Deciphering the Biosynthesis of TDP-ß-l-oleandrose in Avermectin.

Avermectin (AVM) refers to eight macrolides containing a common l-oleandrosyl disaccharide chain indispensable to their antiparasitic bioactivities. We delineated the biosynthetic pathway of TDP-ß-l-oleandrose (1), the sugar donor of AVM, by characterizing AveBVIII, AveBV, and AveBVII as TDP-sugar 3-ketoreductase, 5-epimerase, and 3-O-methyltransferase, respectively. On the basis of this pathway, we successfully reconstituted the biosynthesis of 1 in Escherichia coli. Our work completes the biosynthetic pathway of AVM and lays a solid foundation for further studies.

Biochemical Characterization of ß-Amino Acid Incorporation in Fluvirucin†B2 Biosynthesis.

Naturally occurring lactams, such as the polyketide-derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. Although ß-amino acid loading in the fluvirucin B2 polyketide pathway was proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. Here we elucidate the complete biosynthetic pathway of the ß-amino acid loading pathway in fluvirucin B2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non-native acyl transferases to selectively transfer ß-amino acids onto a polyketide synthase (PKS) loading platform. The results presented here provide a detailed biochemical description of ß-amino acid selection and will further aid in future efforts to develop engineered lactam-producing PKS platforms.

Another Brick in the Wall: a Rhamnan Polysaccharide Trapped inside Peptidoglycan of Lactococcus lactis.

Polysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In Lactococcus lactis, a polysaccharide pellicle (PSP) forms a layer at the cell surface. The PSP structure varies among lactococcal strains; in L. lactis MG1363, the PSP is composed of repeating hexasaccharide phosphate units. Here, we report the presence of an additional neutral polysaccharide in L. lactis MG1363 that is a rhamnan composed of α-l-Rha trisaccharide repeating units. This rhamnan is still present in mutants devoid of the PSP, indicating that its synthesis can occur independently of PSP synthesis. High-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) analysis of whole bacterial cells identified a PSP at the surface of wild-type cells. In contrast, rhamnan was detected only at the surface of PSP-negative mutant cells, indicating that rhamnan is located underneath the surface-exposed PSP and is trapped inside peptidoglycan. The genetic determinants of rhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery, except the gene tagO encoding the initiating glycosyltransferase. We present a model of rhamnan biosynthesis based on an ABC transporter-dependent pathway. Conditional mutants producing reduced amounts of rhamnan exhibit strong morphological defects and impaired division, indicating that rhamnan is essential for normal growth and division. Finally, a mutation leading to reduced expression of lcpA, encoding a protein of the LytR-CpsA-Psr (LCP) family, was shown to severely affect cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, rhamnan was detected by HR-MAS NMR, suggesting that LcpA participates in the attachment of rhamnan to peptidoglycan.IMPORTANCE In the cell wall of Gram-positive bacteria, the peptidoglycan sacculus is considered the major structural component, maintaining cell shape and integrity. It is decorated with other glycopolymers, including polysaccharides, the roles of which are not fully elucidated. In the ovococcus Lactococcus lactis, a polysaccharide with a different structure between strains forms a layer at the bacterial surface and acts as the receptor for various bacteriophages that typically exhibit a narrow host range. The present report describes the identification of a novel polysaccharide in the L. lactis cell wall, a rhamnan that is trapped inside the peptidoglycan and covalently bound to it. We propose a model of rhamnan synthesis based on an ABC transporter-dependent pathway. Rhamnan appears as a conserved component of the lactococcal cell wall playing an essential role in growth and division, thus highlighting the importance of polysaccharides in the cell wall integrity of Gram-positive ovococci.

One-Pot Combinatorial Biosynthesis of Glycosylated Anthracyclines by Cocultivation of Streptomyces Strains Producing Aglycones and Nucleotide Deoxysugars.

Anthracyclines, such as doxorubicin, are effective anticancer drugs composed of a tetracyclic polyketide aglycone and one or more deoxysugar moieties, which play a critical role in their biological activity. A facile one-pot combinatorial biosynthetic system was developed for the generation of a range of glycosylated derivatives of anthracyclines. Cocultivation of Streptomyces venezuelae mutants producing two anthracycline aglycones with eight different nucleotide deoxysugar-producing S. venezuelae mutants that coexpress a substrate-flexible glycosyltransferase led to the generation of 16 aklavinone or ε-rhodomycinone glycosides containing diverse deoxysugar moieties, seven of which are new. This demonstrates the potential of the one-pot combinatorial biosynthetic system based on cocultivation as a facile biological tool capable of combining diverse aglycones and deoxysugars to generate structurally diverse polyketides carrying engineered sugars for drug discovery and development.

A Single Gene Cluster for Chalcomycins and Aldgamycins: Genetic Basis for Bifurcation of Their Biosynthesis.

Aldgamycins are 16-membered macrolide antibiotics with a rare branched-chain sugar d-aldgarose or decarboxylated d-aldgarose at C-5. In our efforts to clone the gene cluster for aldgamycins from a marine-derived Streptomyces sp. HK-2006-1 capable of producing both aldgamycins and chalcomycins, we found that both are biosynthesized from a single gene cluster. Whole-genome sequencing combined with gene disruption established the entire gene cluster of aldgamycins: nine new genes are incorporated with the previously identified chalcomycin gene cluster. Functional analysis of these genes revealed that almDI/almDII, (encoding α/ß subunits of pyruvate dehydrogenase) triggers the biosynthesis of aldgamycins, whereas almCI (encoding an oxidoreductase) initiates chalcomycins biosynthesis. This is the first report that aldgamycins and chalcomycins are derived from a single gene cluster and of the genetic basis for bifurcation in their biosynthesis.

Identification of the Fluvirucin B2 (Sch 38518) Biosynthetic Gene Cluster from Actinomadura fulva subsp. indica ATCC 53714: substrate Specificity of the ß-Amino Acid Selective Adenylating Enzyme FlvN.

Fluvirucins are 14-membered macrolactam polyketides that show antifungal and antivirus activities. Fluvirucins have the ß-alanine starter unit at their polyketide skeletons. To understand the construction mechanism of the ß-alanine moiety in fluvirucin biosyntheses, we have identified the biosynthetic cluster of fluvirucin B2 produced from Actinomadura fulva subsp. indica ATCC 53714. The identified gene cluster contains three polyketide synthases, four characteristic ß-amino acid-carrying enzymes, one decarboxylase, and one amidohydrolase. We next investigated the activity of the adenylation enzyme FlvN, which is a key enzyme for the selective incorporation of a ß-amino acid substrate. FlvN showed strong preference for l-aspartate over other amino acids such as ß-alanine. Based on these results, we propose a biosynthetic pathway for fluvirucin B2.

Novel process for producing 6-deoxy monosaccharides from l-fucose by coupling and sequential enzymatic method.

We biosynthesized 6-deoxy-L-talose, 6-deoxy-L-sorbose, 6-deoxy-L-gulose, and 6-deoxy-L-idose, which rarely exist in nature, from L-fucose by coupling and sequential enzymatic reactions. The first product, 6-deoxy-L-talose, was directly produced from L-fucose by the coupling reactions of immobilized D-arabinose isomerase and immobilized L-rhamnose isomerase. In one-pot reactions, the equilibrium ratio of L-fucose, L-fuculose, and 6-deoxy-L-talose was 80:9:11. In contrast, 6-deoxy-L-sorbose, 6-deoxy-L-gulose, and 6-deoxy-L-idose were produced from L-fucose by sequential enzymatic reactions. D-Arabinose isomerase converted L-fucose into L-fuculose with a ratio of 88:12. Purified L-fuculose was further epimerized into 6-deoxy-L-sorbose by D-allulose 3-epimerase with a ratio of 40:60. Finally, purified 6-deoxy-L-sorbose was isomerized into both 6-deoxy-L-gulose with an equilibrium ratio of 40:60 by L-ribose isomerase, and 6-deoxy-L-idose with an equilibrium ratio of 73:27 by D-glucose isomerase. Based on the amount of L-fucose used, the production yields of 6-deoxy-L-talose, 6-deoxy-L-sorbose, 6-deoxy-L-gulose, and 6-deoxy-L-idose were 7.1%, 14%, 2%, and 2.4%, respectively.

Optimal production of 4-deoxy-L-erythro-5-hexoseulose uronic acid from alginate for brown macro algae saccharification by combining endo- and exo-type alginate lyases.

Algae are considered as third-generation biomass, and alginate is the main component of brown macroalgae. Alginate can be enzymatically depolymerized by alginate lyases into uronate monomers, such as mannuronic acid and guluronic acid, which are further nonenzymatically converted to 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). We have optimized an enzymatic saccharification process using two recombinant alginate lyases, endo-type Alg7D and exo-type Alg17C, for the efficient production of DEH from alginate. When comparing the sequential and simultaneous additions of Alg7D and Alg17C, it was found that the final yield of DEH was significantly higher when the enzymes were added sequentially. The progress of saccharification reactions and production of DEH were verified by thin layer chromatography and gas chromatography-mass spectrometry, respectively. Our results showed that the two recombinant enzymes could be exploited for the efficient production of DEH that is the key substrate for producing biofuels from brown macro algal biomass.

Production of a novel N-monomethylated dideoxysugar.

The importance of unusual deoxysugars in biology has become increasingly apparent over the past decade. Some, for example, play key roles in the physiological activities of the natural products to which they are attached. Here we describe a study of TylM1, a dimethyltransferase from Streptomyces fradiae involved in the production of dTDP-mycaminose. From this investigation, the manner in which the enzyme binds its dimethylated product has been revealed. More significantly, by providing the enzyme with an alternative substrate, it was possible to produce a monomethylated product not observed in nature. This has important ramifications for the production of unique carbohydrates that may prove useful in drug design.

C-glycosylation of anhydrotetracycline scaffold with SsfS6 from the SF2575 biosynthetic pathway.

Glycosylation with deoxysugar is a common strategy used by nature to introduce structural diversity and biological activities among natural products. In this study, we biochemically confirmed the activities of SsfS6, a C-glycosyltransferase in the SF2575 biosynthetic pathway, as a regioselective D-olivose transferase that acts on the C-9 position of an anhydrotetracycline aglycon. To perform the glycosyl transfer reaction using Escherichia coli as a whole-cell biocatalyst, we reconstituted the biosynthesis of TDP-D-olivose using a heterologous pathway. Under in vivo conditions, SsfS6 transferred multiple endogenous sugar substrates, in addition to D-olivose, to the anhydrotetracycline substrate, demonstrating broad substrate tolerance and potential as a tetracycline-diversifying enzyme.

Structural and biochemical characterization of a bifunctional ketoisomerase/N-acetyltransferase from Shewanella denitrificans.

Unusual N-acetylated sugars have been observed on the O-antigens of some Gram-negative bacteria and on the S-layers of both Gram-positive and Gram-negative bacteria. One such sugar is 3-acetamido-3,6-dideoxy-α-d-galactose or Fuc3NAc. The pathway for its production requires five enzymes with the first step involving the attachment of dTMP to glucose-1-phosphate. Here, we report a structural and biochemical characterization of a bifunctional enzyme from Shewanella denitificans thought to be involved in the biosynthesis of dTDP-Fuc3NAc. On the basis of a bioinformatics analysis, the enzyme, hereafter referred to as FdtD, has been postulated to catalyze the third and fifth steps in the pathway, namely, a 3,4-keto isomerization and an N-acetyltransferase reaction. For the X-ray analysis reported here, the enzyme was crystallized in the presence of dTDP and CoA. The crystal structure shows that FdtD adopts a hexameric quaternary structure with 322 symmetry. Each subunit of the hexamer folds into two distinct domains connected by a flexible loop. The N-terminal domain adopts a left-handed ß-helix motif and is responsible for the N-acetylation reaction. The C-terminal domain folds into an antiparallel flattened ß-barrel that harbors the active site responsible for the isomerization reaction. Biochemical assays verify the two proposed catalytic activities of the enzyme and reveal that the 3,4-keto isomerization event leads to the inversion of configuration about the hexose C-4' carbon.

Cooperation of two bifunctional enzymes in the biosynthesis and attachment of deoxysugars of the antitumor antibiotic mithramycin.

Two bifunctional enzymes cooperate in the assembly and the positioning of two sugars, D-olivose and D-mycarose, of the anticancer antibiotic mithramycin. MtmC finishes the biosynthesis of both sugar building blocks depending on which MtmGIV activity is supported. MtmGIV transfers these two sugars onto two structurally distinct acceptor substrates. The dual function of these enzymes explains two essential but previously unidentified activities.

Characterization of the lipopolysaccharide from Pasteurella multocida Heddleston serovar 9: identification of a proposed bi-functional dTDP-3-acetamido-3,6-dideoxy-α-D-glucose biosynthesis enzyme.

Pasteurella multocida strains are classified into 16 different lipopolysaccharide (LPS) serovars using the Heddleston serotyping scheme. Ongoing studies in our laboratories on the LPS aim to determine the core oligosaccharide (OS) structures expressed by each of the Heddleston type strains and identify the genes and transferases required for the biosynthesis of the serovar-specific OSs. In this study, we have determined the core OS of the LPS expressed by the Heddleston serovar 9 type strain, P2095. Structural information was established by a combination of monosaccharide and methylation analyses, nuclear magnetic resonance spectroscopy and mass spectrometry revealing the following structure: . The serovar 9 OS contains an inner core that is conserved among P. multocida strains with an elaborate outer core extension containing rhamnose (Rha), a D-glycero-D-manno isomer of heptose, and the unusual deoxyamino sugar, 3-acetamido-3,6-dideoxy-α-D-glucose (Qui3NAc). Genetic analyses of the LPS outer core biosynthesis locus revealed that in addition to the glycosyltransferases predicted to transfer the sugars to the nascent LPS molecule, the locus also contained the complete set of genes required for the biosynthesis of the nucleotide sugar donors dTDP-Rha and dTDP-Qui3NAc. One of the genes identified as part of the dTDP-Qui3NAc biosynthesis pathway, qdtD, encodes a proposed bi-functional enzyme with N-terminal amino acid identity to dTDP-4-oxo-6-deoxy-D-glucose-3,4-oxoisomerase and C-terminal amino acid identity to dTDP-3-oxo-6-deoxy-α-D-glucose transacetylase.

Characterization of the GDP-D-mannose biosynthesis pathway in Coxiella burnetii: the initial steps for GDP-ß-D-virenose biosynthesis.

Coxiella burnetii, the etiologic agent of human Q fever, is a gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars ß-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-ß-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames (CBU0671, CBU0294 and CBU0689) annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is critical to fully understand the biosynthesic pathway of GDP-ß-D-virenose and LPS structure in C. burnetii.

Investigating Mithramycin deoxysugar biosynthesis: enzymatic total synthesis of TDP-D-olivose.

Mix'n'match: Enzymatic total synthesis of TDP-D-olivose was achieved, starting from TDP-4-keto-6-deoxy-D-glucose, by combining three pathway enzymes with one cofactor-regenerating enzyme. The results also revealed that MtmC is a bifunctional enzyme that can perform a 4-ketoreduction necessary for D-olivose biosynthesis besides the previously found C-methyltransfer for D-mycarose biosynthesis.

Mechanisms and structures of vitamin B(6)-dependent enzymes involved in deoxy sugar biosynthesis.

PLP is well-regarded for its role as a coenzyme in a number of diverse enzymatic reactions. Transamination, deoxygenation, and aldol reactions mediated by PLP-dependent enzymes enliven and enrich deoxy sugar biosynthesis, endowing these compounds with unique structures and contributing to their roles as determinants of biological activity in many natural products. The importance of deoxy aminosugars in natural product biosynthesis has spurred several recent structural investigations of sugar aminotransferases. The structure of a PMP-dependent enzyme catalyzing the C-3 deoxygenation reaction in the biosynthesis of ascarylose was also determined. These studies, and the crystal structures they have provided, offer a wealth of new insights regarding the enzymology of PLP/PMP-dependent enzymes in deoxy sugar biosynthesis. In this review, we consider these recent achievements in the structural biology of deoxy sugar biosynthetic enzymes and the important implications they hold for understanding enzyme catalysis and natural product biosynthesis in general. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

Biochemical analysis of the biosynthetic pathway of an anticancer tetracycline SF2575.

SF2575 1 is a tetracycline polyketide produced by Streptomyces sp. SF2575 and displays exceptionally potent anticancer activity toward a broad range of cancer cell lines. The structure of SF2575 is characterized by a highly substituted tetracycline aglycon. The modifications include methylation of the C-6 and C-12a hydroxyl groups, acylation of the 4-(S)-hydroxyl with salicylic acid, C-glycosylation of the C-9 of the D-ring with D-olivose and further acylation of the C4'-hydroxyl of D-olivose with the unusual angelic acid. Understanding the biosynthesis of SF2575 can therefore expand the repertoire of enzymes that can modify tetracyclines, and facilitate engineered biosynthesis of SF2575 analogues. In this study, we identified, sequenced, and functionally analyzed the ssf biosynthetic gene cluster which contains 40 putative open reading frames. Genes encoding enzymes that can assemble the tetracycline aglycon, as well as installing these unique structural features, are found in the gene cluster. Biosynthetic intermediates were isolated from the SF2575 culture extract to suggest the order of pendant-group addition is C-9 glycosylation, C-4 salicylation, and O-4' angelylcylation. Using in vitro assays, two enzymes that are responsible for C-4 acylation of salicylic acid were identified. These enzymes include an ATP-dependent salicylyl-CoA ligase SsfL1 and a putative GDSL family acyltransferase SsfX3, both of which were shown to have relaxed substrate specificity toward substituted benzoic acids. Since the salicylic acid moiety is critically important for the anticancer properties of SF2575, verification of the activities of SsfL1 and SsfX3 sets the stage for biosynthetic modification of the C-4 group toward structure-activity relationship studies of SF2575. Using heterologous biosynthesis in Streptomyces lividans, we also determined that biosynthesis of the SF2575 tetracycline aglycon 8 parallels that of oxytetracycline 4 and diverges after the assembly of 4-keto-anhydrotetracycline 51. The minimal ssf polyketide synthase together with the amidotransferase SsfD produced the amidated decaketide backbone that is required for the formation of 2-naphthacenecarboxamide skeleton. Additional enzymes, such as cyclases C-6 methyltransferase and C-4/C-12a dihydroxylase, were functionally reconstituted.

Enzymatic synthesis of TDP-deoxysugars.

Many biologically active bacterial natural products contain highly modified deoxysugar residues that are often critical for the activity of the parent compounds. Most of these deoxysugars are secondary metabolites that are biosynthesized in the form of nucleotide diphosphate (NDP) sugars prior to their transfer to natural product aglycones by glycosyltransferases. Over the past decade, many biosynthetic pathways that lead to the formation of these unusual sugars have been unraveled, and the mechanisms of many key enzymatic transformations involved in these pathways have been elucidated. However, obtaining workable quantities of NDP-deoxysugars for in vitro studies is often a difficult task. This limitation has hindered an in-depth investigation of the substrate specificity of deoxysugar biosynthetic enzymes, many of which are promiscuous with respect to their NDP-sugar substrates and are, thus, potentially useful catalysts for natural product glycoengineering. Presented in this review are procedures for the enzymatic synthesis and purification of a variety of NDP-deoxysugars, including some early intermediates in NDP-deoxysugar biosynthetic pathways, and highly modified NDP-deoxysugars that are late intermediates in their respective biosynthetic pathways. The procedures described herein could be used as general guidelines for the development of specific protocols for the synthesis of other NDP-deoxysugars.

Glycosylation engineering of spinosyn analogues containing an L-olivose moiety.

Biosynthetic genes encoding proteins involved in the first steps of deoxyhexose biosynthesis from D-glucose-1-phosphate were expressed in Saccharopolyspora erythraea. The resulting mutant was able to accumulate and utilise TDP-L-olivose. Co-expression of the spinosyn glycosyl transferase SpnP in the resulting mutant endowed upon it the ability to biotransform exogenously added spinosyn aglycones to yield novel spinosyn analogues.

Chapter 11. Sugar biosynthesis and modification.

Many bioactive compounds contain as part of their molecules one or more deoxysugar units. Their presence in the final compound is generally necessary for biological activity. These sugars derive from common monosaccharides, like d-glucose, which have lost one or more hydroxyl groups (monodeoxysugars, dideoxysugars, trideoxysugars) during their biosynthesis. These deoxysugars are transferred to the final molecule by the action of a glycosyltransferase. Here, we first summarize the different biosynthetic steps required for the generation of the different families of deoxysugars, including those containing extra methyl or amino groups, or tailoring modifications of the glycosylated compounds. We then give examples of several strategies for modification of the glycosylation pattern of a given bioactive compound: inactivation of genes involved in the biosynthesis of deoxysugars; heterologous expression of genes for the biosynthesis or transfer of a specific deoxysugar; and combinatorial biosynthesis (including the use of gene cassette plasmids). Finally, we report techniques for the isolation and detection of the new glycosylated derivatives generated using these strategies.