation markers, such as IL-8 and LDH, and provide a new biomarker endpoint for mechanistic studies in toxicity of air pollution exposure.
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http://dx.doi.org/10.3109/08958378.2014.987361DOI Listing
January 2015