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HtPIP: High-throughput phage isolation platform increases diversity and reduces isolation time using multiple bacteria
Bacteriophages are ubiquitous in nature, but relatively few have been isolated and characterized compared to the number of bacterial strains. Phage biotechnology applications benefit from a diverse library of isolated phages to kill or transfer genetic material to a bacterium of interest. However, scaling up phage discovery for diverse bacterial hosts can be time-consuming and costly. Here, we developed an approach to capture novel phages for multiple bacterial strains in parallel from an environmental sample using commercially available 0.2-μM filter plates. Using this High-throughput Phage Isolation Platform (HtPIP), 12 novel phages were isolated spanning 9 diverse bacterial host genera. Eleven of the isolated phages define new phage species, with nine also defining new genera. The HtPIP was used to discover both DNA and RNA phages, including a Tectiviridae infecting Pseudomonas putida mt-2 and a Leviviricetes infecting a Microbacterium isolate, which represents the first cultured RNA phage infecting a host outside of Proteobacteria. Using a metagenomic approach, we demonstrate that the HtPIP captures a higher proportion of novel phages compared to traditional low-throughput methods.
Analysis of individual biological particles by mass spectrometry
A method is developed for the detection and identification of biological particles introduced in aerosol form into a quadrupole mass spectrometer. The bacterial aerosol is generated by nebulizing an ethanol suspension. The particles are introduced into the ion source of the mass spectrometer in the form of a beam, where they are individually volatilized on a V-type rhenium filament and ionized by electron impaction. It is shown that the average intensity of a mass peak is obtained from the pulse height distribution of about a thousand ion pulses from different particles. Pseudomonas putida, Bacillus subtilis, and Bacillus cereus are used in the studies. Differences between the relative intensities of mass peaks in the spectra from P. putida and B. subtilis are found and may provide a method for differentiation of microorganisms. The results for the two species agree reasonably well with those reported by Kistemaker et al. (1975) and Schulten et al. (1973). However, there exist some differences between the two spectra in the high mass range due to the difference in the pyrolysis conditions.
Monitoring of bacteria growth using a wireless, remote query resonant-circuit sensor: application to environmental sensing
A new technique is presented for in-vivo remote query measurement of the complex permittivity spectra of a biological culture solution. A sensor comprised of a printed inductor-capacitor resonant-circuit is placed within the culture solution of interest, with the impedance spectrum of the sensor measured using a remotely located loop antenna; the complex permittivity spectra of the culture is calculated from the measured impedance spectrum. The remote query nature of the sensor platform enables, for example, the in-vivo real-time monitoring of bacteria or yeast growth from within sealed opaque containers. The wireless monitoring technique does not require a specific alignment between sensor and antenna. Results are presented for studies conducted on laboratory strains of Bacillus subtilis, Escherichia coli JM109, Pseudomonas putida and Saccharomyces cerevisiae.
Economic and Environmental Assessment of Biological Conversions of Agile BioFoundry (ABF) Bio-Derived Chemicals
Bio-derived chemicals are an essential part of the growing bioeconomy. They possess the potential to boost a new domestic bioproduct industry, improve the sustainability of integrated biomanufacturing, and reduce U.S. dependence on fossil energy. The Agile BioFoundry (ABF) consortium, a Department of Energy (DOE)-sponsored collaboration, is investigating biobased pathways to produce advantaged products including advanced biofuels, fuel intermediates, and bioproducts. ABF is integrating advanced computational tools for biological engineering, process design and data analysis, and economic/sustainability modeling tools into a comprehensive and dynamic platform for biomanufacturing of microbes and using them to produce key metabolic intermediates or beachhead molecules which may be derivatized into several distinct bioproducts of industrial interest. In this presentation, we first discuss a methodology to select a single exemplar product molecule to represent each beachhead pathway based on similarities with other end-molecule options (yields/titers/rates, fermentation operation mode, oxygen requirements, general separations challenges, etc.), in order to maintain a reasonable number of cases for rigorous process simulation. We then use techno-economic analysis (TEA) and life-cycle analysis (LCA) to highlight sensitivities and trends around economic performance and environmental footprint, reflecting examples for two selected ABF technology pathways to bio-derived chemicals: 1) adipic acid production via muconic acid fermentation from mixed sugars with Pseudomonas putida and 2) cineole via geranyl diphosphate with Rhodosporidium toruloides. We present multi-variable scan plots highlighting key drivers on costs and greenhouse gas emissions in order to identify the parameter space in which each pathway could ultimately achieve economic and environmental sustainability meeting or exceeding commodity product benchmarks, thus prioritizing future R&D focus areas.
Bioconversion of Thermochemical Intermediates
Thermochemical (TC) biofuels production via both pyrolysis and hydrothermal liquefaction produces aqueous waste streams, typically with organic compounds at concentrations of -50-100 g/L. These streams represent a wastewater treatment cost and carbon loss for the TC biorefinery, but the concentration range for these compounds is ideal for bioconversion. To that end, the Bioconversion of Thermochemical Intermediates (BTI) project is developing advanced analytics and engineered microbes to convert these waste streams to co-products, with the overall aim of improving the economics and carbon conversion efficiency of TC biorefining. To date, we have primarily focused on development of advanced analytical chemistry approaches to fully characterize TC aqueous streams and engineering of Pseudomonas putida for conversion of non-conventional substrates, including methylated phenolics, cyclic ketones, furans, and C1-C3 light oxygenates, into atom-efficient products. Two primary challenges are the rapid deployment of aqueous-compatible analytics to changing upstream conditions and dealing with the toxicity of the feed streams to engineered microbes. The project efforts have resulted in engineered strains of P. putida able to consume 90% of the organic compounds in aqueous waste streams from catalytic fast pyrolysis, more than 300-fold toxicity tolerance improvements in P. putida, and carbon closures exceeding 90% for TC wastewater streams across multiple processing technologies.
Biological Lignin Valorization
Given lignin's heterogeneity, catalytic depolymerization results in aromatic compound mixtures, and conversion of this complex substrate to a single product is challenging. To that end, the Biological Lignin Valorization (BLV) project is pursuing biological funneling, wherein aromatic catabolic microbes are engineered to convert a mixture of lignin-derived compounds to a single product. Namely, we employ Pseudomonas putida and pursue atom-efficient products, such as muconic acid, which can be further converted to direct replacements or used in performance-advantaged bioproducts. Overall, biological lignin conversion can make major contributions to reduce the minimum fuel selling price of the integrated biorefinery. Early industrial efforts in this area are also leading to value-added products, including in collaboration with the BLV project. Primary challenges associated with BLV efforts include accessing bio-available monomers from lignin (with the Lignin Utilization project), enabling commercial titers, rates, and yields of bioproducts from lignin-derived compounds, and overcoming substrate and product toxicity. To date, we have 1) demonstrated 49 g/L of muconate from aromatic compounds and 4 g/L of muconate from lignin, 2) improved the toxicity tolerance of P. putida to key aromatic substrates, 3) debottlenecked biological funneling for higher rates, and 4) engineered P. putida to convert S, G, and H-type lignin-derived compounds to a single product.
2.3.2.100 - Biological Lignin Valorization (BLV)
The Biological Lignin Valorization (BLV) project develops microbial strains and associated bioprocesses to convert lignin-derived aromatic compounds into value-added bioproducts. Our main objective in the BLV project is to achieve industrially relevant bioproduction metrics that can directly contribute to the economic viability and improved sustainability of the integrated lignocellulosic biorefinery, in collaboration with complementary BETO-funded lignin valorization projects. Specifically, the BLV project works closely with the BETO-funded Lignin Utilization project, which provides bio-available aromatic compounds from chemo-catalytic lignin depolymerization. We use the robust soil bacterium, Pseudomonas putida, as our primary microbial host for the conversion of lignin-derived compounds to bioproducts. To date, we have focused on atom-efficient bioproducts that can be used as either direct replacement chemicals or performance-advantaged bioproducts, including cis,cis-muconic acid, beta-ketoadipic acid, and 2-pyrone-4,6-dicarboxylic acid. From model aromatic substrates, we have achieved titers of each of these compounds approaching 40 g/L and productivity values ranging from 0.5 to over 1 g/L/hr, all at 90% molar yield or higher. From real lignin streams, we have thus far achieved 24 g/L, 0.66 g/L/hr, and theoretical yield of beta-ketoadipic acid. A major pursuit now is to reach industrially relevant performance metrics on an expanded slate of lignin-derived streams.
Reversibly soluble bases for lignin oxidative depolymerization
Disclosed herein are lignin valorization strategies that integrate thermochemical pretreatment strategies. Using methods disclosed herein, yields of greater than 40% of usable monomers are obtained from lignin in biomass. The monomers can be assimilated by strains of Pseudomonas putida.
Maximizing microbial bioproduction from sustainable carbon sources using iterative systems engineering
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Precise Genomic Riboregulator Control of Metabolic Flux in Microbial Systems
Not Available
Experimental and Analytical Approaches for Improving the Resolution of Randomly Barcoded Transposon Insertion Sequencing (RB-TnSeq) Studies
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Bioconversion of wastewater-derived cresols to methyl muconic acids for use in performance-advantaged bioproducts
We combine metabolic engineering and polymer chemistry to convert cresols, a main component of biomass-derived catalytic fast pyrolysis wastewater, to methyl muconic acids that have performance-advantaged properties in nylons and plasticizers.
Discovery, characterization, and metabolic engineering of Rieske non-heme iron monooxygenases for guaiacol O-demethylation
Discovery, characterization, and metabolic engineering of Rieske non-heme iron monooxygenases for guaiacol O-demethylation
Rieske non-heme iron monooxygenase for guaiacol O-demethylation
PDB file results of X-ray crystallography data
Article including Gene lists, and clone information
Supplementary information includes genelists and strain ids
Proteome results.
Proteome results. Protein abundance was determined for N. aromaticivorans DSM12444 wild-type and strain JMN2 during growth with glucose, and for strain JMN2 during growth with guaiacol.