Challenges and opportunities in biological funneling of heterogeneous and toxic substrates beyond lignin.

Significant developments in the understanding and manipulation of microbial metabolism have enabled the use of engineered biological systems toward a more sustainable energy and materials economy. While developments in metabolic engineering have primarily focused on the conversion of carbohydrates, substantial opportunities exist for using these same principles to extract value from more heterogeneous and toxic waste streams, such as those derived from lignin, biomass pyrolysis, or industrial waste. Funneling heterogeneous substrates from these streams toward valuable products, termed biological funneling, presents new challenges in balancing multiple catabolic pathways competing for shared cellular resources and engineering against perturbation from toxic substrates. Solutions to many of these challenges have been explored within the field of lignin valorization. This perspective aims to extend beyond lignin to highlight the challenges and discuss opportunities for use of biological systems to upgrade previously inaccessible waste streams.

Biological upgrading of pyrolysis-derived wastewater: Engineering Pseudomonas putida for alkylphenol, furfural, and acetone catabolism and (methyl)muconic acid production.

While biomass-derived carbohydrates have been predominant substrates for biological production of renewable fuels, chemicals, and materials, organic waste streams are growing in prominence as potential alternative feedstocks to improve the sustainability of manufacturing processes. Catalytic fast pyrolysis (CFP) is a promising approach to generate biofuels from lignocellulosic biomass, but it generates a complex, carbon-rich, and toxic wastewater stream that is challenging to process catalytically but could be biologically upgraded to valuable co-products. In this work, we implemented modular, heterologous catabolic pathways in the Pseudomonas putida KT2440-derived EM42 strain along with the overexpression of native toxicity tolerance machinery to enable utilization of 89% (w/w) of carbon in CFP wastewater. The dmp monooxygenase and meta-cleavage pathway from Pseudomonas putida CF600 were constitutively expressed to enable utilization of phenol, cresols, 2- and 3-ethyl phenol, and methyl catechols, and the native chaperones clpB, groES, and groEL were overexpressed to improve toxicity tolerance to diverse aromatic substrates. Next, heterologous furfural and acetone utilization pathways were incorporated, and a native alcohol dehydrogenase was overexpressed to improve methanol utilization, generating reducing equivalents. All pathways (encoded by genes totaling ~30 kilobases of DNA) were combined into a single strain that can catabolize a mock CFP wastewater stream as a sole carbon source. Further engineering enabled conversion of all aromatic compounds in the mock wastewater stream to (methyl)muconates with a ~90% (mol/mol) yield. Biological upgrading of CFP wastewater as outlined in this work provides a roadmap for future applications in valorizing other heterogeneous waste streams.

Structural Determination of a New Peptidolipid Family from Rhodococcus opacus and the Pathogen Rhodococcus equi by Multiple Stage Mass Spectrometry.

The cell walls of the genus Rhodococcus including the pathogenic bacterium Rhodococcus equi (R. equi) and biotechnologically important bacterium Rhodococcus opacus (R. opacus) contain an abundant peptidolipid (or termed lipopeptide) family whose structures have not been reported previously. Here, we describe a linear ion-trap multiple-stage mass spectrometric (LIT MSn) approach with high resolution mass spectrometry (HRMS), in conjunction with NMR spectroscopy, chemical reactions, and GC/MS analysis to define the structures of these compounds. We employed LIT MSn (n = 2-8) on the [M + Na]+ ion species to establish the peptide sequence, the identity of the fatty acyl substituent, and its location within the molecule, while NMR spectroscopy and GC/MS were used to recognize the Leu and Ile moieties. The major new lipopeptide found in R. opacus is defined as C17H35CH(OH)CH2CO-NHLeu-Ser-Leu-Ile-Thr-Ile-PheCOOH, where a ß-OH fatty acyl (C18-C22) substituent is attached to the N-terminal of the LSLITIF peptide chain via a NH-CO bond. By contrast, the main peptidolipids found in R. equi belong to the cyclopeptidolipid family, which possesses the same peptide sequence and lipid chain, but the ß-OH group of the fatty acyl moiety and the C-terminus of the peptide (i.e., the -COOH) are cyclized by an ester bond formation to a lactone, with a structure similar to iturin-A (Peypoux, F. et al. Biochemistry 1978, 17, 3992-3996). The antibiotic activity test of these new lipids did not reveal an activity against any of seven microorganisms tested.

A concerted systems biology analysis of phenol metabolism in Rhodococcus opacus PD630.

Rhodococcus opacus PD630 metabolizes aromatic substrates and naturally produces branched-chain lipids, which are advantageous traits for lignin valorization. To provide insights into its lignocellulose hydrolysate utilization, we performed 13C-pathway tracing, 13C-pulse-tracing, transcriptional profiling, biomass composition analysis, and metabolite profiling in conjunction with 13C-metabolic flux analysis (13C-MFA) of phenol metabolism. We found that 1) phenol is metabolized mainly through the ortho-cleavage pathway; 2) phenol utilization requires a highly active TCA cycle; 3) NADPH is generated mainly via NADPH-dependent isocitrate dehydrogenase; 4) active cataplerotic fluxes increase plasticity in the TCA cycle; and 5) gluconeogenesis occurs partially through the reversed Entner-Doudoroff pathway (EDP). We also found that phenol-fed R. opacus PD630 generally has lower sugar phosphate concentrations (e.g., fructose 1,6-bisphosphatase) compared to metabolite pools in 13C-glucose-fed Escherichia coli (set as internal standards), while its TCA metabolites (e.g., malate, succinate, and α-ketoglutarate) accumulate intracellularly with measurable succinate secretion. In addition, we found that phenol utilization was inhibited by benzoate, while catabolite repressions by other tested carbon substrates (e.g., glucose and acetate) were absent in R. opacus PD630. Three adaptively-evolved strains display very different growth rates when fed with phenol as a sole carbon source, but they maintain a conserved flux network. These findings improve our understanding of R. opacus' metabolism for future lignin valorization.

Multi-omic elucidation of aromatic catabolism in adaptively evolved Rhodococcus opacus.

Lignin utilization has been identified as a key factor in biorefinery profitability. However, lignin depolymerization generates heterogeneous aromatic mixtures that inhibit microbial growth and the conversion of lignocellulose to biochemicals. Rhodococcus opacus is a promising aromatic-catabolizing, oleaginous bacterium, but mechanisms for its aromatic tolerance and utilization remain undercharacterized. To better understand these mechanisms, we adaptively evolved R. opacus for improved utilization of 32 combinations of diverse aromatic compounds. Evolved R. opacus mutants showed up to 1900% growth improvement in the utilization of phenol, guaiacol, 4-hydroxybenzoate, vanillate, and benzoate compared to the wild-type strain. Whole genome sequencing revealed several redox-related genes with mutations shared across multiple adapted mutants. PVHG6, the mutant with the most improved growth on a mixture of multiple aromatic compounds, showed 56% lower superoxide dismutase activity than the wild-type strain, suggesting that redox reactions are important for aromatic tolerance and utilization. Comparative transcriptomics revealed by-product detoxification pathways and five aromatic funneling pathways that were upregulated in response to specific aromatic compounds. Gene knockout experiments confirmed the two degradation routes of the ß-ketoadipate pathway for five aromatic compounds. These results provide an improved understanding of aromatic bioconversion and facilitate development of R. opacus as a biorefinery host.

Molecular Toolkit for Gene Expression Control and Genome Modification in Rhodococcus opacus PD630.

Rhodococcus opacus PD630 is a non-model Gram-positive bacterium that possesses desirable traits for lignocellulosic biomass conversion. In particular, it has a relatively rapid growth rate, exhibits genetic tractability, produces high quantities of lipids, and can tolerate and consume toxic lignin-derived aromatic compounds. Despite these unique, industrially relevant characteristics, R. opacus has been underutilized because of a lack of reliable genetic parts and engineering tools. In this work, we developed a molecular toolbox for reliable gene expression control and genome modification in R. opacus. To facilitate predictable gene expression, a constitutive promoter library spanning ∼45-fold in output was constructed. To improve the characterization of available plasmids, the copy numbers of four heterologous and nine endogenous plasmids were determined using quantitative PCR. The molecular toolbox was further expanded by screening a previously unreported antibiotic resistance marker (HygR) and constructing a curable plasmid backbone for temporary gene expression (pB264). Furthermore, a system for genome modification was devised, and three neutral integration sites were identified using a novel combination of transcriptomic data, genomic architecture, and growth rate analysis. Finally, the first reported system for targeted, tunable gene repression in Rhodococcus was developed by utilizing CRISPR interference (CRISPRi). Overall, this work greatly expands the ability to manipulate and engineer R. opacus, making it a viable new chassis for bioproduction from renewable feedstocks.

Lipid metabolism of phenol-tolerant Rhodococcus opacus strains for lignin bioconversion.

BACKGROUND: Lignin is a recalcitrant aromatic polymer that is a potential feedstock for renewable fuel and chemical production. Rhodococcus opacus PD630 is a promising strain for the biological upgrading of lignin due to its ability to tolerate and utilize lignin-derived aromatic compounds. To enhance its aromatic tolerance, we recently applied adaptive evolution using phenol as a sole carbon source and characterized a phenol-adapted R. opacus strain (evol40) and the wild-type (WT) strain by whole genome and RNA sequencing. While this effort increased our understanding of the aromatic tolerance, the tolerance mechanisms were not completely elucidated. RESULTS: We hypothesize that the composition of lipids plays an important role in phenol tolerance. To test this hypothesis, we applied high-resolution mass spectrometry analysis to lipid samples obtained from the WT and evol40 strains grown in 1 g/L glucose (glucose), 0.75 g/L phenol (low phenol), or 1.5 g/L phenol (high phenol, evol40 only) as a sole carbon source. This analysis identified > 100 lipid species of mycolic acids, phosphatidylethanolamines (PEs), phosphatidylinositols (PIs), and triacylglycerols. In both strains, mycolic acids had fewer double bond numbers in phenol conditions than the glucose condition, and evol40 had significantly shorter mycolic acid chain lengths than the WT strain in phenol conditions. These results indicate that phenol adaptation affected mycolic acid membrane composition. In addition, the percentage of unsaturated phospholipids decreased for both strains in phenol conditions compared to the glucose condition. Moreover, the PI content increased for both strains in the low phenol condition compared to the glucose condition, and the PI content increased further for evol40 in the high phenol condition relative to the low phenol condition. CONCLUSIONS: This work represents the first comprehensive lipidomic study on the membrane of R. opacus grown using phenol as a sole carbon source. Our results suggest that the alteration of the mycolic acid and phospholipid membrane composition may be a strategy of R. opacus for phenol tolerance.

Development of Chemical and Metabolite Sensors for Rhodococcus opacus PD630.

Rhodococcus opacus PD630 is a nonmodel, Gram-positive bacterium that possesses desirable traits for biomass conversion, including consumption capabilities for lignocellulose-based sugars and toxic lignin-derived aromatic compounds, significant triacylglycerol accumulation, relatively rapid growth rate, and genetic tractability. However, few genetic elements have been directly characterized in R. opacus, limiting its application for lignocellulose bioconversion. Here, we report the characterization and development of genetic tools for tunable gene expression in R. opacus, including: (1) six fluorescent reporters for quantifying promoter output, (2) three chemically inducible promoters for variable gene expression, and (3) two classes of metabolite sensors derived from native R. opacus promoters that detect nitrogen levels or aromatic compounds. Using these tools, we also provide insights into native aromatic consumption pathways in R. opacus. Overall, this work expands the ability to control and characterize gene expression in R. opacus for future lignocellulose-based fuel and chemical production.

Dynamics of sequestration-based gene regulatory cascades.

Gene regulatory cascades are ubiquitous in biology. Because regulatory cascades are integrated within complex networks, their quantitative analysis is challenging in native systems. Synthetic biologists have gained quantitative insights into the properties of regulatory cascades by building simple circuits, but sequestration-based regulatory cascades remain relatively unexplored. Particularly, it remains unclear how the cascade components collectively control the output dynamics. Here, we report the construction and quantitative analysis of the longest sequestration-based cascade in Escherichia coli. This cascade consists of four Pseudomonas aeruginosa protein regulators (ExsADCE) that sequester their partner. Our computational analysis showed that the output dynamics are controlled in a complex way by the concentration of the unbounded transcriptional activator ExsA. By systematically varying the cascade length and the synthesis rate of each regulator, we experimentally verified the computational prediction that ExsC plays a role in rapid circuit responses by sequestering the anti-activator ExsD, while ExsD increases response times by decreasing the free ExsA concentration. In contrast, when additional ExsD was introduced to the cascade via indirect negative feedback, the response time was significantly reduced. Sequestration-based regulatory cascades with negative feedback are often found in biology, and thus our finding provides insights into the dynamics of this recurring motif.

Comparative transcriptomics elucidates adaptive phenol tolerance and utilization in lipid-accumulating Rhodococcus opacus PD630.

Lignin-derived (e.g. phenolic) compounds can compromise the bioconversion of lignocellulosic biomass to fuels and chemicals due to their toxicity and recalcitrance. The lipid-accumulating bacterium Rhodococcus opacus PD630 has recently emerged as a promising microbial host for lignocellulose conversion to value-added products due to its natural ability to tolerate and utilize phenolics. To gain a better understanding of its phenolic tolerance and utilization mechanisms, we adaptively evolved R. opacus over 40 passages using phenol as its sole carbon source (up to 373% growth improvement over wild-type), and extensively characterized two strains from passages 33 and 40. The two adapted strains showed higher phenol consumption rates (∼20 mg/l/h) and ∼2-fold higher lipid production from phenol than the wild-type strain. Whole-genome sequencing and comparative transcriptomics identified highly-upregulated degradation pathways and putative transporters for phenol in both adapted strains, highlighting the important linkage between mechanisms of regulated phenol uptake, utilization, and evolved tolerance. Our study shows that the R. opacus mutants are likely to use their transporters to import phenol rather than export them, suggesting a new aromatic tolerance mechanism. The identified tolerance genes and pathways are promising candidates for future metabolic engineering in R. opacus for improved lignin conversion to lipid-based products.

Rapid metabolic analysis of Rhodococcus opacus PD630 via parallel 13C-metabolite fingerprinting.

For rapid analysis of microbial metabolisms, (13)C-fingerprinting employs a set of tracers to generate unique labeling patterns in key amino acids that can highlight active pathways. In contrast to rigorous (13)C-metabolic flux analysis ((13)C-MFA), this method aims to provide metabolic insights without expensive flux measurements. Using (13)C-fingerprinting, we investigated the metabolic pathways in Rhodococcus opacus PD630, a promising biocatalyst for the conversion of lignocellulosic feedstocks into value-added chemicals. Specifically, seven metabolic insights were gathered as follows: (1) glucose metabolism mainly via the Entner-Doudoroff (ED) pathway; (2) lack of glucose catabolite repression during phenol co-utilization; (3) simultaneous operation of gluconeogenesis and the ED pathway for the co-metabolism of glucose and phenol; (4) an active glyoxylate shunt in acetate-fed culture; (5) high flux through anaplerotic pathways (e.g., malic enzyme and phosphoenolpyruvate carboxylase); (6) presence of alternative glycine synthesis pathway via glycine dehydrogenase; and (7) utilization of preferred exogenous amino acids (e.g., phenylalanine). Additionally, a (13)C-fingerprinting kit was developed for studying the central metabolism of non-model microbial species. This low-cost kit can be used to characterize microbial metabolisms and facilitate the design-build-test-learn cycle during the development of microbial cell factories.

Robust, tunable genetic memory from protein sequestration combined with positive feedback.

Natural regulatory networks contain many interacting components that allow for fine-tuning of switching and memory properties. Building simple bistable switches, synthetic biologists have learned the design principles of complex natural regulatory networks. However, most switches constructed so far are so simple (e.g. comprising two regulators) that they are functional only within a limited parameter range. Here, we report the construction of robust, tunable bistable switches in Escherichia coli using three heterologous protein regulators (ExsADC) that are sequestered into an inactive complex through a partner swapping mechanism. On the basis of mathematical modeling, we accurately predict and experimentally verify that the hysteretic region can be fine-tuned by controlling the interactions of the ExsADC regulatory cascade using the third member ExsC as a tuning knob. Additionally, we confirm that a dual-positive feedback switch can markedly increase the hysteretic region, compared to its single-positive feedback counterpart. The dual-positive feedback switch displays bistability over a 10(6)-fold range of inducer concentrations, to our knowledge, the largest range reported so far. This work demonstrates the successful interlocking of sequestration-based ultrasensitivity and positive feedback, a design principle that can be applied to the construction of robust, tunable, and predictable genetic programs to achieve increasingly sophisticated biological behaviors.

Linker-free deposition and adhesion of Photosystem I onto nanostructured TiO2 for biohybrid photoelectrochemical cells.

Photosystem I (PSI) from oxygenic photosynthetic organisms is an attractive sensitizer for nano-biohybrid solar cells as it has a combined light-harvesting and reaction center in one protein complex and operates at a quantum yield close to one in biological systems. Using a linker-free deposition technique enabled by an electrospray system, PSI was coupled to 1-D nanostructured titanium dioxide thin films to fabricate an electrode for a photoelectrochemical cell. After deposition, the surfactant in the PSI aggregate was dissolved in the surfactant-free electrolyte, ensuring that partly hydrophobic PSI was not resuspended and stayed in contact with titanium dioxide. A maximum current density of 4.15 mA cm(-2) was measured after 10 min of electrospray deposition, and this is the highest current density reported so far for PSI-based photoelectrochemical cells. The high current is attributed to 1D nanostructure of titanium dioxide and orientation of the PSI onto the surface, which allows easy transfer of electrons.

High-speed microscopic imaging of flagella motility and swimming in Giardia lamblia trophozoites.

We report, in this paper, several findings about the swimming and attachment mechanisms of Giardia lamblia trophozoites. These data were collected using a combination of a high-contrast CytoViva imaging system and a particle image velocimetry camera, which can capture images at speeds greater than 800 frames/s. Using this system, we discovered that, during rapid swimming of Giardia trophozoites, undulations of the caudal region contributed to forward propulsion combined with the beating of the flagella pairs. It was also discovered, in contrast to previous studies with 10 times slower image sampling technique, that the anterior and posterolateral flagella beat with a clearly defined power stroke and not symmetrical undulations. During the transition from free swimming to attachment, trophozoites modified their swimming behavior from a rapid rotating motion to a more stable planar swimming. While using this planar swimming motion, the trophozoites used the flagella for propulsion and directional control. In addition to examination of the posterolateral and anterior flagella, a model to describe the motion of the ventral flagella was derived, indicating that the ventral flagella beat in an expanding sine wave. In addition, the structure of the ventrocaudal groove creates boundary conditions that determine the form of beating of the ventral flagella. The results from this study indicate that Giardia is able to simultaneously generate both ciliary beating and typical eukaryotic flagellar beating using different pairs of flagella.

Nanofibers and nanoparticles from the insect-capturing adhesive of the Sundew (Drosera) for cell attachment.

BACKGROUND: The search for naturally occurring nanocomposites with diverse properties for tissue engineering has been a major interest for biomaterial research. In this study, we investigated a nanofiber and nanoparticle based nanocomposite secreted from an insect-capturing plant, the Sundew, for cell attachment. The adhesive nanocomposite has demonstrated high biocompatibility and is ready to be used with minimal preparation. RESULTS: Atomic force microscopy (AFM) conducted on the adhesive from three species of Sundew found that a network of nanofibers and nanoparticles with various sizes existed independent of the coated surface. AFM and light microscopy confirmed that the pattern of nanofibers corresponded to Alcian Blue staining for polysaccharide. Transmission electron microscopy identified a low abundance of nanoparticles in different pattern form AFM observations. In addition, energy-dispersive X-ray spectroscopy revealed the presence of Ca, Mg, and Cl, common components of biological salts. Study of the material properties of the adhesive yielded high viscoelasticity from the liquid adhesive, with reduced elasticity observed in the dried adhesive. The ability of PC12 neuron-like cells to attach and grow on the network of nanofibers created from the dried adhesive demonstrated the potential of this network to be used in tissue engineering, and other biomedical applications. CONCLUSIONS: This discovery demonstrates how a naturally occurring nanofiber and nanoparticle based nanocomposite from the adhesive of Sundew can be used for tissue engineering, and opens the possibility for further examination of natural plant adhesives for biomedical applications.