A Defined and Flexible Pocket Explains Aryl Substrate Promiscuity of the Cahuitamycin Starter Unit-Activating Enzyme CahJ.

Cahuitamycins are biofilm inhibitors assembled by a convergent nonribosomal peptide synthetase pathway. Previous genetic analysis indicated that a discrete enzyme, CahJ, serves as a gatekeeper for cahuitamycin structural diversification. Here, the CahJ protein was probed structurally and functionally to guide the formation of new analogues by mutasynthetic studies. This analysis enabled the in vivo production of a new cahuitamycin congener through targeted precursor incorporation.

Resistance to ketolide antibiotics by coordinated expression of rRNA methyltransferases in a bacterial producer of natural ketolides.

Ketolides are promising new antimicrobials effective against a broad range of Gram-positive pathogens, in part because of the low propensity of these drugs to trigger the expression of resistance genes. A natural ketolide pikromycin and a related compound methymycin are produced by Streptomyces venezuelae strain ATCC 15439. The producer avoids the inhibitory effects of its own antibiotics by expressing two paralogous rRNA methylase genes pikR1 and pikR2 with seemingly redundant functions. We show here that the PikR1 and PikR2 enzymes mono- and dimethylate, respectively, the N6 amino group in 23S rRNA nucleotide A2058. PikR1 monomethylase is constitutively expressed; it confers low resistance at low fitness cost and is required for ketolide-induced activation of pikR2 to attain high-level resistance. The regulatory mechanism controlling pikR2 expression has been evolutionary optimized for preferential activation by ketolide antibiotics. The resistance genes and the induction mechanism remain fully functional when transferred to heterologous bacterial hosts. The anticipated wide use of ketolide antibiotics could promote horizontal transfer of these highly efficient resistance genes to pathogens. Taken together, these findings emphasized the need for surveillance of pikR1/pikR2-based bacterial resistance and the preemptive development of drugs that can remain effective against the ketolide-specific resistance mechanism.

Chemoenzymatic Total Synthesis and Structural Diversification of Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring, and C-H Functionalization.

Polyketide synthases (PKSs) represent a powerful catalytic platform capable of effecting multiple carbon-carbon bond forming reactions and oxidation state adjustments. We explored the functionality of two terminal PKS modules that produce the 16-membered tylosin macrocycle, using them as biocatalysts in the chemoenzymatic synthesis of tylactone and its subsequent elaboration to complete the first total synthesis of the juvenimicin, M-4365, and rosamicin classes of macrolide antibiotics via late-stage diversification. Synthetic chemistry was employed to generate the tylactone hexaketide chain elongation intermediate that was accepted by the juvenimicin (Juv) ketosynthase of the penultimate JuvEIV PKS module. The hexaketide is processed through two complete modules (JuvEIV and JuvEV) in vitro, which catalyze elongation and functionalization of two ketide units followed by cyclization of the resulting octaketide into tylactone. After macrolactonization, a combination of in vivo glycosylation, selective in vitro cytochrome P450-mediated oxidation, and chemical oxidation was used to complete the scalable construction of a series of macrolide natural products in as few as 15 linear steps (21 total) with an overall yield of 4.6%.

An overview of rapamycin: from discovery to future perspectives.

Rapamycin is an immunosuppressive metabolite produced from several actinomycete species. Besides its immunosuppressive activity, rapamycin and its analogs have additional therapeutic potentials, including antifungal, antitumor, neuroprotective/neuroregenerative, and lifespan extension activities. The core structure of rapamycin is derived from (4R,5R)-4,5-dihydrocyclohex-1-ene-carboxylic acid that is extended by polyketide synthase. The resulting linear polyketide chain is cyclized by incorporating pipecolate and further decorated by post-PKS modification enzymes. Herein, we review the discovery and biological activities of rapamycin as well as its mechanism of action, mechanistic target, biosynthesis, and regulation. In addition, we introduce the many efforts directed at enhancing the production of rapamycin and generating diverse analogs and also explore future perspectives in rapamycin research. This review will also emphasize the remarkable pilot studies on the biosynthesis and production improvement of rapamycin by Dr. Demain, one of the world's distinguished scientists in industrial microbiology and biotechnology.

The biosynthetic pathway of FK506 and its engineering: from past achievements to future prospects.

FK506, a 23-membered macrolide produced by several Streptomyces species, is an immunosuppressant widely used to prevent the rejection of transplanted organs. In addition, FK506 and its analogs possess numerous promising therapeutic potentials including antifungal, neuroprotective, and neuroregenerative activities. Herein, we introduce the biological activities and mechanisms of action of FK506 and discuss recent progress made in understanding its biosynthetic pathway, improving production, and in the mutasynthesis of diverse analogs. Perspectives highlighting further strain improvement and structural diversification aimed at generating more analogs with improved pharmaceutical properties will be emphasized.

Evolution of Efficient Modular Polyketide Synthases by Homologous Recombination.

The structural scaffolds of many complex natural products are produced by multifunctional type I polyketide synthase (PKS) enzymes that operate as biosynthetic assembly lines. The modular nature of these mega-enzymes presents an opportunity to construct custom biocatalysts built in a lego-like fashion by inserting, deleting, or exchanging native or foreign domains to produce targeted variants of natural polyketides. However, previously engineered PKS enzymes are often impaired resulting in limited production compared to native systems. Here, we show a versatile method for generating and identifying functional chimeric PKS enzymes for synthesizing custom macrolactones and macrolides. PKS genes from the pikromycin and erythromycin pathways were hybridized in Saccharomyces cerevisiae to generate hybrid libraries. We used a 96-well plate format for plasmid purification, transformations, sequencing, protein expression, in vitro reactions and analysis of metabolite formation. Active chimeric enzymes were identified with new functionality. Streptomyces venezuelae strains that expressed these PKS chimeras were capable of producing engineered macrolactones. Furthermore, a macrolactone generated from selected PKS chimeras was fully functionalized into a novel macrolide analogue. This method permits the engineering of PKS pathways as modular building blocks for the production of new antibiotic-like molecules.

2-Deoxystreptamine-containing aminoglycoside antibiotics: recent advances in the characterization and manipulation of their biosynthetic pathways.

The 2-deoxystreptamine-containing aminoglycosides, such as neomycin, kanamycin and gentamicin, are an important class of antibiotics. A detailed understanding of the complete biosynthetic pathway of aminoglycosides and their biosynthetic enzymes will allow us to not only generate more robust antibiotic agents or drugs with other altered biological activities, but also to produce clinically important semi-synthetic antibiotics by direct fermentation. This Highlight focuses on recent advances in the characterization of their biosynthetic enzymes and pathway as well as some chemo-enzymatic and metabolic engineering approaches for the biological production of natural, semi-synthetic, and novel aminoglycosides.

Discovery of parallel pathways of kanamycin biosynthesis allows antibiotic manipulation.

Kanamycin is one of the most widely used antibiotics, yet its biosynthetic pathway remains unclear. Current proposals suggest that the kanamycin biosynthetic products are linearly related via single enzymatic transformations. To explore this system, we have reconstructed the entire biosynthetic pathway through the heterologous expression of combinations of putative biosynthetic genes from Streptomyces kanamyceticus in the non-aminoglycoside-producing Streptomyces venezuelae. Unexpectedly, we discovered that the biosynthetic pathway contains an early branch point, governed by the substrate promiscuity of a glycosyltransferase, that leads to the formation of two parallel pathways in which early intermediates are further modified. Glycosyltransferase exchange can alter flux through these two parallel pathways, and the addition of other biosynthetic enzymes can be used to synthesize known and new highly active antibiotics. These results complete our understanding of kanamycin biosynthesis and demonstrate the potential of pathway engineering for direct in vivo production of clinically useful antibiotics and more robust aminoglycosides.

Achievements and impacts of glycosylation reactions involved in natural product biosynthesis in prokaryotes.

Bioactive natural products, such as polyketides, flavonoids, glycopeptides, and aminoglycosides, have been used as therapeutic agents. Many of them contain structurally diverse sugar moieties attached to the aglycone core structures. Glycosyltransferases (GTs) catalyze the attachment of nucleotide-activated sugar substrates to acceptor aglycones. Because these sugar moieties are usually essential for biological activity, in vivo pathway engineering in prokaryotic hosts and in vitro enzymatic approaches coupled with GT engineering are currently being used to synthesize novel glycosylated derivatives, and some of them exhibited improved biological activities compared to the parent molecules. Therefore, harnessing the potential of diverse glycosylation reactions in prokaryotes will increase the structural diversity of natural products and the possibility to generate new bioactive products.

Engineered biosynthesis of glycosylated derivatives of narbomycin and evaluation of their antibacterial activities.

A 14-membered macrolide antibiotic narbomycin produced from Streptomyces venezuelae ATCC 15439 is composed of polyketide macrolactone ring and D-desosamine as a deoxysugar moiety, which acts as an important determinant of its antibacterial activity. In order to generate diverse glycosylated derivatives of narbomycin, expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into S. venezuelae YJ003 mutant strain bearing a deletion of thymidine-5'-diphospho-D-desosamine biosynthetic gene cluster. The resulting recombinants of S. venezuelae produced a range of new analogs of narbomycin, which possess unnatural sugar moieties instead of native deoxysugar D-desosamine. The structures of narbomycin derivatives were determined through nuclear magnetic resonance spectroscopy and mass spectrometry analyses and their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with L-rhamnose or 3-O-demethyl-D-chalcose was demonstrated to exhibit greater antibacterial activity than narbomycin and the clinically relevant erythromycin. This work provides new insight into the functions of deoxysugar biosynthetic enzymes and structure-activity relationships of the sugar moieties attached to the macrolides and demonstrate the potential of combinatorial biosynthesis for the generation of new macrolides carrying diverse sugars with increased antibacterial activities.

Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds through a dedicated polyketide synthase and facilitates the mutasynthesis of analogues.

The allyl moiety of the immunosuppressive agent FK506 is structurally unique among polyketides and critical for its potent biological activity. Here, we detail the biosynthetic pathway to allylmalonyl-coenzyme A (CoA), from which the FK506 allyl group is derived, based on a comprehensive chemical, biochemical, and genetic interrogation of three FK506 gene clusters. A discrete polyketide synthase (PKS) with noncanonical domain architecture presumably in coordination with the fatty acid synthase pathway of the host catalyzes a multistep enzymatic reaction to allylmalonyl-CoA via trans-2-pentenyl-acyl carrier protein. Characterization of this discrete pathway facilitated the engineered biosynthesis of novel allyl group-modified FK506 analogues, 36-fluoro-FK520 and 36-methyl-FK506, the latter of which exhibits improved neurite outgrowth activity. This unique feature of FK506 biosynthesis, in which a dedicated PKS provides an atypical extender unit for the main modular PKS, illuminates a new strategy for the combinatorial biosynthesis of designer macrolide scaffolds as well as FK506 analogues.

Microbial transformation of trichostatin A to 2,3-dihydrotrichostatin A.

A new reduced hydroxamate, 2,3-dihydrotrichostatin A, was created from trichostatin A by employing a recombinant strain of Streptomyces venezuelae as a microbial catalyst. Compared with trichostatin A, 2,3-dihydrotrichostatin A showed similar antifungal activity against Saccharomyces cerevisiae, but, interestingly, approximately twice the cytostatic activity against human small-cell lung cancer cells. The production of 2,3-dihydrotrichostatin A via microbial biotransformation demonstrates that the regiospecific and substrate-flexible hydrogenation by S. venezuelae provides a new approach for creating natural product analogues with improved bioactive properties.

A combined approach of classical mutagenesis and rational metabolic engineering improves rapamycin biosynthesis and provides insights into methylmalonyl-CoA precursor supply pathway in Streptomyces hygroscopicus ATCC 29253.

Rapamycin is a macrocyclic polyketide with immunosuppressive, antifungal, and anticancer activity produced by Streptomyces hygroscopicus ATCC 29253. Rapamycin production by a mutant strain (UV2-2) induced by ultraviolet mutagenesis was improved by approximately 3.2-fold (23.6 mg/l) compared to that of the wild-type strain. The comparative analyses of gene expression and intracellular acyl-CoA pools between wild-type and the UV2-2 strains revealed that the increased production of rapamycin in UV2-2 was due to the prolonged expression of rapamycin biosynthetic genes, but a depletion of intracellular methylmalonyl-CoA limited the rapamycin biosynthesis of the UV2-2 strain. Therefore, three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA were evaluated to identify the effective precursor supply pathway that can support the high production of rapamycin: propionyl-CoA carboxylase (PCC), methylmalonyl-CoA mutase, and methylmalonyl-CoA ligase. Among them, only the PCC pathway along with supplementation of propionate was found to be effective for an increase in intracellular pool of methylmalonyl-CoA and rapamycin titers in UV2-2 strain (42.8 mg/l), indicating that the PCC pathway is a major methylmalonyl-CoA supply pathway in the rapamycin producer. These results demonstrated that the combined approach involving traditional mutagenesis and metabolic engineering could be successfully applied to the diagnosis of yield-limiting factors and the enhanced production of industrially and clinically important polyketide compounds.

Genetic dissection of the biosynthetic route to gentamicin A2 by heterologous expression of its minimal gene set.

Since the first use of streptomycin as an effective antibiotic drug in the treatment of tuberculosis, aminoglycoside antibiotics have been widely used against a variety of bacterial infections for over six decades. However, the pathways for aminoglycoside biosynthesis still remain unclear, mainly because of difficulty in genetic manipulation of actinomycetes producing this class of antibiotics. Gentamicin belongs to the group of 4,6-disubstituted aminoglycosides containing a characteristic core aminocyclitol moiety, 2-deoxystreptamine (2-DOS), and the recent discovery of its biosynthetic gene cluster in Micromonospora echinospora has enabled us to decipher its biosynthetic pathway. To determine the minimal set of genes and their functions for the generation of gentamicin A(2), the first pseudotrisaccharide intermediate in the biosynthetic pathway for the gentamicin complex, various sets of candidate genes from M. echinospora and other related aminoglycoside-producing strains were introduced into a nonaminoglycoside producing strain of Streptomyces venezuelae. Heterologous expression of different combinations of putative 2-DOS biosynthetic genes revealed that a subset, gtmB-gtmA-gacH, is responsible for the biosynthesis of this core aminocyclitol moiety of gentamicin. Expression of gtmG together with gtmB-gtmA-gacH led to production of 2'-N-acetylparomamine, demonstrating that GtmG acts as a glycosyltransferase that adds N-acetyl-d-glucosamine (GLcNA) to 2-DOS. Expression of gtmM in a 2'-N-acetylparomamine-producing recombinant S. venezuelae strain generated paromamine. Expression of gtmE in an engineered paromamine-producing strain of S. venezuelae successfully generated gentamicin A(2), indicating that GtmE is another glycosyltransferase that attaches d-xylose to paromamine. These results represent in vivo evidence elucidating the complete biosynthetic pathway of the pseudotrisaccharide aminoglycoside.

Genetic engineering of macrolide biosynthesis: past advances, current state, and future prospects.

Polyketides comprise one of the major families of natural products. They are found in a wide variety of bacteria, fungi, and plants and include a large number of medically important compounds. Polyketides are biosynthesized by polyketide synthases (PKSs). One of the major groups of polyketides are the macrolides, the activities of which are derived from the presence of a macrolactone ring to which one or more 6-deoxysugars are attached. The core macrocyclic ring is biosynthesized from acyl-CoA precursors by PKS. Genetic manipulation of PKS-encoding genes can result in predictable changes in the structure of the macrolactone component, many of which are not easily achieved through standard chemical derivatization or total synthesis. Furthermore, many of the changes, including post-PKS modifications such as glycosylation and oxidation, can be combined for further structural diversification. This review highlights the current state of novel macrolide production with a focus on the genetic engineering of PKS and post-PKS tailoring genes. Such engineering of the metabolic pathways for macrolide biosynthesis provides attractive alternatives for the production of diverse non-natural compounds. Other issues of importance, including the engineering of precursor pathways and heterologous expression of macrolide biosynthetic genes, are also considered.

Biosynthesis of plant-specific flavones and flavonols in Streptomyces venezuelae.

Recently, recombinant Streptomyces venezuelae has been established as a heterologous host for microbial production of flavanones and stilbenes, a class of plant-specific polyketides. In the present work, we expanded the applicability of the S. venezuelae system to the production of more diverse plant polyketides including flavones and flavonols. A plasmid with the synthetic codon-optimized flavone synthase I gene from Petroselium crispum was introduced to S. venezuelae DHS2001 bearing a deletion of the native pikromycin polyketide synthase gene, and the resulting strain generated flavones from exogenously fed flavanones. In addition, a recombinant S. venezuelae mutant expressing a codon-optimized flavanone 3beta-hydroxylase gene from Citrus siensis and a flavonol synthase gene from Citrus unshius also successfully produced flavonols.

Liquid chromatography-mass spectrometry characterization of FK506 biosynthetic intermediates in Streptomyces clavuligerus KCTC 10561BP.

The development of an efficient analytical method for the reliable detection and identification of the biosynthetic intermediates found in microbial cultures, which usually produce complex intermediates of the metabolites of interest, is essential for further biosynthetic investigations. This study developed a simple and highly selective method for detecting the biosynthetic intermediates involved in the FK506 pathway of Streptomyces clavuligerus KCTC 10561BP involving a cleanup procedure using a solid-phase extraction technique to provide reliable extraction of FK506-related compounds from a cell culture broth and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to separate and detect the FK506-related intermediates at concentrations as low as 0.2 microg/L in the broth. This method enabled the analytical profiling of the intermediates formed during the biosynthesis of FK506 in this S. clavuligerus strain, which produced FK506 as a main product. Eight FK506 intermediates--FK520, 37,38-dihydroFK506, prolylFK506, 9-decarbonyl-9-hydroxylFK506, 9-deoxoFK506, desmethylFK520, prolylFK520, and 9-deoxoFK520--were identified. This is the first report of the LC-ESI-MS/MS characterization of a wide range of FK506 analogs from a bacterial fermentation broth. The protocol employed in this study may be useful for estimating the structure of the metabolites without the need for a time-consuming isolation process and nuclear magnetic resonance (NMR) spectroscopy.

A Review of the Microbial Production of Bioactive Natural Products and Biologics.

A variety of organisms, such as bacteria, fungi, and plants, produce secondary metabolites, also known as natural products. Natural products have been a prolific source and an inspiration for numerous medical agents with widely divergent chemical structures and biological activities, including antimicrobial, immunosuppressive, anticancer, and anti-inflammatory activities, many of which have been developed as treatments and have potential therapeutic applications for human diseases. Aside from natural products, the recent development of recombinant DNA technology has sparked the development of a wide array of biopharmaceutical products, such as recombinant proteins, offering significant advances in treating a broad spectrum of medical illnesses and conditions. Herein, we will introduce the structures and diverse biological activities of natural products and recombinant proteins that have been exploited as valuable molecules in medicine, agriculture and insect control. In addition, we will explore past and ongoing efforts along with achievements in the development of robust and promising microorganisms as cell factories to produce biologically active molecules. Furthermore, we will review multi-disciplinary and comprehensive engineering approaches directed at improving yields of microbial production of natural products and proteins and generating novel molecules. Throughout this article, we will suggest ways in which microbial-derived biologically active molecular entities and their analogs could continue to inspire the development of new therapeutic agents in academia and industry.

Enhanced heterologous production of desosaminyl macrolides and their hydroxylated derivatives by overexpression of the pikD regulatory gene in Streptomyces venezuelae.

To elevate the production level of heterologous polyketide in Streptomyces venezuelae, an additional copy of the positive regulatory gene pikD was introduced into the pikromycin (Pik) polyketide synthase (PKS) deletion mutant of S. venezuelae ATCC 15439 expressing tylosin PKS genes. The resulting mutant strain showed enhanced production of both tylactone (TL) and desosaminyl tylactone (DesTL) of 2.7- and 17.1-fold, respectively. The notable increase in DesTL production strongly suggested that PikD upregulates the expression of the desosamine (des) biosynthetic gene cluster. In addition, two hydroxylated forms of DesTL were newly detected from the extract of this mutant. These hydroxylated forms presumably resulted from a PikD-dependent increase in expression of the pikC gene that encodes P450 hydroxylase. Gene expression analysis by reverse transcriptase PCR and bioconversion experiments of 10-deoxymethynolide, narbonolide, and TL into the corresponding desosaminyl macrolides indicated that PikD is a positive regulator of the des and pikC genes, as well as the Pik PKS genes. These results demonstrate the role of PikD as a pathway-specific positive regulator of the entire Pik biosynthetic pathway and its usefulness in the development of a host-vector system for efficient heterologous production of desosaminyl macrolides and novel hydroxylated compounds.

Heterologous production of epothilones B and D in Streptomyces venezuelae.

Epothilones, produced from the myxobacterium Sorangium cellulosum, are potential anticancer agents that stabilize microtubules in a similar manner to paclitaxel. The entire epothilone biosynthetic gene cluster was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin polyketide synthase gene cluster. The resulting strains produced approximately 0.1 microg/l of epothilone B as a sole product after 4 days cultivation. Deletion of an epoF encoding the cytochrome P450 epoxidase gave rise to a mutant that selectively produces 0.4 microg/l of epothilone D. To increase the production level of epothilones B and D, an additional copy of the positive regulatory gene pikD was introduced into the chromosome of both S. venezuleae mutant strains. The resulting strains showed enhanced production of corresponding compounds (approximately 2-fold). However, deletion of putative transport genes, orf3 and orf14 in the epothilone D producing S. venezuelae mutant strain, led to an approximately 3-fold reduction in epothilone D production. These results introduce S. venezuelae as an alternative heterologous host for the production of these valuable anticancer agents and demonstrate the possibility of engineering this strain as a generic heterologous host for the production of polyketides and hybrid polyketide-nonribosomal peptides.