@article{zeldes_loder_counts_haque_widney_keller_albers_kelly_2019, title={Determinants of sulphur chemolithoautotrophy in the extremely thermoacidophilic Sulfolobales}, volume={21}, ISSN={["1462-2920"]}, DOI={10.1111/1462-2920.14712}, abstractNote={SummarySpecies in the archaeal order Sulfolobales thrive in hot acid and exhibit remarkable metabolic diversity. Some species are chemolithoautotrophic, obtaining energy through the oxidation of inorganic substrates, sulphur in particular, and acquiring carbon through the 3‐hydroxypropionate/4‐hydroxybutyrate (3‐HP/4‐HB) CO2‐fixation cycle. The current model for sulphur oxidation in the Sulfolobales is based on the biochemical analysis of specific proteins from Acidianus ambivalens, including sulphur oxygenase reductase (SOR) that disproportionates S° into H2S and sulphite (SO32−). Initial studies indicated SOR catalyses the essential first step in oxidation of elemental sulphur, but an ancillary role for SOR as a ‘recycle’ enzyme has also been proposed. Here, heterologous expression of both SOR and membrane‐bound thiosulphate‐quinone oxidoreductase (TQO) from Sulfolobus tokodaii ‘restored’ sulphur oxidation capacity in Sulfolobus acidocaldarius DSM639, but not autotrophy, although earlier reports indicate this strain was once capable of chemolithoautotrophy. Comparative transcriptomic analyses of Acidianus brierleyi, a chemolithoautotrophic sulphur oxidizer, and S. acidocaldarius DSM639 showed that while both share a strong transcriptional response to elemental sulphur, S. acidocaldarius DSM639 failed to upregulate key 3‐HP/4‐HB cycle genes used by A. brierleyi to drive chemolithoautotrophy. Thus, the inability for S. acidocaldarius DSM639 to grow chemolithoautotrophically may be rooted more in gene regulation than the biochemical capacity.}, number={10}, journal={ENVIRONMENTAL MICROBIOLOGY}, author={Zeldes, Benjamin M. and Loder, Andrew J. and Counts, James A. and Haque, Mashkurul and Widney, Karl A. and Keller, Lisa M. and Albers, Sonja-Verena and Kelly, Robert M.}, year={2019}, month={Oct}, pages={3696–3710} }
 @article{khatibi_chou_loder_zurawski_adams_kelly_2017, title={Impact of growth mode, phase, and rate on the metabolic state of the extremely thermophilic archaeon Pyrococcus furiosus}, volume={114}, ISSN={["1097-0290"]}, DOI={10.1002/bit.26408}, abstractNote={AbstractThe archaeon Pyrococcus furiosus is emerging as a metabolic engineering platform for production of fuels and chemicals, such that more must be known about this organism's characteristics in bioprocessing contexts. Its ability to grow at temperatures from 70 to greater than 100°C and thereby avoid contamination, offers the opportunity for long duration, continuous bioprocesses as an alternative to batch systems. Toward that end, we analyzed the transcriptome of P. furiosus to reveal its metabolic state during different growth modes that are relevant to bioprocessing. As cells progressed from exponential to stationary phase in batch cultures, genes involved in biosynthetic pathways important to replacing diminishing supplies of key nutrients and genes responsible for the onset of stress responses were up‐regulated. In contrast, during continuous culture, the progression to higher dilution rates down‐regulated many biosynthetic processes as nutrient supplies were increased. Most interesting was the contrast between batch exponential phase and continuous culture at comparable growth rates (∼0.4 hr−1), where over 200 genes were differentially transcribed, indicating among other things, N‐limitation in the chemostat and the onset of oxidative stress. The results here suggest that cellular processes involved in carbon and electron flux in P. furiosus were significantly impacted by growth mode, phase and rate, factors that need to be taken into account when developing successful metabolic engineering strategies.}, number={12}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Khatibi, Piyum A. and Chou, Chung-jung and Loder, Andrew J. and Zurawski, Jeffrey V. and Adams, Michael W. W. and Kelly, Robert M.}, year={2017}, month={Dec}, pages={2947–2954} }
 @article{lian_zeldes_lipscomb_hawkins_han_loder_nishiyama_adams_kelly_2016, title={Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus}, volume={113}, number={12}, journal={Biotechnology and Bioengineering}, author={Lian, H. and Zeldes, B. M. and Lipscomb, G. L. and Hawkins, A. B. and Han, Y. J. and Loder, A. J. and Nishiyama, D. and Adams, M. W. W. and Kelly, R. M.}, year={2016}, pages={2652–2660} }
 @article{loder_han_hawkins_lian_lipscomb_schut_keller_adams_kelly_2016, title={Reaction kinetic analysis of the 3-hydroxypropionate/4-hydroxybutyrate CO2 fixation cycle in extremely thermoacidophilic archaea}, volume={38}, ISSN={["1096-7184"]}, DOI={10.1016/j.ymben.2016.10.009}, abstractNote={The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle fixes CO2 in extremely thermoacidophilic archaea and holds promise for metabolic engineering because of its thermostability and potentially rapid pathway kinetics. A reaction kinetics model was developed to examine the biological and biotechnological attributes of the 3HP/4HB cycle as it operates in Metallosphaera sedula, based on previous information as well as on kinetic parameters determined here for recombinant versions of five of the cycle enzymes (malonyl-CoA/succinyl-CoA reductase, 3-hydroxypropionyl-CoA synthetase, 3-hydroxypropionyl-CoA dehydratase, acryloyl-CoA reductase, and succinic semialdehyde reductase). The model correctly predicted previously observed features of the cycle: the 35–65% split of carbon flux through the acetyl-CoA and succinate branches, the high abundance and relative ratio of acetyl-CoA/propionyl-CoA carboxylase (ACC) and MCR, and the significance of ACC and hydroxybutyryl-CoA synthetase (HBCS) as regulated control points for the cycle. The model was then used to assess metabolic engineering strategies for incorporating CO2 into chemical intermediates and products of biotechnological importance: acetyl-CoA, succinate, and 3-hydroxypropionate.}, journal={METABOLIC ENGINEERING}, author={Loder, Andrew J. and Han, Yejun and Hawkins, Aaron B. and Lian, Hong and Lipscomb, Gina L. and Schut, Gerrit J. and Keller, Matthew W. and Adams, Michael W. W. and Kelly, Robert M.}, year={2016}, month={Nov}, pages={446–463} }
 @article{loder_zeldes_garrison_lipscomb_adams_kelly_2015, title={Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes}, volume={81}, ISSN={["1098-5336"]}, DOI={10.1128/aem.02028-15}, abstractNote={ABSTRACT
          
            n
            -Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. A synthetic pathway for
            n
            -butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled
            in vitro
            from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Recombinant versions of eight thermophilic enzymes (β-ketothiolase [Thl], 3-hydroxybutyryl-CoA dehydrogenase [Hbd], and 3-hydroxybutyryl-CoA dehydratase [Crt] from
            Caldanaerobacter subterraneus
            subsp.
            tengcongensis
            ;
            trans
            -2-enoyl-CoA reductase [Ter] from
            Spirochaeta thermophila
            ; bifunctional acetaldehyde dehydrogenase/alcohol dehydrogenase [AdhE] from
            Clostridium thermocellum
            ; and AdhE, aldehyde dehydrogenase [Bad], and butanol dehydrogenase [Bdh] from
            Thermoanaerobacter
            sp. strain X514) were utilized to examine three possible pathways for
            n
            -butanol. These pathways differed in the two steps required to convert butyryl-CoA to
            n
            -butanol: Thl-Hbd-Crt-Ter-AdhE (
            C. thermocellum
            ), Thl-Hbd-Crt-Ter-AdhE (
            Thermoanaerobacter
            X514), and Thl-Hbd-Crt-Ter-Bad-Bdh.
            n
            -Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to
            in vitro
            experiments, was used to determine relative enzyme ratios needed to maximize
            n
            -butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to
            n
            -butanol was observed
            in vitro
            , but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to
            n
            -butanol is predicted, with a 19% increase in pathway flux. Thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic
            n
            -butanol production at elevated temperatures.
          }, number={20}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Loder, Andrew J. and Zeldes, Benjamin M. and Garrison, G. Dale, II and Lipscomb, Gina L. and Adams, Michael W. W. and Kelly, Robert M.}, year={2015}, month={Oct}, pages={7187–7200} }
 @article{hawkins_lian_zeldes_loder_lipscomb_schut_keller_adams_kelly_2015, title={Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO2-based 3-hydroxypropionate production}, volume={112}, ISSN={["1097-0290"]}, DOI={10.1002/bit.25584}, abstractNote={ABSTRACTMetabolically engineered strains of the hyperthermophile Pyrococcus furiosus (Topt 95–100°C), designed to produce 3‐hydroxypropionate (3HP) from maltose and CO2 using enzymes from the Metallosphaera sedula (Topt 73°C) carbon fixation cycle, were examined with respect to the impact of heterologous gene expression on metabolic activity, fitness at optimal and sub‐optimal temperatures, gas‐liquid mass transfer in gas‐intensive bioreactors, and potential bottlenecks arising from product formation. Transcriptomic comparisons of wild‐type P. furiosus, a genetically‐tractable, naturally‐competent mutant (COM1), and COM1‐based strains engineered for 3HP production revealed numerous differences after being shifted from 95°C to 72°C, where product formation catalyzed by the heterologously‐produced M. sedula enzymes occurred. At 72°C, significantly higher levels of metabolic activity and a stress response were evident in 3HP‐forming strains compared to the non‐producing parent strain (COM1). Gas–liquid mass transfer limitations were apparent, given that 3HP titers and volumetric productivity in stirred bioreactors could be increased over 10‐fold by increased agitation and higher CO2 sparging rates, from 18 mg/L to 276 mg/L and from 0.7 mg/L/h to 11 mg/L/h, respectively. 3HP formation triggered transcription of genes for protein stabilization and turnover, RNA degradation, and reactive oxygen species detoxification. The results here support the prospects of using thermally diverse sources of pathways and enzymes in metabolically engineered strains designed for product formation at sub‐optimal growth temperatures. Biotechnol. Bioeng. 2015;112: 1533–1543. © 2015 Wiley Periodicals, Inc.}, number={8}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Hawkins, Aaron B. and Lian, Hong and Zeldes, Benjamin M. and Loder, Andrew J. and Lipscomb, Gina L. and Schut, Gerrit J. and Keller, Matthew W. and Adams, Michael W. W. and Kelly, Robert M.}, year={2015}, month={Aug}, pages={1533–1543} }
 @misc{zeldes_keller_loder_straub_adams_kelly_2015, title={Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals}, volume={6}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2015.01209}, abstractNote={Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point that the use of these microorganisms as metabolic engineering platforms has become possible. While in its early days, complex metabolic pathways have been altered or engineered into recombinant extreme thermophiles, such that the production of fuels and chemicals at elevated temperatures has become possible. Not only does this expand the thermal range for industrial biotechnology, it also potentially provides biodiverse options for specific biotransformations unique to these microorganisms. The list of extreme thermophiles growing optimally between 70 and 100°C with genetic toolkits currently available includes archaea and bacteria, aerobes and anaerobes, coming from genera such as Caldicellulosiruptor, Sulfolobus, Thermotoga, Thermococcus, and Pyrococcus. These organisms exhibit unusual and potentially useful native metabolic capabilities, including cellulose degradation, metal solubilization, and RuBisCO-free carbon fixation. Those looking to design a thermal bioprocess now have a host of potential candidates to choose from, each with its own advantages and challenges that will influence its appropriateness for specific applications. Here, the issues and opportunities for extremely thermophilic metabolic engineering platforms are considered with an eye toward potential technological advantages for high temperature industrial biotechnology.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Zeldes, Benjamin M. and Keller, Matthew W. and Loder, Andrew J. and Straub, Christopher T. and Adams, Michael W. W. and Kelly, Robert M.}, year={2015}, month={Nov} }
 @article{keller_lipscomb_loder_schut_kelly_adams_2015, title={A hybrid synthetic pathway for butanol production by a hyperthermophilic microbe}, volume={27}, ISSN={["1096-7184"]}, DOI={10.1016/j.ymben.2014.11.004}, abstractNote={Biologically produced alcohols are of great current interest for renewable solvents and liquid transportation fuels. While bioethanol is now produced on a massive scale, butanol has superior fuel characteristics and an additional value as a solvent and chemical feedstock. Butanol production has been demonstrated at ambient temperatures in metabolically-engineered mesophilic organisms, but the ability to engineer a microbe for in vivo high-temperature production of commodity chemicals has several distinct advantages. These include reduced contamination risk, facilitated removal of volatile products, and a wide temperature range to modulate and balance both the engineered pathway and the host׳s metabolism. We describe a synthetic metabolic pathway assembled from genes obtained from three different sources for conversion of acetyl-CoA to 1-butanol, and 1-butanol generation from glucose was demonstrated near 70 °C in a microorganism that grows optimally near 100 °C. The module could also be used in thermophiles capable of degrading plant biomass.}, journal={METABOLIC ENGINEERING}, author={Keller, Matthew W. and Lipscomb, Gina L. and Loder, Andrew J. and Schut, Gerrit J. and Kelly, Robert M. and Adams, Michael W. W.}, year={2015}, month={Jan}, pages={101–106} }
 @article{lewis_notey_chandrayan_loder_lipscomb_adams_kelly_2015, title={A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus, lacking flagella, has unusual growth physiology}, volume={19}, ISSN={["1433-4909"]}, DOI={10.1007/s00792-014-0712-3}, abstractNote={A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus DSM3638 exhibited an extended exponential phase and atypical cell aggregation behavior. Genomic DNA from the mutant culture was sequenced and compared to wild-type (WT) DSM3638, revealing 145 genes with one or more insertions, deletions, or substitutions (12 silent, 33 amino acid substitutions, and 100 frame shifts). Approximately, half of the mutated genes were transposases or hypothetical proteins. The WT transcriptome revealed numerous changes in amino acid and pyrimidine biosynthesis pathways coincidental with growth phase transitions, unlike the mutant whose transcriptome reflected the observed prolonged exponential phase. Targeted gene deletions, based on frame-shifted ORFs in the mutant genome, in a genetically tractable strain of P. furiosus (COM1) could not generate the extended exponential phase behavior observed for the mutant. For example, a putative radical SAM family protein (PF2064) was the most highly up-regulated ORF (>25-fold) in the WT between exponential and stationary phase, although this ORF was unresponsive in the mutant; deletion of this gene in P. furiosus COM1 resulted in no apparent phenotype. On the other hand, frame-shifting mutations in the mutant genome negatively impacted transcription of a flagellar biosynthesis operon (PF0329-PF0338).Consequently, cells in the mutant culture lacked flagella and, unlike the WT, showed minimal evidence of exopolysaccharide-based cell aggregation in post-exponential phase. Electron microscopy of PF0331-PF0337 deletions in P. furiosus COM1 showed that absence of flagella impacted normal cell aggregation behavior and, furthermore, indicated that flagella play a key role, beyond motility, in the growth physiology of P. furiosus.}, number={2}, journal={EXTREMOPHILES}, author={Lewis, Derrick L. and Notey, Jaspreet S. and Chandrayan, Sanjeev K. and Loder, Andrew J. and Lipscomb, Gina L. and Adams, Michael W. W. and Kelly, Robert M.}, year={2015}, month={Mar}, pages={269–281} }