Filter by type:

A thermochemical–biochemical hybrid processing of lignocellulosic biomass for producing fuels and chemicals

Journal Article
Yanwen Shen, Laura Jarboe, Robert Brown, Zhiyou Wen
Biotechnology Advances, Volume 33, Issue 8, Pages 1799-1813
Publication year: 2015

Abstract

Thermochemical–biological hybrid processing uses thermochemical decomposition of lignocellulosic biomass to produce a variety of intermediate compounds that can be converted into fuels and chemicals through microbial fermentation. It represents a unique opportunity for biomass conversion as it mitigates some of the deficiencies of conventional biochemical (pretreatment–hydrolysis–fermentation) and thermochemical (pyrolysis or gasification) processing. Thermochemical–biological hybrid processing includes two pathways: (i) pyrolysis/pyrolytic substrate fermentation, and (ii) gasification/syngas fermentation. This paper provides a comprehensive review of these two hybrid processing pathways, including the characteristics of fermentative substrates produced in the thermochemical stage and microbial utilization of these compounds in the fermentation stage. The current challenges of these two biomass conversion pathways include toxicity of the crude pyrolytic substrates, the inhibition of raw syngas contaminants, and the mass-transfer limitations in syngas fermentation. Possible approaches for mitigating substrate toxicities are discussed. The review also provides a summary of the current efforts to commercialize hybrid processing.

Transcriptomic Analysis of Carboxylic Acid Challenge in Escherichia coli: Beyond Membrane Damage

Journal Article
Liam A. Royce, Erin Boggess, Yao Fu, Ping Liu, Jacqueline V. Shanks, Julie Dickerson, Laura R. Jarboe
PLoS ONE (9)2: e89580
Publication year: 2014

Abstract

Carboxylic acids are an attractive biorenewable chemical. Enormous progress has been made in engineering microbes for production of these compounds though titers remain lower than desired. Here we used transcriptome analysis of Escherichia coli during exogenous challenge with octanoic acid (C8) at pH 7.0 to probe mechanisms of toxicity. This analysis highlights the intracellular acidification and membrane damage caused by C8 challenge. Network component analysis identified transcription factors with altered activity including GadE, the activator of the glutamate-dependent acid resistance system (AR2) and Lrp, the amino acid biosynthesis regulator. The intracellular acidification was quantified during exogenous challenge, but was not observed in a carboxylic acid producing strain, though this may be due to lower titers than those used in our exogenous challenge studies. We developed a framework for predicting the proton motive force during adaptation to strong inorganic acids and carboxylic acids. This model predicts that inorganic acid challenge is mitigated by cation accumulation, but that carboxylic acid challenge inverts the proton motive force and requires anion accumulation. Utilization of native acid resistance systems was not useful in terms of supporting growth or alleviating intracellular acidification. AR2 was found to be non-functional, possibly due to membrane damage. We proposed that interaction of Lrp and C8 resulted in repression of amino acid biosynthesis. However, this hypothesis was not supported by perturbation of lrp expression or amino acid supplementation. E. coli strains were also engineered for altered cyclopropane fatty acid content in the membrane, which had a dramatic effect on membrane properties, though C8 tolerance was not increased. We conclude that achieving higher production titers requires circumventing the membrane damage. As higher titers are achieved, acidification may become problematic.

Technoeconomic evaluation of bio-based styrene production by engineered Escherichia coli

Journal Article
Joshua T. Claypool, D. Raj Raman, Laura R. Jarboe, David R. Nielsen
Journal of Industrial Microbiology & Biotechnology
Publication year: 2014

Abstract

Styrene is an important commodity chemical used in polymers and resins, and is typically produced from the petrochemical feedstocks benzene and ethylene. Styrene has recently been produced biosynthetically for the first time using engineered Escherichia coli, and this bio-based route may represent a lower energy and renewable alternative to petroleum-derived styrene. However, the economics of such an approach has not yet been investigated. Using an early-stage technoeconomic evaluation tool, a preliminary economic analysis of bio-based styrene from C6-sugar feedstock has been conducted. Owing to styrene’s limited water solubility, it was assumed that the resulting fermentation broth would spontaneously form two immiscible liquid phases that could subsequently be decanted. Assuming current C6 sugar prices and industrially achievable biokinetic parameter values (e.g., product yield, specific growth rate), commercial-scale bio-based styrene has a minimum estimated selling price (MESP) of 1.90 USD kg−1 which is in the range of current styrene prices. A Monte Carlo analysis revealed a potentially large (0.45 USD kg−1) standard deviation in the MESP, while a sensitivity analysis showed feedstock price and overall yield as primary drivers of MESP.

Flow cytometry is a promising and rapid method for differentiating between freely suspended Escherichia coli and E. coli attached to clay particles

Journal Article
X. Liang, M.L. Soupir, S. Rigby, L.R. Jarboe, W. Zhang
Journal of Applied Microbiology, Volume 117, Issue 6, Pages 1730-1739
Publication year: 2014

Abstract

Flow cytometry is a rapid and culture‐independent method for differentiating between attached and unattached micro‐organisms.

A standard procedure does not exist to distinguish between attached and unattached micro‐organisms. In this study, we compared two methods to quantify between Escherichia coli attached to clay particles and E. coli freely suspended in solution: flow cytometry (attachment assay and viability assay) and settling (or centrifugation followed by settling).

Methods were tested using three environmental strains collected from swine facilities (A, B and C) and one purchased modified pathogenic strain (ATCC 43888); four clay particles: Hectorite, Kaolinite, Ca‐Montmorillonite, Montmorillonite K‐10; and a range of surface area ratios (particle surface area to E. coli surface area). When comparing the two methods, the per cent attached obtained from the flow cytometry was lower, but not significantly different from the per cent attached obtained from the settling method for all conditions except when the particle was Hectorite or Montmorillonite K‐10; when the strain was C; and when the surface area ratio was below 100. Differences between the methods are likely because traditional culture‐based methods cannot detect the viable but nonculturable (VBNC) population, whereas flow cytometry can detect the fraction of VBNC with intact membranes.

The damaging effects of short chain fatty acids on Escherichia coli membranes

Journal Article
Liam A. Royce, Ping Liu, Matthew J. Stebbins, Benjamin C. Hanson, Laura R. Jarboe
Applied Microbiology and Biotechnology, Volume 97, Issue 18, Pages 8317-8327
Publication year: 2013

Abstract

Carboxylic acids are an attractive biorenewable chemical. However, like many other fermentatively produced compounds, they are inhibitory to the biocatalyst. An understanding of the mechanism of toxicity can aid in mitigating this problem. Here, we show that hexanoic and octanoic acids are completely inhibitory to Escherichia coli MG1655 in minimal medium at a concentration of 40 mM, while decanoic acid was inhibitory at 20 mM. This growth inhibition is pH-dependent and is accompanied by a significant change in the fluorescence polarization (fluidity) and integrity. This inhibition and sensitivity to membrane fluidization, but not to damage of membrane integrity, can be at least partially mitigated during short-term adaptation to octanoic acid. This short-term adaptation was accompanied by a change in membrane lipid composition and a decrease in cell surface hydrophobicity. Specifically, the saturated/unsaturated lipid ratio decreased and the average lipid length increased. A fatty acid-producing strain exhibited an increase in membrane leakage as the product titer increased, but no change in membrane fluidity. These results highlight the importance of the cell membrane as a target for future metabolic engineering efforts for enabling resistance and tolerance of desirable biorenewable compounds, such as carboxylic acids. Knowledge of these effects can help in the engineering of robust biocatalysts for biorenewable chemicals production.

Membrane stress caused by octanoic acid in Saccharomyces cerevisiae

Journal Article
Ping Liu, Andriy Chernyshov, Tarek Najdi, Yao Fu, Julie Dickerson, Suzanne Sandmeyer, Laura Jarboe
Applied Microbiology and Biotechnology, Volume 97, Issue 7, Pages 3239-3251
Publication year: 2013

Abstract

In order to compete with petroleum-based fuel and chemicals, engineering a robust biocatalyst that can convert renewable feedstocks into biorenewable chemicals, such as carboxylic acids, is increasingly important. However, product toxicity is often problematic. In this study, the toxicity of the carboxylic acids hexanoic, octanoic, and decanoic acid on Saccharomyces cerevisiae was investigated, with a focus on octanoic acid. These compounds are completely inhibitory at concentrations of magnitude 1 mM, and the toxicity increases as chain length increases and as media pH decreases. Transciptome analysis, reconstruction of gene regulatory network, and network component analysis suggested decreased membrane integrity during challenge with octanoic acid. This was confirmed by quantification of dose-dependent and chain length-dependent induction of membrane leakage, though membrane fluidity was not affected. This induction of membrane leakage could be significantly decreased by a period of pre-adaptation, and this pre-adaptation was accompanied by increased oleic acid content in the membrane, significantly increased production of saturated lipids relative to unsaturated lipids, and a significant increase in the average lipid chain length in the membrane. However, during adaptation cell surface hydrophobicity was not altered. The supplementation of oleic acid to the medium not only elevated the tolerance of yeast cells to octanoic acid but also attenuated the membrane leakiness. However, while attempts to mimic the oleic acid supplementation effects through expression of the Trichoplusia ni acyl-CoA Δ9 desaturase OLE1(TniNPVE desaturase) were able to increase the oleic acid content, the magnitude of the increase was not sufficient to reproduce the supplementation effect and increase octanoic acid tolerance. Similarly, introduction of cyclopropanated fatty acids through expression of the Escherichia coli cfa gene was not helpful for tolerance. Thus, we have provided quantitative evidence that carboxylic acids damage the yeast membrane and that manipulation of the lipid content of the membrane can increase tolerance, and possibly production, of these valuable products.

Identification of Mutations in Evolved Bacterial Genomes

Book Chapter
Liam Royce, Erin Boggess, Tao Jin, Julie Dickerson, Laura Jarboe
Systems Metabolic Engineering, Pages 249-267
Publication year: 2013

Abstract

Directed laboratory evolution is a common technique to obtain an evolved bacteria strain with a desired phenotype. This technique is especially useful as a supplement to rational engineering for complex phenotypes such as increased biocatalyst tolerance to toxic compounds. However, reverse engineering efforts are required in order to identify the mutations that occurred, including single nucleotide polymorphisms (SNPs), insertions/deletions (indels), duplications, and rearrangements. In this protocol, we describe the steps to (1) obtain and sequence the genomic DNA, (2) process and analyze the genomic DNA sequence data, and (3) verify the mutations by Sanger resequencing.

Metabolic Engineering of biocatalysts for carboxylic acids production

Journal Article
Ping Liu, Laura R. Jarboe
Computational and Structural Biotechnology Journal, Volume 3, Issue 4
Publication year: 2012

Abstract

Fermentation of renewable feedstocks by microbes to produce sustainable fuels and chemicals has the potential to replace petrochemical-based production. For example, carboxylic acids produced by microbial fermentation can be used to generate primary building blocks of industrial chemicals by either enzymatic or chemical catalysis. In order to achieve the titer, yield and productivity values required for economically viable processes, the carboxylic acid-producing microbes need to be robust and well-performing. Traditional strain development methods based on mutagenesis have proven useful in the selection of desirable microbial behavior, such as robustness and carboxylic acid production. On the other hand, rationally-based metabolic engineering, like genetic manipulation for pathway design, has becoming increasingly important to this field and has been used for the production of several organic acids, such as succinic acidmalic acid and lactic acid. This review investigates recent works on Saccharomyces cerevisiae and Escherichia coli, as well as the strategies to improve tolerance towards these chemicals.

Growth condition optimization for docosahexaenoic acid (DHA) production by Moritella marina MP-1

Journal Article
Kumar B. Kautharapu, John Rathmacher, Laura R. Jarboe
Applied Microbiology and Biotechnology, Volume 97, Issue 7, Pages 2859-2866
Publication year: 2012

Abstract

The marine organism Moritella marina MP-1 produces the polyunsaturated fatty acid docosahexaenoic acid (DHA). While the basic metabolic pathway for DHA production in this organism has been identified, the impact of growth conditions on DHA production is largely unknown. This study examines the effect of supplemental carbon, nitrogen and salts, growth temperature and media composition and pH on DHA and biomass production and the fatty acid profile. The addition of supplemental nitrogen significantly increased the overall DHA titer via an increase in biomass production. Supplemental glucose or glycerol increased biomass production, but decreased the amount of DHA per biomass, resulting in no net change in the DHA titer. Acidification of the baseline media pH to 6.0 increased DHA per biomass. Changes in growth temperature or provision of supplemental sodium or magnesium chloride did not increase DHA titer. This organism was also shown to grow on defined minimal media. For both media types, glycerol enabled more DHA production per biomass than glucose. Combination of these growth findings into marine broth supplemented with glycerol, yeast extract, and tryptone at pH 6.0 resulted in a final titer of 82 ± 5 mg/L, a nearly eightfold increase relative to the titer of 11 ± 1 mg/L seen in the unsupplemented marine broth. The relative distribution of other fatty acids was relatively robust to growth condition, but the presence of glycerol resulted in a significant increase in myristic acid (C14:0) and decrease in palmitic acid (C16:0). In summary, DHA production by M. marina MP-1 can be increased more than fivefold by changing the growth media. Metabolic engineering of this organism to increase the amount of DHA produced per biomass could result in additional increases in titer.

Hybrid thermochemical processing: fermentation of pyrolysis-derived bio-oil

Journal Article
Laura R. Jarboe, Zhiyou Wen, DongWon Choi, Robert C. Brown
Applied Microbiology and Biotechnology
Publication year: 2011

Abstract

Thermochemical processing of biomass by fast pyrolysis provides a nonenzymatic route for depolymerization of biomass into sugars that can be used for the biological production of fuels and chemicals. Fermentative utilization of this bio-oil faces two formidable challenges. First is the fact that most bio-oil-associated sugars are present in the anhydrous form. Metabolic engineering has enabled utilization of the main anhydrosugar, levoglucosan, in workhorse biocatalysts. The second challenge is the fact that bio-oil is rich in microbial inhibitors. Collection of bio-oil in distinct fractions, detoxification of bio-oil prior to fermentation, and increased robustness of the biocatalyst have all proven effective methods for addressing this inhibition.

Engineering inhibitor tolerance for the production of biorenewable fuels and chemicals

Journal Article
Laura R Jarboe Ping Liu, Liam A Royce
Current Opinion in Chemical Engineering, Volume 1, Issue 1, Pages 38-42
Publication year: 2011

Abstract

Inhibition of bacterial metabolism hinders production of biorenewable compounds. Transcriptome analysis can be used to identify the mechanism of bacterial inhibition. Randomly selected tolerant strains can be reverse engineered to find key mutations. When the mechanism of inhibition is known it can be rationally alleviated. Efflux pumps and alteration of the cell membrane are increasingly important.

Metabolic Engineering has enabled the production of biorenewable fuels and chemicals from biomass using recombinant bacteria. The economic viability of these processes is often limited by inhibition of the biocatalyst by the metabolic product, such as a carboxylic acid or alcohol, or by contaminant compounds in the biomass-derived sugars, such as acetic acid or furans. Historically, selection-based methods have been used to improve biocatalyst tolerance to these inhibitors. But recently, genome-wide analysis has been used to both identify the mechanism of inhibition and reverse engineer inhibitor-tolerant strains, enabling the rational, predictive manipulation of bacteria in order to increase inhibitor tolerance. Here we review recent work in this area, particularly in relation to carboxylic acids, furfural and butanol.

Engineering ethanologenic Escherichia coli for levoglucosan utilization

Journal Article
Donovan S. Layton, Avanthi Ajjarapu, DongWon Choi, Laura R.Jarboe
Bioresource Technology, Volume 102, Issue 17, Pages 8318-8322
Publication year: 2011

Abstract

Levoglucosan is a major product of biomass pyrolysis. While this pyrolyzed biomass, also known as bio-oil, contains sugars that are an attractive fermentation substrate, commonly-used biocatalysts, such as Escherichia coli, lack the ability to metabolize this anhydrosugar. It has previously been shown that recombinant expression of the levoglucosan kinase enzyme enables use of levoglucosan as carbon and energy source. Here, ethanologenic E. coli KO11 was engineered for levoglucosan utilization by recombinant expression of levoglucosan kinase from Lipomyces starkeyi. Our engineering strategy uses a codon-optimized gene that has been chromosomally integrated within the pyruvate to ethanol (PET) operon and does not require additional antibiotics or inducers. Not only does this engineered strain use levoglucosan as sole carbon source, but it also ferments levoglucosan to ethanol. This work demonstrates that existing biocatalysts can be easily modified for levoglucosan utilization.

Association of Antibiotic Resistance in Agricultural Escherichia coli Isolates with Attachment to Quartz

Journal Article
Ping Liu, Michelle L. Soupir, Martha Zwonitzer, Bridgette Huss and Laura R. Jarboe
Applied and Environmental Microbiology, Volume 77, Issue 19, Pages 6945-6953
Publication year: 2011

Abstract

Surface water can be contaminated by bacteria from various sources, including manure from agricultural facilities. Attachment of these bacteria to soil and organic particles contributes to their transport through the environment, though the mechanism of attachment is unknown. As bacterial attachment to human tissues is known to be correlated with antibiotic resistance, we have investigated here the relationship between bacterial attachment to environmental particles and antibiotic resistance in agricultural isolates. We evaluated 203 Escherichia coli isolates collected from swine facilities for attachment to quartz, resistance to 13 antibiotics, and the presence of genes encoding 13 attachment factors. The genes encoding type I, EcpA, P pili, and Ag43 were detected, though none was significantly related to attachment. Quartz attachment was positively and significantly (P < 0.0038) related to combined resistance to amoxicillin/streptomycin/tetracycline/sulfamethazine/tylosin/chlortetracycline and negatively and significantly (P < 0.0038) related to combined resistance to nalidixic acid/kanamycin/neomycin. These results provide clear evidence for a link between antibiotic resistance and attachment to quartz in agricultural isolates. We propose that this may be due to encoding by the responsible genes on a mobile genetic element. Further exploration of the relationship between antibiotic resistance and attachment to environmental particles will improve the understanding and modeling of environmental transport processes, with the goal of preventing human exposure to antibiotic-resistant or virulent microorganisms.

YqhD: a broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals

Journal Article
Laura R. Jarboe
Applied Microbiology and Biotechnology, Volume 89, Issue 2, Pages 249-257
Publication year: 2010

Abstract

The Escherichia coli NADPH-dependent aldehyde reductase YqhD has contributed to a variety of metabolic engineering projects for production of biorenewable fuels and chemicals. As a scavenger of toxic aldehydes produced by lipid peroxidation, YqhD has reductase activity for a broad range of short-chain aldehydes, including butyraldehyde, glyceraldehyde, malondialdehyde, isobutyraldehyde, methylglyoxal, propanealdehyde, acrolein, furfural, glyoxal, 3-hydroxypropionaldehyde, glycolaldehyde, acetaldehyde, and acetol. This reductase activity has proven useful for the production of biorenewable fuels and chemicals, such as isobutanol and 1,3- and 1,2-propanediol; additional capability exists for production of 1-butanol, 1-propanol, and allyl alcohol. A drawback of this reductase activity is the diversion of valuable NADPH away from biosynthesis. This YqhD-mediated NADPH depletion provides sufficient burden to contribute to growth inhibition by furfural and 5-hydroxymethyl furfural, inhibitory contaminants of biomass hydrolysate. The structure of YqhD has been characterized, with identification of a Zn atom in the active site. Directed engineering efforts have improved utilization of 3-hydroxypropionaldehyde and NADPH. Most recently, two independent projects have demonstrated regulation of yqhD by YqhC, where YqhC appears to function as an aldehyde sensor.

YqhC regulates transcription of the adjacent Escherichia coli genes yqhD and dkgA that are involved in furfural tolerance

Journal Article
Peter C. Turner, Elliot N. Miller, Laura R. Jarboe, Christy L. Baggett, K. T. Shanmugam, Lonnie O. Ingram
Journal of Industrial Microbiology & Biotechnology, Volume 38, Issue 3, Pages 431-439
Publication year: 2010

Abstract

Previous results have demonstrated that the silencing of adjacent genes encoding NADPH-dependent furfural oxidoreductases (yqhD dkgA) is responsible for increased furfural tolerance in an E. coli strain EMFR9 [Miller et al., Appl Environ Microbiol 75:4315–4323, 2009]. This gene silencing is now reported to result from the spontaneous insertion of an IS10 into the coding region of yqhC, an upstream gene. YqhC shares homology with transcriptional regulators belonging to the AraC/XylS family and was shown to act as a positive regulator of the adjacent operon encoding YqhD and DkgA. Regulation was demonstrated by constructing a chromosomal deletion of yqhC, a firefly luciferase reporter plasmid for yqhC, and by a direct comparison of furfural resistance and NADPH-dependent furfural reductase activity. Closely related bacteria contain yqhCyqhD, and dkgA orthologs in the same arrangement as in E. coli LY180. Orthologs of yqhC are also present in more distantly related Gram-negative bacteria. Disruption of yqhCoffers a useful approach to increase furfural tolerance in bacteria.

Metabolic Engineering for Production of Biorenewable Fuels and Chemicals: Contributions of Synthetic Biology

Journal Article
Laura R. Jarboe, Xueli Zhang, Xuan Wang, Jonathan C. Moore, K. T. Shanmugam, and Lonnie O. Ingram
Journal of Biomedicine and Biotechnology, Volume 2010, Article ID 761042, 18 Pages
Publication year: 2010

Abstract

Production of fuels and chemicals through microbial fermentation of plant material is a desirable alternative to petrochemical-based production. Fermentative production of biorenewable fuels and chemicals requires the engineering of biocatalysts that can quickly and efficiently convert sugars to target products at a cost that is competitive with existing petrochemical-based processes. It is also important that biocatalysts be robust to extreme fermentation conditions, biomass-derived inhibitors, and their target products. Traditional metabolic engineering has made great advances in this area, but synthetic biology has contributed and will continue to contribute to this field, particularly with next-generation biofuels. This work reviews the use of metabolic engineering and synthetic biology in biocatalyst engineering for biorenewable fuels and chemicals production, such as ethanol, butanol, acetate, lactate, succinate, alanine, and xylitol. We also examine the existing challenges in this area and discuss strategies for improving biocatalyst tolerance to chemical inhibitors.

Genetic changes that increase 5-hydroxymethyl furfural resistance in ethanol-producing Escherichia coli LY180

Journal Article
E. N. Miller, P. C. Turner, L. R. Jarboe, L. O. Ingram
Biotechnology Letters, Volume 32, Issue 5, Pages 661-667
Publication year: 2010

Abstract

The ability of a biocatalyst to tolerate furan inhibitors present in hemicellulose hydrolysates is important for the production of renewable chemicals. This study shows EMFR9, a furfural-tolerant mutant of ethanologenic E. coli LY180, has also acquired tolerance to 5-hydroxymethyl furfural (5-HMF). The mechanism of action of 5-HMF and furfural appear similar. Furan tolerance results primarily from lower expression of yqhD and dkgA, two furan reductases with a low Km for NADPH. Furan tolerance was also increased by adding plasmids encoding a NADPH/NADH transhydrogenase (pntAB). Together, these results support the hypothesis that the NADPH-dependent reduction of furans by YqhD and DkgA inhibits growth by competing with biosynthesis for this limiting cofactor.

Systems Approaches to Unraveling Nitric Oxide Response Networks in Prokaryotes

Book Chapter
Jarboe L. R., D. R. Hyduke, J. C. Liao
Nitric Oxide (Second Edition), Biology and Pathobiology 2010, Pages 103-136
Publication year: 2009

Abstract

This chapter describes various components of reactive nitrogen species (RNS) response networks and their associated regulatory circuitry. It focuses on how these components are used to identify additional regulatory components and elucidate RNS response networks as a function of RNS source and growth conditions. It summarizes the complementary nature of traditional biochemical analysis and systems-wide analysis in identification and characterization of the bacterial response to RNS. Bacteria have evolved complex regulatory networks for dealing with a variety of chemical stressors. The bacterial response to RNS is relevant to both pathogenesis and denitrification and thus has been extensively characterized. Traditional biochemical analysis has identified and characterized many of the key RNS response elements, such as flavohemoglobin hmpA and response regulator NorR. Additionally, systems-wide analysis aids in the identification of additional network components and determination of their contribution to the overall network behavior. These systems-wide analyses have led to the identification of response regulator NsrR, Fe-S cluster repair agent YtfE, and the critical NO target. The extensive body of knowledge regarding the bacterial RNS response and RNS chemistry makes the RNS response network an excellent example of the combined power of traditional biochemical analysis and systems-wide analysis.

Silencing of NADPH-Dependent Oxidoreductase Genes (yqhD and dkgA) in Furfural-Resistant Ethanologenic Escherichia coli

Journal Article
E. N. Miller, L. R. Jarboe, L. P. Yomano, S. W. York, K. T. Shanmugam and L. O. Ingram
Applied and Environmental Microbiology, Volume 75, Issue 13, Pages 4315-4323
Publication year: 2009

Abstract

Low concentrations of furfural are formed as a side product during the dilute acid hydrolysis of hemicellulose. Growth is inhibited by exposure to furfural but resumes after the complete reduction of furfural to the less toxic furfuryl alcohol. Growth-based selection was used to isolate a furfural-resistant mutant of ethanologenic Escherichia coli LY180, designated strain EMFR9. Based on mRNA expression levels in the parent and mutant in response to furfural challenge, genes encoding 12 oxidoreductases were found to vary by more than twofold (eight were higher in EMFR9; four were higher in the parent). All 12 genes were cloned. When expressed from plasmids, none of the eight genes in the first group increased furfural tolerance in the parent (LY180). Expression of three of the silenced genes (yqhDdkgA, and yqfA) in EMFR9 was found to decrease furfural tolerance compared to that in the parent. Purified enzymes encoded by yqhD and dkgA were shown to have NADPH-dependent furfural reductase activity. Both exhibited low Km values for NADPH (8 μM and 23 μM, respectively), similar to those of biosynthetic reactions. Furfural reductase activity was not associated with yqfA. Deleting yqhD and dkgA in the parent (LY180) increased furfural tolerance, but not to the same extent observed in the mutant EMFR9. Together, these results suggest that the process of reducing furfural by using an enzyme with a low Km for NADPH rather than a direct inhibitory action is the primary cause for growth inhibition by low concentrations of furfural.

Furfural Inhibits Growth by Limiting Sulfur Assimilation in Ethanologenic Escherichia coli Strain LY180

Journal Article
Elliot N. Miller, Laura R. Jarboe, Peter C. Turner, Priti Pharkya, Lorraine P. Yomano, Sean W. York, David Nunn, K. T. Shanmugam and Lonnie O. Ingram
Applied and Environmental Microbiology, Volume 75, Issue 19, Pages 6132-6141
Publication year: 2009

Abstract

A wide variety of commercial products can be potentially made from monomeric sugars produced by the dilute acid hydrolysis of lignocellulosic biomass. However, this process is accompanied by side products such as furfural that hinder microbial growth and fermentation. To investigate the mechanism of furfural inhibition, mRNA microarrays of an ethanologenic strain of Escherichia coli (LY180) were compared immediately prior to and 15 min after a moderate furfural challenge. Expression of genes and regulators associated with the biosynthesis of cysteine and methionine was increased by furfural, consistent with a limitation of these critical metabolites. This was in contrast to a general stringent response and decreased expression of many other biosynthetic genes. Of the 20 amino acids individually tested as supplements (100 μM each), cysteine and methionine were the most effective in increasing furfural tolerance with serine (precursor of cysteine), histidine, and arginine of lesser benefit. Supplementation with other reduced sulfur sources such as D-cysteine and thiosulfate also increased furfural tolerance. In contrast, supplementation with taurine, a sulfur source that requires 3 molecules of NADPH for sulfur assimilation, was of no benefit. Furfural tolerance was also increased by inserting a plasmid encoding pntAB, a cytoplasmic NADH/NADPH transhydrogenase. Based on these results, a model is proposed for the inhibition of growth in which the reduction of furfural by YqhD, an enzyme with a low Km for NADPH, depletes NADPH sufficiently to limit the assimilation of sulfur into amino acids (cysteine and methionine) by CysIJ (sulfite reductase).

The development of ethanologenic bacteria for fuel ethanol production

Book Chapter
Luli, G.W., L. R. Jarboe , L. O. Ingram
Bioenergy, Chapter 10, Pages 129-137
Publication year: 2008

Abstract

This chapter focuses on the development of two related bacteria that have proven to be effective in a variety of physical and chemical processes:  and . Development of recombinant microbes that utilize a variety of sugars for ethanol production in laboratory media under optimum growth conditions has been repeated by several laboratories around the world. The use of dilute acid at temperatures above 140ºC is effective for the hydrolysis of hemicellulose in bagasse without significant loss of sugars or the production of degraded by-products. Dilute acid hydrolysis of hemicellulose has been used in order to produce high concentrations of hemicellulose sugars for fermentation by  strain KO11-RD1. The yield of ethanol from acidic hydrolysis of cellulose is limited due to the poor recovery of glucose during the acid hydrolysis process. The degradation of glucose occurs very rapidly under conditions necessary for cellulose hydrolysis. Therefore, the use of cellulolytic enzymes has been pursued for several decades as a means of increasing the ethanol yield from cellulose. The simultaneous saccharification and fermentation (SSF) model has the following advantages over the sequential hydrolysis and fermentation process model: (i) lower enzyme dosages required for efficient conversion, (ii) compatibility with coproduction of enzymes during ethanol fermentation, and (iii) lower free-sugar concentrations during the SSF process.

Integrated network analysis identifies nitric oxide response networks and dihydroxyacid dehydratase as a crucial target in Escherichia coli

Journal Article
Daniel R. Hyduke, Laura R. Jarboe, Linh M. Tran, Katherine J. Y. Chou, and James C. Liao
PNAS, Volume 104, Issue 20, Pages 8484-8489
Publication year: 2007

Abstract

Nitric oxide (NO) is used by mammalian immune systems to counter microbial invasions and is produced by bacteria during denitrification. As a defense, microorganisms possess a complex network to cope with NO. Here we report a combined transcriptomic, chemical, and phenotypic approach to identify direct NO targets and construct the biochemical response network. In particular, network component analysis was used to identify transcription factors that are perturbed by NO. Such information was screened with potential NO reaction mechanisms and phenotypic data from genetic knockouts to identify active chemistry and direct NO targets in Escherichia coli. This approach identified the comprehensive E. coli NO response network and evinced that NO halts bacterial growth via inhibition of the branched-chain amino acid biosynthesis enzyme dihydroxyacid dehydratase. Because mammals do not synthesize branched-chain amino acids, inhibition of dihydroxyacid dehydratase may have served to foster the role of NO in the immune arsenal.

Ethanol Encyclopedia of Microbiology (3rd Ed)

Book Chapter
Jarboe, L. R., K. T. Shanmugam, L. O. Ingram
Ed. D. Majumder-Russell. Elsevier
Publication year: 2007

Abstract

N/A

Development of Ethanologenic Bacteria

Journal Article
L. R. Jarboe, T. B. Grabar, L. P. Yomano, K. T. Shanmugan, L. O. Ingram
Biofuels, Pages 327-261
Publication year: 2007

Abstract

The utilization of lignocellulosic biomass as a petroleum alternative faces many challenges. This work reviews recent progress in the engineering of Escherichia coli and Klebsiella oxytoca to produce ethanol from biomass with minimal nutritional supplementation. A combination of directed engineering and metabolic evolution has resulted in microbial biocatalysts that produce up to 45 g L−1 ethanol in 48 h in a simple mineral salts medium, and convert various lignocellulosic materials to ethanol. Mutations contributing to ethanologenesis are discussed. The ethanologenic biocatalyst design approach was applied to other commodity chemicals, including optically pure d(−)- and l(+)-lactic acid, succinate and l-alanine with similar success. This review also describes recent progress in growth medium development, the reduction of hemicellulose hydrolysate toxicity and reduction of the demand for fungal cellulases.

Transcriptional Responses of Escherichia coli to S-Nitrosoglutathione under Defined Chemostat Conditions Reveal Major Changes in Methionine Biosynthesis

Journal Article
Janet Flatley, Jason Barrett, Steven T. Pullan, Martin N. Hughes, Jeffrey Green and Robert K. Poole
Journal of Biological Chemistry
Publication year: 2005

Abstract

Nitric oxide and nitrosating agents exert powerful antimicrobial effects and are central to host defense and signal transduction. Nitric oxide and S-nitrosothiols can be metabolized by bacteria, but only a few enzymes have been shown to be important in responses to such stresses. Glycerol-limited chemostat cultures in defined medium of Escherichia coli MG1655 were used to provide bacteria in defined physiological states before applying nitrosative stress by addition of S-nitrosoglutathione (GSNO). Exposure to 200 μM GSNO for 5 min was sufficient to elicit an adaptive response as judged by the development of NO-insensitive respiration. Transcriptome profiling experiments were used to investigate the transcriptional basis of the observed adaptation to the presence of GSNO. In aerobic cultures, only 17 genes were significantly up-regulated, including genes known to be involved in NO tolerance, particularly hmp (encoding the NO-consuming flavohemoglobin Hmp) and norV (encoding flavorubredoxin). Significantly, none of the up-regulated genes were members of the Fur regulon. Six genes involved in methionine biosynthesis or regulation were significantly up-regulated; metNmetI, and metR were shown to be important for GSNO tolerance, because mutants in these genes exhibited GSNO growth sensitivity. Furthermore, exogenous methionine abrogated the toxicity of GSNO supporting the hypothesis that GSNO nitrosates homocysteine, thereby withdrawing this intermediate from the methionine biosynthetic pathway. Anaerobically, 10 genes showed significant up-regulation, of which norVhcpmetR, and metB were also up-regulated aerobically. The data presented here reveal new genes important for nitrosative stress tolerance and demonstrate that methionine biosynthesis is a casualty of nitrosative stress.

Stochastic Modeling of the Phase-Variable pap Operon Regulation in Uropathogenic Escherichia coli

Journal Article
Laura R. Jarboe, David Beckwith, James C. Liao
Biotechnology Bioengineering, Volume 88, Issue 2, Pages 189-203
Publication year: 2004

Abstract

Regulation of the pap operon in uropathogenic Escherichia coli is phase variable. This phase variation arises from competition between regulatory proteins at two sites within the regulatory region, GATCdist and GATCprox. We have used the available literature data to design a stochastic model of the molecular interactions of pap regulation and expression during growth in a non-glucose environment at 37jC. The resulting wild-type model is consistent with reported data. The wild-type model served as a basis for two ‘‘in silico’’ mutant models for investigating the role of key regulatory components, the GATCdist binding site and the PapI interaction with Lrp at the GATCprox site. Our results show that competition at GATCdist is required for phase variation, as previously reported. However, our results suggest that removal of competition at GATCdist does not affect initial state dependence. Additionally, the PapI involvement in Lrp translocation from GATCprox to GATCdist is required for the initial state dependence but not for phase variation. Our results also predict that pap expression is maximized at low growth rates and minimized at high growth rates. These predictions provide a basis for further experimental investigation.