Functional identification of the gene encoding the enzyme involved in mannosylation in ramoplanin biosynthesis in Actinoplanes sp.

Ramoplanin is a lipopeptide antibiotic active against multi-drug-resistant, Gram-positive pathogens. Structurally, it contains a di-mannose moiety attached to the peptide core at Hpg¹¹. The biosynthetic gene cluster of ramoplanin has already been reported and the assembly of the depsipeptide has been elucidated but the mechanism of transferring sugar moiety to the peptide core remains unclear. Sequence analysis of the biosynthetic gene cluster indicated ramo-orf29 was a mannosyltransferase candidate. To investigate the involvement of ramo-orf29 in ramoplanin biosynthesis, gene inactivation and complementation have been conducted in Actinoplanes sp. ATCC 33076 by homologous recombination. Metabolite analysis revealed that the ramo-orf29 inactivated mutant produced no ramoplanin but the ramoplanin aglycone. Thus, ramo-orf29 codes for the mannosyltransferase in the ramoplanin biosynthesis pathway. This lays the foundation for further exploitation of the ramoplanin mannosyltransferase and aglycone in combinatorial biosynthesis.     

Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene

The introduction or creation of metabolic pathways in microbial hosts has allowed for the production of complex chemicals of therapeutic and industrial importance. However, these pathways rarely function optimally when first introduced into the host organism and can often deleteriously affect host growth, resulting in suboptimal yields of the desired product. Common methods used to improve production from engineered biosynthetic pathways include optimizing codon usage, enhancing production of rate-limiting enzymes, and eliminating the accumulation of toxic intermediates or byproducts to improve cell growth. We have employed these techniques to improve production of amorpha-4,11-diene (amorphadiene), a precursor to the anti-malarial compound artemisinin, by an engineered strain of Escherichia coli. First we developed a simple cloning system for expression of the amorphadiene biosynthetic pathway in E. coli, which enabled the identification of two rate-limiting enzymes (mevalonate kinase (MK) and amorphadiene synthase (ADS)). By optimizing promoter strength to balance expression of the encoding genes we alleviated two pathway bottlenecks and improved production five fold. When expression of these genes was further increased by modifying plasmid copy numbers, a seven-fold increase in amorphadiene production over that from the original strain was observed. The methods demonstrated here are applicable for identifying and eliminating rate-limiting steps in other constructed biosynthetic pathways.     

Engineered biosynthesis of the peptide antibiotic bacitracin in the surrogate host Bacillus subtilis.

Nonribosomal peptides are processed on multifunctional enzymes called nonribosomal peptide synthetases (NRPSs), whose modular multidomain arrangement allowed the rational design of new peptide products. However, the lack of natural competence and efficient transformation methods for most of nonribosomal peptide producer strains prevented the in vivo manipulation of these biosynthetic gene clusters. In this study, we present methods for the construction of a genetically engineered Bacillus subtilis surrogate host for the integration and heterologous expression of foreign NRPS genes. In the B. subtilis surrogate host, we deleted the resident 26-kilobase srfA gene cluster encoding the surfactin synthetases and subsequently used the same chromosomal location for integration of the entire 49-kilobase bacitracin biosynthetic gene cluster from Bacillus licheniformis by a stepwise homologous recombination method. Synthesis of the branched cyclic peptide antibiotic bacitracin in the engineered B. subtilis strain was achieved at high level, indicating a functional production and proper posttranslational modification of the bacitracin synthetases BacABC, as well as the expression of the associated bacitracin self-resistance genes. This engineered and genetically amenable B. subtilis strain will facilitate the rational design of new bacitracin derivatives.     

Recent developments towards the heterologous expression of complex bacterial natural product biosynthetic pathways

The heterologous expression of natural product biosynthetic pathways is of increasing interest in biotechnology and drug discovery. It enables the (over)production of structurally complex substances through transfer of the biosynthetic genes from the original producer to more amenable heterologous hosts, and provides the basis to generate novel analogs through biosynthetic engineering. Furthermore, the lateral transfer of 'silent' (not expressed under standard laboratory conditions) secondary metabolite pathways or metagenomic DNA into surrogate host strains is expected to yield new, potentially bioactive compounds. This review discusses recent reports on the heterologous production of natural products with emphasis on polyketide and nonribosomally biosynthesized peptide compounds.     

Chemistry and biology of the ramoplanin family of peptide antibiotics

The peptide antibiotic ramoplanin factor A2 is a promising clinical candidate for treatment of Gram-positive bacterial infections that are resistant to antibiotics such as glycopeptides, macrolides, and penicillins. Since its discovery in 1984, no clinical or laboratory-generated resistance to this antibiotic has been reported. The mechanism of action of ramoplanin involves sequestration of peptidoglycan biosynthesis Lipid intermediates, thus physically occluding these substrates from proper utilization by the late-stage peptidoglycan biosynthesis enzymes MurG and the transglycosylases (TGases). Ramoplanin is structurally related to two cell wall active lipodepsipeptide antibiotics, janiemycin, and enduracidin, and is functionally related to members of the lantibiotic class of antimicrobial peptides (mersacidin, actagardine, nisin, and epidermin) and glycopeptide antibiotics (vancomycin and teicoplanin). Peptidomimetic chemotherapeutics derived from the ramoplanin sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and related pathogens. Here we review the chemistry and biology of the ramoplanins including its discovery, structure elucidation, biosynthesis, antimicrobial activity, mechanism of action, and total synthesis.     

Combinatorial biosynthesis of antimicrobials and other natural products

Combinatorial biosynthesis utilizes the enzymes from antibiotic (and other natural product) biosynthetic pathways to create novel chemical structures. The manipulation of modular polyketide synthases (PKSs) has been the major focus of this effort and has led to the production of, for example, several erythromycin analogs. Many new tools for manipulating and studying these multifunctional enzymes have been developed. These include multiple hosts and expression systems, enzymology tools for in vitro study, and ways to engineer pre-PKS and post-PKS pathways. The result is more rational and faster methods of engineering new compounds for the development of chemotherapeutic agents from natural products. The most significant recent advances in combinatorial biosynthesis are outlined.     

Reconstitution of the peptidoglycan cytoplasmic precursor biosynthetic pathway in cell-free system and rapid screening of antisense oligonucleotides for Mur enzymes

Bacterial peptidoglycan is the cell wall component responsible for various biological activities. Its cytoplasmic precursor UDP-N-acetylmuramyl pentapeptide is biosynthesized by the first six enzymes of peptidoglycan synthetic pathways (Mur enzymes), which are all proved to be important targets for antibiotic screening. In our present work, the genes encoding Mur enzymes from Escherichia coli were co-expressed in the cell-free protein synthesis (CFPS) system, and the activities of Mur enzymes derived from CFPS system were validated by the synthesis of the final product UDP-N-acetylmuramyl pentapeptide. Then this in vitro reconstituted Mur biosynthetic pathway was used to screen a panel of specific antisense oligonucleotides for MurA and MurB. The selected oligonucleotides were proved to eliminate the expression of Mur enzymes, and thus inhibit the Mur biosynthetic pathway. The present work not only developed a rapid method to reconstruct and regulate a biosynthetic pathway in vitro, but also may provide insight into the development of novel antibiotics targeting on peptidoglycan biosynthetic pathway.     

Lantibiotic engineering: molecular characterization and exploitation of lantibiotic-synthesizing enzymes for peptide engineering

Lanthionine-containing peptide antibiotics called lantibiotics are produced by a large number of Gram-positive bacteria. Nukacin ISK-1 produced by Staphylococcus warneri ISK-1 is type-A(II) lantibiotic. Ribosomally synthesized nukacin ISK-1 prepeptide (NukA) consists of an N-terminal leader peptide followed by a C-terminal propeptide moiety that undergoes several post-translational modification events including unusual amino acid formation by the modification enzyme NukM, cleavage of leader peptide and export by the dual functional ABC transporter NukT, finally yielding a biologically active peptide. Unusual amino acids in lantibiotics contribute to biological activity and also structural stability against proteases. Thus, lantibiotic-synthesizing enzymes have a high potentiality for peptide engineering by introduction of unusual amino acids into desired peptides with altering biological and physicochemical properties, e.g., activity and stability, termed lantibiotic engineering. We report the establishment of a heterologous expression of nukacin ISK-1 biosynthetic gene cluster by the nisin-controlled expression system and discuss our recent progress in understanding of the biosynthetic enzymes for nukacin ISK-1 such as localization, molecular interaction in biophysical and biochemical aspects. Substrate specificity of the lantibiotic-synthesizing enzymes was evaluated by complementation of the biosynthetic enzymes (LctM and LctT) of closely related lantibiotic lacticin 481 for nukacin ISK-1 biosynthesis. We further explored a rapid and powerful tool for introduction of unusual amino acids by co-expression of hexa-histidine-tagged NukA and NukM in Escherichia coli.     

Lessons learned from the transformation of natural product discovery to a genome-driven endeavor

Natural product discovery is currently undergoing a transformation from a phenotype-driven field to a genotype-driven one. The increasing availability of genome sequences, coupled with improved techniques for identifying biosynthetic gene clusters, has revealed that secondary metabolomes are strikingly vaster than previously thought. New approaches to correlate biosynthetic gene clusters with the compounds they produce have facilitated the production and isolation of a rapidly growing collection of what we refer to as “reverse-discovered” natural products, in analogy to reverse genetics. In this review, we present an extensive list of reverse-discovered natural products and discuss seven important lessons for natural product discovery by genome-guided methods: structure prediction, accurate annotation, continued study of model organisms, avoiding genome-size bias, genetic manipulation, heterologous expression, and potential engineering of natural product analogs.     

A functional screen for recovery of 4'-phosphopantetheinyl transferase and associated natural product biosynthesis genes from metagenome libraries

The single-module non-ribosomal peptide synthetase BpsA from Streptomyces lavendulae has the unique ability to autonomously synthesize a coloured product (indigoidine) from a single substrate (l-glutamine), conditional upon activation by a 4'-phosphopantetheinyl transferase (PPTase) partner. We show that bpsA can be expressed in an entD PPTase gene deleted mutant of Escherichia coli to yield a sensitive reporter strain for recovery of PPTase genes from metagenome libraries. We also show that recombinant bpsA constructs, generated by substitution of the native peptidyl carrier protein domain followed by directed evolution to restore function, can be used to increase the diversity of PPTase genes recovered from a sample. As PPTases are essential for activation of non-ribosomal peptide synthetase and polyketide synthase enzymes, they are frequently associated with secondary metabolite gene clusters. Nearly half of the PPTases recovered in our screening of two small-insert soil metagenome libraries were genetically linked to recognizable secondary metabolite biosynthetic genes, demonstrating that PPTase-targeting functional screens can be used for efficient recovery of natural product gene clusters from metagenome libraries. The plasticity and portability of bpsA reporter genes can potentially be exploited to maximize recovery and expression of PPTase-bearing clones in a wide range of hosts.     

Stapled NRPS enhances the production of valinomycin in Escherichia coli

Nonribosomal peptides (NRPs) are a large family of secondary metabolites with notable bioactivities, which distribute widely in natural resources across microbes and plants. To obtain these molecules, heterologous production of NRPs in robust surrogate hosts like Escherichia coli represent a feasible approach. However, reconstitution of the full biosynthetic pathway in a host often leads to low productivity, which is at least in part due to the low efficiency of enzyme interaction in vivo except for the well-known reasons of metabolic burden (e.g., expression of large NRP synthetases—NRPSs with molecular weights of >100 kDa) and cellular toxicity on host cells. To enhance the catalytic efficiency of large NRPSs in vivo, here we propose to staple NRPS enzymes by using short peptide/protein pairs (e.g., SpyTag/SpyCatcher) for enhanced NRP production. We achieve this goal by introducing a stapled NRPS system for the biosynthesis of the antibiotic NRP valinomycin in E. coli. The results indicate that stapled valinomycin synthetase (Vlm1 and Vlm2) enables higher product accumulation than those two free enzymes (e.g., the maximum improvement is nearly fourfold). After further optimization by strain and bioprocess engineering, the final valinomycin titer maximally reaches about 2800 µg/L, which is 73 times higher than the initial titer of 38 µg/L. We expect that stapling NRPS enzymes will be a promising catalytic strategy for high-level biosynthesis of NRP natural products.     

The cyclic peptide synthetase catalyzing HC-toxin production in the filamentous fungus Cochliobolus carbonum is encoded by a 15.7-kilobase open reading frame.

Race 1 of Cochliobolus carbonum, a fungal plant pathogen, owes its exceptional virulence on certain genotypes of maize to the production of HC-toxin, a cyclic tetrapeptide. Production of HC-toxin is controlled by a single known gene, TOX2. Race 1, but not races that do not make HC-toxin, contains two copies of a 22-kilobase (kb) region of chromosomal DNA that is required for HC-toxin biosynthesis and hence virulence. We have sequenced this 22-kb region and here show that it contains an open reading frame of 15.7 kb that encodes a multifunctional cyclic peptide synthetase of potential M(r)574,620. This gene, called HTS1, apparently contains no introns. The predicted gene product, HC-toxin synthetase (HTS), contains four amino acid-binding (adenylate-forming) domains that are highly similar to those found in other cyclic peptide synthetases and other adenylate-binding enzymes. The DNA sequence encodes tryptic peptides derived from two HC-toxin biosynthetic enzymes, HC-toxin synthetase 1 (HTS-1) and HC-toxin synthetase 2 (HTS-2), indicating that these two enzymes exist in vivo as part of a single polypeptide. Consistent with this, in some enzyme preparations antibodies against the enzyme HTS-2, which was originally purified as a protein with a subunit M(r) of 160,000, recognize a protein with an estimated subunit M(r) greater than 480,000.     

Chemoenzymatic synthesis of cryptophycin/arenastatin natural products

Microbially derived modular polyketide synthase and nonribosomal peptide synthetase biosynthetic pathways are a rich source of novel natural products. Development of these systems for the engineered biosynthesis of diverse secondary metabolites continues to progress as a robust source of chemical diversity. Recent efforts that employ individual enzymes and catalytic domains for the production or modification of small molecules have met with growing success. In this study, the thioesterase domain from the cryptophycin biosynthetic pathway was isolated and its function evaluated with a series of linear chain elongation intermediates in developing a novel chemoenzymatic synthesis of the cryptophycin/arenastatin class of antitumor agents. The results show the high efficiency of the thioesterase in generating the 16-membered depsipeptide ring of this important natural product system. Moreover, analysis of selected substrates revealed considerable tolerance for structural variation within the seco-cryptophycin unit C beta-alanine residue, but strict structural requirements at the phenyl group position of the unit A delta-hydroxy octadienoate chain elongation intermediates.     

Site-specific observation of acyl intermediate processing in thiotemplate biosynthesis by fourier transform mass spectrometry: the polyketide module of yersiniabactin synthetase

Complex arrays of thioester bound intermediates are present on 100-700 kDa enzymes during the biogenesis of diverse types of pharmacophores and natural product drugs. These multidomain enzymes, known as nonribosomal peptide synthetases and polyketide synthases (NRPSs and PKSs, respectively), synthesize from simple, physiologically available substrates bioactive compounds that can be further tailored by a host of modifying domains (e.g., methylation, cyclization, and epimerization) to increase the complexity of the mature final product. Interrogation of such covalent intermediates using mass spectrometry (MS) presents an underutilized method for understanding the covalent catalysis executed by NRPS and PKS enzymes. For the PKS module (205 kDa) from the yersiniabactin (Ybt) gene cluster of Yersinia pestis, limited proteolysis afforded a key 11 kDa peptide from the acyl-carrier protein (ACP) domain upon which at least five covalent intermediates could be detected (42, 70, 86, 330, and 358 Da). The isotopic resolution achieved by Fourier transform mass spectrometry (FTMS) allowed for the incorporation of substrates with stable isotopes to confirm the structural assignments of three intermediates (86, 330, and 358 Da) on the Ybt biosynthetic pathway to within 1 Da. Approximately 75% of the enzyme capacity is lost to unproductive decarboxylation of malonyl-S-ACP partly constraining the 1.4 min(-)(1) rate of Ybt production in vitro. Acyl transfer to the ACP domain (on the Ybt pathway) was promoted by a factor of approximately 10 over unproductive CO(2) loss in the presence of the cosubstrate S-adenosylmethionine (SAM), with S-adenosylhomocysteine unable to restore the condensation yield observed with SAM. The data are consistent with Claisen condensation from KS to the ACP carrier site being reversible, with the absence of downstream methylation providing more opportunity for unproductive CO(2) loss. Extension of such FTMS-based studies will allow the direct visualization of multiple intermediates in determining the catalytic order of events and kinetics of NRPS and PKS systems.     

NisT, the transporter of the lantibiotic nisin, can transport fully modified, dehydrated, and unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides

Lantibiotics are lanthionine-containing peptide antibiotics. Nisin, encoded by nisA, is a pentacyclic lantibiotic produced by some Lactococcus lactis strains. Its thioether rings are posttranslationally introduced by a membrane-bound enzyme complex. This complex is composed of three enzymes: NisB, which dehydrates serines and threonines; NisC, which couples these dehydrated residues to cysteines, thus forming thioether rings; and the transporter NisT. We followed the activity of various combinations of the nisin enzymes by measuring export of secreted peptides using antibodies against the leader peptide and mass spectroscopy for detection. L. lactis expressing the nisABTC genes efficiently produced fully posttranslationally modified prenisin. Strikingly, L. lactis expressing the nisBT genes could produce dehydrated prenisin without thioether rings and a dehydrated form of a non-lantibiotic peptide. In the absence of the biosynthetic NisBC enzymes, the NisT transporter was capable of excreting unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides. Our data show that NisT specifies a broad spectrum (poly)peptide transporter that can function either in conjunction with or independently from the biosynthetic genes. NisT secretes both unmodified and partially or fully posttranslationally modified forms of prenisin and non-lantibiotic peptides. These results open the way for efficient production of a wide range of peptides with increased stability or novel bioactivities.     

游动放线菌中orf20基因敲除对雷莫拉宁合成的影响

雷莫拉宁生物合成基因簇中orf20与已知的卤化酶基因高度同源.本研究在大肠杆菌中构建同框敲除质粒pSM-3,将其转入游动放线菌Actinoplanes sp.ATCC 33076,通过同源重组双交换的方法将orf20基因内部编码64个氨基酸的序列敲除,筛选得到双交换阻断突变株SIPI-A.2001 dorf20(Δor...     

The enzymology of combinatorial biosynthesis

Combinatorial biosynthesis involves the genetic manipulation of natural product biosynthetic enzymes to produce potential new drug candidates that would otherwise be difficult to obtain. In either a theoretical or practical sense, the number of combinations possible from different types of natural product pathways ranges widely. Enzymes that have been the most amenable to this technology synthesize the polyketides, nonribosomal peptides, and hybrids of the two. The number of polyketide or peptide natural products theoretically possible is huge, but considerable work remains before these large numbers can be realized. Nevertheless, many analogs have been created by this technology, providing useful structure-activity relationship data and leading to a few compounds that may reach the clinic in the next few years. In this review the focus is on recent advances in our understanding of how different enzymes for natural product biosynthesis can be used successfully in this technology.     

Adenosylation reactions catalyzed by the radical S-adenosylmethionine superfamily enzymes

The radical S-adenosylmethionine (SAM) superfamily enzymes reductively cleave SAM to produce a highly reactive 5ˊ-deoxyadenosyl (dAdo) radical, which in most cases abstracts a hydrogen from the substrate and initiates highly diverse reactions. In rare cases, the dAdo radical can add to a sp² carbon to result in the production an adenosylated product. These radical SAM-dependent adenosylation reactions are present in natural product biosynthetic pathways and can be achieved by using unnatural substrate analogs containing olefin or aryl moieties. This Opinion provides a focused perspective on this emerging type of biochemistry and discusses its potential use in bioengineering and biocatalysis.