Cannabinoid Biosynthesis Using Noncanonical Cannabinoid Synthases.

We report enzymes from the berberine bridge enzyme (BBE) superfamily that catalyze the oxidative cyclization of the monoterpene moiety in cannabigerolic acid (CBGA) to form cannabielsoin (CBE). The enzymes are from a variety of organisms and are previously uncharacterized. Out of 232 homologues chosen from the enzyme superfamily, four orthologues were shown to accept CBGA as a substrate and catalyze the biosynthesis of CBE. The four enzymes discovered in this study were recombinantly expressed and purified in Pichia pastoris. These enzymes are the first report of heterologous expression of BBEs that did not originate from the Cannabis plant that catalyze the production of cannabinoids using CBGA as substrate. This study details a new avenue for discovering and producing natural and unnatural cannabinoids.

Engineered Nucleotide Chemicapacitive Microsensor Array Augmented with Physics-Guided Machine Learning for High-Throughput Screening of Cannabidiol.

The recent legalization of cannabidiol (CBD) to treat neurological conditions such as epilepsy has sparked rising interest across global pharmaceuticals and synthetic biology industries to engineer microbes for sustainable synthetic production of medicinal CBD. Since the process involves screening large amounts of samples, the main challenge is often associated with the conventional screening platform that is time consuming, and laborious with high operating costs. Here, a portable, high-throughput Aptamer-based BioSenSing System (ABS3 ) is introduced for label-free, low-cost, fully automated, and highly accurate CBD concentrations' classification in a complex biological environment. The ABS3 comprises an array of interdigitated microelectrode sensors, each functionalized with different engineered aptamers. To further empower the functionality of the ABS3 , unique electrochemical features from each sensor are synergized using physics-guided multidimensional analysis. The capabilities of this ABS3 are demonstrated by achieving excellent CBD concentrations' classification with a high prediction accuracy of 99.98% and a fast testing time of 22 µs per testing sample using the optimized random forest (RF) model. It is foreseen that this approach will be the key to the realistic transformation from fundamental research to system miniaturization for diagnostics of disease biomarkers and drug development in the field of chemical/bioanalytics.

COVID-19 endocrinopathy with hindsight from SARS.

The current COVID-19 pandemic is probably the worst the world has ever faced since the start of the new millennium. Although the respiratory system is the most prominent target of SARS-CoV-2 (the contagion of COVID-19), extrapulmonary involvement are emerging as important contributors of its morbidity and lethality. This article summarizes the impact of SARS-CoV and SARS-CoV-2 on the endocrine system to facilitate our understanding of the nature of coronavirus-associated endocrinopathy. Although new data are rapidly accumulating on this novel infection, many of the endocrine manifestations of COVID-19 remain incompletely elucidated. We, hereby, summarize various endocrine dysfunctions including coronavirus-induced new onset diabetes mellitus, hypocortisolism, thyroid hormone, and reproductive system aberrations so that clinicians armed with such insights can potentially benefit patients with COVID-19 at the bedside.

Structure of a Minimal α-Carboxysome-Derived Shell and Its Utility in Enzyme Stabilization.

Bacterial microcompartments are proteinaceous shells that encase specialized metabolic processes in bacteria. Recent advances in simplification of these intricate shells have encouraged bioengineering efforts. Here, we construct minimal shells derived from the Halothiobacillus neapolitanus α-carboxysome, which we term Cso-shell. Using cryogenic electron microscopy, the atomic-level structures of two shell forms were obtained, reinforcing notions of evolutionarily conserved features in bacterial microcompartment shell architecture. Encapsulation peptide sequences that facilitate loading of heterologous protein cargo within the shells were identified. We further provide a first demonstration in utilizing minimal bacterial microcompartment-derived shells for hosting heterologous enzymes. Cso-shells were found to stabilize enzymatic activities against heat shock, presence of methanol co-solvent, consecutive freeze-thawing, and alkaline environments. This study yields insights into α-carboxysome assembly and advances the utility of synthetic bacterial microcompartments as nanoreactors capable of stabilizing enzymes with varied properties and reaction chemistries.

A Novel Lipase from Lasiodiplodia theobromae Efficiently Hydrolyses C8-C10 Methyl Esters for the Preparation of Medium-Chain Triglycerides' Precursors.

Medium-chain triglycerides (MCTs) are an emerging choice to treat neurodegenerative disorders such as Alzheimer's disease. They are triesters of glycerol and three medium-chain fatty acids, such as capric (C8) and caprylic (C10) acids. The availability of C8-C10 methyl esters (C8-C10 ME) from vegetable oil processes has presented an opportunity to use methyl esters as raw materials for the synthesis of MCTs. However, there are few reports on enzymes that can efficiently hydrolyse C8-C10 ME to industrial specifications. Here, we report the discovery and identification of a novel lipase from Lasiodiplodia theobromae fungus (LTL1), which hydrolyses C8-C10 ME efficiently. LTL1 can perform hydrolysis over pH ranges from 3.0 to 9.0 and maintain thermotolerance up to 70 °C. It has high selectivity for monoesters over triesters and displays higher activity over commercially available lipases for C8-C10 ME to achieve 96.17% hydrolysis within 31 h. Structural analysis by protein X-ray crystallography revealed LTL1's well-conserved lipase core domain, together with a partially resolved N-terminal subdomain and an inserted loop, which may suggest its hydrolytic preference for monoesters. In conclusion, our results suggest that LTL1 provides a tractable route towards to production of C8-C10 fatty acids from methyl esters for the synthesis of MCTs.

The Divergent Immunomodulatory Effects of Short Chain Fatty Acids and Medium Chain Fatty Acids.

Fatty acids are derived from diet and fermentative processes by the intestinal flora. Two to five carbon chain fatty acids, termed short chain fatty acids (SCFA) are increasingly recognized to play a role in intestinal homeostasis. However, the characteristics of slightly longer 6 to 10 carbon, medium chain fatty acids (MCFA), derived primarily from diet, are less understood. Here, we demonstrated that SCFA and MCFA have divergent immunomodulatory propensities. SCFA down-attenuated host pro-inflammatory IL-1ß, IL-6, and TNFα response predominantly through the TLR4 pathway, whereas MCFA augmented inflammation through TLR2. Butyric (C4) and decanoic (C10) acid displayed most potent modulatory effects within the SCFA and MCFA, respectively. Reduction in TRAF3, IRF3 and TRAF6 expression were observed with butyric acid. Decanoic acid induced up-regulation of GPR84 and PPARγ and altered HIF-1α/HIF-2α ratio. These variant immune characteristics of the fatty acids which differ by just several carbon atoms may be attributable to their origins, with SCFA being primarily endogenous and playing a physiological role, and MCFA exogenously from the diet.

Genetically Encodable Scaffolds for Optimizing Enzyme Function.

Enzyme engineering is an indispensable tool in the field of synthetic biology, where enzymes are challenged to carry out novel or improved functions. Achieving these goals sometimes goes beyond modifying the primary sequence of the enzyme itself. The use of protein or nucleic acid scaffolds to enhance enzyme properties has been reported for applications such as microbial production of chemicals, biosensor development and bioremediation. Key advantages of using these assemblies include optimizing reaction conditions, improving metabolic flux and increasing enzyme stability. This review summarizes recent trends in utilizing genetically encodable scaffolds, developed in line with synthetic biology methodologies, to complement the purposeful deployment of enzymes. Current molecular tools for constructing these synthetic enzyme-scaffold systems are also highlighted.

Biosynthesis of Nature-Inspired Unnatural Cannabinoids.

Natural products make up a large proportion of medicine available today. Cannabinoids from the plant Cannabis sativa is one unique class of meroterpenoids that have shown a wide range of bioactivities and recently seen significant developments in their status as therapeutic agents for various indications. Their complex chemical structures make it difficult to chemically synthesize them in efficient yields. Synthetic biology has presented a solution to this through metabolic engineering in heterologous hosts. Through genetic manipulation, rare phytocannabinoids that are produced in low yields in the plant can now be synthesized in larger quantities for therapeutic and commercial use. Additionally, an exciting avenue of exploring new chemical spaces is made available as novel derivatized compounds can be produced and investigated for their bioactivities. In this review, we summarized the biosynthetic pathways of phytocannabinoids and synthetic biology efforts in producing them in heterologous hosts. Detailed mechanistic insights are discussed in each part of the pathway in order to explore strategies for creating novel cannabinoids. Lastly, we discussed studies conducted on biological targets such as CB1, CB2 and orphan receptors along with their affinities to these cannabinoid ligands with a view to inform upstream diversification efforts.

Engineering microbes for targeted strikes against human pathogens.

Lack of pathogen specificity in antimicrobial therapy causes non-discriminant microbial cell killing that disrupts the microflora present. As a result, potentially helpful microbial cells are killed along with the pathogen, altering the biodiversity and dynamic interactions within the population. Moreover, the unwarranted exposure of antibiotics to microbes increases the likelihood of developing resistance and perpetuates the emergence of multidrug resistance. Synthetic biology offers an alternative solution where specificity can be conferred to reduce the non-specific, non-targeted activity of currently available antibiotics, and instead provides targeted therapy against specific pathogens and minimising collateral damage to the host's inherent microbiota. With a greater understanding of the microbiome and the available genetic engineering tools for microbial cells, it is possible to devise antimicrobial strategies for novel antimicrobial therapy that are able to precisely and selectively remove infectious pathogens. Herein, we review the strategies developed by unlocking some of the natural mechanisms used by the microbes and how these may be utilised in targeted antimicrobial therapy, with the promise of reducing the current global bane of multidrug antimicrobial resistance.

Mutational Mtc6p attenuates autophagy and improves secretory expression of heterologous proteins in Kluyveromyces marxianus.

BACKGROUND: The yeast Kluyveromyces marxianus is an emerging cell factory for heterologous protein biosynthesis and its use holds tremendous advantages for multiple applications. However, which genes influence the productivity of desired proteins in K. marxianus has so far been investigated by very few studies. RESULTS: In this study, we constructed a K. marxianus recombinant (FIM1/Est1E), which expressed the heterologous ruminal feruloyl esterase Est1E as reporter. UV-60Co-γ irradiation mutagenesis was performed on this recombinant, and one mutant (be termed as T1) was screened and reported, in which the productivity of heterologous Est1E was increased by at least tenfold compared to the parental FIM1/Est1E recombinant. Transcriptional perturbance was profiled and presented that the intracellular vesicle trafficking was enhanced while autophagy be weakened in the T1 mutant. Moreover, whole-genome sequencing combined with CRISPR/Cas9 mediated gene-editing identified a novel functional protein Mtc6p, which was prematurely terminated at Tyr251 by deletion of a single cytosine at 755 loci of its ORF in the T1 mutant. We found that deleting C755 of MTC6 in FIM1 led to 4.86-fold increase in the production of Est1E compared to FIM1, while the autophagy level decreased by 47%; on the contrary, when reinstating C755 of MTC6 in the T1 mutant, the production of Est1E decreased by 66% compared to T1, while the autophagy level increased by 124%. Additionally, in the recombinant with attenuated autophagy (i.e., FIM1 mtc6C755Δ and T1) or interdicted autophagy (i.e., FIM1 atg1Δ and T1 atg1Δ), the productivity of three other heterologous proteins was also increased, specifically the heterologous mannase Man330, the ß-1,4-endoxylanase XynCDBFV or the conventional EGFP. CONCLUSIONS: Our results demonstrated that Mtc6p was involved in regulating autophagy; attenuating or interdicting autophagy would dramatically improve the yields of desired proteins in K. marxianus, and this modulation could be achieved by focusing on the premature mutation of Mtc6p target.

Reprogrammable microbial cell-based therapeutics against antibiotic-resistant bacteria.

The discovery of antimicrobial drugs and their subsequent use has offered an effective treatment option for bacterial infections, reducing morbidity and mortality over the past 60 years. However, the indiscriminate use of antimicrobials in the clinical, community and agricultural settings has resulted in selection for multidrug-resistant bacteria, which has led to the prediction of possible re-entrance to the pre-antibiotic era. The situation is further exacerbated by significantly reduced antimicrobial drug discovery efforts by large pharmaceutical companies, resulting in a steady decline in the number of new antimicrobial agents brought to the market in the past several decades. Consequently, there is a pressing need for new antimicrobial therapies that can be readily designed and implemented. Recently, it has become clear that the administration of broad-spectrum antibiotics can lead to collateral damage to the human commensal microbiota, which plays several key roles in host health. Advances in genetic engineering have opened the possibility of reprogramming commensal bacteria that are in symbiotic existence throughout the human body to implement antimicrobial drugs with high versatility and efficacy against pathogenic bacteria. In this review, we discuss recent advances and potentialities of engineered bacteria in providing a novel antimicrobial strategy against antibiotic resistance.

Loss of quaternary structure is associated with rapid sequence divergence in the OSBS family.

The rate of protein evolution is determined by a combination of selective pressure on protein function and biophysical constraints on protein folding and structure. Determining the relative contributions of these properties is an unsolved problem in molecular evolution with broad implications for protein engineering and function prediction. As a case study, we examined the structural divergence of the rapidly evolving o-succinylbenzoate synthase (OSBS) family, which catalyzes a step in menaquinone synthesis in diverse microorganisms and plants. On average, the OSBS family is much more divergent than other protein families from the same set of species, with the most divergent family members sharing <15% sequence identity. Comparing 11 representative structures revealed that loss of quaternary structure and large deletions or insertions are associated with the family's rapid evolution. Neither of these properties has been investigated in previous studies to identify factors that affect the rate of protein evolution. Intriguingly, one subfamily retained a multimeric quaternary structure and has small insertions and deletions compared with related enzymes that catalyze diverse reactions. Many proteins in this subfamily catalyze both OSBS and N-succinylamino acid racemization (NSAR). Retention of ancestral structural characteristics in the NSAR/OSBS subfamily suggests that the rate of protein evolution is not proportional to the capacity to evolve new protein functions. Instead, structural features that are conserved among proteins with diverse functions might contribute to the evolution of new functions.

Exploiting the Biosynthetic Potential of Type III Polyketide Synthases.

Polyketides are structurally and functionally diverse secondary metabolites that are biosynthesized by polyketide synthases (PKSs) using acyl-CoA precursors. Recent studies in the engineering and structural characterization of PKSs have facilitated the use of target enzymes as biocatalysts to produce novel functionally optimized polyketides. These compounds may serve as potential drug leads. This review summarizes the insights gained from research on type III PKSs, from the discovery of chalcone synthase in plants to novel PKSs in bacteria and fungi. To date, at least 15 families of type III PKSs have been characterized, highlighting the utility of PKSs in the development of natural product libraries for therapeutic development.

Glycine decarboxylase is an unusual amino acid decarboxylase involved in tumorigenesis.

Glycine decarboxylase (GLDC) is a metabolic oncogene that links glycine metabolism with tumorigenesis. In humans, GLDC is part of a multienzyme complex (which includes the lipoyl-containing H-protein) that couples the decarboxylation of glycine to the biosynthesis of serine. Details of the GLDC-catalyzed glycine decarboxylation reaction are critical to drug development but remain elusive. This is the first report on the mechanism of the GLDC-catalyzed reaction and shows that GLDC is an unusual PLP-containing α-amino acid decarboxylase that removes carbon dioxide from the glycine substrate without releasing the expected amine (methylamine, a metabolic precursor of toxic formaldehyde) as a product. In an unusual decarboxylation mechanism, the resulting aminomethyl moiety is instead transferred to an accessory H-protein. This study defines the role of H-protein in GLDC-catalyzed glycine decarboxylation. (1) H-Protein is not required for glycine decarboxylation but, instead, is required for the release of the aminomethyl moiety from the quinonoid adduct. (2) Glycine decarboxylation is reversible and presumably proceeds through a stable quinonoid intermediate. (3) The physiological product of glycine decarboxylation is H-protein-S-aminomethyl dihydrolipoyllysine and not methylamine (in the absence of H-protein, the aminomethyl moiety remains as a quinonoid adduct). Mechanistic insights obtained from this study will inform future efforts for targeted anticancer therapeutic development.

Disruption of biofilm formation by the human pathogen Acinetobacter baumannii using engineered quorum-quenching lactonases.

Acinetobacter baumannii is a major human pathogen associated with multidrug-resistant nosocomial infections; its virulence is attributed to quorum-sensing-mediated biofilm formation, and disruption of biofilm formation is an attractive antivirulence strategy. Here, we report the first successful demonstration of biofilm disruption in a clinical isolate of A. baumannii S1, using a quorum-quenching lactonase obtained by directed evolution; this engineered lactonase significantly reduced the biomass of A. baumannii-associated biofilms, demonstrating the utility of this antivirulence strategy.

Engineering a Metal Reductase for the Bioremediation of Anthropogenic Electronic Wastes: From Hg(II) to Au(III) and Ag(I) Enzymatic Reduction.

Recovering precious metals from electronic waste (e-waste) using microbes presents a sustainable methodology that can contribute toward the maintenance of planetary health. To better realize the potential of bioremediation using engineered microbes, enzymes that mediate the reduction of Au(III) to Au(0) have been the subject of intense research. In this study, we report the successful engineering of a metal reductase, MerA, whose cognate substrate is mercury(II), toward other precious metals such as Au(III) and Ag(I). The engineered variant, G415I, exhibited a 15-fold increase in catalytic efficiency (k cat/K M) in Au(III) reduction to Au(0) and a 200-fold increase in catalytic efficiency in Ag(I) reduction to Ag(0) with respect to the wild-type enzyme. The apparent shift in preference toward noncognate metal ions may be attributed to the energetics of valency preference. The improved Au(III) reductase has an apparent increased preference toward monovalent cations such as Au(I) and Ag(I), with respect to divalent cations such as Hg(II), the cognate substrate of the progenitor MerA (an increase in K M of 5.0-fold for Hg(II), compared to a decrease in K M of 5.8-fold for Au(III) and 1.8-fold for Ag(I), respectively). This study further extends the mechanistic understanding of Au(III) bioreduction that could proceed through the stabilization of Au(I) en route to Au(0) and suggests that the biosynthesis of Au nanoparticles with high efficiency can be realized through the engineering of promiscuous metal reductases for precious metal recovery from e-wastes.

Structural evidence of a productive active site architecture for an evolved quorum-quenching GKL lactonase.

The in vitro evolution and engineering of quorum-quenching lactonases with enhanced reactivities was achieved using a thermostable GKL enzyme as a template, yielding the E101G/R230C GKL mutant with increased catalytic activity and a broadened substrate range [Chow, J. Y., Xue, B., Lee, K. H., Tung, A., Wu, L., Robinson, R. C., and Yew, W. S. (2010) J. Biol. Chem. 285, 40911-40920]. This enzyme possesses the (ß/α)8-barrel fold and is a member of the PLL (phosphotriesterase-like lactonase) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acyl-homoserine lactones, which mediate the quorum-sensing pathways of bacteria. The structure of the evolved N-butyryl-l-homoserine lactone (substrate)-bound E101G/R230C GKL enzyme was determined, in the presence of the inactivating D266N mutation, to a resolution of 2.2 Å to provide an explanation for the observed rate enhancements. In addition, the substrate-bound structure of the catalytically inactive E101N/D266N mutant of the manganese-reconstituted enzyme was determined to a resolution of 2.1 Å and the structure of the ligand-free, manganese-reconstituted E101N mutant to a resolution of 2.6 Å, and the structures of ligand-free zinc-reconstituted wild-type, E101N, R230D, and E101G/R230C mutants of GKL were determined to resolutions of 2.1, 2.1, 1.9, and 2.0 Å, respectively. In particular, the structure of the evolved E101G/R230C mutant of GKL provides evidence of a catalytically productive active site architecture that contributes to the observed enhancement of catalysis. At high concentrations, wild-type and mutant GKL enzymes are differentially colored, with absorbance maxima in the range of 512-553 nm. The structures of the wild-type and mutant GKL provide a tractable link between the origins of the coloration and the charge-transfer complex between the α-cation and Tyr99 within the enzyme active site. Taken together, this study provides evidence of the modulability of enzymatic catalysis through subtle changes in enzyme active site architecture.

Development of quorum-based anti-virulence therapeutics targeting Gram-negative bacterial pathogens.

Quorum sensing is a cell density-dependent signaling phenomenon used by bacteria for coordination of population-wide phenotypes, such as expression of virulence genes, antibiotic resistance and biofilm formation. Lately, disruption of bacterial communication has emerged as an anti-virulence strategy with enormous therapeutic potential given the increasing incidences of drug resistance in pathogenic bacteria. The quorum quenching therapeutic approach promises a lower risk of resistance development, since interference with virulence generally does not affect the growth and fitness of the bacteria and, hence, does not exert an associated selection pressure for drug-resistant strains. With better understanding of bacterial communication networks and mechanisms, many quorum quenching methods have been developed against various clinically significant bacterial pathogens. In particular, Gram-negative bacteria are an important group of pathogens, because, collectively, they are responsible for the majority of hospital-acquired infections. Here, we discuss the current understanding of existing quorum sensing mechanisms and present important inhibitory strategies that have been developed against this group of pathogenic bacteria.

Increasing lysergic acid levels for ergot alkaloid biosynthesis: Directing catalysis via the F-G loop of Clavine oxidases.

Most ergot alkaloid drugs are semi-synthetically derived from the natural product lysergic acid, a valuable precursor for the development of novel ergot alkaloid drugs. Clavine oxidase (CloA) is a putative cytochrome P450, identified in the ergot alkaloid biosynthesis pathway, and a key enzyme that catalyzes the formation of lysergic acid from the precursor alkaloid agroclavine in a two-step oxidation reaction. We demonstrated in this study that Saccharomyces cerevisiae can be used as a viable host for the functional expression of CloA from Claviceps purpurea and its orthologs. We also showed that CloA orthologs differ in their ability to oxidize the substrate agroclavine, with some orthologs only able to perform the first oxidation reaction to produce elymoclavine. Of particular note, we identified a region between the F-G helices of the enzyme that may be involved in directing oxidation of agroclavine by substrate recognition and uptake. Using this knowledge, engineered CloAs were shown to produce lysergic acid at levels exceeding that of wildtype CloA orthologs; a CloA variant, chimeric AT5 9Hypo CloA, increased production levels of lysergic acid to 15 times higher as compared to the wildtype enzyme, demonstrating future utility for the industrial production of ergot alkaloids using biosynthetic routes.

A biological camera that captures and stores images directly into DNA.

The increasing integration between biological and digital interfaces has led to heightened interest in utilizing biological materials to store digital data, with the most promising one involving the storage of data within defined sequences of DNA that are created by de novo DNA synthesis. However, there is a lack of methods that can obviate the need for de novo DNA synthesis, which tends to be costly and inefficient. Here, in this work, we detail a method of capturing 2-dimensional light patterns into DNA, by utilizing optogenetic circuits to record light exposure into DNA, encoding spatial locations with barcoding, and retrieving stored images via high-throughput next-generation sequencing. We demonstrate the encoding of multiple images into DNA, totaling 1152 bits, selective image retrieval, as well as robustness to drying, heat and UV. We also demonstrate successful multiplexing using multiple wavelengths of light, capturing 2 different images simultaneously using red and blue light. This work thus establishes a 'living digital camera', paving the way towards integrating biological systems with digital devices.