Synthetic biology and metabolic engineering of actinomycetes for natural product discovery

Actinomycetes are one of the most valuable sources of natural products with industrial and medicinal importance. After more than half a century of exploitation, it has become increasingly challenging to find novel natural products with useful properties as the same known compounds are often repeatedly re-discovered when using traditional approaches. Modern genome mining approaches have led to the discovery of new biosynthetic gene clusters, thus indicating that actinomycetes still harbor a huge unexploited potential to produce novel natural products. In recent years, innovative synthetic biology and metabolic engineering tools have greatly accelerated the discovery of new natural products and the engineering of actinomycetes. In the first part of this review, we outline the successful application of metabolic engineering to optimize natural product production, focusing on the use of multi-omics data, genome-scale metabolic models, rational approaches to balance precursor pools, and the engineering of regulatory genes and regulatory elements. In the second part, we summarize the recent advances of synthetic biology for actinomycetal metabolic engineering including cluster assembly, cloning and expression, CRISPR/Cas9 technologies, and chassis strain development for natural product overproduction and discovery. Finally, we describe new advances in reprogramming biosynthetic pathways through polyketide synthase and non-ribosomal peptide synthetase engineering. These new developments are expected to revitalize discovery and development of new natural products with medicinal and other industrial applications.     

Probing light chain mutation effects on thrombin via molecular dynamics simulations and machine learning

Thrombin is a key component for chemotherapeutic and antithrombotic therapy development. As the physiologic and pathologic roles of the light chain still remain vague, here, we continue previous efforts to understand the impacts of the disease-associated single deletion of LYS9 in the light chain. By combining supervised and unsupervised machine learning methodologies and more traditional structural analyses on data from 10 μs molecular dynamics simulations, we show that the conformational ensemble of the ΔK9 mutant is significantly perturbed. Our analyses consistently indicate that LYS9 deletion destabilizes both the catalytic cleft and regulatory functional regions and result in some conformational changes that occur in tens to hundreds of nanosecond scaled motions. We also reveal that the two forms of thrombin each prefer a distinct binding mode of a Na⁺ ion. We expand our understanding of previous experimental observations and shed light on the mechanisms of the LYS9 deletion associated bleeding disorder by providing consistent but more quantitative and detailed structural analyses than early studies in literature. With a novel application of supervised learning, i.e. the decision tree learning on the hydrogen bonding features in the wild-type and ΔK9 mutant forms of thrombin, we predict that seven pairs of critical hydrogen bonding interactions are significant for establishing distinct behaviors of wild-type thrombin and its ΔK9 mutant form. Our calculations indicate the LYS9 in the light chain has both localized and long-range allosteric effects on thrombin, supporting the opinion that light chain has an important role as an allosteric effector.     

Carbohydrate-based heteronuclear complexes as topoisomerase Iα inhibitor: approach toward anticancer chemotherapeutics

Due to the critical role of cellular enzymes necessary for cell proliferation by deciphering topological hurdles in the process of DNA replication, topoisomerases have been one of the major targets in the anticancer drug development area. A need, therefore, arises for new metallodrugs that specifically recognizes DNA and inhibits the activity of topoisomerase enzymes, herein, we report the synthesis and characterization of new metal-based glycoconjugate entities containing heterobimetallic core Cu^II–Sn^IV (1) and Ni^II–Sn^IV (2) derived from N-glycoside ligand (L). The optimized structure of complex 1 and other significant vibrational modes have been explained using dispersion corrected B3LYP/DFT calculations. In vitro DNA binding profile of the L and both the complexes 1 and 2 were done by various biophysical studies. Complex 1 breaks pBR322 DNA via a hydrolytic means which was validated by T4 DNA enzymatic assay. To get a mechanistic insight of mode of action topoisomerase I (Topo I) inhibition assay was carried out. Also, we have taken the help of molecular modeling studies in accordance with experimental findings. In vitro cytotoxicity of the complex 1 was evaluated against a panel of cancer cells which exhibited remarkably good anticancer activity (GI₅₀ values <10 μg/ml). Moreover, intracellular localization of the complex 1 was visualized by confocal microscopy against HeLa cells.     

Functional divergence analysis of vertebrate neuronal nicotinic acetylcholine receptor subunits

Nicotinic acetylcholine receptors (nAChRs) are pentamers formed by subunits from a large multigene family and are highly variable in kinetic, electrophysiological and pharmacological properties. Due to the essential roles of nAChRs in many physiological procedures and diversity in function, identifying the function-related sites specific to each subunit is not only necessary to understand the properties of the receptors but also useful to design potential therapeutic compounds that target these macromolecules for treating a series of central neuronal disorders. By conducting a detailed function divergence analysis on nine neuronal nAChR subunits from representative vertebrate species, we revealed the existence of significant functional variation between most subunit pairs. Specifically, 44 unique residues were identified for the α7 subunit, while another 22 residues that were likely responsible for the specific features of other subunits were detected. By mapping these sites onto the 3?D structure of the human α7 subunit, a structure-function relationship profile was revealed. Our results suggested that the functional divergence related sites clustered in the ligand binding domain, the β2–β3 linker close to the N-terminal α-helix, the intracellular linkers between transmembrane domains, and the “transition zone” may have experienced altered evolutionary rates. The former two regions may be potential binding sites for the α7* subtype-specific allosteric modulators, while the latter region is likely to be subtype-specific allosteric modulations of the heteropentameric descendants such as the α4β2* nAChRs.

Communicated by Ramaswamy H. Sarma     

Affinity maturation and characterization of the ofatumumab monoclonal antibody

CD20 molecule, a phosphoprotein with 297 amino acids and four transmembrane domains, is a member of MS4A protein family. Anti-CD20 antibodies such as ofatumumab, which have been developed for cancer treatment and has demonstrated efficacy in relapsed/refractory chronic lymphocytic leukemia, are among the most successful therapies to date. Rational engineering methods can be applied with reasonable success to improve functional characteristics of antibodies. Considering the importance of this issue, we have used in silico modeling approach for the improvement of ofatumumab monoclonal antibody. Four mutated variants of ofatumumab were developed and expressed in Chinese hamster ovary (CHO) cells along with the unmodified antibody. Analysis of affinity of the purified antibodies with CD20 showed significant improvement in antigen-binding characteristics of one of the variants compared with the control antibody. This study represents the first step toward development of the second generation ofatumumab antibody with improved affinity.     

Mechanistic elucidation of amphetamine metabolism by tyramine oxidase from human gut microbiota using molecular dynamics simulations

The human gut harbors diverse bacterial species in the gut, which play an important role in the metabolism of food and host health. Recent studies have also revealed their role in altering the pharmacological properties and efficacy of oral drugs through promiscuous metabolism. However, the atomistic details of the enzyme-drug interactions of gut bacterial enzymes which can potentially carry out the metabolism of drug molecules are still scarce. A well-known example is the FDA drug amphetamine (a central nervous system stimulant), which has been predicted to undergo promiscuous metabolism by gut bacteria. Therefore, to understand the atomistic details and energy landscape of the gut microbial enzyme-mediated metabolism of this drug, molecular dynamics studies were performed. It was observed that amphetamine binds to tyramine oxidase from the Escherichia coli strain present in the human gut microbiota at the binding site harboring polar and nonpolar amino acids. The stability analysis of amphetamine at the binding site showed that the binding is stable and the free energy for the binding of amphetamine was found to be ~ −51.71 kJ/mol. The insights provided by this study on promiscuous metabolism of amphetamine by a gut enzyme will be very useful to improve the efficacy of the drug.