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.

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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.

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.cell motility | cytoskeleton | swimming microorganism | ciliary and flagellar motion | bio-robotics

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.

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 106-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.

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.

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.