Molecular engineering of bacterial natural product biosynthetic pathways via Red/ET recombineering

Bacterial genome-sequencing projects have revealed that a large number of natural product biosynthetic pathways presented in genomes are cryptic. Cloning, engineering and expression of a cryptic biosynthetic pathway in a well-characterized heterologous host to discover and optimize its product is an effective alternate to traditional approaches. We developed RecE/RecT mediated linear-linear homologous recombination (LLHR) for direct cloning of ten unknown NRPS/PKS gene clusters from the genomic DNA of Photorhabdus luminescens into expression vectors in E. coli. Coupled with robust heterologous expression in E. coli, the products of plu3263 and plu1881-plu1877, luminmides A-G and luminmycins A-C, were determined, respectively. By site-directed modification of the third adenylation domain of NRPS Plu3263, we efficiently created mutants using ccdB counterselection recombineering, which led to a significant alteration of relative yields of luminmides A and B. Partial syringolin biosynthetic pathway (sylCDE) from Pseudomonas syringae was cloned by LLHR and expressed in E. coli resulting in the identification of two new syringolins. Intact syringolin pathway was reassembled by addition of sylAB and engineering of promoter via Red/ET recombineering. The varying production distribution of syringolins showed the different efficiencies of native and synthetic promoters in E. coli.Bakterielle Genom-Sequenzierungsprojekte zeigten, dass zahlreiche im Genom vorhandene Sekundärmetabolit Biosynthesewege kryptisch sind. Um die Produkte zu entdecken und optimieren, ist Klonierung, Engineering und Expression von diesen Biosynthesewegen in einem gut charakterisierten heterologen System eine effektive Alternative zu traditionellen Ansätzen. Wir entwickelten die von RecE/RecT vermittelten linear-linear homologen Rekombination (LLHR) für die direkte Klonierung von zehn unbekannten NRPS/PKS-Genen aus Genom von Photorhabdus luminescens in E. coli-Expressionsvektoren. Mittels robuster heterologer Expression in E. coli wurden die Produkte von plu3263 und plu1881-plu1877, Luminmide A-G und Luminmycin A-C, jeweils identifiziert. Die \u27\u27site-directed\u27\u27 Modifikation der dritten Adenylierungsdomaine von NRPS Plu3263 wurde effizient durch ccdB Conter-selektion Rekombination erstellt. Das führte dazu, dass sich die relative Ausbeute von Luminmides A und B stark veränderten. Der partielle Syringolin Biosyntheseweg (sylCDE) aus Pseudomonas syringae wurde durch LLHR kloniert und die Expression in E. coli versursachte die Identifizierung von zwei neuen Syringolinen. Der intakte Syringolin Biosynthesesweg wurde durch Zugabe von sylAB und Promotor-Engineering via Red/ET-Rekombination wieder zusammengesetzt. Die variierende Verteilung der Produktion aller Syringolin-derivate zeigte die unterschiedliche Effizienzen zwischen nativen und synthetischen Promotoren in E. coli

Leader peptide removal in lasso peptide biosynthesis based on penultimate isoleucine residue

Lasso peptides are ribosomally synthesized peptides that undergo post-translational modifications including leader peptide removal by B (or the segregated B1 and B2) proteins and core peptide macrolactamization by C proteins to form a unique lariat topology. A conserved threonine residue at the penultimate position of leader peptide is hitherto found in lasso peptide precursors and shown to be a critical recognition element for effective enzymatic processing. We identified a lasso peptide biosynthetic gene cluster (bsf) from Bradymonas sediminis FA350, a Gram-negative and facultatively prey-dependent bacterium that belongs to a novel bacterial order Bradymonadales in the class Deltaproteobacteria. The kinase BsfK specifically catalyzes the phosphorylation of the precursor peptide BsfA on the Ser3 residue. BsfB1 performs dual functions to accelerate the post-translational phosphorylation and assist BsfB2 in leader peptide removal. Most importantly, the penultimate residue of leader peptide is an isoleucine rather than the conserved threonine and this isoleucine has a marked impact on the phosphorylation of Ser3 as well as leader peptide removal, implying that BsfB1 and BsfB2 exhibit a new substrate selectivity for leader peptide binding and excision. This is the first experimentally validated penultimate isoleucine residue in a lasso peptide precursor to our knowledge. In silico analysis reveals that the leader peptide Ile/Val(-2) residue is rare but not uncommon in phosphorylated lasso peptides, as this residue is also discovered in Acidobacteriaceae and Sphingomonadales in addition to Bradymonadales

Gaze Patterns in Auditory-Visual Perception of Emotion by Children with Hearing Aids and Hearing Children

This study investigated eye-movement patterns during emotion perception for children with hearing aids and hearing children. Seventy-eight participants aged from 3 to 7 were asked to watch videos with a facial expression followed by an oral statement, and these two cues were either congruent or incongruent in emotional valence. Results showed that while hearing children paid more attention to the upper part of the face, children with hearing aids paid more attention to the lower part of the face after the oral statement was presented, especially for the neutral facial expression/neutral oral statement condition. These results suggest that children with hearing aids have an altered eye contact pattern with others and a difficulty in matching visual and voice cues in emotion perception. The negative cause and effect of these gaze patterns should be avoided in earlier rehabilitation for hearing-impaired children with assistive devices

Biosynthesis of Fungal Natural Products Involving Two Separate Pathway Crosstalk

Fungal natural products (NPs) usually possess complicated structures, exhibit satisfactory bioactivities, and are an outstanding source of drug leads, such as the cholesterol-lowering drug lovastatin and the immunosuppressive drug mycophenolic acid. The fungal NPs biosynthetic genes are always arranged within one single biosynthetic gene cluster (BGC). However, a rare but fascinating phenomenon that a crosstalk between two separate BGCs is indispensable to some fungal dimeric NPs biosynthesis has attracted increasing attention. The hybridization of two separate BGCs not only increases the structural complexity and chemical diversity of fungal NPs, but also expands the scope of bioactivities. More importantly, the underlying mechanism for this hybridization process is poorly understood and needs further exploration, especially the determination of BGCs for each building block construction and the identification of enzyme(s) catalyzing the two biosynthetic precursors coupling processes such as Diels–Alder cycloaddition and Michael addition. In this review, we summarized the fungal NPs produced by functional crosstalk of two discrete BGCs, and highlighted their biosynthetic processes, which might shed new light on genome mining for fungal NPs with unprecedented frameworks, and provide valuable insights into the investigation of mysterious biosynthetic mechanisms of fungal dimeric NPs which are constructed by collaboration of two separate BGCs

2-[(E)-({3-[(E)-(2-Hydroxybenzylidene)aminomethyl]-1,4-dioxaspiro[4.5]decan-2-yl}methyl)iminomethyl]phenol

In the title compound, C24H28N2O4, the dioxalane ring has an envelope conformation. The cyclohexane ring adopts a chair conformation. The dihedral angle between the benzene rings is 72.5 (3)°. The molecular conformation is stabilized by two intramolecular O—H...N hydrogen-bonding interactions with an S(6) graph-set motif. The crystal structure is stabilized by van der Waals interactions

Microbial chassis engineering drives heterologous production of complex secondary metabolites

The cryptic secondary metabolite biosynthetic gene clusters (BGCs) far outnumber currently known secondary metabolites. Heterologous production of secondary metabolite BGCs in suitable chassis facilitates yield improvement and discovery of new-to-nature compounds. The two juxtaposed conventional model microorganisms, Escherichia coli, Saccharomyces cerevisiae, have been harnessed as microbial chassis to produce a bounty of secondary metabolites with the help of certain host engineering. In last decade, engineering non-model microbes to efficiently biosynthesize secondary metabolites has received increasing attention due to their peculiar advantages in metabolic networks and/or biosynthesis. The state-of-the-art synthetic biology tools lead the way in operating genetic manipulation in non-model microorganisms for phenotypic optimization or yields improvement of desired secondary metabolites. In this review, we firstly discuss the pros and cons of several model and non-model microbial chassis, as well as the importance of developing broader non-model microorganisms as alternative programmable heterologous hosts to satisfy the desperate needs of biosynthesis study and industrial production. Then we highlight the lately advances in the synthetic biology tools and engineering strategies for optimization of non-model microbial chassis, in particular, the successful applications for efficient heterologous production of multifarious complex secondary metabolites, e.g., polyketides, nonribosomal peptides, as well as ribosomally synthesized and post-translationally modified peptides. Lastly, emphasis is on the perspectives of chassis cells development to access the ideal cell factory in the artificial intelligence-driven genome era. © 2022 Elsevier Inc.National Natural Science Foundation of China; Natural Science Foundation of Shandong Province; National Key Research and Development Program of China: This work was supported by the National Key R&D Program of China (Grant nos. 2021YFC2100500 , 2019YFA0905700 ), National Natural Science Foundation of China (Grant nos. 32070060 , 32161133013 ), Shandong Provincial Natural Science Foundation (Grant no. ZR2019JQ11 , ZR2019ZD18 )

Physicochemical Properties, in Vitro Antioxidant Activities and Inhibitory Potential against α-Glucosidase of Polysaccharides from Ampelopsis grossedentata Leaves and Stems

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Unusual Post-Translational Modifications in the Biosynthesis of Lasso Peptides

Lasso peptides are a subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs) and feature the threaded, lariat knot-like topology. The basic post-translational modifications (PTMs) of lasso peptide contain two steps, including the leader peptide removal of the ribosome-derived linear precursor peptide by an ATP-dependent cysteine protease, and the macrolactam cyclization by an ATP-dependent macrolactam synthetase. Recently, advanced bioinformatic tools combined with genome mining have paved the way to uncover a rapidly growing number of lasso peptides as well as a series of PTMs other than the general class-defining processes. Despite abundant reviews focusing on lasso peptide discoveries, structures, properties, and physiological functionalities, few summaries concerned their unique PTMs. In this review, we summarized all the unique PTMs of lasso peptides uncovered to date, shedding light on the related investigations in the future

Heterologous expression of bacterial natural product biosynthetic pathways.

Covering: 2013 to June 2018 Heterologous expression of natural product biosynthetic pathways is of increasing interest in microbial biotechnology, drug discovery and optimization. It empowers not only the robust production of valuable biomolecules in more amenable heterologous hosts but also permits the generation of novel analogs through biosynthetic engineering. This strategy also facilitates the discovery of novel bioactive compounds following the functional expression of cryptic biosynthetic gene clusters (BGCs) from fastidious original producers or metagenomic DNA in surrogate hosts, thus facilitating genome mining in the post-genomic era. This review discusses recent advances and trends pertaining to the heterologous production of bacterial natural products, with an emphasis on new techniques, heterologous hosts, and novel chemistry since 2013