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This work shows the integration of reductive lignin depolymerization and microbial funneling with Novosphingobium aromaticivorans to produce 2-pyrone-4,6-dicarboxylic acid (PDC) in a lignocellulosic biorefinery.
The paper presents fabrication methodologies that integrate silicon components into soft microfluidic devices to perform microbial cell lysis for biological applications.
Strain engineering has advanced in the past decade. Herein, we review the importance of growth coupling, growth decoupling, regulatory control and medium optimization for microbial bioproduction to provide stable conversion over a longer period.
Hydrogen derived from bio-based sources, or biohydrogen (bioH 2 ), has the potential to reduce GHG emissions from industrial and transportation sectors, owing to the low carbon footprint and myriad applications like refinery operation, ammonia production, steel production, fuel cell, etc. To evaluate the commercialization potential of bioH 2 production, we modeled bioH 2 production and conducted techno-economic analysis (TEA) and life cycle analysis (LCA) of two facilities producing 50 metric tonnes of bioH 2 per day from cheese whey (CW) and solid food waste (SFW) through the integration of dark fermentation (DF) and microbial electrolysis cell (MEC) technologies. LCA results showed that CW and SFW can produce carbon-negative bioH 2 , with emissions of −8.6 and −8.0 kg GHG kg −1 bioH 2 with carbon sequestration and renewable electricity resources, respectively, making bioH 2 potentially eligible for a tax credit of $\$3$ kg −1 H 2 based on provision 45 V of the U.S. Inflation Reduction Act (IRA). In this study, bioH 2 production treats waste streams to generate fresh water, thus, potentially can receive waste water treatment fee that varies with regions. The MEC capital cost dominates the bioH 2 cost, which is mainly determined by current density. With a current density of 20 A m −2 , the production cost for CW input varied between $\$17$ and $\$24$ kg −1 bioH 2 , while that for SFW input ranged from $\$29$ to $\$30$ kg −1 bioH 2 under different operating conditions, considering the 45 V tax credit, waste water treatment fee and production revenue. If the current density increases to 100 A m −2 , the bioH 2 cost decreases to a range of $\$4.0$–$\$6.9$ for CW and $\$5$–$\$6$ for SFW scenarios. This study also shows that low-cost bioH 2 can be produced using CW waste stream as feedstock.
Replacing biorefinery designs that use stepwise fractionation, depolymerization, and conversion processes with designed tandem process steps can greatly reduce a biorefinery's operating costs and environmental impact. Reductive Catalytic Fractionation (RCF) is a highly efficient lignin-first approach that combines biomass fractionation and lignin depolymerization to generate a hydrogenolysis oil and pulp. The oil is composed of a complex mixture of phenolic monomers, dimers, and oligomers. Intergrating this chemical deconstruction process with microbial funneling of phenolics can simplify the product mixture and make high-value products. We applied RCF to poplar biomass in a biomass-to-bioproduct processing chain in which the phenolics were funneled to 2-pyrone-4,6-dicarboxylic acid (PDC) by an engineered strain of Novosphingobium aromaticivorans DSM12444. The pulp was enzymatically digested and the glucose and xylose was funneled to ethanol by an engineered strain of Saccharomyces cerevisiae GLBRCY945. By combining biomass fractionation and lignin depolymerization we removed a costly processing step that directly translated into a 29% reduction in the minimum selling price of PDC. This work combines experimentation with process modeling of an integrated biorefinery design to show how utilizing tandem process steps can significantly reduce operating expenses and environmental impact of upgrading lignocellulosic biomass to a portfolio of high value products.
Low-boiling alkylamines such as butylamine offer promise as effective biomass pretreatment solvents that can be readily recovered and recycled; however, their capability to support microbial conversion of nutrients present in hydrolysates represents an important area for investigation. Here we employed butylamine to pretreat poplar biomass and characterize its effects on the release of fermentable sugars after solvent removal and enzymatic hydrolysis, as well as the biocompatibility of the produced hydrolysates with three organisms commonly used as bioconversion hosts. We observed that residual butylamine and the derivative butylacetamide were present in high enough concentrations to exert toxicity to strains of Aspergillus niger, Pseudomonas putida, and Rhodosporidium toruloides that produce malic acid, isoprenol and bisabolene, respectively. Removal of the toxic compounds by charcoal filtration and nutrient supplementation resulted in a hydrolysate containing >100 g L −1 of sugars that enabled strong growth, substrate consumption and bioproduct accumulation, outperforming defined cultivation media. This is the first demonstration of a butylamine-based deconstruction process for poplar biomass at a pilot-scale to achieve conversion of high sugar concentrations to valuable bioproducts with engineered microbes.
Significance Synthetic biology has entered an era in which reading and writing DNA sequences are no longer rate-limiting steps in microbial strain engineering. Indeed, analytical methods measuring the resulting metabolic outcomes of specific gene edits have lagged behind the ability to generate new recombinant strains. Herein, we report a mass spectrometry strategy to accelerate these analytical workflows by directly analyzing metabolites and molecules produced from engineered microorganisms in a multiplexed process. Using untargeted acquisitions and unsupervised analytics, we assess the molecular features that change across discrete strains including primary target species, secondary products, and species outside the engineered fatty acid biosynthesis pathway.
Evolutionary pressure on microbial communities propagating under extreme environmental conditions often results in unique structural adaptations to promote cell survival. In this work, we report an investigation of AbpX, a biomatrix protein identified in cultures of the hyperthermophilic archaeon Pyrodictium abyssi. Under ex vivo and in vitro conditions, AbpX assembles into a paracrystalline lattice composed of semiflexible fibrils. CryoEM analysis of recombinant AbpX fibrils reveals that the precursor protein polymerizes through donor strand complementation (DSC), a process previously reported for chaperone-usher fimbriae in Gram-negative bacteria. Unlike the latter DSC protein polymers, AbpX undergoes chaperone-free polymerization in the presence of calcium ions, which are sequestered at the donor strand-acceptor groove interface between protomers in the fibril. Using a combination of cryoEM and crystallographic information, a structural model is proposed for the AbpX lattice that provides insight into its potential role in biofilm formation. These findings suggest that calcium ion coordination may contribute to fibril assembly and preorganize fibrils for incorporation into the protein lattice. Bioinformatic analysis indicates that AbpX exemplifies a distinct and broadly distributed clade of calcium ion responsive biomatrix proteins within the TasA superfamily that can be fabricated into hydrogel biomaterials in vitro under environmentally benign conditions.
Microbial electrosynthesis (MES) is an emerging technology that can convert carbon dioxide (CO 2 ) into value-added organic carbon compounds using electrons supplied from a cathode. However, MES is affected by low product formation due to limited extracellular electron uptake by microbes. Herein, a novel cathode was developed from chemically synthesized magnetite nanoparticles and reduced graphene oxide nanocomposite (rGO-MNPs). This nanocomposite was electrochemically deposited on carbon felt (CF/rGO-MNPs), and the modified material was used as a cathode for MES production. The bioplastic, polyhydroxybutyrate (PHB) produced by Rhodopseudomonas palustris TIE-1 (TIE-1), was measured from reactors with modified and unmodified cathodes. Results demonstrate that the magnetite nanoparticle anchored graphene cathode (CF/rGO-MNPs) exhibited higher PHB production (91.31 ± 0.9 mg l -1 ). This is ~4.2 times higher than unmodified carbon felt (CF), and 20 times higher than previously reported using graphite. This modified cathode enhanced electron uptake to -11.7 ± 0.1 μ A cm -2 , ~5 times higher than CF cathode (-2.3 ± 0.08 μ A cm -2 ). The faradaic efficiency of the modified cathode was ~2 times higher than the unmodified cathode. Electrochemical analysis and scanning electron microscopy suggest that rGO-MNPs facilitated electron uptake and improved PHB production by TIE-1. Overall, the nanocomposite (rGO-MNPs) cathode modification enhances MES efficiency.
Abstract Cellular populations assume an incredible variety of shapes ranging from circular molds to irregular tumors. While we understand many of the mechanisms responsible for these spatial patterns, little is known about how the shape of a population influences its ecology and evolution. Here, we investigate this relationship in the context of microbial colonies grown on hard agar plates. This a well-studied system that exhibits a transition from smooth circular disks to more irregular and rugged shapes as either the nutrient concentration or cellular motility is decreased. Starting from a mechanistic model of colony growth, we identify two dimensionless quantities that determine how morphology and genetic diversity of the population depend on the model parameters. Our simulations further reveal that population dynamics cannot be accurately described by the commonly-used surface growth models. Instead, one has to explicitly account for the emergent growth instabilities and demographic fluctuations. Overall, our work links together environmental conditions, colony morphology, and evolution. This link is essential for a rational design of concrete, biophysical perturbations to steer evolution in the desired direction.
Increasingly, national space agencies are expanding their goals to include Mars exploration with sample return. To better protect Earth and its biosphere from potential extraterrestrial sources of contamination, as set forth in the Outer Space Treaty of 1967, international efforts to develop planetary protection measures strive to understand the danger of cross-contamination processes in Mars sample return missions. We aim to better understand the impact of the martian surface on microbial dormancy and survivability. Radiation resistance of microbes is a key parameter in considering survivability of microbes over geologic times on the frigid, arid surface of Mars that is bombarded by solar and galactic cosmic radiation. We tested the influence of desiccation and freezing on the ionizing radiation survival of six model microorganisms: vegetative cells of two bacteria (Deinococcus radiodurans, Escherichia coli) and a strain of budding yeast (Saccharomyces cerevisiae); and vegetative cells and endospores of three Bacillus bacteria (B. subtilis, B. megaterium, B. thuringiensis). Desiccation and freezing greatly increased radiation survival of vegetative polyploid microorganisms when applied separately, and when combined, desiccation and freezing increased radiation survival even more so. Thus, the radiation survival threshold of polyploid D. radiodurans cells can be extended from the already high value of 25 kGy in liquid culture to an astonishing 140 kGy when the cells are both desiccated and frozen. However, such synergistic radioprotective effects of desiccation and freezing were not observed in monogenomic or digenomic Bacillus cells and endospores, which are generally sterilized by 12 kGy. This difference is associated with a critical requirement for survivability under radiation, that is, repair of genome damage caused by radiation. Deinococcus radiodurans and S. cerevisiae accumulate similarly high levels of the Mn antioxidants that are required for extreme radiation resistance, as do endospores, though they greatly exceed spores in radioresistance because they contain multiple identical genome copies, which in D. radiodurans are joined by persistent Holliday junctions. We estimate ionizing radiation survival limits of polyploid DNA-based life-forms to be hundreds of millions of years of background radiation while buried in the martian subsurface. Our findings imply that forward contamination of Mars will essentially be permanent, and backward contamination is a possibility if life ever existed on Mars.
Summary GenomeDepot is an open-source web-based platform for annotation, management, and comparative analysis of microbial genomic sequences and associated data including ortholog families, protein domains, operons, regulatory interactions, strain taxonomy, and sample metadata. GenomeDepot supports rapid creation of websites for user-defined genome collections that include bioinformatic tools for interactive genome browsing, Basic Local Alignment Search Tool (BLAST) search, annotation search, comparative genomic neighborhood visualization, and sequence download. Gene function annotations are generated by a customizable annotation pipeline. The pipeline runs annotation tools in Conda environments and can be easily extended with additional user-specified tools. Availability and implementation GenomeDepot is open source and distributed under the GNU General Public License via GitHub (https://github.com/aekazakov/genome-depot). GenomeDepot is implemented in Python and was tested in Ubuntu Linux. Full installation instructions and documentation are available at https://aekazakov.github.io/genome-depot/. GenomeDepot demo server is freely accessible at https://iseq.lbl.gov/demogd/.
Microbial natural products represent a major source of bioactive compounds for drug discovery. Among these molecules, nonribosomal peptides (NRPs) represent a diverse class that include antibiotics, immunosuppressants, anticancer agents, toxins, siderophores, pigments, and cytostatics. The discovery of novel NRPs remains a laborious process because many NRPs consist of nonstandard amino acids that are assembled by nonribosomal peptide synthetases (NRPSs). Adenylation domains (A-domains) in NRPSs are responsible for selection and activation of monomers appearing in NRPs. During the past decade, several support vector machine-based algorithms have been developed for predicting the specificity of the monomers present in NRPs. These algorithms utilize physiochemical features of the amino acids present in the A-domains of NRPSs. In this article, we benchmarked the performance of various machine learning algorithms and features for predicting specificities of NRPSs and we showed that the extra trees model paired with one-hot encoding features outperforms the existing approaches. Moreover, we show that unsupervised clustering of 453560 A-domains reveals many clusters that correspond to potentially novel amino acids. While it is challenging to predict the chemical structure of these amino acids, we developed novel techniques to predict their various properties, including polarity, hydrophobicity, charge, and presence of aromatic rings, carboxyl, and hydroxyl groups.
Abstract Soil microbial communities perform critical ecosystem services through the collective metabolic activities of numerous individual organisms. Most microbes use corrinoids, a structurally diverse family of cofactors related to vitamin B12. Corrinoid structure influences the growth of individual microbes, yet how these growth responses scale to the community level remains unknown. Analysis of metagenome-assembled genomes suggests that corrinoids are supplied to the community by members of the archaeal and bacterial phyla Thermoproteota, Actinobacteria, and Proteobacteria. Corrinoids were found largely adhered to the soil matrix in a grassland soil, at levels exceeding those required by cultured bacteria. Enrichment cultures and soil microcosms seeded with different corrinoids showed distinct shifts in bacterial community composition, supporting the hypothesis that corrinoid structure can shape communities. Environmental context influenced both community- and taxon-specific responses to specific corrinoids. These results implicate corrinoids as key determinants of soil microbiome structure and suggest that environmental micronutrient reservoirs promote community stability.
Seasonal variation in algae biomass productivity is a significant obstacle to the economical production of fuels and chemicals from algae biomass. Long-term storage is necessary to guarantee a constant supply to a conversion facility and harvested microalgae biomass (20% solids) is subject to degradation immediately upon harvesting as a result of aerobic instability. Ensiling has been successfully used to preserve microalgal biomass, but biomass attributes that contribute to successful storage are poorly understood. Storage studies of Monoraphidium sp. biomass indicate a strong correlation between nitrogen management in algae cultivation and stability of post-harvest algae biomass in 30 days of anaerobic storage. Algae cultivated with periodic nitrogen addition stored poorly (> 20% loss, dry basis), while algae biomass from cultures that were allowed to continue growing after nitrogen depletion was more stable in storage (8% loss, dry basis). A follow-up study aimed at exploring the relationship between nitrogen management in cultivation and stability of post-harvest algae biomass stored Monoraphidium biomass cultivated in nitrogen deplete and nitrogen replete conditions, as well as with blends of each in varying ratios. Monoraphidium biomass from nitrogen replete cultivations experienced the largest degradation (24%, dry basis), while nitrogen deplete biomass experienced the least (10%, dry basis). Dry matter loss experienced during storage of blends was positively correlated with the amount of nitrogen replete biomass that a blend contained. Further, the composition of the post-storage algae microbial community was also strongly affected by cultivation conditions, with Clostridia species being more prevalent in stored biomass obtained from nitrogen replete cultivations. Nitrogen management has long been known to influence algae biomass productivity and biochemical composition; here, we demonstrate that it also strongly influences the stability of post-harvest algae biomass in anaerobic storage.
To realize the full potential of biomanufacturing, the breadth of industrial microbes used to consume diverse feedstock and generate bioproducts needs to expand. As such, portable tools are required that can be used by multiple hosts for straightforward genomic manipulation and precise gene expression. Here, we demonstrate the co-utilization of two synthetic biology tools to achieve these goals: cis-repressors (CRs) and serine recombinase-assisted genome engineering (SAGE). CRs are small, noncoding RNAs that are placed upstream of the target gene to modulate bacterial translation rates at varying, discrete levels. SAGE uses site-specific serine recombinases to catalyze highly efficient, unidirectional insertion of DNA into the chromosome of diverse organisms. We used SAGE to integrate a suite of CRs into the industrially relevant hosts Pseudomonas putida, Corynebacterium glutamicum, and Cupriavidus necator. Using a fluorescent reporter as a readout of CR functionality, we found that CR performance across these backgrounds was similar—providing a range of translational repression up to 100-fold. Overall, these results demonstrate the high portability of CRs across bacterial genetic backgrounds, which ideally can be used in future microbial engineering efforts pertinent to biomanufacturing.
Abstract The Novel Metagenome Protein Families Database (NMPFamsDB) is a database of metagenome- and metatranscriptome-derived protein families, whose members have no hits to proteins of reference genomes or Pfam domains. Each protein family is accompanied by multiple sequence alignments, Hidden Markov Models, taxonomic information, ecosystem and geolocation metadata, sequence and structure predictions, as well as 3D structure models predicted with AlphaFold2. In its current version, NMPFamsDB hosts over 100 000 protein families, each with at least 100 members. The reported protein families significantly expand (more than double) the number of known protein sequence clusters from reference genomes and reveal new insights into their habitat distribution, origins, functions and taxonomy. We expect NMPFamsDB to be a valuable resource for microbial proteome-wide analyses and for further discovery and characterization of novel functions. NMPFamsDB is publicly available in http://www.nmpfamsdb.org/ or https://bib.fleming.gr/NMPFamsDB.
Abstract The microbiome is a complex community of microorganisms, encompassing prokaryotic (bacterial and archaeal), eukaryotic, and viral entities. This microbial ensemble plays a pivotal role in influencing the health and productivity of diverse ecosystems while shaping the web of life. However, many software suites developed to study microbiomes analyze only the prokaryotic community and provide limited to no support for viruses and microeukaryotes. Previously, we introduced the Viral Eukaryotic Bacterial Archaeal (VEBA) open-source software suite to address this critical gap in microbiome research by extending genome-resolved analysis beyond prokaryotes to encompass the understudied realms of eukaryotes and viruses. Here we present VEBA 2.0 with key updates including a comprehensive clustered microeukaryotic protein database, rapid genome/protein-level clustering, bioprospecting, non-coding/organelle gene modeling, genome-resolved taxonomic/pathway profiling, long-read support, and containerization. We demonstrate VEBA’s versatile application through the analysis of diverse case studies including marine water, Siberian permafrost, and white-tailed deer lung tissues with the latter showcasing how to identify integrated viruses. VEBA represents a crucial advancement in microbiome research, offering a powerful and accessible software suite that bridges the gap between genomics and biotechnological solutions.