Search

Efficient co‐utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose−xylose mixtures. This study focuses on enhancing phenylalanine production by engineering Escherichia coli to efficiently co‐utilize glucose and xylose. Flux balance analysis identified E4P flux as a bottleneck which could be alleviated by increasing the xylose‐to‐glucose flux ratio. A mutant copy of the xylose‐specific activator (XylR) was then introduced into the phenylalanine‐overproducing E. coli NST74, which relieved carbon catabolite repression and enabled efficient glucose−xylose co‐utilization. Carbon contribution analysis through 13C‐fingerprinting showed a higher preference for xylose in the engineered strain (NST74X), suggesting superior catabolism of xylose relative to glucose. As a result, NST74X produced 1.76 g/L phenylalanine from a model glucose−xylose mixture; a threefold increase over NST74. Then, using biomass‐derived sugars, NST74X produced 1.2 g/L phenylalanine, representing a 1.9‐fold increase over NST74. Notably, and consistent with the carbon contribution analysis, the xylR* mutation resulted in a fourfold greater maximum rate of xylose consumption without significantly impeding the maximum rate of total sugar consumption (0.87 vs. 0.70 g/L‐h). This study presents a novel strategy for enhancing phenylalanine production through the co‐utilization of glucose and xylose in aerobic E. coli cultures, and highlights the potential synergistic benefits associated with using substrate mixtures over single substrates when targeting specific products. [ABSTRACT FROM AUTHOR]

Phenol is an important petrochemical that is conventionally used as a precursor for synthesizing an array of plastics and fine chemicals. As an emerging alternative to its traditional petrochemical production, multiple enzyme pathways have been engineered to date to enable its renewable biosynthesis from biomass feedstocks, each incorporating unique enzyme chemistries and intermediate molecules. Leveraging all three of the unique phenol biosynthesis pathways reported to date, a series of synthetic 'metabolic funnels' was engineered, each with the goal of maximizing net precursor assimilation and flux towards phenol via the parallel co-expression of multiple distinct pathways within the same Escherichia coli host. By constructing and evaluating all possible binary and tertiary pathway combinations, one 'funnel' was ultimately identified, which supported enhanced phenol production relative to all three individual pathways by 16 to 69%. Further host engineering to increase endogenous precursor availability then allowed for 26% greater phenol production, reaching a final titer of 554 ± 19 mg/L and 28.8 ± 0.34 mg/g yield on glucose. Lastly, using a diphasic culture including dibutyl phthalate for in situ phenol extraction, final titers were further increased to a maximum of 812 ± 145 mg/L at a yield of 40.6 ± 7.2 mg/g. The demonstrated 'funneling' pathway holds similar promise in support of phenol production by other, non-E. coli hosts, while this general approach can be readily extended towards a diversity of other value-added bioproducts of interest. [ABSTRACT FROM AUTHOR]

The global transcriptional response of Escherichia coli to styrene and potential influence of exposure source was determined by performing RNA sequencing (RNA-seq) analysis on both styrene-producing and styrene-exposed cells. In both cases, styrene exposure appears to cause both cell envelope and DNA damage, to which cells respond by down-regulating key genes/pathways involved in DNA replication, protein production, and cell wall biogenesis. Among the most significantly up-regulated genes were those involved with phage shock protein response (e.g. pspABCDE/G), general stress regulators (e.g. marA, rpoH), and membrane-altering genes (notably, bhsA, ompR, ldtC), whereas efflux transporters were, surprisingly, unaffected. Subsequent studies with styrene addition demonstrate how strains lacking ompR [involved in controlling outer membrane (OM) composition/osmoregulation] or any of tolQ, tolA, or tolR (involved in OM constriction) each displayed over 40% reduced growth relative to wild-type. Conversely, despite reducing basal fitness, overexpression of plsX (involved in phospholipid biosynthesis) led to 70% greater growth when styrene exposed. These collective differences point to the likely importance of OM properties in controlling native styrene tolerance. Overall, the collective behaviours suggest that, regardless of source, prolonged exposure to inhibitory styrene levels causes cells to shift from'growth mode' to 'survival mode', redistributing cellular resources to fuel native tolerance mechanisms. [ABSTRACT FROM AUTHOR]

Aromatic compounds, which are traditionally derived from petroleum feedstocks, represent a diverse class of molecules with a wide range of industrial and commercial applications. Significant progress has been made to alternatively and sustainably produce many aromatics from renewable substrates using microbial biocatalysts. While the construction of both natural and non‐natural pathways has expanded the number and diversity of aromatic bioproducts, pathway modularization in both single‐ and multi‐strain systems continues to support the enhancement of key production metrics towards economically‐viable levels. Emerging tools for implementing more precise metabolic control (e.g. CRISPRi, sRNA) as well as the engineering of novel high‐throughput screening platforms utilizing in vivo aromatic biosensors, meanwhile, continue to facilitate further optimization of both pathways and hosts. While product toxicity persists as a key challenge limiting the production of many aromatics, various successful strategies have been demonstrated towards improving tolerance, including via membrane and efflux pump engineering as well as by exploiting alternative production hosts. Finally, as a further step towards sustainable and economical aromatic bioproduction, non‐model substrates including lignin‐derived compounds continue to emerge as viable feedstocks. This review highlights recent and notable achievements related to such efforts while offering future outlooks towards engineering microbial cell factories for aromatic production. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

ABSTRACT The non-renewable petrochemical phenol is used as a precursor to produce numerous fine and commodity chemicals, including various pharmaceuticals and phenolic resins. Microbial phenol biosynthesis has previously been established, stemming from endogenous tyrosine via tyrosine phenol lyase (TPL). TPL, however, suffers from feedback inhibition and equilibrium limitations, both of which contribute to reduced flux through the overall pathway. To address these limitations, two novel and non-natural phenol biosynthesis pathways, both stemming instead from chorismate, were constructed and comparatively evaluated. The first proceeds to phenol in one heterologous step via the intermediate p-hydroxybenzoic acid, while the second involves two heterologous steps and the associated intermediates isochorismate and salicylate. Maximum phenol titers achieved via these two alternative pathways reached as high as 377 ± 14 and 259 ± 31 mg/L in batch shake flask cultures, respectively. In contrast, under analogous conditions, phenol production via the established TPL-dependent route reached 377 ± 23 mg/L, which approaches the maximum achievable output reported to date under batch conditions. Additional strain development and optimization of relevant culture conditions with respect to each individual pathway is ultimately expected to result in further improved phenol production. Biotechnol. Bioeng. 2016;113: 1745-1754. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Ionicliquids (ILs) are ionic compounds that are liquid at roomtemperature. We studied the spontaneous mixing behavior between twoILs, ethylammonium nitrate (EAN) and 1-butyl-3-methylimidazolium hexafluorophosphate([BMIM][PF6]), and observed notable phenomena. Experimentalstudies showed that the interface between the two ILs was unusuallylong-lived, despite the ILs being miscible with one another. Moleculardynamics (MD) simulations supported these findings and provided insightinto the micromixing behavior of the ILs. We found that not only didthe ions experience slow diffusion as they mix but also exhibitedsignificant ordering into distinct regions. We suspect that this orderingdisrupted concentration gradients in the direction normal to the interface,thus hindering diffusion in this direction and allowing the macroscopicinterface to remain for long periods of time. Intermolecular interactionsresponsible for this behavior included the O–NH interactionbetween the EAN ions and the carbon chain–carbon chain interactionsbetween the [BMIM]+cations, which associate more stronglyin the mixed state than in the pure IL state. [ABSTRACT FROM AUTHOR]

Recently, a number of publications have suggested thationic liquids(ILs) can absorb solid particles. This development may have implicationsin fields likeoil sand processing, oil spill beach cleanup, and water treatment.In this Article, we provide a computational investigation of thisphenomenon via molecular dynamics simulations. Two particle surfacechemistries were investigated: (1) hydrocarbon-saturated and (2) silanol-saturated,representing hydrophobic and hydrophilic particles, respectively.Employing 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) as a model IL, these nanoparticles were allowed to equilibrateat the IL/water and IL/hexane interfaces to observe the interfacialself-assembled structures. At the IL/water interface, the hydrocarbon-basednanoparticles were nearly completely absorbed by the IL, while thesilica nanoparticles maintained equal volume in both phases. At theIL/hexane interface, the hydrocarbon nanoparticles maintained minimalinteractions with the IL, whereas the silica nanoparticles were nearlycompletely absorbed by it. Studies of these two types of nanoparticlesimmersed in the bulk IL indicate that the surface chemistry has agreat effect on the corresponding IL liquid structure. These effectsinclude layering of the ions, hydrogen bonding, and irreversible absorptionof some ions to the silica nanoparticle surface. We quantify theseeffects with respect to each nanoparticle. The results suggest thatILs likely exhibit this absorption capability because they can formsolvation layers with reduced dynamics around the nanoparticles. [ABSTRACT FROM AUTHOR]

Although biological upgrading of lignocellulosic sugars represents a promising and sustainable route to bioplastics, diverse and variable feedstock compositions (e.g., glucose from the cellulose fraction and xylose from the hemicellulose fraction) present several complex challenges. Specifically, sugar mixtures are often incompletely metabolized due to carbon catabolite repression while composition variability further complicates the optimization of co-utilization rates. Benefiting from several unique features including division of labor, increased metabolic diversity, and modularity, synthetic microbial communities represent a promising platform with the potential to address persistent bioconversion challenges. In this work, two unique and catabolically orthogonal Escherichia coli co-cultures systems were developed and used to enhance the production of D-lactate and succinate (two bioplastic monomers) from glucose-xylose mixtures (100 g L -1 total sugars, 2:1 by mass). In both cases, glucose specialist strains were engineered by deleting xylR (encoding the xylose-specific transcriptional activator, XylR) to disable xylose catabolism, whereas xylose specialist strains were engineered by deleting several key components involved with glucose transport and phosphorylation systems (i.e., ptsI , ptsG , galP , glk ) while also increasing xylose utilization by introducing specific xylR mutations. Optimization of initial population ratios between complementary sugar specialists proved a key design variable for each pair of strains. In both cases, ∼91% utilization of total sugars was achieved in mineral salt media by simple batch fermentation. High product titer (88 g L -1 D-lactate, 84 g L -1 succinate) and maximum productivity (2.5 g L -1 h -1 D-lactate, 1.3 g L -1 h -1 succinate) and product yield (0.97 g g-total sugar -1 for D-lactate, 0.95 g g-total sugar -1 for succinate) were also achieved., (Copyright © 2020 Flores, Choi, Martinez, Onyeabor, Ayla, Godar, Machas, Nielsen and Wang.)

Muconic acid is a promising platform biochemical and precursor to adipic acid, which can be used to synthesize various plastics and polymers. In this study, the systematic construction and comparative evaluation of a modular network of non-natural pathways for muconic acid biosynthesis was investigated in Escherichia coli, including via three distinct and novel pathways proceeding via phenol as a common intermediate. However, poor recombinant activity and high promiscuity of phenol hydroxylase ultimately limited "phenol-dependent" muconic acid production. A fourth pathway proceeding via p-hydroxybenzoate, protocatechuate, and catechol was accordingly developed, though with muconic acid titers by this route reaching just 819 mg/L, its performance lagged behind that of the established, "3-dehydroshikimiate-derived" route. Finally, these two most promising pathways were coexpressed in parallel to create a synthetic "metabolic funnel" that, by enabling maximal net precursor assimilation and flux while preserving native chorismate biosynthesis, nearly doubled muconic acid production to up to >3.1 g/L at a glucose yield of 158 mg/g while introducing only a single auxotrophy. This generalizable, "funneling" strategy is expected to have broad applications in metabolic engineering for further enhancing production of muconic acid, as well as other important bioproducts of interest.