Insights to Phenylalanine Ammonia Lyase (PAL) and Secondary Metabolism in Orchids: An in silico Approach.

The phenylalanine ammonia lyase (PAL) catalyses the first step of phenylpropanoid metabolic pathway which leads to the biosynthesis of a diverse group of secondary metabolites. Orchids serve as a rich source of metabolites and the availability of genome or transcriptome for selected orchid species provides an opportunity to analyse the PAL genes in orchids. In the present study, 21 PAL genes were characterized using bioinformatics tools in nine orchid species (Apostasia shenzhenica, Cypripedium formosanum, Dendrobium catenatum, Phalaenopsis aphrodite, Phalaenopsis bellina, Phalaenopsis equestris, Phalaenopsis lueddemanniana, Phalaenopsis modesta and Phalaenopsis schilleriana). Multiple sequence alignment confirmed the presence of PAL-specific conserved domains (N-terminal, MIO, core, shielding and C-terminal domain). All these proteins were predicted to be hydrophobic in nature and to have cytoplasmic localisation. Structural modelling depicted the presence of alpha helices, extended strands, beta turns and random coils in their structure. Ala-Ser-Gly triad known for substrate binding and catalysis of MIO-domain was found to be completely conserved in all the proteins. Phylogenetic study showed that the PALs of pteridophytes, gymnosperms and angiosperms clustered together in separate clades. Expression profiling showed tissue-specific expression for all the 21 PAL genes in the various reproductive and vegetative tissues which suggested their diverse role in growth and development. This study provides insights to the molecular characterization of PAL genes which may help in developing biotechnological strategies to enhance the synthesis of phenylpropanoids in orchids and other heterologous systems for pharmaceutical applications.

Vaccine production and supply need a paradigm change.

We discuss the impact of COVID-19, the journey towards developing vaccines against the disease, and how biomanufacturing should evolve in order to meet similar challenges in the future.

A Biogenic Photovoltaic Material.

A proof-of-concept for the fabrication of genetically customizable biogenic materials for photovoltaic applications is presented. E. coli is first genetically engineered to heterologously express the carotenoid biosynthetic pathway from plants. This modification yields a strain that overproduces the photoactive pigment lycopene. The pigment-producing cells are then coated with TiO2 nanoparticles via a tryptophan-mediated supramolecular interface, and subsequent incorporation of the resulting biogenic material (cells@TiO2 ) as an anode in an I- /I3- -based dye-sensitized solar cell yields an excellent photovoltaic (PV) response. This work lays strong foundations for the development of bio-PV materials and next-generation organic optoelectronics that are green, inexpensive, and easy to manufacture.

Cheminformatic Analysis of Antimalarial Chemical Space Illuminates Therapeutic Mechanisms and Offers Strategies for Therapy Development.

The clear and present danger of malaria, which has been amplified in recent years by climate change, and the progressive thinning of our drug arsenal over the past two decades raise uncomfortable questions about the current state and future of antimalarial drug development. Besides suffering from many of the same technical challenges that affect drug development in other disease areas, the quest for new antimalarial therapies is also hindered by the complex, dynamic life cycle of the malaria parasite, P. falciparum, in its mosquito and human hosts, and its role thereof in the elicitation of drug resistance. New strategies are needed in order to ensure economical and expeditious development of new, more efficacious treatments. In the present study, we employ open-source cheminformatics tools to analyze the chemical space traversed by approved antimalarial drugs and promising candidates at various stages of development to uncover insights that could shape future endeavors in the field. Our scaffold-centric analysis reveals that the antimalarial chemical space is disjointed and segregated into a few dominant structural groups. In fact, the structures of antimalarial drugs and drug candidates are distributed according to Pareto's principle. This structural convergence can potentially be exploited for future drug discovery by incorporating it into bioinformatics workflows that are typically employed for solving problems in structural biology. Significantly, we demonstrate how molecular scaffold hunting can be applied to unearth putative mechanisms of action of drugs whose activities remain a mystery, and how scaffold-centric analysis of drug space can also provide a recipe for combination therapies that minimize the likelihood of emergence of drug resistance, as well as identify areas on which to focus efforts. Finally, we also observe that over half of the molecules in the antimalarial space bear no resemblance to other molecules in the collection, which suggests that the pharmacobiology of antimalarial drugs has not been entirely surveyed.

Towards Precision Engineering of Canonical Polyketide Synthase Domains: Recent Advances and Future Prospects.

Modular polyketide synthases (mPKSs) build functionalized polymeric chains, some of which have become blockbuster therapeutics. Organized into repeating clusters (modules) of independently-folding domains, these assembly-line-like megasynthases can be engineered by introducing non-native components. However, poor introduction points and incompatible domain combinations can cause both unintended products and dramatically reduced activity. This limits the engineering and combinatorial potential of mPKSs, precluding access to further potential therapeutics. Different regions on a given mPKS domain determine how it interacts both with its substrate and with other domains. Within the assembly line, these interactions are crucial to the proper ordering of reactions and efficient polyketide construction. Achieving control over these domain functions, through precision engineering at key regions, would greatly expand our catalogue of accessible polyketide products. Canonical mPKS domains, given that they are among the most well-characterized, are excellent candidates for such fine-tuning. The current minireview summarizes recent advances in the mechanistic understanding and subsequent precision engineering of canonical mPKS domains, focusing largely on developments in the past year.

The mechanical effects of chemical stimuli on neurospheres.

The formulation of more accurate models to describe tissue mechanics necessitates the availability of tools and instruments that can precisely measure the mechanical response of tissues to physical loads and other stimuli. In this regard, neuroscience has trailed other life sciences owing to the unavailability of representative live tissue models and deficiency of experimentation tools. We previously addressed both challenges by employing a novel instrument called the cantilevered-capillary force apparatus (CCFA) to elucidate the mechanical properties of mouse neurospheres under compressive forces. The neurospheres were derived from murine stem cells, and our study was the first of its kind to investigate the viscoelasticity of living neural tissues in vitro. In the current study, we demonstrate the utility of the CCFA as a broadly applicable tool to evaluate tissue mechanics by quantifying the effect that oxidative stress has on the mechanical properties of neurospheres. We treated mouse neurospheres with non-cytotoxic levels of hydrogen peroxide and subsequently evaluated the storage and loss moduli of the tissues under compression and tension. We observed that the neurospheres exhibit viscoelasticity consistent with neural tissue and show that elastic modulus decreases with increasing size of the neurosphere. Our study yields insights for establishing rheological measurements as biomarkers by laying the groundwork for measurement techniques and showing that the influence of a particular treatment may be misinterpreted if the size dependence is ignored.

Polyketide synthases (PKSs) of secondary metabolism: in silico identification and characterization in orchids.

Type III polyketide synthases (PKSs) catalyse the formation of an array of polyketides with diverse structures that play an important role in secondary metabolism in plants. This group of enzymes is encoded by a multigene family, the Type III polyketide synthase (PKS) gene family. Vast reserves of secondary metabolites in orchids make these plants suitable candidates for research in the area. In this study, genome-wide searches lead to the identification of five PeqPKS, eight DcaPKS and six AshPKS genes in Phalaenopsis equestris, Dendrobium catenatum and Apostasia shenzhenica, respectively. All the members showed the presence of two characteristic conserved domains (Chal_sti_synt_N and Chal_sti_synt_C) and were generally localised in the cytoplasm. The phylogenetic analysis led to the classification of these proteins into two groups: CHS (chalcone synthase (CHS) and non-CHS. A single protein in P. equestris and two proteins each in D. catenatum and A. shenzhenica clustered within the CHS clade. The majority of the genes exhibited similar structural patterns with a single intron. Expression profiling revealed the tissue-specific expression of these genes with high expression in reproductive tissues for most genes. A number of stress-responsive cis-regulatory elements were predicted, noteworthy amongst these are, ABRE and CGTCA that are chiefly responsible for responding to abscisic acid and methyl jasmonate, respectively. Our study provides a reference framework for future studies involving functional elucidation of PKS genes and biotechnological production of polyketides.Communicated by Ramaswamy H. Sarma.

Identification of five PeqPKS, eight DcaPKS and six AshPKS genes in Phalaenopsis equestris, Dendrobium catenatum and Apostasia shenzhenica based on genome-wide analysisPresence of characteristic conserved domains (Chal_sti_synt_N, Chal_sti_synt_C) with cytological localisationPhylogenetic clustering into two groups, CHS chalcone synthase (CHS) and non-CHSExpression profiling revealing high expression in reproductive tissuesPrediction of stress-responsive cis-regulatory elements like ABRE and CGTCA.

Green synthesis of lignin-based nanoparticles as a bio-carrier for targeted delivery in cancer therapy.

Polymeric magnetic nanoparticles have shown higher efficacy in cancer diagnosis and treatment than conventional chemotherapies. Lignin is an abundantly available natural polymer that can be selectively modified using a rapidly expanding toolkit of biocatalytic and chemical reactions to yield 'intelligent' theranostic-nanoprobes. We aim to valorize lignin to develop a natural polymeric-magnetic-nano-system for the targeted delivery of methotrexate. In the current study, we synthesized nanoparticles of lignin and iron oxide with methotrexate using a new approach of anti-solvent precipitation with ultrasonication. The ensuing nanoparticles are magnetic, smooth, polyhedral with characteristic dimension of 110-130 nm. The drug loading and encapsulation efficiencies were calculated to be 66.06 % and 64.88 %, respectively. The nanoparticles exhibit a concentration-dependent release of methotrexate for the initial 24 h, followed by sustained release. Moreover, formulation is non-hemolytic and scavenges radicals owing to the antioxidant property of lignin. Additionally, methotrexate delivered using the nanoparticles exhibited higher cytotoxicity in cellular-viability assays employing breast cancer and macrophage cell lines compared to the pure form of the drug. Synergistic action of lignin, iron oxide, and methotrexate contribute to enhanced caspase-3 activity and reduced glutathione levels in the breast cancer cells, as well as elevated internalization of the drug on account of increased receptor-mediated endocytosis.

The future of metabolic engineering and synthetic biology: towards a systematic practice.

Industrial biotechnology promises to revolutionize conventional chemical manufacturing in the years ahead, largely owing to the excellent progress in our ability to re-engineer cellular metabolism. However, most successes of metabolic engineering have been confined to over-producing natively synthesized metabolites in E. coli and S. cerevisiae. A major reason for this development has been the descent of metabolic engineering, particularly secondary metabolic engineering, to a collection of demonstrations rather than a systematic practice with generalizable tools. Synthetic biology, a more recent development, faces similar criticisms. Herein, we attempt to lay down a framework around which bioreaction engineering can systematize itself just like chemical reaction engineering. Central to this undertaking is a new approach to engineering secondary metabolism known as 'multivariate modular metabolic engineering' (MMME), whose novelty lies in its assessment and elimination of regulatory and pathway bottlenecks by re-defining the metabolic network as a collection of distinct modules. After introducing the core principles of MMME, we shall then present a number of recent developments in secondary metabolic engineering that could potentially serve as its facilitators. It is hoped that the ever-declining costs of de novo gene synthesis; the improved use of bioinformatic tools to mine, sort and analyze biological data; and the increasing sensitivity and sophistication of investigational tools will make the maturation of microbial metabolic engineering an autocatalytic process. Encouraged by these advances, research groups across the world would take up the challenge of secondary metabolite production in simple hosts with renewed vigor, thereby adding to the range of products synthesized using metabolic engineering.

A Pichia biosensor for high-throughput analyses of compounds that can influence mosquito behavior.

Mosquitoes utilize their sense of smell to locate prey and feed on their blood. Repellents interfere with the biochemical cascades that detect odors. Consequently, repellants are highly effective and resource-efficient alternatives for controlling the spread of mosquito-borne illnesses. Unfortunately, the discovery of repellents is slow, laborious, and error-prone. To this end, we have taken a giant stride toward improving the speed and accuracy of repellant discovery by constructing a prototypical whole-cell biosensor for accurate detection of mosquito behavior-modifying compounds such as repellants. As a proof-of-concept, we genetically engineered Pichia pastoris to express the olfactory receptor co-receptor (Orco) of Anopheles gambiae mosquitoes. This transmembrane protein behaves like a cationic channel upon activation by stimulatory odorants. When the engineered Pichia cells are cultured in calcium-containing Hank's buffer, induction of the medium with a stimulatory odorant results in an influx of calcium ions into the cells, and the stimulatory effect is quantifiable using the calcium-sequestering fluorescent dye, fluo-4-acetoxymethyl ester. Moreover, the stimulatory effect can be titrated by adjusting either the concentration of calcium ions in the medium or the level of induction of the stimulatory odorant. Subsequent exposure of the activated Pichia cells to a repellant molecule inhibits the stimulatory effect and quenches the fluorescent signal, also in a titratable manner. Significantly, the modular architecture of the biosensor allows easy and efficient expansion of its detection range by co-expressing Orco with other olfactory receptors. The high-throughput assay is also compatible with robotic screening infrastructure, and our development represents a paradigm change for the discovery of mosquito repellants.

Transcriptome Analysis of Environmental Pseudomonas Isolates Reveals Mechanisms of Biodegradation of Naphthenic Acid Fraction Compounds (NAFCs) in Oil Sands Tailings.

Naphthenic acid fraction compounds (NAFCs) are highly recalcitrant constituents of oil sands tailings. Although some microorganisms in the tailings can individually and synergistically metabolize NAFCs, the biochemical mechanisms that underpin these processes are hitherto unknown. To this end, we isolated two microorganisms, Pseudomonas protegens and Pseudomonas putida, from oils sands tailings and analyzed their transcriptomes to shed light on the metabolic processes employed by them to degrade and detoxify NAFCs. We identified 1048, 521 and 1434 genes that are upregulated in P. protegens, P. putida and a 1:1 co-culture of the strains, respectively. We subsequently enumerated the biochemical activities of enriched genes and gene products to reveal the identities of the enzymes that are associated with NAFC degradation. Separately, we analyzed the NAFCs that are degraded by the two pseudomonads and their 1:1 co-culture and determined the composition of the molecules using mass spectrometry. We then compared these molecular formulas to those of the cognate substrates of the enriched enzymes to chart the metabolic network and understand the mechanisms of degradation that are employed by the microbial cultures. Not only does the consortium behave differently than the pure cultures, but our analysis also revealed the mechanisms responsible for accelerated rate of degradation of NAFCs by the co-culture. Our findings provide new directions for engineering or evolving microorganisms and their consortia for degrading NAFCs more stably and aggressively.

Optogenetic Control of Heterologous Metabolism in E. coli.

Multiobjective optimization of microbial chassis for the production of xenobiotic compounds requires the implementation of metabolic control strategies that permit dynamic distribution of cellular resources between biomass and product formation. We addressed this need in a previous study by engineering the T7 RNA polymerase to be thermally responsive. The modified polymerase is activated only after the temperature of the host cell falls below 18 °C, and Escherichia coli cells that employ the protein to transcribe the heterologous lycopene biosynthetic pathway exhibit impressive improvements in productivity. We have expanded our toolbox of metabolic switches in the current study by engineering a version of the T7 RNA polymerase that drives the transition between biomass and product formation upon stimulation with red light. The engineered polymerase is expressed as two distinct polypeptide chains. Each chain comprises one of two photoactive components from Arabidopsis thaliana, phytochrome B (PhyB) and phytochrome-integrating factor 3 (PIF3), as well as the N- or C-terminus domains of both, the vacuolar ATPase subunit (VMA) intein of Saccharomyces cerevisiae and the polymerase. Red light drives photodimerization of PhyB and PIF3, which then brings together the N- and C-terminus domains of the VMA intein. Trans-splicing of the intein follows suit and produces an active form of the polymerase that subsequently transcribes any sequence that is under the control of a T7 promoter. The photodimerization also involves a third element, the cyanobacterial chromophore phycocyanobilin (PCB), which too is expressed heterologously by E. coli. We deployed this version of the T7 RNA polymerase to control the production of lycopene in E. coli and observed tight control of pathway expression. We tested a variety of expression configurations to identify one that imposes the lowest metabolic burden on the strain, and we subsequently optimized key parameters such as the source, moment, and duration of photostimulation. We also identified targets for future refinement of the circuit. In summary, our work is a significant advance for the field and greatly expands on previous work by other groups that have used optogenetic circuits to control heterologous metabolism in prokaryotic hosts.

Engineering Microbes for Remediation of Oil Sands Tailings.

Synthetic biology and adaptive laboratory evolution are key tools for developing biotechnology platforms for the remediation of oil sands tailings. However, field deployment and subsequent regulation of engineered and/or evolved strains is rife with uncertainties and risks. Here, we detail an innovation strategy to derisk and deploy engineered bioremediation platforms.

The production of fuels and chemicals in the new world: critical analysis of the choice between crude oil and biomass vis-à-vis sustainability and the environment.

ABSTRACT: Energy and the environment are intimately related and hotly debated issues. Today's crude oil-based economy for the manufacture of fuels, chemicals and materials will not have a sustainable future. The over-use of oil products has done a great damage to the environment. Faced with the twin challenges of sustaining socioeconomic development and shrinking the environmental footprint of chemicals and fuel manufacturing, a major emphasis is on either converting biomass into low-value, high-volume biofuels or refining it into a wide spectrum of products. Using carbon for fuel is a flawed approach and unlikely to achieve any nation's socioeconomic or environmental targets. Biomass is chemically and geographically incompatible with the existing refining and pipeline infrastructure, and biorefining and biofuels production in their current forms will not achieve economies of scale in most nations. Synergistic use of crude oil, biomass, and shale gas to produce fuels, value-added chemicals, and commodity chemicals, respectively, can continue for some time. However, carbon should not be used as a source of fuel or energy but be valorized to other products. In controlling CO2 emissions, hydrogen will play a critical role. Hydrogen is best suited for converting waste biomass and carbon dioxide emanated from different sources, whether it be fossil fuel-derived carbon or biomass-derived carbon, into fuels and chemicals as well as it will also lead, on its own as energy source, to the carbon negative scenario in conjunction with other renewable non-carbon sources. This new paradigm for production of fuels and chemicals not only offers the greatest monetization potential for biomass and shale gas, but it could also scale down output and improve the atom and energy economies of oil refineries. We have also highlighted the technology gaps with the intention to drive R&D in these directions. We believe  this article will generate a considerable debate in energy sector and lead to better energy and material policy across the world.

Improving the Rate of Translation of Tissue Engineering Products.

Over 100 000 research articles and 9000 patents have been published on tissue engineering (TE) in the past 20 years. Yet, very few TE products have made their way to the market during the same period. Experts have proposed a variety of strategies to address the lack of translation of TE products. However, since these proposals are guided by qualitative insights, they are limited in scope and impact. Machine learning is utilized in the current study to analyze the entire body of patents that have been published over the past twenty years and understand patenting trends, topics, areas of application, and exemplifications. This analysis yields surprising and little-known insights about the differences in research priorities and perceptions of innovativeness of tissue engineers in academia and industry, as well as aids to chart true advances in the field during the past twenty years. It is hoped that this analysis and subsequent proposal to improve translational rates of TE products will spur much needed dialogue about this important pursuit.

Brain Organoids: A New, Transformative Investigational Tool for Neuroscience Research.

Brain organoids are self-assembled, three-dimensionally structured tissues that are typically derived from pluripotent stem cells. They are multicellular aggregates that more accurately recapitulate the tissue microenvironment compared to the other cell culture systems and can also reproduce organ function. They are promising models for evaluating drug leads, particularly those that target neurodegeneration, since they are genetically and phenotypically stable over prolonged durations of culturing and they reasonably reproduce critical physiological phenomena such as biochemical gradients and responses by the native tissue to stimuli. Beyond drug discovery, the use of brain organoids could also be extended to investigating early brain development and identifying the mechanisms that elicit neurodegeneration. Herein, the current state of the fabrication and use of brain organoids in drug development and medical research is summarized. Although the use of brain organoids represents a quantum leap over existing investigational tools used by the pharmaceutical industry, they are nonetheless imperfect systems that could be greatly improved through bioengineering. To this end, some key scientific challenges that would need to be addressed in order to enhance the relevance of brain organoids as model tissue are listed. Potential solutions to these challenges, including the use of bioprinting, are highlighted thereafter.

Bionic Manufacturing: Towards Cyborg Cells and Sentient Microbots.

Bio-inspired engineering applies biological design principles towards developing engineering solutions but is not practical as a manufacturing paradigm. We advocate 'bionic manufacturing', a synergistic fusion of biotic and abiotic components, to transition away from bio-inspiration toward bio-augmentation to address current limitations in bio-inspired manufacturing.

Metagenomic discovery of a novel transaminase for valorization of monoaromatic compounds.

The profitability of next-generation biorefineries is acutely contingent on the discovery and utilization of biocatalysts that can valorize lignin. To this end, the metabolic catalogues of diverse microbiota have been mined previously using functional metagenomics in order to identify biocatalysts that can selectively degrade lignin into monoaromatic compounds. Herein, we have further improved the valorization factor of biorefining by deploying functional metagenomics toward the identification of a novel transaminase that can selectively functionalize lignin-derived monoaromatics to produce value-added feedstocks for pharmaceutical synthesis. We implemented a high-throughput colorimetric assay using o-xylylenediamine as the amino donor and successfully identified a transaminase that utilizes the canonical cofactor, pyridoxal 5'-phosphate, to aminate as many as 14 monoaromatic aldehydes and ketones. We subsequently identified the optimal conditions for enzyme activity towards the most favoured amino acceptor, benzaldehyde, including temperature, pH and choice of co-solvent. We also evaluated the specificity of the enzyme towards a variety of amino donors, as well as the optimal concentration of the most favoured amino donor. Significantly, the novel enzyme is markedly smaller than typical transaminases, and it is stably expressed in E. coli without any modifications to its amino acid sequence. Finally, we developed and implemented a computational methodology to assess the activity of the novel transaminase. The methodology is generalizable for assessing any transaminase and facilitates in silico screening of enzyme-substrate combinations in order to develop efficient biocatalytic routes to value-added amines. The computational pipeline is an ideal complement to metagenomics and opens new possibilities for biocatalyst discovery.

An Improved Whole-Cell Biosensor for the Discovery of Lignin-Transforming Enzymes in Functional Metagenomic Screens.

The discovery and utilization of biocatalysts that selectively valorize lignocellulose is critical to the profitability of next-generation biorefineries. Here, we report the development of a refactored, whole-cell, GFP-based biosensor for high-throughput identification of biocatalysts that transform lignin into specialty chemicals from environmental DNA of uncultivable archaea and bacteria. The biosensor comprises the transcriptional regulator and promoter of the emrRAB operon of E. coli, and the configuration of the biosensor was tuned with the aid of mathematical model. The biosensor sensitively and selectively detects vanillin and syringaldehyde, and responds linearly over a wide detection range. We employed the biosensor to screen 42 520 fosmid clones comprising environmental DNA isolated from two coal beds and successfully identified 147 clones that transform hardwood kraft lignin to vanillin and syringaldehyde.

A stimulus-responsive, in situ-forming, nanoparticle-laden hydrogel for ocular drug delivery.

Most medications targeting optic neuropathies are administered as eye drops. However, their corneal penetration efficiencies are typically < 5%. There is a clear, unmet need for novel transcorneal drug delivery vehicles. To this end, we have developed a stimulus-responsive, in situ-forming, nanoparticle-laden hydrogel for controlled release of poorly bioavailable drugs into the aqueous humor of the eye. The hydrogel is formulated as a composite of hyaluronic acid (HA) and methylcellulose (MC). The amphiphilic nanoparticles are composed of poly(ethylene oxide) (PEO) and poly(lactic acid) (PLA). Experimental design aided the identification of hydrogel composition and nanoparticle content in the formulation, and the formulation reliably switched between thixotropy and temperature-dependent rheopexy when it was tested in a rheometer under conditions that simulate the ocular surface, including blinking. These properties should ensure that the formulation coats the cornea through blinking of the eyelid and facilitate application of the medication as an eye drop immediately prior to the patient's bedtime. We subsequently tested the efficacy of our formulation in whole-eye experiments by loading the nanoparticles with cannabigerolic acid (CBGA). Our formulation exhibits over a 300% increase in transcorneal penetration over control formulations. This work paves the way for the introduction of novel products targeting ocular diseases to the market.