Harnessing the chemical activation inherent to carrier protein-bound thioesters for the characterization of lipopeptide fatty acid tailoring enzymes

Here, we report a new experimental approach utilizing an amide ligation reaction for the characterization of acyl carrier protein (ACP)-bound reaction intermediates, which are otherwise difficult to analyze by traditional biochemical methods. To explore fatty acid tailoring enzymes of the calcium-dependent antibiotic (CDA) biosynthetic pathway, this strategy enabled the transformation of modified fatty acids, covalently bound as thioesters to an ACP, into amide ligation products that can be directly analyzed and compared to synthetic standards by HPLC-MS. The driving force of the amide formation is the thermodynamic activation inherent to thioester-bound compounds. Using this novel method, we were able to characterize the ACP-mediated biosynthesis of the unique 2,3-epoxyhexanoyl moiety of CDA, revealing a new type of FAD-dependent oxidase HxcO with intrinsic enoyl-ACP epoxidase activity, as well as a second enoyl-ACP epoxidase, HcmO. In general, our approach should be widely applicable for the in vitro characterization of other biosynthetic systems acting on carrier proteins, such as integrated enzymes from NRPS and PKS assembly lines or tailoring enzymes of fatty and amino acid precursor synthesis.     

Heterologous Co-expression of accA, fabD, and Thioesterase Genes for Improving Long-Chain Fatty Acid Production in Pseudomonas aeruginosa and Escherichia coli

The goal of the present study was to increase the content of intracellular long-chain fatty acids in two bacterial strains, Pseudomonas aeruginosa PA14 and Escherichia coli K-12 MG1655, by co-overexpressing essential enzymes that are involved in the fatty acid synthesis metabolic pathway. Recently, microbial fatty acids and their derivatives have been receiving increasing attention as an alternative source of fuel. By introducing two genes (accA and fabD) of P. aeruginosa into the two bacterial strains and by co-expressing with them the fatty acyl?Cacyl carrier protein thioesterase gene of Streptococcus pyogenes (strain MGAS10270), we have engineered recombinant strains that are efficient producers of long-chain fatty acids (C16 and C18). The recombinant strains exhibit a 1.3?C1.7-fold increase in the production of long-chain fatty acids over the wild-type strains. To enhance the production of total long-chain fatty acids, we researched the carbon sources for optimized culture conditions and results were used for post-culture incubation period. E. coli SGJS17 (containing the accA, fabD, and thioesterase genes) produced the highest content of intracellular total fatty acids; in particular, the unsaturated fatty acid content was about 20-fold higher than that in the wild-type E. coli.     

Isolation, enzyme-bound structure, and activity of platensimycin A1 from Streptomyces platensis

Inhibition of fatty acid synthesis is emerging as a valuable target for antibacterial agents. Platensimycin and platencin are novel natural products that were reported recently to inhibit the FabF and FabF/FabH condensing enzymes, respectively, present in the fatty acid biosynthetic pathway. Selective inhibition of these enzymes by platensimycin and platencin accounts for their potent antibiotic activity. We have continued our quest to find additional members of this class of compounds leading to discovery of platensimycin A₁, a hydroxylated congener. We report herein the isolation, structure, antibacterial and enzymatic activities, and co-crystal structure bound to Escherichia coli FabF. The lower activity of platensimycin A₁ suggests that substitution at C-14 is detrimental for the activity.     

Structure,biosynthetic origin,and engineered biosynthesis of calcium-dependent antibiotics from Streptomyces coelicolor

The calcium-dependent antibiotic (CDA), from Streptomyces coelicolor, is an acidic lipopeptide comprising an N-terminal 2,3-epoxyhexanoyl fatty acid side chain and several nonproteinogenic amino acid residues. S. coelicolor grown on solid media was shown to produce several previously uncharacterized peptides with C-terminal Z-dehydrotryptophan residues. The CDA biosynthetic gene cluster contains open reading frames encoding nonribosomal peptide synthetases, fatty acid synthases, and enzymes involved in precursor supply and tailoring of the nascent peptide. On the basis of protein sequence similarity and chemical reasoning, the biosynthesis of CDA is rationalized. Deletion of SCO3229 (hmaS), a putative 4-hydroxymandelic acid synthase-encoding gene, abolishes CDA production. The exogenous supply of 4-hydroxymandelate, 4-hydroxyphenylglyoxylate, or 4-hydroxyphenylglycine re-establishes CDA production by the DeltahmaS mutant. Feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues.     

Rational pathway engineering of type I fatty acid synthase allows the biosynthesis of triacetic acid lactone from D-glucose in vivo

Metabolic pathway engineering is a powerful tool to synthesize structurally diverse and complex chemicals via genetic manipulation of multistep catalytic systems involved in cell metabolism. Here, we report the rational design of a fatty acid biosynthetic pathway, Brevibacterium ammoniagenes fatty acid synthase B (FAS-B), that allows the microbial synthesis of triacetic acid lactone (TAL) from an inexpensive feedstock, d-glucose. TAL can be chemically converted to phloroglucinol, which is a core structure for the synthesis of various high value bioactive compounds and energetic compounds such as 1,3,5-triamino-2,4,6-trinitrobenzene (TATB). Synthesis of phloroglucinol from d-glucose using this combined biological and chemical synthesis may offer significant advantages over the current phloroglucinol manufacture, including environmental friendliness and reduction in the cost of phloroglucinol. More importantly, it represents a novel strategy for the benzene-free synthesis of aromatic chemicals.     

Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes

HSAF was isolated from Lysobacter enzymogenes , a bacterium used in the biological control of fungal diseases of plants. Structurally, it is a tetramic acid-containing macrolactam fused to a tricyclic system. HSAF exhibits a novel mode of action by disrupting sphingolipids important to the polarized growth of filamentous fungi. Here we describe the HSAF biosynthetic gene cluster, which contains only a single-module polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS), although the biosynthesis of HSAF apparently requires two separate polyketide chains that are linked together by one amino acid (ornithine) via two amide bonds. Flanking the PKS/NRPS are six genes that encoding a cascade of four tightly clustered redox enzymes on one side and a sterol desaturase/fatty acid hydroxylase and a ferredoxin reductase on the other side. The genetic data demonstrate that the four redox genes, in addition to the PKS/NRPS gene and the sterol desaturase/fatty acid hydroxylase gene, are required for HSAF production. The biochemical data show that the adenylation domain of the NRPS specifically activates L-ornithine and that the four-domain NRPS is able to catalyze the formation of a tetramic acid-containing product from acyl-S-ACP and ornithinyl-S-NRPS. These results reveal a previously unrecognized biosynthetic mechanism for hybrid PK/NRP in prokaryotic organisms.     

Effect of Salicylic Acid on the Activity of PAL and PHB Geranyltransferase and Shikonin Derivatives Production in Cell Suspension Cultures of Arnebia euchroma (Royle) Johnst—a Medicinally Important Plant Species

Cell suspension cultures of Arnebia euchroma were established from the friable callus on liquid Murashige and Skoog medium supplemented with 6-benzylaminopurine (10.0 μM) and indole-3-butyric acid (5.0 μM). Salicylic acid was used to study its effect on the enzymes which participate in shikonin biosynthesis with respect to metabolite (shikonin) content in the cell suspension culture of A. euchroma. In our study, phenylalanine ammonia lyase and PHB geranyltransferase were selected from the entire biosynthetic pathway. Results showed that phenylalanine ammonia lyase is responsible for growth and PHB geranyltransferase for metabolite production. Salicylic acid exhibited an inverse relationship with the metabolite content (shikonin); salicylic acid (100 μM) completely inhibited shikonin biosynthesis. The results presented in the current study can be successfully employed for the metabolic engineering of its biosynthetic pathway for the enhancement of shikonin, which will not only help in meeting its industrial demand but also lead to the conservation of species in its natural habitat.     

Long‐Chain Alkyl Cyanides: Unprecedented Volatile Compounds Released by Pseudomonas and Micromonospora Bacteria

The analysis of volatiles from bacterial cultures revealed long‐chain aliphatic nitriles, a new class of natural products. Such nitriles are produced by both Gram‐positive Micromonospora echinospora and Gram‐negative Pseudomonas veronii bacteria, although the structures differ. A variable sequence of chain elongation and dehydration in the fatty acid biosynthesis leads to either unbranched saturated or unsaturated nitriles with an ω−7 double bond, such as (Z )‐11‐octadecenenitrile, or methyl‐branched unsaturated nitriles with the double bond located at C‐3, such as (Z )‐13‐methyltetradec‐3‐enenitrile. The nitrile biosynthesis starts from fatty acids, which are converted into their amides and finally dehydrated. The structures and biosyntheses of the 19 naturally occurring compounds were elucidated by mass spectrometry, synthesis, and feeding experiments with deuterium‐labeled precursors. Some of the nitriles showed antimicrobial activity, for example, against multiresistant Staphylococcus aureus strains.     

Biosynthesis of L-p-hydroxyphenylglycine, a non-proteinogenic amino acid constituent of peptide antibiotics

BACKGROUND: The non-proteinogenic amino acid p-hydroxyphenylglycine is a crucial component of certain peptidic natural products synthesized by a non-ribosomal peptide synthetase mechanism. In particular, for the vancomycin group of antibiotics p-hydroxyphenylglycine plays a structural role in formation of the rigid conformation of the central heptapeptide aglycone in addition to being the site of glycosylation. Initial labeling studies suggested tyrosine was a precursor of p-hydroxyphenylglycine but the specific steps in p-hydroxyphenylglycine biosynthesis remained unknown. Recently, the sequencing of the chloroeremomycin gene cluster from Amycolatopsis orientalis gave new insights into the biosynthetic pathway and allowed for the prediction of a four enzyme pathway leading to L-p-hydroxyphenylglycine from the common metabolite prephenate. RESULTS: We have characterized three of the four proposed enzymes of the L-p-hydroxyphenylglycine biosynthetic pathway. The three enzymes are encoded by open reading frames (ORFs) 21, 22 and 17 (ORF21: [PCZA361.1, O52791, CAA11761]; ORF22: [PCZA361. 2, O52792, CAA11762]; ORF17: [PCZA361.25, O52815, CAA11790]), of the chloroeremomycin biosynthetic gene cluster and we show they have p-hydroxymandelate synthase, p-hydroxymandelate oxidase and L-p-hydroxyphenylglycine transaminase activities, respectively. CONCLUSIONS: The L-p-hydroxyphenylglycine biosynthetic pathway shown here is proposed to be the paradigm for how this non-proteinogenic amino acid is synthesized by microorganisms incorporating it into peptidic natural products. This conclusion is supported by the finding of homologs for the four L-p-hydroxyphenylpyruvate biosynthetic enzymes in four organisms known to synthesize peptidic natural products that contain p-hydroxyphenylglycine. Three of the enzymes are proposed to function in a cyclic manner in vivo with L-tyrosine being both the amino donor for L-p-hydroxyphenylglycine and a source of p-hydroxyphenylpyruvate, an intermediate in the biosynthetic pathway.     

Structure of homoplatensimide A: a potential key biosynthetic intermediate of platensimycin isolated from Streptomyces platensis

Platensimycin and platencin are novel natural product antibiotics that inhibit bacterial growth by inhibiting fatty acid biosynthesis enzymes FabF and FabF/FabH, respectively. Continued search for the natural congeners for structure activity relationship studies led to the isolation of a congener which possesses all of the twenty carbons of diterpenoid unit, a potential biosynthetic intermediate of platensic acid unit of platensimycin. Isolation, structure, and activity of homoplatensimide A and biosynthetic relationship to platensimycin have been described.     

Isolation, structure, and absolute stereochemistry of platensimycin, a broad spectrum antibiotic discovered using an antisense differential sensitivity strategy

Fatty acids are essential for survival of bacteria and are synthesized by a series of enzymes including the elongation enzymes, beta-ketoacyl acyl carrier protein synthase I/II (FabF/B). Inhibition of fatty acid synthesis is one of the new targets for the discovery and development of antibacterial agents. Platensimycin (1a) is a novel broad spectrum Gram-positive antibiotic produced by Streptomyces platensis. It was discovered by target-based whole-cell screening strategy using antisense differential sensitivity assay. It inhibits bacterial growth by selectively inhibiting condensing enzyme FabF of the fatty acid synthesis pathway and was isolated by a two-step process, a capture step followed by reversed-phase HPLC. The structure was elucidated by 2D NMR methods and confirmed by X-ray crystallographic analysis of a bromo derivative. It was determined that potential reactivity of the enone moiety does not play a key role in the biological activity of platensimycin. However, cyclohexenone ring conformation renders for the stronger binding interaction with the enzyme. The isolation, structure elucidation, derivatization, and biological activity of 6,7-dihydroplatensimycin are described.     

Elucidation of the Streptomyces coelicolor pathway to 2-undecylpyrrole, a key intermediate in undecylprodiginine and streptorubin B biosynthesis

The red gene cluster of Streptomyces coelicolor directs production of undecylprodiginine. Here we report that this gene cluster also directs production of streptorubin B and show that 2-undecylpyrrole (UP) is an intermediate in the biosynthesis of undecylprodiginine and streptorubin B. The redPQRKL genes are involved in UP biosynthesis. RedL and RedK are proposed to generate UP from dodecanoic acid or a derivative. A redK(-) mutant produces a hydroxylated undecylprodiginine derivative, whereas redL(-) and redK(-) mutants require addition of chemically synthesized UP for production of undecylprodiginine and streptorubin B. Fatty acid biosynthetic enzymes can provide dodecanoic acid, but efficient and selective prodiginine biosynthesis requires RedPQR. Deletion of redP, redQ, or redR leads to an 80%-95% decrease in production of undecylprodiginine and an array of prodiginine analogs with varying alkyl chains. In a redR(-) mutant, the ratio of these can be altered in a logical manner by feeding various fatty acids.     

Frontiers and opportunities in chemoenzymatic synthesis

Natural product biosynthetic pathways have evolved enzymes with myriad activities that represent an expansive array of chemical transformations for constructing secondary metabolites. Recently, harnessing the biosynthetic potential of these enzymes through chemoenzymatic synthesis has provided a powerful tool that often rivals the most sophisticated methodologies in modern synthetic chemistry and provides new opportunities for accessing chemical diversity. Herein, we describe our research efforts with enzymes from a broad collection of biosynthetic systems, highlighting recent progress in this exciting field.     

Bacterial beta-ketoacyl-acyl carrier protein synthases as targets for antibacterial agents

As a result of increasing drug resistance in pathogenic bacteria, there is a critical need for novel broad-spectrum antibacterial agents. As fatty acid synthesis (FAS) in bacteria is an essential process for cell survival, the enzymes involved in the FAS pathway have emerged as promising targets for antimicrobial agents. Several lines of evidence have indicated that bacterial condensing enzymes are central to the initiation and elongation steps in bacterial fatty acid synthesis and play a pivotal role in the regulation of the entire fatty acid synthesis pathway. beta-ketoacyl-acyl carrier protein (ACP) synthases (KAS) from various bacterial species have been cloned, expressed and purified in large quantities for detailed enzymological, structural and screening studies. Availability of purified KAS from a variety of bacteria, along with a combination of techniques, including combinatorial chemistry, high-throughput screening, and rational drug design based on crystal structures, will undoubtedly aid in the discovery and development of much needed potent and broad-spectrum antibacterial agents. In this review we summarize the biochemical, biophysical and inhibition properties of beta-ketoacyl-ACP synthases from a variety of bacterial species.     

Construction and evaluation of nagR-nagAa::lux fusion strains in biosensing for salicylic acid derivatives

The NagR protein is a response regulatory protein found in the bacterium Ralstonia sp. U2 that is involved in sensing for salicylic acid and the subsequent induction of the operon just upstream of its gene. The genes encoded for in this operon are involved in the degradation of salicylic acid. Escherichia coli strain RFM443 carrying a fusion of the Photorhabdus luminescens luxCDABE operon with the nagR gene and upstream region of the nagAa gene was constructed and characterized with respect to its optimum temperature, its response time and kinetics, and its ability to detect numerous benzoic acid derivatives. Although capable of detecting 0.5 mM salicylic acid at any temperature between 28 and 40 degrees C, this E. coli strain, labeled DNT5, showed its greatest relative activity at 30 degrees C, i.e., the temperature at which the largest induction was seen. Furthermore, experiments done with numerous benzoic acid derivatives found the NagR protein to be responsive to only a few of the compounds tested, including salicylic acid and 3-methyl salicylic acid, and acetyl salicylic acid was the strongest inducer. The lower limits of detection for these compounds with E. coli strain DNT5 were also established, with the native inducer, salicylic acid, giving the most sensitive response and detectable down to a concentration of about 2 microM. A second lux fusion plasmid was also constructed and transformed into an NahR background, Pseudomonas putida KCTC1768. Within this strain, NAGK-1768, the supplemental activity of the NahR protein on the nagAa promoter, was shown to extend both the range of chemicals detected and the sensitivity.     

Biosynthesis of violacein: a genuine intermediate, protoviolaceinic acid, produced by VioABDE, and insight into VioC function

A biosynthetic intermediate of violacein produced by the mixed enzymes of VioABDE was elucidated to be 5-(5-hydroxy-1H-indol-3-yl)-3-(1H-indol-3-yl)-1H-pyrrole-2-carboxylic acid, named protoviolaceinic acid, indicating that VioC, responsible for the final biosynthetic step, works to oxygenate at the 2-position of the right side indole ring, and that the oxygenation reaction to form the central pyrrolidone core proceeds in a non-enzymatic fashion.     

Engineering of an active animal fatty acid synthase dimer with only one competent subunit

Animal fatty acid synthases are large polypeptides containing seven functional domains that are active only in the dimeric form. Inactivity of the monomeric form has long been attributed to the obligatory participation of domains from both subunits in catalysis of substrate loading and condensation reactions. However, we have engineered a fatty acid synthase containing one wild-type subunit and one subunit compromised by mutations in all seven functional domains that is active in fatty acid synthesis. This finding indicates that a single subunit, in the context of a dimer, is able to catalyze the entire biosynthetic pathway and suggests that, in the natural complex, each of the two subunits forms a scaffold that optimizes the conformation of the companion subunit.     

Microbial synthesis of the energetic material precursor 1,2,4-butanetriol

The lack of a route to precursor 1,2,4-butanetriol that is amenable to large-scale synthesis has impeded substitution of 1,2,4-butanetriol trinitrate for nitroglycerin. To identify an alternative to the current commercial synthesis of racemic d,l-1,2,4-butanetriol involving NaBH4 reduction of esterified d,l-malic acid, microbial syntheses of d- and l-1,2,4-butanetriol have been established. These microbial syntheses rely on the creation of biosynthetic pathways that do not exist in nature. Oxidation of d-xylose by Pseudomonas fragi provides d-xylonic acid in 70% yield. Escherichia coli DH5alpha/pWN6.186A then catalyzes the conversion of d-xylonic acid into d-1,2,4-butanetriol in 25% yield. P. fragi is also used to oxidize l-arabinose to a mixture of l-arabino-1,4-lactone and l-arabinonic acid in 54% overall yield. After hydrolysis of the lactone, l-arabinonic acid is converted to l-1,2,4-butanetriol in 35% yield using E. coli BL21(DE3)/pWN6.222A. As a catalytic route to 1,2,4-butanetriol, microbial synthesis avoids the high H2 pressures and elevated temperatures required by catalytic hydrogenation of malic acid.     

基于子空间夹角判据监测阿司匹林合成体系中的水杨酸和阿司匹林

利用水杨酸、阿司匹林、乙酸酐和乙酸的多波长紫外光谱作为本底光谱库和样本光谱库,建立一种基于子空间夹角判据的快速定量分析阿司匹林合成体系中水杨酸和阿司匹林的方法。水杨酸、阿司匹林质量浓度范围分别在1.16～34.684,9.84～188.928μg/mL内,所测得水杨酸和阿司匹林检验集的R分别为0.999 7和0.999 1,RMSE分别为0.509 5,2.624 0μg/mL,效果优于普通波峰点最小二乘法和偏最小二乘法。水杨酸和阿司匹林的加标回收率在94.01%～106.44%之间,测定结果的相对标准偏差分别为3.45%和4.80%(n=7)。该方法可用于监测阿司匹林合成体系中的水杨酸和阿司匹林。     

An Unprecedented Cyclization Mechanism in the Biosynthesis of Carbazole Alkaloids in Streptomyces

Carquinostatin A (CQS), a potent neuroprotective substance, is a unique carbazole alkaloid with both an ortho‐quinone function and an isoprenoid moiety. We identified the entire gene cluster responsible for CQS biosynthesis in Streptomyces exfoliatus through heterologous production of CQS and gene deletion. Biochemical characterization of seven CQS biosynthetic gene products (CqsB1–7) established the total biosynthetic pathway of CQS. Reconstitution of CqsB1 and CqsB2 showed that the synthesis of the carbazole skeleton involves CqsB1‐catalyzed decarboxylative condensation of an α‐hydroxyl‐β‐keto acid intermediate with 3‐hydroxybutyryl‐ACP followed by CqsB2‐catalyzed oxidative cyclization. Based on crystal structures and mutagenesis‐based biochemical assays, a detailed mechanism for the unique deprotonation‐initiated cyclization catalyzed by CqsB2 is proposed. Finally, analysis of the substrate specificity of the biosynthetic enzymes led to the production of novel carbazoles.