Recent Publications

Lipid membrane remodeling and metabolic response during isobutanol and ethanol exposure in Zymomonas mobilis

Julio Rivera Vazquez, Edna Trujillo, Jonathan Williams, Fukang She, Fitsum Getahun Melanie M. Callaghan, Joshua J. Coon, and Daniel Amador‑Noguez


We utilized a systems-level approach comprising LC-MS/MS-based lipidomics, metabolomics, and shotgun proteomics, to investigate how exposure to ethanol and isobutanol impact the lipid membrane composition and overall physiology of Z. mobilis. Our analysis revealed significant and distinct alterations in membrane phospholipid and fatty acid composition resulting from ethanol and isobutanol exposure. Notably, ethanol exposure increased membrane cyclopropane fatty acid content and expression of cyclopropane fatty acid (CFA) synthase. Surprisingly, isobutanol decreased cyclopropane fatty acid content despite robust upregulation of CFA synthase. Overexpression of the native Z. mobilis’ CFA synthase increased cyclopropane fatty acid content in all phospholipid classes and was associated with a significant improvement in growth rates in the presence of added ethanol and isobutanol. Heterologous expression of CFA synthase from Clostridium acetobutylicum resulted in a near complete replacement of unsaturated fatty acids with cyclopropane fatty acids, affecting all lipid classes. However, this did not translate to improved growth rates under isobutanol exposure. Correlating with its greater susceptibility to isobutanol, Z. mobilis exhibited more pronounced alterations in its proteome, metabolome, and overall cell morphology-including cell swelling and formation of intracellular protein aggregates -when exposed to isobutanol compared to ethanol. Isobutanol triggered a broad stress response marked by the upregulation of heat shock proteins, efflux transporters, DNA repair systems, and the downregulation of cell motility proteins. Isobutanol also elicited widespread dysregulation of Z. mobilis’ primary metabolism evidenced by increased levels of nucleotide degradation intermediates and the depletion of biosynthetic and glycolytic intermediates. This study provides a comprehensive, systems-level evaluation of the impact of ethanol and isobutanol exposure on the lipid membrane composition and overall physiology of Z. mobilis. These findings will guide engineering of Z. mobilis towards the creation of isobutanol-tolerant strains that can serve as robust platforms for the industrial production of isobutanol from lignocellulosic sugars.

Fig 1: A Anaerobic growth of Z. mobilis ZM4 in minimal media containing varying amounts of added ethanol (0.3, 0.6, or 0.8 M) or isobutanol (0.1 and 0.15 M). Z. mobilis was more susceptible to growth inhibition by isobutanol than ethanol. Each data point represents the average of four biological replicates, with error bars indicating the standard deviation. Refer to Table S2 for doubling times and methods section for cell density measurements. B Bright-field microscopy evaluation of gross morphological alterations when Z mobilis was grown in minimal media supplemented with either ethanol (0.3, 0.6, or 0.8 M) or isobutanol (0.10 or 0.15 M)

Deuterated water as a substrate-agnostic isotope tracer for investigating reversibility and thermodynamics of reactions in central carbon metabolism

Melanie M. Callaghan, Eashant Thusoo, Bishal D. Sharma, Fitsum Getahun, David M. Stevenson, Costas Maranas, Daniel G. Olson, Lee R. Lynd, Daniel Amador-Noguez


Stable isotope tracers are a powerful tool for the quantitative analysis of microbial metabolism, enabling pathway elucidation, metabolic flux quantification, and assessment of reaction and pathway thermodynamics. 13C and 2H metabolic flux analysis commonly relies on isotopically labeled carbon substrates, such as glucose. However, the use of 2H-labeled nutrient substrates faces limitations due to their high cost and limited availability in comparison to 13C-tracers. Furthermore, isotope tracer studies in industrially relevant bacteria that metabolize complex substrates such as cellulose, hemicellulose, or lignocellulosic biomass, are challenging given the difficulty in obtaining these as isotopically labeled substrates. In this study, we examine the potential of deuterated water (2H2O) as an affordable, substrate-neutral isotope tracer for studying central carbon metabolism. We apply 2H2O labeling to investigate the reversibility of glycolytic reactions across three industrially relevant bacterial species -C. thermocellum, Z. mobilis, and E. coli harboring distinct glycolytic pathways with unique thermodynamics. We demonstrate that 2H2O labeling recapitulates previous reversibility and thermodynamic findings obtained with established 13C and 2H labeled nutrient substrates. Furthermore, we exemplify the utility of this 2H2O labeling approach by applying it to high substrate C. thermocellum fermentations -a setting in which the use of conventional tracers is impractical-thereby identifying the glycolytic enzyme phosphofructokinase as a major bottleneck during high-substrate fermentations and unveiling critical insights that will steer future engineering efforts to enhance ethanol production in this cellulolytic organism. This study demonstrates the utility of deuterated water as a substrate-agnostic isotope tracer for examining flux and reversibility of central carbon metabolic reactions, which yields biological insights comparable to those obtained using costly 2H-labeled nutrient substrates.

Fig 7: High Solids (100g/L cellobiose) fermentation in 25% 2H2O in Clostridium thermocellum engineered strain


Systematic Analysis of Metabolic Bottlenecks in the Methylerythritol 4-Phosphate (MEP) Pathway of Zymomonas mobilis

Daven B Khana, Mehmet Tatli, Julio Rivera Vazquez, Sarathi M Weraduwage, Noah Stern, Alexander S Hebert, Edna Angelica Trujillo, David M Stevenson, Joshua J Coon, Thomas D Sharky, Daniel Amador-Noguez



Zymomonas mobilis is an industrially relevant aerotolerant anaerobic bacterium that can convert up to 96% of consumed glucose to ethanol. This highly catabolic metabolism could be leveraged to produce isoprenoid-based bioproducts via the methylerythritol 4-phosphate (MEP) pathway, but we currently have limited knowledge concerning the metabolic constraints of this pathway in Z. mobilis. Here, we performed an initial investigation of the metabolic bottlenecks within the MEP pathway of Z. mobilis using enzyme overexpression strains and quantitative metabolomics. Our analysis revealed that 1-deoxy-d-xylulose 5-phosphate synthase (DXS) represents the first enzymatic bottleneck in the Z. mobilis MEP pathway. DXS overexpression triggered large increases in the intracellular levels of the first five MEP pathway intermediates, of which the buildup in 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) was the most substantial. The combined overexpression of DXS, 4-hydroxy-3-methylbut-2-enyl diphosphate (HMBDP) synthase (IspG), and HMBDP reductase (IspH) mitigated the bottleneck at MEcDP and mobilized carbon to downstream MEP pathway intermediates, indicating that IspG and IspH activity become the primary pathway constraints during DXS overexpression. Finally, we overexpressed DXS with other native MEP enzymes and a heterologous isoprene synthase and showed that isoprene can be used as a carbon sink in the Z. mobilis MEP pathway. By revealing key bottlenecks within the MEP pathway of Z. mobilis, this study will aid future engineering efforts aimed at developing this bacterium for industrial isoprenoid production.



Fig. 3 The overexpression of MEP enzymes can elicit major changes to the levels of MEP pathway metabolites.


Listeria monocytogenes GlmR Is an Accessory Uridyltransferase Essential for Cytosolic Survival and Virulence

Daniel A Pensinger, Kimberly V Gutierrez, Hans B Smith, William J B Vincent, David S Stevenson, Katherine A Black, Krizia M Perez-Medina, Joseph P Dillard, Kyu Y Rhee, Daniel Amador-Noguez, TuAnh N Huynh, John-Demian Sauer


The cytosol of eukaryotic host cells is an intrinsically hostile environment for bacteria. Understanding how cytosolic pathogens adapt to and survive in the cytosol is critical to developing novel therapeutic interventions against these pathogens. The cytosolic pathogen Listeria monocytogenes requires glmR (previously known as yvcK), a gene of unknown function, for resistance to cell-wall stress, cytosolic survival, inflammasome avoidance, and, ultimately, virulence in vivo. In this study, a genetic suppressor screen revealed that blocking utilization of UDP N-acetylglucosamine (UDP-GlcNAc) by a nonessential wall teichoic acid decoration pathway restored resistance to lysozyme and partially restored virulence of ΔglmR mutants. In parallel, metabolomic analysis revealed that ΔglmR mutants are impaired in the production of UDP-GlcNAc, an essential peptidoglycan and wall teichoic acid (WTA) precursor. We next demonstrated that purified GlmR can directly catalyze the synthesis of UDP-GlcNAc from GlcNAc-1P and UTP, suggesting that it is an accessory uridyltransferase. Biochemical analysis of GlmR orthologues suggests that uridyltransferase activity is conserved. Finally, mutational analysis resulting in a GlmR mutant with impaired catalytic activity demonstrated that uridyltransferase activity was essential to facilitate cell-wall stress responses and virulence in vivo. Taken together, these studies indicate that GlmR is an evolutionary conserved accessory uridyltransferase required for cytosolic survival and virulence of L. monocytogenes.


Fig. 1 Inhibition of GlcNAc WTA modification suppresses ΔglmR mutant phenotypes.

Rapid Targeted Quantitation of Protein Overexpression with Direct Infusion Shotgun Proteome Analysis (DISPA-PRM)

Edna A Trujillo, Alexander S Hebert, Julio C Rivera Vazquez, Dain R Brademan, Mehmet Tatli, Daniel Amador-Noguez, Jesse G Meyer, Joshua J Coon


While much effort has been placed on comprehensive quantitative proteome analysis, certain applications demand the measurement of only a few target proteins from complex systems. Traditional approaches to targeted proteomics rely on nanoliquid chromatography (nLC) and targeted mass spectrometry (MS) methods, e.g., parallel reaction monitoring (PRM). However, the time requirement for nLC can limit the throughput of targeted proteomics. To achieve rapid and high-throughput targeted methods, here we show that nLC separations can be eliminated and replaced with direct infusion shotgun proteome analysis (DISPA) using high-field asymmetric waveform ion mobility spectrometry (FAIMS) with PRM. We demonstrate the application of DISPA-PRM for rapid targeted quantification of bacterial enzymes utilized in the production of biofuels by monitoring temporal expression in 72 metabolically engineered bacterial cultures in less than 2.5 h, with a measured dynamic range >1200-fold. We conclude that DISPA-PRM presents a valuable innovative tool with results comparable to nLC-MS/MS, enabling fast and rapid detection of targeted proteins in complex mixtures.

Fig 1. Qualitative comparison of DISPA-PRM signals for peptides from IspG and IspH proteins.


Assessing the impact of substrate-level enzyme regulations limiting ethanol titer in Clostridium thermocellum using a core kinetic model

Charles Foster, Veda Sheersh Boorla, Satyakam Dash, Saratram Gopalakrishnan, Tyler B Jacobson, Daniel G Olson, Daniel Amador-Noguez, Lee R Lynd, Costas D Maranas


Clostridium thermocellum is a promising candidate for consolidated bioprocessing because it can directly ferment cellulose to ethanol. Despite significant efforts, achieved yields and titers fall below industrially relevant targets. This implies that there still exist unknown enzymatic, regulatory, and/or possibly thermodynamic bottlenecks that can throttle back metabolic flow. By (i) elucidating internal metabolic fluxes in wild-type C. thermocellum grown on cellobiose via 13C-metabolic flux analysis (13C-MFA), (ii) parameterizing a core kinetic model, and (iii) subsequently deploying an ensemble-docking workflow for discovering substrate-level regulations, this paper aims to reveal some of these factors and expand our knowledgebase governing C. thermocellum metabolism. Generated 13C labeling data were used with 13C-MFA to generate a wild-type flux distribution for the metabolic network. Notably, flux elucidation through MFA alluded to serine generation via the mercaptopyruvate pathway. Using the elucidated flux distributions in conjunction with batch fermentation process yield data for various mutant strains, we constructed a kinetic model of C. thermocellum core metabolism (i.e. k-ctherm138). Subsequently, we used the parameterized kinetic model to explore the effect of removing substrate-level regulations on ethanol yield and titer. Upon exploring all possible simultaneous (up to four) regulation removals we identified combinations that lead to many-fold model predicted improvement in ethanol titer. In addition, by coupling a systematic method for identifying putative competitive inhibitory mechanisms using K-FIT kinetic parameterization with the ensemble-docking workflow, we flagged 67 putative substrate-level inhibition mechanisms across central carbon metabolism supported by both kinetic formalism and docking analysis.

Fig. 1. Ensemble docking workflow for inferring the binding energy of hypothetical substrate-level regulations.


Novel computational and experimental approaches for investigating the thermodynamics of metabolic networks

Daven B Khana, Melanie M Callaghan, Daniel Amador-Noguez


Thermodynamic analysis of metabolic networks has emerged as a useful new tool for pathway design and metabolic engineering. Understanding the relationship between the thermodynamic driving force of biochemical reactions and metabolic flux has generated new insights regarding the design principles of microbial carbon metabolism. This review summarizes the various lessons that can be obtained from the thermodynamic analysis of metabolic pathways, illustrates concepts of computational thermodynamic tools, and highlights recent applications of thermodynamic analysis to pathway design in industrially relevant microbes.

Fig. 1 The Flux-Force Efficacy equation and the relationship between free energies, metabolic fluxes, and enzyme efficiency.

Novel computational and experimental approaches for investigating the thermodynamics of metabolic networks

Khana DB, Callaghan MM, Amador-Noguez D.


Thermodynamic analysis of metabolic networks has emerged as a useful new tool for pathway design and metabolic engineering. Understanding the relationship between the thermodynamic driving force of biochemical reactions and metabolic flux has generated new insights regarding the design principles of microbial carbon metabolism. This review summarizes the various lessons that can be obtained from the thermodynamic analysis of metabolic pathways, illustrates concepts of computational thermodynamic tools, and highlights recent applications of thermodynamic analysis to pathway design in industrially relevant microbes.

Fig. 1. The Flux-Force Efficacy equation and the relationship between free energies, metabolic fluxes, and enzyme efficiency.


Dominant Bacterial Phyla from the Human Gut Show Widespread Ability To Transform and Conjugate Bile Acids

Lucas LN, Barrett K, Kerby RL, Zhang Q, Cattaneo LE, Stevenson D, Rey FE, Amador-Noguez D.


Gut bacteria influence human physiology by chemically modifying host-synthesized primary bile acids. These modified bile acids, known as secondary bile acids, can act as signaling molecules that modulate host lipid, glucose, and energy metabolism and affect gut microbiota composition via selective antimicrobial properties. However, knowledge regarding the bile acid-transforming capabilities of individual gut microbes remains limited. To help address this knowledge gap, we screened 72 bacterial isolates, spanning seven major phyla commonly found in the human gut, for their ability to chemically modify unconjugated bile acids. We found that 43 isolates, representing 41 species, were capable of in vitro modification of one or more of the three most abundant unconjugated bile acids in humans: cholic acid, chenodeoxycholic acid, and deoxycholic acid. Of these, 32 species have not been previously described as bile acid transformers. The most prevalent bile acid transformations detected were oxidation of 3α-, 7α-, or 12α-hydroxyl groups on the steroid core, a reaction catalyzed by hydroxysteroid dehydrogenases. In addition, we found 7α-dehydroxylation activity to be distributed across various bacterial genera, and we observed several other complex bile acid transformations. Finally, our screen revealed widespread bacterial conjugation of primary and secondary bile acids to glycine, a process that was thought to only occur in the liver, and to 15 other amino acids, resulting in the discovery of 44 novel microbially conjugated bile acids.

Fig. 1. Bile acid production and enterohepatic circulation.


Metabolic Remodeling during Nitrogen Fixation in Zymomonas mobilis

Martien JI, Trujillo EA, Jacobson TB, Tatli M, Hebert AS, Stevenson DM, Coon JJ, Amador-Noguez D.


Zymomonas mobilis is an ethanologenic bacterium currently being developed for production of advanced biofuels. Recent studies have shown that Z. mobilis can fix dinitrogen gas (N2) as a sole nitrogen source. During N2 fixation, Z. mobilis exhibits increased biomass-specific rates of ethanol production. In order to better understand the physiology of Z. mobilis during N2 fixation and during changes in ammonium (NH4+) availability, we performed liquid chromatography-mass spectrometry (LC-MS)-based targeted metabolomics and shotgun proteomics under three regimes of nitrogen availability: continuous N2 fixation, gradual NH4+ depletion, and acute NH4+ addition to N2-fixing cells. We report dynamic changes in abundance of proteins and metabolites related to nitrogen fixation, motility, ammonium assimilation, amino acid biosynthesis, nucleotide biosynthesis, isoprenoid biosynthesis, and Entner-Doudoroff (ED) glycolysis, providing insight into the regulatory mechanisms that control these processes in Z. mobilis. Our analysis identified potential physiological mechanisms that may contribute to increased specific ethanol production during N2 fixation, including decreased activity of biosynthetic pathways, increased protein abundance of alcohol dehydrogenase (ADHI), and increased thermodynamic favorability of the ED pathway. Of particular relevance to advanced biofuel production, we found that intermediates in the methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis were depleted during N2 fixation, coinciding with decreased protein abundance of deoxyxylulose 5-phosphate synthase (DXS), the first enzyme in the pathway. This implies that DXS protein abundance serves as a native control point in regulating MEP pathway activity in Z. mobilis. The results of this study will inform metabolic engineering to further develop Z. mobilis as a platform organism for biofuel production.

Fig. 1. Schematic of experimental design


Hostile Takeover: How Viruses Reprogram Prokaryotic Metabolism

Jacobson TB, Callaghan MM, Amador-Noguez D.


To reproduce, prokaryotic viruses must hijack the cellular machinery of their hosts and redirect it toward the production of viral particles. While takeover of the host replication and protein synthesis apparatus has long been considered an essential feature of infection, recent studies indicate that extensive reprogramming of host primary metabolism is a widespread phenomenon among prokaryotic viruses that is required to fulfill the biosynthetic needs of virion production. In this review we provide an overview of the most significant recent findings regarding virus-induced reprogramming of prokaryotic metabolism and suggest how quantitative systems biology approaches may be used to provide a holistic understanding of metabolic remodeling during lytic viral infection.

In Vivo Thermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using 13 C and 2 H Tracers

Jacobson TB, Korosh TK, Stevenson DM, Foster C, Maranas C, Olson DG, Lynd LR, Amador-Noguez D.


Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic anaerobic bacteria with complementary metabolic capabilities that utilize distinct glycolytic pathways for the conversion of cellulosic sugars to biofuels. We integrated quantitative metabolomics with 2H and 13C metabolic flux analysis to investigate the in vivo reversibility and thermodynamics of the central metabolic networks of these two microbes. We found that the glycolytic pathway in C. thermocellum operates remarkably close to thermodynamic equilibrium, with an overall drop in Gibbs free energy 5-fold lower than that of T. saccharolyticum or anaerobically grown Escherichia coli. The limited thermodynamic driving force of glycolysis in C. thermocellum could be attributed in large part to the small free energy of the phosphofructokinase reaction producing fructose bisphosphate. The ethanol fermentation pathway was also substantially more reversible in C. thermocellum than in T. saccharolyticum. These observations help explain the comparatively low ethanol titers of C. thermocellum and suggest engineering interventions that can be used to increase its ethanol productivity and glycolytic rate. In addition to thermodynamic analysis, we used our isotope tracer data to reconstruct the T. saccharolyticum central metabolic network, revealing exclusive use of the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis, a bifurcated tricarboxylic acid (TCA) cycle, and a sedoheptulose bisphosphate bypass active within the pentose phosphate pathway.

Fig. 2. EMP is the primary glycolytic route in T. saccharolyticum.


Metabolic flux analysis and fluxomics-driven determination of reaction free energy using multiple isotopes

Xu J, Martien J, Gilbertson C, Ma J, Amador-Noguez D, Park JO.


Metabolite concentrations, fluxes, and free energies constitute the basis for understanding and controlling metabolism. Mass spectrometry and stable isotopes are integral tools in quantifying these metabolic features. For absolute metabolite concentration and flux measurement, 13C internal standards and tracers have been the gold standard. In contrast, no established methods exist for comprehensive thermodynamic quantitation under physiological environments. Recently, using high-resolution mass spectrometry and multi-isotope tracing, flux quantitation has been increasingly adopted in broader metabolism. The improved flux quantitation led to determination of Gibbs free energy of reaction (ΔG) in central carbon metabolism using a relationship between reaction reversibility and thermodynamic driving force. Here we highlight recent advances in multi-isotope tracing for metabolic flux and free energy analysis.

Fig. 1. Graphical Abstract


The nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p)ppGpp

Yang J, Anderson BW, Turdiev A, Turdiev H, Stevenson DM, Amador-Noguez D, Lee VT, Wang JD.


The alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly referred to as (p)ppGpp, regulate bacterial responses to nutritional and other stresses. There is evidence for potential existence of a third alarmone, guanosine-5′-monophosphate-3′-diphosphate (pGpp), with less-clear functions. Here, we demonstrate the presence of pGpp in bacterial cells, and perform a comprehensive screening to identify proteins that interact respectively with pGpp, ppGpp and pppGpp in Bacillus species. Both ppGpp and pppGpp interact with proteins involved in inhibition of purine nucleotide biosynthesis and with GTPases that control ribosome assembly or activity. By contrast, pGpp interacts with purine biosynthesis proteins but not with the GTPases. In addition, we show that hydrolase NahA (also known as YvcI) efficiently produces pGpp by hydrolyzing (p)ppGpp, thus modulating alarmone composition and function. Deletion of nahA leads to reduction of pGpp levels, increased (p)ppGpp levels, slower growth recovery from nutrient downshift, and loss of competitive fitness. Our results support the existence and physiological relevance of pGpp as a third alarmone, with functions that can be distinct from those of (p)ppGpp.

Fig. 2. NahA (YvcI) produces pGpp via (p)ppGpp hydrolysis.

2 H and 13 C metabolic flux analysis elucidates in vivo thermodynamics of the ED pathway in Zymomonas mobilis

Jacobson TB, Adamczyk PA, Stevenson DM, Regner M, Ralph J, Reed JL, Amador-Noguez D.


Zymomonas mobilis is an industrially relevant bacterium notable for its ability to rapidly ferment simple sugars to ethanol using the Entner-Doudoroff (ED) glycolytic pathway, an alternative to the well-known Embden-Meyerhof-Parnas (EMP) pathway used by most organisms. Recent computational studies have predicted that the ED pathway is substantially more thermodynamically favorable than the EMP pathway, a potential factor explaining the high glycolytic rate in Z. mobilis. Here, to investigate the in vivo thermodynamics of the ED pathway and central carbon metabolism in Z. mobilis, we implemented a network-level approach that integrates quantitative metabolomics with 2H and 13C metabolic flux analysis to estimate reversibility and Gibbs free energy (ΔG) of metabolic reactions. This analysis revealed a strongly thermodynamically favorable ED pathway in Z. mobilis that is nearly twice as favorable as the EMP pathway in E. coli or S. cerevisiae. The in vivo step-by-step thermodynamic profile of the ED pathway was highly similar to previous in silico predictions, indicating that maximizing ΔG for each pathway step likely constitutes a cellular objective in Z. mobilis. Our analysis also revealed novel features of Z. mobilis metabolism, including phosphofructokinase-like enzyme activity, tricarboxylic acid cycle anaplerosis via PEP carboxylase, and a metabolic imbalance in the pentose phosphate pathway resulting in excretion of shikimate pathway intermediates. The integrated approach we present here for in vivo ΔG quantitation may be applied to the thermodynamic profiling of pathways and metabolic networks in other microorganisms and will contribute to the development of quantitative models of metabolism.

Fig. 1. Entner-Doudoroff (ED) and Embden-Meyerhof-Parnas (EMP) glycolytic pathways.


Systems-Level Analysis of Oxygen Exposure in Zymomonas mobilis: Implications for Isoprenoid Production

Martien JI, Hebert AS, Stevenson DM, Regner MR, Khana DB, Coon JJ, Amador-Noguez D.


Zymomonas mobilis is an aerotolerant anaerobe and prolific ethanologen with attractive characteristics for industrial bioproduct generation. However, there is currently insufficient knowledge of the impact that environmental factors have on flux through industrially relevant biosynthetic pathways. Here, we examined the effect of oxygen exposure on metabolism and gene expression in Z. mobilis by combining targeted metabolomics, mRNA sequencing, and shotgun proteomics. We found that exposure to oxygen profoundly influenced metabolism, inducing both transient metabolic bottlenecks and long-term metabolic remodeling. In particular, oxygen induced a severe but temporary metabolic bottleneck in the methyl erythritol 4-phosphate pathway for isoprenoid biosynthesis caused by oxidative damage to the iron-sulfur cofactors of the final two enzymes in the pathway. This bottleneck was resolved with minimal changes in expression of isoprenoid biosynthetic enzymes. Instead, it was associated with pronounced upregulation of enzymes related to iron-sulfur cluster maintenance and biogenesis (i.e., flavodoxin reductase and the suf operon). We also detected major changes in glucose utilization in the presence of oxygen. Specifically, we observed increased gluconate production following exposure to oxygen, accounting for 18% of glucose uptake. Our results suggest that under aerobic conditions, electrons derived from the oxidation of glucose to gluconate are diverted to the electron transport chain, where they can minimize oxidative damage by reducing reactive oxygen species such as H2O2. This model is supported by the simultaneous upregulation of three membrane-bound dehydrogenases, cytochrome c peroxidase, and a cytochrome bd oxidase following exposure to oxygen.

Fig. 3. Intermediates of the MEP pathway during exposure to oxygen.

OptSSeq explores enzyme expression and function landscapes to maximize isobutanol production rate

Ghosh IN, Martien J, Hebert AS, Zhang Y, Coon JJ, Amador-Noguez D, Landick R.


Efficient microbial production of the next-generation biofuel isobutanol (IBA) is limited by metabolic bottlenecks. Overcoming these bottlenecks will be aided by knowing the optimal ratio of enzymes for efficient flux through the IBA biosynthetic pathway. OptSSeq (Optimization by Selection and Sequencing) accomplishes this goal by tracking growth rate-linked selection of optimal expression elements from a combinatorial library. The 5-step pathway to IBA consists of Acetolactate synthase (AlsS), Keto-acid reductoisomerase (KARI), Di-hydroxy acid dehydratase (DHAD), Ketoisovalerate decarboxylase (Kivd) and Alcohol dehydrogenase (Adh). Using OptSSeq, we identified gene expression elements leading to optimal enzyme levels that enabled theoretically maximal productivities per cell biomass in Escherichia coli. We identified KARI as the rate-limiting step, requiring the highest levels of enzymes expression, followed by AlsS and AdhA. DHAD and Kivd required relatively lower levels of expression for optimal IBA production. OptSSeq also enabled the identification of an Adh enzyme variant capable of an improved rate of IBA production. Using models that predict impacts of enzyme synthesis costs on cellular growth rates, we found that optimum levels of pathway enzymes led to maximal IBA production, and that additional limitations lie in the E. coli metabolic network. Our optimized constructs enabled the production of ~3 g IBA per hour per gram dry cell weight and was achieved with 20 % of the total cell protein devoted to IBA-pathway enzymes in the molar ratio 2.5:6.7:2:1:5.2 (AlsS:IlvC:IlvD:Kivd:AdhA). These enzyme levels and ratios optimal for IBA production in E. coli provide a useful starting point for optimizing production of IBA in diverse microbes and fermentation conditions.

Fig. 1. Strategy for optimizing expression levels in IBA pathway.


Fecal Aliquot Straw Technique (FAST) allows for easy and reproducible subsampling: assessing interpersonal variation in trimethylamine-N-oxide (TMAO) accumulation

Romano KA, Dill-McFarland KA, Kasahara K, Kerby RL, Vivas EI, Amador-Noguez D, Herd P, Rey FE.


Convenient, reproducible, and rapid preservation of unique biological specimens is pivotal to their use in microbiome analyses. As an increasing number of human studies incorporate the gut microbiome in their design, there is a high demand for streamlined sample collection and storage methods that are amenable to different settings and experimental needs. While several commercial kits address collection/shipping needs for sequence-based studies, these methods do not preserve samples properly for studies that require viable microbes. We describe the Fecal Aliquot Straw Technique (FAST) of fecal sample processing for storage and subsampling. This method uses a straw to collect fecal material from samples recently voided or preserved at low temperature but not frozen (i.e., 4 °C). Different straw aliquots collected from the same sample yielded highly reproducible communities as disclosed by 16S rRNA gene sequencing; operational taxonomic units that were lost, or gained, between the two aliquots represented very low-abundance taxa (i.e., < 0.3% of the community). FAST-processed samples inoculated into germ-free animals resulted in gut communities that retained on average ~ 80% of the donor’s bacterial community. Assessment of choline metabolism and trimethylamine-N-oxide accumulation in transplanted mice suggests large interpersonal variation. Overall, FAST allows for repetitive subsampling without thawing of the specimens and requires minimal supplies and storage space, making it convenient to utilize both in the lab and in the field. FAST has the potential to advance microbiome research through easy, reproducible sample processing.

                                                                                                                                                                 Fig. 2. FAST subsamples capture reproducible communities.