Biosynthetic Engineering Research Articles

Secondary metabolites overproduction through transcriptional gene cluster refactoring

We present a random rational approach enabling the construction of overproducing strains in two steps. The approach first involves creating a library of clusters of interest, in which native promoters are substituted with randomly generated constitutive synthetic promoters, and then expressing this library in an appropriate host strain. This strategy is fast, easy to use, accounts for the architecture of a cluster and completely decouples the expression of a gene cluster from complex native regulatory networks. The strategy was applied to improve the production of a macrocyclic peptide, bottromycin, which possesses antibacterial activity against multidrug-resistant bacteria and is a blueprint for a new class of antibacterials. We successfully optimized the expression of genes in operons and created several variants of the bottromycin gene cluster that provide 5–50 fold higher titres of bottromycin than the natural one, thus resulting in the identification of several new bottromycin derivatives not previously described. Moreover, due to the higher bottromycin yield, bottromycin derivatization was performed via the biosynthetic engineering of the gene cluster. The abovementioned features make this generic strategy a promising tool for the overproduction of known secondary metabolites and the activation of silent secondary metabolites in Actinobacteria.

P450-Catalyzed Tailoring Steps in Leinamycin Biosynthesis Featuring Regio- and Stereoselective Hydroxylations and Substrate Promiscuities.

Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. Both in vivo and in vitro characterization of the LNM biosynthetic machinery have established the formation of the 18-membered macrolactam backbone and the C-3 alkyl branch; the nascent product, LNM E1, of the hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS); and the generation of the thiol moiety at C-3 of LNM E1. However, the tailoring steps converting LNM E1 to LNM are still unknown. Based on gene inactivation and chemical investigation of three mutant strains, we investigated the tailoring steps catalyzed by two cytochromes P450 (P450s), LnmA and LnmZ, in LNM biosynthesis. Our studies revealed that (i) LnmA and LnmZ regio- and stereoselectively hydroxylate the C-8 and C-4' positions, respectively, on the scaffold of LNM; (ii) both LnmA and LnmZ exhibit substrate promiscuity, resulting in multiple LNM analogs from several shunt pathways; and (iii) the C-8 and C-4' hydroxyl groups play important roles in the cytotoxicity of LNM analogs against different cancer cell lines, shedding light on the structure-activity relationships of the LNM scaffold and the LNM-type natural products in general. These studies set the stage for future biosynthetic pathway engineering and combinatorial biosynthesis of the LNM family of natural products for structure diversity and drug discovery.

Lysoquinone-TH1, a New Polyphenolic Tridecaketide Produced by Expressing the Lysolipin Minimal PKS II in Streptomyces albus.

The structural repertoire of bioactive naphthacene quinones is expanded by engineering Streptomyces albus to express the lysolipin minimal polyketide synthase II (PKS II) genes from Streptomyces tendae Tü 4042 (llpD-F) with the corresponding cyclase genes llpCI-CIII. Fermentation of the recombinant strain revealed the two new polyaromatic tridecaketides lysoquinone-TH1 (7, identified) and TH2 (8, postulated structure) as engineered congeners of the dodecaketide lysolipin (1). The chemical structure of 7, a benzo[a]naphthacene-8,13-dione, was elucidated by NMR and HR-MS and confirmed by feeding experiments with [1,2-13C2]-labeled acetate. Lysoquinone-TH1 (7) is a pentangular polyphenol and one example of such rare extended polyaromatic systems of the benz[a]napthacene quinone type produced by the expression of a minimal PKS II in combination with cyclases in an artificial system. While the natural product lysolipin (1) has antimicrobial activity in nM-range, lysoquinone-TH1 (7) showed only minor potency as inhibitor of Gram-positive microorganisms. The bioactivity profiling of lysoquinone-TH1 (7) revealed inhibitory activity towards phosphodiesterase 4 (PDE4), an important target for the treatment in human health like asthma or chronic obstructive pulmonary disease (COPD). These results underline the availability of pentangular polyphenolic structural skeletons from biosynthetic engineering in the search of new chemical entities in drug discovery.

Biosynthesis and Heterologous Production of Vioprolides: Rational Biosynthetic Engineering and Unprecedented 4-Methylazetidinecarboxylic Acid Formation.

Vioprolides are a promising class of anticancer and antifungal lead compounds produced by the myxobacterium Cystobacter violaceus Cb vi35. Previously nothing had been reported about their biosynthesis, including the origin of the unusual 4-methylazetidinecarboxylic acid (MAZ) moiety. We describe the vioprolide biosynthetic gene cluster and solve the production obstacle by expression in three heterologous hosts. Starting from unstable production in the wild type at the single-digit mg L-1 scale, we developed a stable host that eventually allowed for yields of up to half a gram per liter in fermenters. Gene inactivations coupled with isotope feeding studies identified an S-adenosylmethionine (SAM)-dependent enzyme and a methyltransferase as being responsible for the generation of the MAZ building block by a proposed mechanism unprecedented in bacteria. Furthermore, nonnatural vioprolide derivatives were generated via rational genetic engineering.

Accessing new chemical diversity from ancient non‐actinobacterial strains

Our work focuses on the discovery of new biosynthetic routes to chemical diversity through the characterization of unexplored polyketide synthase (PKS) gene clusters and enzymes from ancient and diverse phyla. In the first part of this work, two ancient non‐actinobacterial organisms were cultured, and their secondary metabolites extracted, fractionated, and characterized by liquid chromatography mass spectrometry (LC‐MS) in the search for novel molecules and biosynthetic pathways. These data are currently being compared to existing molecular networks in order to identify novel molecules. In a second part of this work, ancient non‐actinobacterial strains are being evaluated as a source of PKS enzymes that can be expressed in tractable heterologous hosts to enable in vitrocharacterization of poorly‐understood PKSs. We have successfully expressed a ketosynthase‐chain length factor (KS‐CLF) heterodimer pair, which is essential in the manufacturing of polyaromatic polyketides, in Escherichia coli, and are currently evaluating its activity in vitro. Results from these studies are expected to lead to the identification of new chemical diversity encoded by nature, as well as novel tools to enable the biosynthetic engineering of hybrid PKSs.Support or Funding InformationWe would like to acknowledge an NSF CAREER Award #R15GM120704 to Louise K. Charkoudian as a source of funding for this project.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Research progress in terms of the biosynthesis and regulation of terpenoids from medicinal plants

Terpenoids from medicinal plants are widespread secondary metabolites in nature and exist as multiple types with different structures that are crucial in plant growth and development. Moreover, terpenoids exert a broad range of biological activities, including antitumor, antioxidation, anti-viral, and so on. In this study, artemisinin, tanshinone, and ginsenoside were selected as three representative examples, and progress in terms of research on biosynthetic genes, transcriptional regulation, metabolic engineering, synthetic biology, biosynthesis and transport was summarized. Future prospects including functional genes excavation and regulation mechanism network of terpenoids were also discussed.

Biosynthetic Pathway and Metabolic Engineering of Plant Dihydrochalcones.

Dihydrochalcones are plant natural products containing the phenylpropanoid backbone and derived from the plant-specific phenylpropanoid pathway. Dihydrochalcone compounds are important in plant growth and response to stresses and, thus, can have large impacts on agricultural activity. In recent years, these compounds have also received increased attention from the biomedical community for their potential as anticancer treatments and other benefits for human health. However, they are typically produced at relatively low levels in plants. Therefore, an attractive alternative is to express the plant biosynthetic pathway genes in microbial hosts and to engineer the metabolic pathway/host to improve the production of these metabolites. In the present review, we discuss in detail the functions of genes and enzymes involved in the biosynthetic pathway of the dihydrochalcones and the recent strategies and achievements used in the reconstruction of multi-enzyme pathways in microorganisms in efforts to be able to attain higher amounts of desired dihydrochalcones.

Three Principles of Diversity-Generating Biosynthesis.

Natural products are significant therapeutic agents and valuable drug leads. This is likely owing to their three-dimensional structural complexity, which enables them to form complex interactions with biological targets. Enzymes from natural product biosynthetic pathways show great potential to generate natural product-like compounds and libraries. Many challenges still remain in biosynthesis, such as how to rationally synthesize small molecules with novel structures and how to generate maximum chemical diversity. In this Account, we describe recent advances from our laboratory in the synthesis of natural product-like libraries using natural biosynthetic machinery. Our work has focused on the pat and tru biosynthetic pathways to patellamides, trunkamide, and related compounds from cyanobacterial symbionts in marine tunicates. These belong to the cyanobactin class of natural products, which are part of the larger group of ribosomally synthesized and post-translationally modified peptides (RiPPs). These results have enabled the synthesis of rationally designed small molecules and libraries covering more than 1 million estimated derivatives. Because the RiPPs are translated on the ribosome and then enzymatically modified, they are highly compatible with recombinant technologies. This is important because it means that the resulting natural products, their derivatives, and wholly new compounds can be synthesized using the tools of genetic engineering. The RiPPs also represent possibly the most widespread group of bioactive natural products, although this is in part because of the broad definition of what constitutes a RiPP. In addition, the underlying ideas may form the basis for broad-substrate biosynthetic pathways beyond the RiPPs. For example, some of the ideas about kinetic ordering of broad substrate pathways may apply to polyketide or nonribosomal peptide biosynthesis as well. While making these products, we have sought to understand what makes biosynthetic pathways plastic and whether there are any rules that might generally apply to plastic biosynthetic pathways. We present three principles of diversity-generating biosynthesis: (1) substrate evolution, in which the substrates change while enzymes remain constant; (2) pairing of recognition sequences on substrates with biosynthetic enzymes; (3) an inverse metabolic flux in comparison to canonical pathways. If these principles are general, they may enable the design of unimagined derivatives using biosynthetic engineering. For example, it is possible to discover substrate evolution directly by examining sequencing data. By shuffling appropriate recognition sequences and biosynthetic enzymes, it has already been possible to make new hybrid products of multiple pathways. While cases so far have been limited, if this is more general, designed synthesis will become routine. Finally, biosynthesis of natural products is regulated in elaborate ways that are just beginning to be understood. If the inverse metabolic flux model is widespread, it potentially informs on what the timing and relative production level of each enzyme in a designer pathway should be in order to optimize the synthesis of new compounds in vivo.

Living GenoChemetics by hyphenating synthetic biology and synthetic chemistry in vivo

Marrying synthetic biology with synthetic chemistry provides a powerful approach toward natural product diversification, combining the best of both worlds: expediency and synthetic capability of biogenic pathways and chemical diversity enabled by organic synthesis. Biosynthetic pathway engineering can be employed to insert a chemically orthogonal tag into a complex natural scaffold affording the possibility of site-selective modification without employing protecting group strategies. Here we show that, by installing a sufficiently reactive handle (e.g., a C–Br bond) and developing compatible mild aqueous chemistries, synchronous biosynthesis of the tagged metabolite and its subsequent chemical modification in living culture can be achieved. This approach can potentially enable many new applications: for example, assay of directed evolution of enzymes catalyzing halo-metabolite biosynthesis in living cells or generating and following the fate of tagged metabolites and biomolecules in living systems. We report synthetic biological access to new-to-nature bromo-metabolites and the concomitant biorthogonal cross-coupling of halo-metabolites in living cultures.

Biosynthetic Insights into Linaridin Natural Products from Genome Mining and Precursor Peptide Mutagenesis.

Linaridin is a small class of peptide natural products belonging to the ribosomally synthesized and post-translationally modified peptides (RiPPs) superfamily. By an extensive genome-wide survey of linaridin biosynthetic genes, we show that this class of natural products is widespread in nature and possesses vast structural diversity. The linaridin precursor peptides are relatively conserved in the N-termini but have diverse sequences in the core region, which appear to have coevolved with the biosynthetic enzymes. Using the prototypic linaridin cypemycin as a model, we have explored the structure-activity relationships involved in precursor peptide maturation and generated a diverse set of novel cypemycin variants, among which the T2S variant exhibits enhanced activity against Micrococcus luteus. Our results reveal valuable insights into linaridin biosynthesis and highlight the potential to explore this class of natural products by genome mining and by biosynthetic engineering studies.

Biosynthesis of the pyrrolidine protein synthesis inhibitor anisomycin involves novel gene ensemble and cryptic biosynthetic steps

The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits diverse biological and pharmacologic activities. Its biosynthetic origin has remained obscure for more than 60 y, however. Here we report the identification of the biosynthetic gene cluster (BGC) of anisomycin in Streptomyces hygrospinosus var. beijingensis by a bioactivity-guided high-throughput screening method. Using a combination of bioinformatic analysis, reverse genetics, chemical analysis, and in vitro biochemical assays, we have identified a core four-gene ensemble responsible for the synthesis of the pyrrolidine system in anisomycin: aniQ, encoding a aminotransferase that catalyzes an initial deamination and a later reamination steps; aniP, encoding a transketolase implicated to bring together an glycolysis intermediate with 4-hydroxyphenylpyruvic acid to form the anisomycin molecular backbone; aniO, encoding a glycosyltransferase that catalyzes a cryptic glycosylation crucial for downstream enzyme processing; and aniN, encoding a bifunctional dehydrogenase that mediates multistep pyrrolidine formation. The results reveal a BGC for pyrrolidine alkaloid biosynthesis that is distinct from known bacterial alkaloid pathways, and provide the signature sequences that will facilitate the discovery of BGCs encoding novel pyrrolidine alkaloids in bacterial genomes. The biosynthetic insights from this study further set the foundation for biosynthetic engineering of pyrrolidine antibiotics.

New insights into bacterial type II polyketide biosynthesis.

Bacterial aromatic polyketides, exemplified by anthracyclines, angucyclines, tetracyclines, and pentangular polyphenols, are a large family of natural products with diverse structures and biological activities and are usually biosynthesized by type II polyketide synthases (PKSs). Since the starting point of biosynthesis and combinatorial biosynthesis in 1984–1985, there has been a continuous effort to investigate the biosynthetic logic of aromatic polyketides owing to the urgent need of developing promising therapeutic candidates from these compounds. Recently, significant advances in the structural and mechanistic identification of enzymes involved in aromatic polyketide biosynthesis have been made on the basis of novel genetic, biochemical, and chemical technologies. This review highlights the progress in bacterial type II PKSs in the past three years (2013–2016). Moreover, novel compounds discovered or created by genome mining and biosynthetic engineering are also included.

Global N-Glycan Site Occupancy of HIV-1 gp120 by Metabolic Engineering and High-Resolution Intact Mass Spectrometry.

A vital step in HIV vaccine development strategies has been the observation that some infected individuals generate broadly neutralizing antibodies that target the glycans on the surface of HIV-1 gp120. These antibodies target glycan epitopes on viral envelope spikes, and yet the positions and degree of occupancy of glycosylation sites is diverse. Therefore, there is a need to understand glycosylation occupancy on recombinant immunogens. The sheer number of potential glycosylation sites and degree of chemical heterogeneity impedes assessing the global sequon occupancy of gp120 glycoforms. Here, we trap the glycan processing of recombinant gp120 to generate homogeneous glycoforms, facilitating occupancy assessment by intact mass spectrometry. We show that gp120 monomers of the BG505 strain contain either fully occupied sequons or missing the equivalent of one and sometimes two glycans across the molecule. This biosynthetic engineering approach enables the analysis of therapeutically important glycoproteins otherwise recalcitrant to analysis by native mass spectrometry.

Biosynthesis of Oxytetracycline by Streptomyces rimosus: Past, Present and Future Directions in the Development of Tetracycline Antibiotics.

Natural tetracycline (TC) antibiotics were the first major class of therapeutics to earn the distinction of 'broad-spectrum antibiotics' and they have been used since the 1940s against a wide range of both Gram-positive and Gram-negative pathogens, mycoplasmas, intracellular chlamydiae, rickettsiae and protozoan parasites. The second generation of semisynthetic tetracyclines, such as minocycline and doxycycline, with improved antimicrobial potency, were introduced during the 1960s. Despite emerging resistance to TCs erupting during the 1980s, it was not until 2006, more than four decades later, that a third--generation TC, named tigecycline, was launched. In addition, two TC analogues, omadacycline and eravacycline, developed via (semi)synthetic and fully synthetic routes, respectively, are at present under clinical evaluation. Interestingly, despite very productive early work on the isolation of a Streptomyces aureofaciens mutant strain that produced 6-demethyl-7-chlortetracycline, the key intermediate in the production of second- and third-generation TCs, biosynthetic approaches in TC development have not been productive for more than 50 years. Relatively slow and tedious molecular biology approaches for the genetic manipulation of TC-producing actinobacteria, as well as an insufficient understanding of the enzymatic mechanisms involved in TC biosynthesis have significantly contributed to the low success of such biosynthetic engineering efforts. However, new opportunities in TC drug development have arisen thanks to a significant progress in the development of affordable and versatile biosynthetic engineering and synthetic biology approaches, and, importantly, to a much deeper understanding of TC biosynthesis, mostly gained over the last two decades.

The Dental Architect

The systematic and simple replacement of missing teeth without radical manipulation of existing sound structure— over preparation, utilizing scaffolding, or a matrix the “Winged Pontic” of prefabricated filled composite resin, may correctly be characterized as bio-synthetic tissue engineering. A new conversation regarding one of the direction of prosthetic dentistry for the 21st Century is offered in Stephan C. Bayne’s review of the state of the art for restorative biomaterials titled Dental Biomaterials: Where Are We and Where Are We Going? The non-invasive, uncomplicated, artistically and professionally rewarding “Winged Pontic” approach is one such arts, synthetic tissue engineering. The case report offered in this manuscript demonstrates the concept of direct and immediate tooth replacement in a step by step methodology that may easily be adapted by the dental surgeon. Fiber reinforcement is unnecessary based upon 25 years of research since it is cumbersome, ineffectual, and disruptive to the filled composite resin matrix. In any instance, the “Winged Pontic” fixed prosthesis may be viewed as a short term, medium term, or definitive restoration. It may also serve as a pediatric tooth space maintainer or as a implant stabilization prosthesis during osseointegration of dental implants by hollowing out the tissue side core of the “Winged Pontic” and placing it over the healing abutment of the dental implant

The effect of biologically and chemically synthesized silver nanoparticles (AgNPs) on biofilm formation

Bionanotechnology has emerged up as integration between biotechnology and nanotechnology for developing biosynthetic and environmental-friendly technology for synthesis of nanomaterials. Different types of nanomaterials like copper, zinc, titanium, magnesium, gold, and silver have applied in the various industries but silver nanoparticles have proved to be most effective against bacteria, viruses and eukaryotic microorganisms. The antimicrobial property of silver nanoparticles are widely known. Due to strong antibacterial property silver nanoparticles are used, e.g. in clothing, food industry, sunscreens, cosmetics and many household and environmental appliances. The aim of the study was to compare the effect of silver nanoparticles (AgNPs) synthesized biologically and chemically on the biofilm formation. The biofilm was formed by the bacteria isolated from the water supply network. The commonly used crystal violet assay (CV) was applied for biofilm analysis. In this study effect of biologically synthesized Ag-NPs on the biofilm formation was evaluated.

Identification and Characterization of the Sulfazecin Monobactam Biosynthetic Gene Cluster

The monobactams, exemplified by the natural product sulfazecin, are the only class of β-lactam antibiotics not inactivated by metallo-β-lactamases, which confer bacteria with extended-spectrum β-lactam resistance. We screened a transposon mutagenesis library from Pseudomonas acidophila ATCC 31363 and isolated a sulfazecin-deficient mutant that revealed a gene cluster encoding two non-ribosomal peptide synthetases (NRPSs), a methyltransferase, a sulfotransferase, and a dioxygenase. Three modules and an aberrant C-terminal thioesterase (TE) domain are distributed across the two NRPSs. Biochemical examination of the adenylation (A) domains provided evidence that L-2,3-diaminopropionate, not L-serine as previously thought, is the direct source of the β-lactam ring of sulfazecin. ATP/PPi exchange assay also revealed an unusual substrate selectivity shift of one A domain when expressed with or without the immediately upstream condensation domain. Gene inactivation analysis defined a cluster of 13 open reading frames sufficient for sulfazecin production, precursor synthesis, self-resistance, and regulation. The identification of a key intermediate supported a proposed NRPS-mediated mechanism of sulfazecin biosynthesis and β-lactam ring formation distinct from the nocardicins, another NRPS-derived subclass of monocyclic β-lactam. These findings will serve as the basis for further biosynthetic research and potential engineering of these important antibiotics.

Artemisinin biosynthesis in Artemisia annua and metabolic engineering: questions, challenges, and perspectives

Artemisinin-based combination therapy (ACT) forms the frontline treatment of malaria. Artemisinin, an endoperoxide sesquiterpenoid lactone biosynthesized by Artemisia annua, is the effective medicine that kills malarial parasites. Due to insufficient production of artemisinin for ACT, millions of people lost their lives in past years worldwide. To solve this severe problem, numerous studies have been undertaken to understand artemisinin biosynthesis and to innovate metabolic engineering technology to increase artemisinin yield. Here, we focus on reviewing progresses achieved in understanding biosynthetic pathway, genetic breeding, metabolic engineering, and synthetic biology. Furthermore, based on current knowledge, we discuss multiple fundamental questions and challenges.

Biosynthetic engineering of nonribosomal peptide synthetases.

From the evolutionary melting pot of natural product synthetase genes, microorganisms elicit antibiotics, communication tools, and iron scavengers. Chemical biologists manipulate these genes to recreate similarly diverse and potent biological activities not on evolutionary time scales but within months. Enzyme engineering has progressed considerably in recent years and offers new screening, modelling, and design tools for natural product designers. Here, recent advances in enzyme engineering and their application to nonribosomal peptide synthetases are reviewed. Among the nonribosomal peptides that have been subjected to biosynthetic engineering are the antibiotics daptomycin, calcium-dependent antibiotic, and gramicidin S. With these peptides, incorporation of unnatural building blocks and modulation of bioactivities via various structural modifications have been successfully demonstrated. Natural product engineering on the biosynthetic level is not a reliable method yet. However, progress in the understanding and manipulation of biosynthetic pathways may enable the routine production of optimized peptide drugs in the near future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Activation of a plasmid-situated type III PKS gene cluster by deletion of a wbl gene in deepsea-derived Streptomyces somaliensis SCSIO ZH66.

BackgroundActinomycete genome sequencing has disclosed a large number of cryptic secondary metabolite biosynthetic gene clusters. However, their unavailable or limited expression severely hampered the discovery of bioactive compounds. The whiB-like (wbl) regulatory genes play important roles in morphological differentiation as well as secondary metabolism; and hence the wblAso gene was probed and set as the target to activate cryptic gene clusters in deepsea-derived Streptomyces somaliensis SCSIO ZH66.ResultswblAso from deepsea-derived S. somaliensis SCSIO ZH66 was inactivated, leading to significant changes of secondary metabolites production in the ΔwblAso mutant, from which α-pyrone compound violapyrone B (VLP B) was isolated. Subsequently, the VLP biosynthetic gene cluster was identified and characterized, which consists of a type III polyketide synthase (PKS) gene vioA and a regulatory gene vioB; delightedly, inactivation of vioB led to isolation of another four VLPs analogues, among which one was new and two exhibited improved anti-MRSA (methicillin-resistant Staphylococcus aureus, MRSA) activity than VLP B. Moreover, transcriptional analysis revealed that the expression levels of whi genes (whiD, whiG, whiH and whiI) and wbl genes (wblC, wblE, wblH, wblI and wblK) were repressed by different degrees, suggesting an intertwined regulation mechanism of wblAso in morphological differentiation and secondary metabolism of S. somaliensis SCSIO ZH66.ConclusionswblA orthologues would be effective targets for activation of cryptic gene clusters in marine-derived Streptomyces strains, notwithstanding the regulation mechanisms might be varied in different strains. Moreover, the availability of the vio gene cluster has enriched the diversity of type III PKSs, providing new opportunities to expand the chemical space of polyketides through biosynthetic engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0515-6) contains supplementary material, which is available to authorized users.