Mining the Biosynthetic Landscape of Lactic Acid Bacteria Unearths a New Family of RiPPs Assembled by a Novel Type of ThiF-like Adenylyltransferases.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are chemically diverse natural products of ribosomal origin. These peptides, which frequently act as signals or antimicrobials, are biosynthesized by conserved enzymatic machinery, making genome mining a powerful strategy for unearthing previously uncharacterized members of their class. Herein, we investigate the untapped biosynthetic potential of Lactobacillales (i.e., lactic acid bacteria), an order of Gram-positive bacteria closely associated with human life, including pathogenic species and industrially relevant fermenters of dairy products. Through genome mining methods, we systematically explored the distribution and diversity of ThiF-like adenylyltransferase-utilizing RiPP systems in lactic acid bacteria and identified a number of unprecedented biosynthetic gene clusters. In one of these clusters, we found a previously undescribed group of macrocyclic imide biosynthetic pathways containing multiple transporters that may be involved in a potential quorum sensing (QS) system. Through in vitro assays, we determined that one such adenylyltransferase specifically catalyzes the intracyclization of its precursor peptide through macrocyclic imide formation. Incubating the enzyme with various primary amines revealed that it could effectively amidate the C-terminus of the precursor peptide. This new transformation adds to the growing list of Nature's peptide macrocyclization strategies and expands the impressive catalytic repertoire of the adenylyltransferase family. The diverse RiPP systems identified herein represent a vast, unexploited landscape for the discovery of a novel class of natural products and QS systems.

Mammalian Commensal Streptococci Utilize a Rare Family of Class VI Lanthipeptide Synthetases to Synthesize Miniature Lanthipeptide-type Ribosomal Peptide Natural Products.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are natural products with remarkable chemical and functional diversities. These peptides are often synthesized as signals or antibiotics and frequently associated with quorum sensing (QS) systems. With the increasing number of available genomes, many hitherto unseen RiPP biosynthetic pathways have been mined, providing new resources for novel bioactive compounds. Herein, we investigated the underexplored biosynthetic potential of Streptococci, prevalent bacteria in mammal-microbiomes that include pathogenic, mutualistic, and commensal members. Using the transcription factor-centric genome mining strategy, we discovered a new family of lanthipeptide biosynthetic loci under the control of potential QS. By in vitro studies, we investigated the reaction of one of these lanthipeptide synthetases and found that it installs only one lanthionine moiety onto its short precursor peptide by connecting a conserved TxxC region. Bioinformatics and in vitro studies revealed that these lanthipeptide synthetases (class VI) are novel lanthipeptide synthetases with a truncated lyase, a kinase, and a truncated cyclase domain. Our data provide important insights into the processing and evolution of lanthipeptide synthetase to tailor smaller substrates. The data are important for obtaining a mechanistic understanding of the post-translational biosynthesis machinery of the growing variety of lanthipeptides.

Bovine lactoferrin inhibits SARS-CoV-2 and SARS-CoV-1 by targeting the RdRp complex and alleviates viral infection in the hamster model.

Breast milk has been found to inhibit coronavirus infection, while the key components and mechanisms are unknown. We aimed to determine the components that contribute to the antiviral effects of breastmilk and explore their potential mechanism. Lactoferrin (Lf) and milk fat globule membrane inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related coronavirus GX_P2V and transcription- and replication-competent SARS-CoV-2 virus-like particles in vitro and block viral entry into cells. We confirmed that bovine Lf (bLf) blocked the binding between human angiotensin-converting enzyme 2 and SARS-CoV-2 spike protein by combining receptor-binding domain (RBD). Importantly, bLf inhibited RNA-dependent RNA polymerase (RdRp) activity of both SARS-CoV-2 and SARS-CoV in vitro in the nanomolar range. So far, no biological macromolecules have been reported to inhibit coronavirus RdRp. Our result indicated that bLf plays a major role in inhibiting viral replication. bLf treatment reduced viral load in lungs and tracheae and alleviated pathological damage. Our study provides evidence that bLf prevents SARS-CoV-2 infection by combining SARS-CoV-2 spike protein RBD and inhibiting coronaviruses' RdRp activity, and may be a promising candidate for the treatment of coronavirus disease 2019.

Antiviral Drugs Screening for Swine Acute Diarrhea Syndrome Coronavirus.

Coronaviruses as possible cross-species viruses have caused several epidemics. The ongoing emergency of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has posed severe threats to the global economy and public health, which has generated great concerns about zoonotic viruses. Swine acute diarrhea syndrome coronavirus (SADS-CoV), an alpha-coronavirus, was responsible for mass piglet deaths, resulting in unprecedented economic losses, and no approved drugs or vaccines are currently available for SADS-CoV infection. Given its potential ability to cause cross-species infection, it is essential to develop specific antiviral drugs and vaccines against SADS-CoV. Drug screening was performed on a total of 3523 compound-containing drug libraries as a strategy of existing medications repurposing. We identified five compounds (gemcitabine, mycophenolate mofetil, mycophenolic acid, methylene blue and cepharanthine) exhibiting inhibitory effects against SADS-CoV in a dose-dependent manner. Cepharanthine and methylene blue were confirmed to block viral entry, and gemcitabine, mycophenolate mofetil, mycophenolic acid and methylene blue could inhibit viral replication after SADS-CoV entry. This is the first report on SADS-CoV drug screening, and we found five compounds from drug libraries to be potential anti-SADS-CoV drugs, supporting the development of antiviral drugs for a possible outbreak of SADS-CoV in the future.

Overview of Breastfeeding Under COVID-19 Pandemic.

During the global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pregnant and lactating women are at higher risk of infection. The potential of viral intrauterine transmission and vertical transmission by breastfeeding has raised wide concerns. Breastmilk is rich in nutrients that contribute to infant growth and development, and reduce the incidence rate of infant illness and death, as well as inhibit pathogens significantly, and protect infants from infection. Although it is controversial whether mothers infected with COVID-19 should continue to breastfeed, many countries and international organizations have provided recommendations and guidance for breastfeeding. This review presents the risks and benefits of breastfeeding for mothers infected with COVID-19, and the reasons for the absence of SARS-CoV-2 active virus in human milk. In addition, the antiviral mechanisms of nutrients in breastmilk, the levels of SARS-CoV-2 specific antibodies in breastmilk from COVID-19 infected mothers and vaccinated mothers are also summarized and discussed, aiming to provide some support and recommendations for both lactating mothers and infants to better deal with the COVID-19 pandemic.

Molnupiravir and Its Antiviral Activity Against COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) constitutes a major worldwide public health threat and economic burden. The pandemic is still ongoing and the SARS-CoV-2 variants are still emerging constantly, resulting in an urgent demand for new drugs to treat this disease. Molnupiravir, a biological prodrug of NHC (ß-D-N(4)-hydroxycytidine), is a novel nucleoside analogue with a broad-spectrum antiviral activity against SARS-CoV, SARS-CoV-2, Middle East respiratory syndrome coronavirus (MERS-CoV), influenza virus, respiratory syncytial virus (RSV), bovine viral diarrhea virus (BVDV), hepatitis C virus (HCV) and Ebola virus (EBOV). Molnupiravir showed potent therapeutic and prophylactic activity against multiple coronaviruses including SARS-CoV-2, SARS-CoV, and MERS-CoV in animal models. In clinical trials, molnupiravir showed beneficial effects for mild to moderate COVID-19 patients with a favorable safety profile. The oral bioavailability and potent antiviral activity of molnupiravir highlight its potential utility as a therapeutic candidate against COVID-19. This review presents the research progress of molnupiravir starting with its discovery and synthesis, broad-spectrum antiviral effects, and antiviral mechanism. In addition, the preclinical studies, antiviral resistance, clinical trials, safety, and drug tolerability of molnupiravir are also summarized and discussed, aiming to expand our knowledge on molnupiravir and better deal with the COVID-19 epidemic.

Discovery and Characterization of Rubrinodin Provide Clues into the Evolution of Lasso Peptides.

Lasso peptides are unique natural products that comprise a class of ribosomally synthesized and post-translationally modified peptides. Their defining three-dimensional structure is a lariat knot, in which the C-terminal tail is threaded through a macrolactam ring formed between the N-terminal amino group and an Asp or Glu side chain (i.e., an isopeptide bond). Recent genome mining strategies have revealed various types of lasso peptide biosynthetic gene clusters and have thus redefined the known chemical space of lasso peptides. To date, over 20 different types of these gene clusters have been discovered, including several different clades from Proteobacteria. Despite the diverse architectures of these gene clusters, which may or may not encode various tailoring enzymes, most currently known lasso peptides are synthesized by two discrete clades defined by the presence of an ATP-binding cassette transporter or its absence and (sometimes) concurrent appearance of an isopeptidase, raising questions about their evolutionary history. Herein, we discovered and characterized the lasso peptide rubrinodin, which is assembled by a gene cluster encoding both an ATP-binding cassette transporter and an isopeptidase. Our bioinformatics analyses of this and other representative cluster types provided new clues into the evolutionary history of lasso peptides. Furthermore, our structural and biochemical investigations of rubrinodin permitted the conversion of this thermolabile lasso peptide into a more thermostable scaffold.

Recent Advances and Perspectives on Expanding the Chemical Diversity of Lasso Peptides.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of natural products that exhibit a range of structures and bioactivities. Initially assembled from the twenty proteinogenic amino acids in a ribosome-dependent manner, RiPPs assume their peculiar bioactive structures through various post-translational modifications. The essential modifications representative of each subfamily of RiPP are performed on a precursor peptide by the so-called processing enzymes; however, various tailoring enzymes can also embellish the precursor peptide or processed peptide with additional functional groups. Lasso peptides are an interesting subfamily of RiPPs characterized by their unique lariat knot-like structure, wherein the C-terminal tail is inserted through a macrolactam ring fused by an isopeptide bond between the N-terminal amino group and an acidic side chain. Until recently, relatively few lasso peptides were found to be tailored with extra functional groups. Nevertheless, the development of new routes to diversify lasso peptides and thus introduce novel or enhanced biological, medicinally relevant, or catalytic properties is appealing. In this review, we highlight several strategies through which lasso peptides have been successfully modified and provide a brief overview of the latest findings on the tailoring of these peptides. We also propose future directions for lasso peptide tailoring as well as potential applications for these peptides in hybrid catalyst design.

ThpacC Acts as a Positive Regulator of Homodimericin A Biosynthesis and Antifungal Activities of Trichoderma harzianum 3.9236.

The Pal/Rim pathway and its key transcription factor PacC play important roles in fungal adaptation to ambient pH regarding growth, secondary metabolism, and virulence. However, the effect of PacC on the secondary metabolism of the important biocontrol fungus Trichoderma harzianum remains elusive. To answer this question, ThpacC deletion (KO-ThpacC) and overexpression (OE-ThpacC) mutants of T. harzianum 3.9236 were constructed. Transcriptomic analysis of T. harzianum and KO-ThpacC suggested that ThpacC acted as both a positive and a negative regulator for secondary metabolite (SM) production. Further investigation revealed that deletion of ThpacC abolished homodimericin A and 8-epi-homodimericin A production. Moreover, ThpacC plays a role in the antagonism of T. harzianum against Sclerotinia sclerotiorum. 8-epi-Homodimericin A demonstrated moderate inhibitory activity against S. sclerotiorum. Our results contribute to a deeper understanding of the ThpacC function on SM production and the antifungal activity of T. harzianum.

Isolation and characterization of a podovirus infecting the opportunist pathogen Vibrio alginolyticus and Vibrio parahaemolyticus.

Bacterial infections have a negative impact on both animal husbandry industry and medicine, and increasing bacterial drug resistance exacerbates this adverse impact. Phages show promise as an alternative to drugs against drug-resistant bacteria. In this study, a novel virulent bacteriophage (phage) vB_ValP_IME234 against Vibrio alginolyticus and Vibrio parahaemolyticus was isolated from freshwater in Beijing, China. Phage vB_ValP_IME234 had an isometric head (59 nm in diameter) and a short tail (10 nm long), belonging to Podoviridae family. Its complete genome is liner double-stranded DNA (dsDNA) with a GC content of 41.6% while encoding 61 putative proteins. Three transfer RNA (tRNA) and no lysogenic gene was detected. vB_ValP_IME234 had a polyvalent infectivity, a burst of 390 PFU/cell, and is stable under different temperatures (4 °C to 50 °C) and pH (6.0 to 10.0) values. Host range test showed that vB_ValP_IME234 has the ability to infect seven strains of Vibrio in total. Phylogenetic analyses based on terminase and capsid suggested that this phage had a close relationship with Vibrio phages. These results indicate that vB_ValP_IME234 could be used as a potential biocontrol agent against V. alginolyticus strains.

Topology engineering via protein catenane construction to strengthen an industrial biocatalyst.

Protein topology engineering has emerged as a new dimension to alter protein stability and function. Inspired by the art of nature, where backbone cyclization is frequently adopted to enhance the stability of natural peptide products and thermostable enzymes; herein, we report protein topology engineering of an industrial thermolabile gamma lactamase via catenation. Two different protein catenanes were successfully constructed via SpyTag/SpyCatcher modules and two different peptide dimer domains. The designed protein catenanes were functionally synthesized in Escherichia coli. A comparison of their biochemical properties revealed that protein topology played a key role in the stability of gamma lactamase. Protein catenation enhanced both the thermo- and proteolytic stabilities of gamma lactamase. Gamma lactamase was stabilized by ∼8 °C in one of the catenated forms. Moreover, Cat1-MhIHL-V54L and Cat2-MhIHL-V54L displayed 1.8- and 2.4-fold higher enzyme efficiencies (Kcat/Km), respectively, than the unattenuated enzyme. Therefore, our results proved that protein catenane construction could be a general strategy to strengthen industrial biocatalysts by mechanisms distinct from those of the conventional direct evolution schemes, whereby our results offer wide applications in the fine chemical industry.

New perspectives on the treatment of mycobacterial infections using antibiotics.

More than 100 years have passed since the discovery of Mycobacterium tuberculosis, in 1882, as the pathogen that causes tuberculosis (TB). However, globally, TB is still one of the leading causes of death by infectious diseases. In 2018, approximately 10.0 million people were diagnosed with TB owing to the development of advanced strategies by M. tuberculosis to resist antibiotics, including the development of a dormant state. The World Health Organization (WHO) and the Sustainable Development Goals (SDGs) are dedicated to ending TB by 2030. However, the development of strategies to discover new TB drugs and new therapies is crucial for the achievement of this goal. Unfortunately, the rapid occurrence of multidrug-resistant strains of M. tuberculosis has worsened the current situation, thereby warranting prioritized discovery of new anti-TB drugs and the development of new treatment regimens in academia and the pharmaceutical industry. In this mini review, we provide a brief overview of the current research and development pipeline for new anti-TB drugs and present our perspective of TB drug innovation. The data presented herein may enable the introduction of more effective medicines and therapeutic regimens into the market.Key Points• The Updated Global New TB Drug Pipelines are briefly summarized.• Novel strategies for the discovery of new TB drugs, including novel sources, bioinformatics, and synthetic biology strategies, are discussed.• New therapeutic options, including living therapeutics and phage therapy, are proposed.

Genome mining integrating semi-rational protein engineering and nanoreactor design: roadmap for a robust biocatalyst for industrial resolution of Vince lactam.

Biomanufacturing of chemicals using biocatalysts is an attractive strategy for the production of valuable pharmaceuticals since it is usually more economical and has a much-reduced environmental impact. However, there are often challenges such as their thermal instability that should be overcome before a newly discovered enzyme is eventually translated into industrial processes. In this work, we describe a roadmap for the development of a robust catalyst for industrial resolution of Vince lactam, a key intermediate for the synthesis of carbocyclic-nucleoside-related pharmaceuticals. By a genome mining strategy, a new (+)-γ-lactamase (MiteL) from Microbacterium testaceum was successfully discovered and biochemically characterized. In vitro studies showed that the enzyme exhibited high activity but poor enantioselectivity (E = 6.3 ± 0.2) toward racemic Vince lactam, and thus, it is not suitable for industrial applications. Based on structural modeling and docking studies, a semi-rational engineering strategy combined with an efficient screening method was then applied to improve the enantioselectivity of MiteL. Several mutants with significant shifting stereoselectivity toward (-)-γ-lactam were obtained by site-saturation mutagenesis. Synergy effects led to the final mutant F14D/Q114R/M117L, which enabled efficient acquisition of (-)-γ-lactam with a high E value (> 200). The mutant was biochemically characterized, and the docking studies suggested a plausible mechanism for its improved selectivity. Finally, a sunflower-like nanoreactor was successfully constructed to improve the mutant's robustness via protein supramolecular self-assembly. Thus, the synergism between semi-rational protein engineering and self-assembling immobilization enabled construction of a nanoreactor with superior properties, which can be used for resolution of Vince lactam in large scale.

Pyrroloindoline cyclization in tryptophan-containing cyclodipeptides mediated by an unprecedented indole C3 methyltransferase from Streptomyces sp. HPH0547.

Diverse bioactive alkaloids with a tryptophan 2,5-diketopiperazine (DKP) core and an annulated structure forming a methylated pyrroloindoline-DKP assembly have been isolated from various microbial sources. However, little is known about their biosynthesis. In this study, a novel indole C3 methyltransferase from Streptomyces sp. HPH0547 was discovered and characterized. Structural elucidation of the products revealed that this enzyme catalyzed unique pyrroloindoline cyclization in tryptophan-containing cyclodipeptides. This is the first C3 methyltransferase reported to catalyze pyrroloindoline cyclization in cyclic dipeptides, which provides a feasible and simple method to access diverse alkaloids.

Lassomycin and lariatin lasso peptides as suitable antibiotics for combating mycobacterial infections: current state of biosynthesis and perspectives for production.

Lasso peptides are ribosomally synthesized and post-translationally modified natural products with a characteristic slipknot-like structure, which confers these peptides remarkable stability and diverse pharmacologically relevant bioactivities. Among all the reported lasso peptides, lassomycin and lariatins are unique lasso peptides that exhibit noticeable anti-tuberculosis (TB) activity. Due to the unique threaded structure and the unusual bactericidal mechanism toward Mycobacterium tuberculosis, these peptides have drawn considerable interest, not only in the field of total synthesis but also in several other fields including biosynthesis, bioengineering, and structure-activity studies. During the past few years, significant progress has been made in understanding the biosynthetic mechanism of these intriguing compounds, which has provided a solid foundation for future work. This review highlights recent achievements in the discovery, structure elucidation, biological activity, and the unique anti-TB mechanism of lasso peptides. Moreover, the discovery of their biosynthetic pathway has laid the foundation for combinatorial biosynthesis of their analogs, which provides new perspectives for the production of novel anti-TB lasso peptides.

Discovery and characterization of a novel C-terminal peptide carboxyl methyltransferase in a lassomycin-like lasso peptide biosynthetic pathway.

Lasso peptides belong to a peculiar family of ribosomally synthesized and post-translationally modified peptides (RiPPs)-natural products with an unusual isopeptide-bonded slipknot structure. Except for assembling of this unusual lasso fold, several further post-translational modifications of lasso peptides, including C-terminal methylation, phosphorylation/poly-phosphorylation, citrullination, and acetylation, have been reported recently. However, most of their biosynthetic logic have not been elucidated except the phosphorylated paeninodin lasso peptide. Herein, we identified two novel lassomycin-like lasso peptide biosynthetic pathways and, for the first time, characterized a novel C-terminal peptide carboxyl methyltransferase involved in these pathways. Our investigations revealed that this new family of methyltransferase could specifically methylate the C terminus of precursor peptide substrates, eventually leading to lassomycin-like C-terminal methylated lasso peptides. Our studies offer another rare insight into the extraordinary strategies of chemical diversification adopted by lasso peptide biosynthetic machinery and predicated two valuable sources for methylated lasso peptide discovery.

Nanoreactor Design Based on Self-Assembling Protein Nanocages.

Self-assembling proteins that form diverse architectures are widely used in material science and nanobiotechnology. One class belongs to protein nanocages, which are compartments with nanosized internal spaces. Because of the precise nanoscale structures, proteinaceous compartments are ideal materials for use as general platforms to create distinct microenvironments within confined cellular environments. This spatial organization strategy brings several advantages including the protection of catalyst cargo, faster turnover rates, and avoiding side reactions. Inspired by diverse molecular machines in nature, bioengineers have developed a variety of self-assembling supramolecular protein cages for use as biosynthetic nanoreactors that mimic natural systems. In this mini-review, we summarize current progress and ongoing efforts creating self-assembling protein based nanoreactors and their use in biocatalysis and synthetic biology. We also highlight the prospects for future research on these versatile nanomaterials.

Dynamic kinetic resolution of Vince lactam catalyzed by γ-lactamases: a mini-review.

γ-Lactamases are versatile enzymes used for enzymatic kinetic resolution of racemic Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) in the industry. Optically pure enantiomers and their hydrolytic products are widely employed as key chemical intermediates for developing a wide range of carbocyclic nucleoside medicines, including US FDA-approved drugs peramivir and abacavir. Owing to the broad applications in the healthcare industry, the resolution process of Vince lactam has witnessed tremendous progress during the past decades. Some of the most important advances are the enzymatic strategies involving γ-lactamases. The strong industrial demand drives the progress in various strategies for discovering novel biocatalysts. In the past few years, several new scientific breakthroughs, including the genome-mining strategy and elucidation of several crystal structures, boosted the research on γ-lactamases. So far, several families of γ-lactamases for resolution of Vince lactam have been discovered, and their number is continuously increasing. The purpose of this mini-review is to describe the discovery strategy and classification of these intriguing enzymes and to cover our current knowledge on their potential biological functions. Moreover, structural properties are described in addition to their possible catalytic mechanisms. Additionally, recent advances in the newest approaches, such as immobilization to increase stability, and other engineering efforts are introduced.

Promiscuous (+)-γ-lactamase activity of an amidase from nitrile hydratase pathway for efficient synthesis of carbocyclic nucleosides intermediate.

Based on bioinformatics analysis, the promiscuous (+)-γ-lactamase activity of an amidase was identified in Rhodococcus erythropolis PR4 and found to be involved in the nitrile hydratase pathway. The amidase is highly enantioselective and can be used in the kinetic resolution of the Vince lactam. The known structure provides a rare insight into the catalytic mechanism of (+)-γ-lactamase with absolute chiral selectivity. This lactamase was cloned, purified, biochemically characterized, and demonstrated to be an ideal catalyst for the preparation of carbocyclic nucleosides of pharmaceutical interest. The chiral selectivity of this enzyme was investigated by molecular docking and site-specific mutagenesis, which provides a foundation for further engineering of these versatile biocatalysts.

Construction of an organelle-like nanodevice via supramolecular self-assembly for robust biocatalysts.

BACKGROUND: When using the microbial cell factories for green manufacturing, several important issues need to be addressed such as how to maintain the stability of biocatalysts used in the bioprocess and how to improve the synthetic efficiency of the biological system. One strategy widely used during natural evolution is the creation of organelles which can be used for regional control. This kind of compartmentalization strategy has inspired the design of artificial organelle-like nanodevice for synthetic biology and "green chemistry". RESULTS: Mimicking the natural concept of functional compartments, here we show that the engineered thermostable ketohydroxyglutarate aldolase from Thermotoga maritima could be developed as a general platform for nanoreactor design via supramolecular self-assembly. An industrial biocatalyst-(+)-γ-lactamase was selected as a model catalyst and successful encapsulated in the nanoreactor with high copies. These nanomaterials could easily be synthesized by Escherichia coli by heterologous expression and subsequently self-assembles into the target organelle-like nanoreactors both in vivo and in vitro. By probing their structural characteristics via transmission electronic microscopy and their catalytic activity under diverse conditions, we proved that these nanoreactors could confer a significant benefit to the cargo proteins. The encapsulated protein exhibits significantly improved stability under conditions such as in the presence of organic solvent or proteases, and shows better substrate tolerance than free enzyme. CONCLUSIONS: Our biodesign strategy provides new methods to develop new catalytically active protein-nanoreactors and could easily be applied into other biocatalysts. These artificial organelles could have widely application in sustainable catalysis, synthetic biology and could significantly improve the performance of microbial cell factories.