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Tyrocidine biosynthetic system. The enzymatic assembly line of the cyclic decapeptide antibiotic tyrocidine consists of three peptide synthetases, TycABC, which are encoded by the polycistronic genes tycABC. The proteins are composed of one, three, and six modules, respectively, each one responsible for the integration of one amino acid into the nascent peptide chain (A). In this study, the first two modules (TycA and TycB1) were used as an artificial bimodular NRPS system for the production of d-Phe-Pro-DKP (B). Different catalytic domains are highlighted: condensation domain (light gray), adenylation domains (medium gray), peptidyl carrier protein domain (dark gray; wavy line, cofactor Ppant), epimerization domain (black), and thioesterase domain (striped).
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Nonribosomal peptide synthetases represent the enzymatic assembly lines for the biosynthesis of pharmacologically relevant natural peptides, e.g., cyclosporine, vancomycin, and penicillin. Due to their modular organization, in which every module accounts for the incorporation of a single amino acid, artificial assembly lines for the production of n...
Citations
... 32,33 To narrow down the remaining 22 candidate positions, their relevance was tested by mutating them to Ala (Supporting Information "Experimental Procedures", Figure S3). Ala mutants were produced in Escherichia coli HM0079 38 Figure S4). Thus, 10 out of 22 positions were added to the priority list based on their significantly lower or higher DKP yield or Val incorporation ( Figure S4A). ...
... Residues F155, N256, and E386 Play Similar Roles in Different C-Domains. To investigate whether the positions identified in GrsB1 would also be determinants of specificity in other NRPSs, we replaced GrsB1 in the GrsA*/GrsB1 model system with TycB1 (native substrate: D-Phe) from the tyrocidine NRPS 38,42 or SrfAC (native substrate: D-Leu) from the surfactin NRPS. 43 TycB1-C and SrfAC-C share 59% and 42% identity with GrsB1-C, respectively ( Figure S9). ...
... We acknowledge donation of strain HM0079 and the pTrc99a and pSU18 vector backbones by D. Hilvert and M. Marahiel. 38 ...
... The genes for the three best C-domain variants from the individual yeast display assays were amplified from the pCT vector by PCR and assembled into the full-length C-A-T-TE SrfA-C module in the pTrc99a plasmid 70 . The C-terminally hexahistidine-tagged holo SrfA-C variants were produced in E. coli HM0079, purified at pH 7.4 and stored at pH 7.25 (see Supplementary Note-Materials and methods for details) 23 . ...
Engineered biosynthetic assembly lines could revolutionize the sustainable production of bioactive natural product analogs. Although yeast display is a proven, powerful tool for altering the substrate specificity of gatekeeper adenylation domains in nonribosomal peptide synthetases (NRPSs), comparable strategies for other components of these megaenzymes have not been described. Here we report a high-throughput approach for engineering condensation (C) domains responsible for peptide elongation. We show that a 120-kDa NRPS module, displayed in functional form on yeast, can productively interact with an upstream module, provided in solution, to produce amide products tethered to the yeast surface. Using this system to screen a large C-domain library, we reprogrammed a surfactin synthetase module to accept a fatty acid donor, increasing catalytic efficiency for this noncanonical substrate >40-fold. Because C domains can function as selectivity filters in NRPSs, this methodology should facilitate the precision engineering of these molecular assembly lines.
... To allow genetic manipulation, the cluster has previously been cloned from the wild-type producer Aneurinibacillus migulanus [8] and plasmid pSU18-grsTAB carrying the gene cluster has been constructed for production of GS in E. coli HM0079. [27][28][29] The highest GS titer of 44 μM was achieved by incubation at 30°C and 300 rpm shaking for 48 h using buffered TB medium ( Figure 1A). After centrifugation of the culture, triple quad LC-MS/MS analysis detected 90 % of the GS in the cell pellet and 10 % in the supernatant ( Figure 1A). ...
Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein‐filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5‐fold, decreases internal stop codons 250‐fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non‐binary sensor will enable more sophisticated structure‐activity studies and new engineering applications in the future.
... The best strategy was found to be using the two plasmids system, followed by the one plasmid strategy and then the fusion strategy. With the two plasmids strategy, an increase of 400% was observed and the production titer reached 9 mg/L [287]. ...
Lipopeptides, biomolecules composed of a hydrophobic fatty acid chain and a hydrophilic peptide moiety, are one of the main promising classes of biosurfactants. Their mode of synthesis, by multienzymatic proteins consisting of repeated modules (nonribosomal peptide synthetases and sometimes polyketide synthases), is responsible for the high biodiversity observed in this family of amphiphilic compounds. This mode of synthesis generates many structural differences in the lipid and peptide moieties. These differences can be linked to multiple biological activities and physicochemical properties including surface-active ones with a wide range of applications in different sectors. One of the main drawbacks of lipopeptides is their production cost and yield. New synthetic biology approaches have been used to build up new lipopeptides, to improve lipopeptide production in the native host and to develop their heterologous production. After describing their mode of synthesis and the high biodiversity observed in surface-active lipopeptides, this chapter develops how this lipopeptide biodiversity can be increased to get new lipopeptides or improve the production of known lipopeptides through synthetic biology. The increasing understanding of the mode of biosynthesis of these lipopeptides and the use of synthetic biology should lead to the development of new cell factories increasing their yields and producing novel lipopeptide derivatives with increased surface-active properties.
... The best strategy was found to be using the two plasmids system, followed by the one plasmid strategy and then the fusion strategy. With the two plasmids strategy, an increase of 400% was observed and the production titer reached 9 mg/L [287]. ...
... Furthermore, it may not provide the required cofactors or partner enzymes (e. g, cytochrome P450 reductase) for enzymatic activity [8]. Many of these challenges can be overcome by carefully choosing the target enzymes, designing the expression constructs, and co-expressing important accessory proteins or pathways [8][9][10]. On the other hand, E. coli hosts have a high growth rate and the ability to yield large amounts of heterologous proteins. ...
... Often, enzymes within a pathway are encoded by colocalized genes in so-called biosynthetic gene clusters (BGCs), which makes it possible to identify them with dedicated bioinformatic tools [12][13][14][15]. Once a target is selected, dedicated E. coli strains or cell-free extracts-optimized for NP production [9,10,[16][17][18]-are used for overexpression (step 2) and characterization of the pathway products (step 3). E. coli is also an ideal host to optimize product yields through modulation of gene expression and enzyme performance by metabolic engineering, directed evolution, and combinatorial biosynthesis (optional step 4). ...
... In the context of NP biosynthesis in E. coli, metabolic engineering has been used to build chassis strains such as HM0079 and BAP1, that are optimized to support the synthesis of complex NPs by expressing phosphopantetheine transferase, an enzyme crucial for the function of the polyketide synthases and nonribosomal peptide synthetases [9,10]. BAP1 and its derivatives are further optimized to support increased pools of building blocks for polyketide biosynthesis, in particular erythromycin and its analogs [62]. ...
New antimicrobials need to be discovered to fight the advance of multidrug-resistant pathogens. A promising approach is the screening for antimicrobial agents naturally produced by living organisms. As an alternative to studying the native producer, it is possible to use genetically tractable microbes as heterologous hosts to aid the discovery process, facilitate product diversification through genetic engineering, and ultimately enable environmentally friendly production. In this mini-review, we summarize the literature from 2017 to 2022 on the application of Escherichia coli and E. coli-based platforms as versatile and powerful systems for the discovery, characterization, and sustainable production of antimicrobials. We highlight recent developments in high-throughput screening methods and genetic engineering approaches that build on the strengths of E. coli as an expression host and that led to the production of antimicrobial compounds. In the last section, we briefly discuss new techniques that have not been applied to discover or engineer antimicrobials yet, but that may be useful for this application in the future.
... In NRPS, dipeptide thioesters are formed during peptide synthesis. A scheme has been proposed in which DKP is produced from dipeptide thioester loaded into NRPS by the intramolecular cyclization reaction (Gruenewald et al. 2004). Using a module containing an epimerization domain in this reaction, DKP containing d-amino acids, which cannot be synthesized by CDPS, can be synthesized. ...
2,5-Diketopiperazine (DKP) is a cyclic peptide composed of two amino acids and has been recently reported to exhibit various biological activities. DKPs have been synthesized using various methods. In chemical synthesis, a multi-step reaction requiring purification and racemization is problematic. Although enzymatic synthesis can overcome these problems, there has been no example of a general-purpose synthesis of DKPs with high titers. Therefore, we propose a chemoenzymatic method that can synthesize DKPs in a general-purpose manner with high efficiency under mild conditions. The adenylation domain of tyrocidine synthetase A (TycA-A) catalyzes the adenylation reaction of amino acids, and various amides can be synthesized by a nucleophilic substitution reaction with any amine. On the other hand, DKPs can be produced via intramolecular cyclization reactions from dipeptide esters. Based on these observations, we expected a one-pot synthesis of DKPs via dipeptide ester synthesis by TycA-A and cyclization reactions. This method enabled the synthesis of more than 128 types of DKPs without racemization. Importantly, the intramolecular cyclization reaction proceeded largely depending on the pH. In particular, the cyclization reaction proceeded well in the pH range of 6.5–9.5. Based on these results, we constructed a bioreactor with pH–stat for purified enzyme reaction; cyclo(l-Trp-l-Pro) was produced at 4.07 mM by controlling the reaction pH over time using this reactor. The DKPs obtained using this method will provide deeper insights into their structures and functions in future studies.
Key points
• Adenylation enzyme enabled one-pot synthesis of arbitrary 2,5-diketopiperazine.
• Little or no racemization occurred during 2,5-diketopiperazine synthesis.
• Bioreactor with pH–stat for purified enzymes improved the reaction rate.
... In case of completely captured BGCs on fosmids such as RiPPs, the respective clones can directly be used for heterologous expression in E. coli. Fosmids can also be transferred into E. coli strains that are more suitable for heterologous expression of natural products such as phosphopantetheinyl transferase (PPtase) carrying E. coli strains (Gruenewald et al. 2004;Jaitzig et al. 2014). Additionally, the fosmid vector backbone can be genetically modified to enable transfer and maintenance of the plasmid in other heterologous hosts. ...
Culture-independent metagenomic approaches offer a promising solution to the discovery of therapeutically relevant compounds such as antibiotics by enabling access to the hidden biosynthetic potential of microorganisms. These strategies, however, often entail laborious, multi-step, and time-consuming procedures to recover the biosynthetic gene clusters (BGCs) from soil metagenomes for subsequent heterologous expression. Here, we developed an efficient method we called single Nanopore read cluster mining (SNRCM), which enables the fast recovery of complete BGCs from a soil metagenome using long- and short-read sequencing. A metagenomic fosmid library of 83,700 clones was generated and sequenced using Nanopore as well as Illumina technologies. Hybrid assembled contigs of the sequenced fosmid library were subsequently analyzed to identify BGCs encoding secondary metabolites. Using SNRCM, we aligned the identified BGCs directly to Nanopore long-reads and were able to detect complete BGCs on single fosmids. This enabled us to select for and recover BGCs of interest for subsequent heterologous expression attempts. Additionally, the sequencing data of the fosmid library and its corresponding metagenomic DNA enabled us to assemble and recover a large nonribosomal peptide synthetase (NRPS) BGC from three different fosmids of our library and to directly amplify and recover a complete lasso peptide BGC from the high-quality metagenomic DNA. Overall, the strategies presented here provide a useful tool for accelerating and facilitating the identification and production of potentially interesting bioactive compounds from soil metagenomes.
Key points
• An efficient approach for the recovery of BGCs from soil metagenomes was developed to facilitate natural product discovery.
• A fosmid library was constructed from soil metagenomic HMW DNA and sequenced via Illumina and Nanopore.
• Nanopore long-reads enabled the direct identification and recovery of complete BGCs on single fosmids.
... The name derives from the gene sfp, which encodes a PPTase involved in the activation of the surfactin synthase in Bacillus subtilis. This enzyme is well expressed in Escherichia coli, where it can be integrated in the genome (Gruenewald et al., 2004), and it exhibits a broad promiscuity toward both CoA and CPs substrates. Therefore, it is widely used for the heterologous expression of NRPS or PKS genes. ...
Nonribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize a diverse variety of peptides. Many of these are currently used as pharmaceuticals, thanks to their activity as antimicrobials (penicillin, vancomycin, daptomycin, echinocandin), immunosuppressant (cyclosporin) and anticancer compounds (bleomycin). Because of their biotechnological potential, NRPSs have been extensively studied in the past decades. In this review, we provide an overview of the main structural and functional features of these enzymes, and we consider the challenges and prospects of engineering NRPSs for the synthesis of novel compounds. Furthermore, we discuss secondary metabolism and NRP synthesis in the filamentous fungus Penicillium rubens and examine its potential for the production of novel and modified β-lactam antibiotics.
... 40 Consequently, the turnover increases with an excess of the second module, as observed in the closely homologous TycA/ TycB1 system. 41 We hypothesized that impaired adenylation in sdV-GrsA might shift this situation. In titration experiments, we investigated the influence of sdV-GrsA/GrsB1 concentration and ratio on the peptide formation rate to find which module limits the rate. ...
